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ABSTRACT

EFFECTS OF CENTRAL ACTING DRUGS AND ER GOT DERIVATIVES
ON PROLACTIN AND GROWTH HORMONE SECRETION,
ON GROWTH OF PITUITARY AND MAMMARY TUMORS
AND ON REPRODUCTION IN OLD RATS
by

Syed Kaleemullah Quadri

1. A Single intraperitoneal injection of the ergot derivatives,
ergocryptine, or LSD (lysergic acid diethylamide), significantly
decreased serum prolactin levels in female rats and blocked the
rise in prolactin that normally occurs in control rats on the afternoon
of proestrus. A single dose of ergocryptine remained effective for
at least 24 hours after it was administered. Ergocryptine was not
able to block the increase in prolactin secretion induced by perphen-
azine, which exerts its effects on the hypothalamus. The primary
action of ergocryptine appears to be directly on the pituitary gland.

2. A Single intraperitoneal injection of l, 8 or 12 mg pyro-
gallol per 100 g body weight caused a significant reduction in serum
prolaCtin levels in female rats. Pyrogallol inhibits degradation of
catecholamines, and an increase in catecholamines results in

r educed prolactin s ecretion.

3. A single intraperitoneal injection of sodium pentobarbital

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produced an initial rise followed by a fall in plasma growth hormone
(GI-I). Ether anesthesia consistently reduced plasma CH levels.
Contrary to their inhibitory effects on CH secretion in rats, stresses
of various kinds stimulate CH release in man and other Species.

4. Daily injections of epinephrine, 1—dOpa or iproniazid for
25 days into 20 to 23 month old constant estrous Sprague-Dawley
rats initiated regular estrous cycles in most rats. On termination
of treatment, a few rats continued to cycle but the majority returned
to a state of constant estrous or went into a prolonged diestrus. It
was concluded that all three drugs restored regular cycling by cor-
recting a catecholamine deficiency in the hypothalamus, thereby
increasing LH and FSH and decreasing release of prolactin.

5. Inbred female Wistar-Furth rats were transplanted with
the mammosomatotropic pituitary tumor, MtT. W15. About 6 weeks
after transplantation, weekly measurements were made of plasma
CH, serum prolactin, tumor size and body weights. A highly
significant and positive correlation was found between tumor size
and prolactin and GH levels, and between tumor size and body weight.
Age of tumor was not always related to the secretory activity or
size of the tumor.

6. ~Daily treatment of rats carrying pituitary tumor trans-
plants with ergocornine produced regression of tumor growth,

degeneration of tumor tissue and suppression of prolactin but not GH

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secretion. Ergocornine is believed to inhibit prolactin secretion by
a direct action on the pituitary tumor. Ergonovine also inhibited
tumor growth but ergocryptine and a thalidomide-related compound,

CG 603, both of which inhibit prolactin release by the normal 3.1. situ

 

pituitary, had no effect on the size or secretory activity of the tumor,

7. Ergocornine and ergocryptine produced marked regres-
sion of spontaneous mammary tumors in old female rats. It was
concluded that the ergot derivatives inhibited mammary tumor growth
by depressing prolactin secretion.

8. The ergot derivatives, ergocornine and LSD, also inhibited
growth of carcinogen-induced mammary tumors. The anticancer
activity of ergocornine was enhanced by Simultaneous treatment
with ovariectomy or large doses of estradiol benzoate which further
decreased prolactin secretion and/or interfered with the peripheral
action of prolactin on mammary tumors, respectively.

9. Drostanolone propionate, a modified testosterone com-
pound, was found to be a potent inhibitor of growth of carcinogen-
induced mammary tumors in rats. However, its inhibitory effects
on tumor growth were completely overcome by simultaneous injec-
tions of prolactin. It is suggested that high doses of androgens, like
high doses of estrogens, inhibit mammary tumor growth by interfer-
ing with the peripheral action of prolactin on tumor parenchyma.

10. Convincing evidence was obtained that mammary tumor

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growth can be altered by altering catecholamine activity in the hypo-

thalamus. Thus treatment with l-dOpa or pargyline, which increases

catecholamines in the hypothalamus and thereby reduce prolactin

release, produced marked regression of established tumors and

complete suppression of new tumor development. By contrast,

methyldopa and haloperidol produced profound stimulation of mam-

mary tumor growth. Both these drugs produce several fold increases

in prolactin secretion by decreasing hypothalamic catecholamine

activity.

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EFFECTS OF CENTRAL ACTING DRUGS AND ER GOT DERIVATIVES
ON PROLACTIN AND GROWTH HORMONE SECRETION,
ON GROWTH OF PITUITARY AND MAMMARY TUMORS
AND ON REPRODUCTION IN OLD RATS

by

Syed Kaleemullah Quadri

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOC TOR OF PHILOSOPHY

Department of Physiolo gy

19 73

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For my parents, brothers, and sisters

and for

Sandy, my companion in my trials and triumphs

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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my academic
advisor, Professor Joseph Meites, without whose help and encourage-
ment this thesis would not have seen the light of the day. He not only
instilled in me a sense of the importance and urgency of my work but
also provided the atmosphere that was most conducive to the creative
process. It was an atmosphere charged with enthusiasm and excite-
ment which generated many a lively discussion on controversial sub-
jects and on the research projects my fellow students and I were
pursuing. He brought to our attention first-hand information from
important laboratories around the world and brought us into close
contact with some of the top scientific minds in our field. His love
and affection for us were surpassed only by his desire to see us
excel in whatever project we were engaged in and by his enormous
trust in our capabilities and intense pride in our accomplishments.
More importantly, we had before us the silent example of his
personality, selfless and completely dedicated to the quest for truth
in biological phenomena. For me, it will always remain a source of
great pride to have been his student.

I wish to extend a special note of appreciation to Mrs. Mabel
Meites, who was such a generous and charming hostess to all of us

on so many occasions. She provided us, who had come from all

iii

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corners of the earth, with many an hour of enjoyment and relaxation
with her kind hospitality.

At this point, when I am about to complete my formal education,
I wish to acknowledge an immense debt of gratitude to my former
academic advisor, Dr. Harold G. Spies, who guided me with great
skill and understanding through my early years in graduate school,
who helped me to develop a taste for research, and who, ever since,
has showered me with his affection and care. It was my great
fortune to have begun my career in research with such a great
scientist and good human being.

I am deeply indebted to Dr. Richard L. Anderson, Associate
Professor, who not only taught me biochemistry but always had time

and a sympathetic ear to listen to my problems. I am likewise

grateful to Dr. Harold A. Hafs, Professor of Reproductive
Physiology, for his extreme kindness and help on many occasions.

I, like all other students in the department of physiology, will always
remain grateful to Dr. E. Paul Reineke, Professor of Physiology,
for his boundless kindness and generous help throughout our stay

in the physiology department. Dr. W. Duane Collings, who served
on my guidance committee along with Drs. Meites, Reineke, Hafs
and Anderson, gave generously of his time and advice, for which I
am grateful. Dr. Francis Haddy, Chairman of the Physiology

Department, made our department a symbol of academic freedom,

iv

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excellence, and efficiency; for this, I shall always be thankful. I
want to make Special mention of Dr. B. H. Selleck, Associate
Professor of Physiology, and Dr. A. J. Morris, Professor of
Biochemistry, for their willingness, even on short notice, to serve
on my examining committee.

Although I am indebted to all secretaries in the physiology
department for their help and kindness, I must mention specifically
Mrs. Amylou Davis, Secretary Graduate Affairs, for her great
skill and understanding in guiding me efficiently and smoothly through
the maze and intricacies of official procedures, and Miss Nancy
Turner, who cheerfully and at a great Speed typed many parts of
this thesis.

Finally, I shall always be grateful for the help and companion-
ship of many of my colleagues, particularly Gary Kledzik, Ed Knecht,
Carol Bradley and Lindsey Grandison. To Gary and Ed especially,

I am indebted for the gift of their friendship and much-needed help.

N
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TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
INTRODUCTION
REVIEW OF LITERATURE
The Beginnings of Neuroendocrinology
Beginnings
Neurosecretion

Hypothalamic Origin of the Posterior Pituitary
Hormones

Vascular Links Between the Hypothalamus and the
Anterior Pituitary

The Chemotransmitte r Hypothe sis

Evidence for Hypothalamic Control of Anterior
Pituitary Function

Hypothalamic Releasing and Inhibiting Factors
Control of Prolactin Secretion

Inhibition of Prolactin Secretion by the Hypothalamus

Prolactin-Inhibiting Factor (PIF)

Control of PIF Activity in the Hypothalamus

Effects of Biogenic Amines on Prolactin Secretion

Effects of Ergot Derivatives on Prolactin Secretion

vi

xiv

xvii

10

12

12

13

14

15

18

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Control of Growth Hormone Secretion

Stimulation of Growth Hormone Secretion by the
Hypothalamus

Growth Hormone Releasing Factor (CH-RF)

Neuroendocrine Changes Associated With Aging in Rats

Changes in Reproductive Cycles
Changes in Neuroendocrine Organs

Reinitiation of Estrous Cycles and Induction of
Ovulation in Old Rats

Mammo s omatotropic Pituita ry Tumor 5

Induction of Pituitary Tumors

Characteristics of Mammosomatotropic Pituitary
Tumors

Hormone Secretion by Mammosomatotropic Pituitary
Tumors

Carcinogenesis

Definition

Classification

Etiology

Mechanism of Carcinogenesis
Chemotherapy of Cancer

Surgery and Radiotherapy

Spontaneous Mammary Tumors in the Rat

vii

Page

20

20
21
22
22

23

24
25

25

27

28
3O
32
33
34
43
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Incidence

Some Factors Affecting Incidence of Breast Cancer
Importance of Spontaneous Mammary Tumors of the Rat
Histological and Growth Characteristics

Neuroendocrine Control of Development and Growth of
Spontaneous Mammary Tumors in the Rat

Role of the Ovaries
Role of Androgens

I Role of the Adrenals
Role of the Pituitary
Role of the Hypothalamus

Induced Mammary Cancers in the Rat

Importance
Induction

Neuroendocrine Control of Development and Growth of
Carcinogen-Induced Mammary Tumors in Rats

Influence of Sex

Role of the Ovaries

Role of Androgens

Role of the Adrenal Glands
Role of the Pituitary Gland
Role of the Hypothalamus

Prolactin Versus Estrogen in Carcinogen-Induced
Mammary Tumorigenesis

GENERAL MATERIALS AND METHODS
Animals

Blood Collection for Radioimmunoassays by Cardiac
Puncture

viii

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Rat Prolactin Radioimmunoassay
Growth Hormone Radioimmunoassay
Transplantation of Pituitary Tumors
Donor Rats
Recipient Rats
Tumor Transplantation
DMBA-Induced Mammary Tumors in Rats
Induction
Measurement
Calculations
EXPERIMENTAL
Inhibition of Prolactin Secretion in Female Sprague-
Dawley Rats by a Single Injection of Lysergic Acid
Diethylamide (LSD)
Objectives
Materials and Methods
Results
Conclusions
Inhibition of Prolactin Secretion in Female Sprague-
Dawley Rats by Ergocryptine; Counteraction by
Perphenazine

Objectives

Materials and Methods

ix

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Results 190
Conclusions 195
Inhibition of Prolactin Secretion in Rats by Pyrogallol 198
Objectives 198
Materials and Methods 198
Results 199
Conclusions 202

Effect of Pentobarbital and Ether on Plasma GH Con-

centration in Sprague-Dawley Rats 208
Objectives 208
Materials and Methods 209
Results 211
Discussion 215

Relation of Size and Age of Pituitary Tumor (MtT. W15)
to Prolactin and Growth Hormone Secretion and to Body

Weight 217
Objectives 217
Materials and Methods 218
Results 221
Conclusions 224

Ergot-Induced Inhibition of Pituitary Tumor Growth
in Rats 230

Objectives 230

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Results 232

Effects of Ergocornine and CG 603 on Blood Prolactin

and GH in Rats Bearing a Pituitary Tumor 238
Objectives 238
Materials and Methods 239
Results 242
Conclusions 251

Reinitiation of Estrous Cycles in Old Constant Estrous

Rats by Central-Acting Drugs 256
Objectives 256
Materials and Methods 258
Results 260
Conclusions 266

Regression of Spontaneous Mammary Tumors in Sprague-

Dawley Female Rats by Ergot Drigs 268
Objectives 268
Materials and Methods 268
Results 269
Conclusions 279

Effects of LSD, Pargyline and Haloperidol on Carcinogen-
Induced Mammary Tumor Growth in Rats 280

Objectives 280

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Conclusions

Androgen-Induced Inhibition of DMBA-Induced Mammary
Tumor Growth in Rats; Counteraction by Prolactin

Objectives

Materials and Methods
Results

Conclusions

Effects of L-Dopa and Methyldopa on Growth of DMBA-
Induced Mammary Tumors in Rats

Objectives

Materials and Methods

Results

Conclusions
Enhanced Regression of Mammary Tumors in Rats by
Combination of Ergocornine and Ovariectomy or High
Levels of Estrogen

Objectives

Materials and Methods

Results

Conclusions

GEN ER A L DISC USSION

xii

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BIBLIOGRAPHY

APPENDIX

xiii

337

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10.

11.

LIST OF TABLES

Effects of a single injection of LSD on serum
prolactin levels in female rats.

Effects of a single injection of ergocryptine on
serum prolactin levels (ng/ml) in female Sprague-
Dawley rats.

Effects of a single injection of ergocryptine on
serum prolactin concentration (ng/ml).

Effect of ergocryptine on perphenazine-induced
increase in serum prolactin.

Effect of a single injection of pyrogallol on serum
prolactin concentrations (ng/ml) in female rats.

Effect of a single injection of pyrogallol on percent
change in serum prolactin concentrations in
female rats.

Effect of sodium pentobarbital (PB) on plasma GH
concentrations (ng/ml) in male rats.

Effect of ether anesthesia on plasma GH concen-
trations (ng/ml) in male rats.

Influence of pituitary tumor size on prolactin and
growth hormone secretion and on body weight of

rats.

Percent changes in pituitary tumor diameters

induced by ergocornine, ergonovine and ergocryptine.

Effect of ergocornine on pituitary tumor size and
body weights of the tumor bearing rats.

xiv

186

193

194

196

205

206

212

214

225

233

237

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12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

 

Effects of ergocornine and CG 603 on serum
prolactin (pg/m1) concentrations in rats carrying
MtT. W15 pituitary tumors.

Effects of ergocornine and CC 603 on plasma GI—I
(pg/m1) in rats carrying MtT. W15 pituitary tumors.

Effects of ergocornine and CC 603 on growth of
MtT.W15 pituitary tumor transplants.

Effects of ergocornine and CG 603 on body weights
of rats carrying MtT. W15 pituitary tumor trans-
plants.

Vaginal patterns in control old constant estrous
female rats.

Effect of epinephrine on vaginal patterns in old
constant estrous rats.

Effect of l-dopa on vaginal patterns in old constant
estrous rats.

Effect of iproniazid on vaginal patterns in old
constant estrous rats.

Effects of ergocornine and ergocryptine on
spontaneous mammary tumors in female rats.

Growth of spontaneous mammary tumors in female
rats during post-treatment period.

Effect of central-acting drugs on growth of DMBA-
induced mammary tumors in rats.

GrOWth 0f DMBA-induced mammary tumors during
pOSt-treatment period.

Effects of dronostanolone propionate (DP), prolactin
(PRL) and both on growth of DMBA-induced mam-
mary tumors in rats.

XV

243

247

250

254

261

262

264

265

272

274

284

286

296

   

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25.

26.

27.

28.

29.

30.

Growth of mammary tumors during post-treatment
period.

Effects of l-dopa and methyldopa on growth of
DMBA-induced mammary tumors.

Effects of l-dopa and methy1d0pa on state of tumors
at end of 4 weeks treatment.

Effects of ergocornine (EC), ovariectomy (OVX),
estradiol benzoate (EB) and combinations on growth
of DMBA-induced mammary tumors in rats by end
of 3 weeks.

Effects of ergocornine (EC), ovariectomy (OVX),
estradiol benzoate (EB) and combinations on growth

of DMBA-induced mammary tumors in rats.

Growth of DMBA-induced mammary tumors during
post-treatment period.

xvi

298

308

312

317

321

323

 

 

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Figure

10.

ll.

12.

13.

LIST OF FIGURES

Biosynthesis of catecholamines
Chemical structure of ergocryptine.

Structural resemblance among carcinogenic
hydrocarbons, aromatic amines, bile acids and
steroids.

Mammary gland from a pregnant rat.
Mammary gland from a lactating rat.

A mammary adenocarcinoma.

A mammary adenocarcinoma in an early stage.

Effect of a single injection of ergocryptine on serum
prolactin levels in female Sprague-Dawley rats.

Effect of a single injection of pyrogallol on serum
prolactin concentrations in female Sprague-Dawley
rats.

Effect of a single injection of pyrogallol on serum
prolactin concentrations in female Sprague-Dawley
rats.

Transplants of "mammosomatotropic" pituitary
tumors in inbred female rats of the Wistar-Furth
strain,

Correlation between serum prolactin concentra—
tions and size of pituitary tumor (MtT.W15) trans-
plants in female Wistar-Furth rats.

Correlation between plasma GH concentrations and
Size of pituitary tumor (MtT. W15) transplants in
female Wistar-Furth rats.

xvii

16

19

41

97

98

99

100

192

201

204

220

223

227

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14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Correlation between body weight of female Wistar-
Furth rats and the size of pituitary tumors (MtT.
W15).

Effects of ergocornine, ergonovine and ergocryptine
on growth of tranSplants of pituitary tumor MtT. W15
in inbred female Wistar-Furth rats.

Effects of daily injections of ergocornine and CG 603
on serum prolactin in inbred female Wistar-Furth
rats carrying a MtT. W15 pituitary tumor transplant.

Effects of daily injections of ergocornine and CG
603 on plasma GH concentrations in inbred female
Wistar-Furth rats carrying a MtT. W15 pituitary
tumor tranSplant.

Effects of daily injections of ergocornine and CG
603 on average tumor diameter of MtT. W15 tumor
transplant in inbred female Wistar-Furth rats.

A spontaneous mammary tumor in an old female
rat of the Sprague-Dawley strain.

Effects of ergocornine on growth of spontaneous
mammary tumors in female Sprague-Dawley rats.

Effects of ergocryptine on growth of Spontaneous
mammary tumors in female Sprague-Dawley rats.

DMBA-induced mammary tumors in female Sprague-
Daney rats.

Effects of LSD, Pargyline and ha10peridol on diameter
0f DMBA-induced mammary tumors in female Sprague-

Dawley rats .

Effects of LSD, Pargyline and haloperidol on number
Of DMBA-induced mammary tumors in female
Sprague-Dawley rats.

xviii

229

236

245

249

253

271

276

278

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288

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25. Effects of drostanolone propionate, prolactin and
their combination on percent change in mean dia—
meter of DMBA—induced mammary cancers in
female Sprague-Dawley rats. 300

26. Effects of drostanolone propionate, prolactin and
their combination on percent change in mean number
of DMBA-induced mammary cancers in female

Sprague-Dawley rats. 303
27. Effects of l—dopa and methyldopa on average per-
and cent change in tumor diameter and average percent
28. change in tumor number. 310
29. Effects of ergocornine, ovariectomy and high levels

of estrogen and their combinations on size of

established 7, 12, dimethylbenz(a)anthracene-

induced mammary tumors in female Sprague-Dawley

rats. 320

30. Effects of ergocornine, ovariectomy and high levels
of estrogen on number of 7, 12, dimethylbenz(a)
anthracene-induced mammary tumors in female
Sprague-Dawley rats. 328

xix

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IN TR ODUC TION

The last two decades have seen a spectacular growth of neuro-
endocrinology. During this period the anterior pituitary was
stripped of some of its former glory as the "master gland" and
reduced to the status of an important manufacturing plant Operating
at the command of the hypothalamus. The recent success on the
structural chemistry and synthesis of hypothalamic hormones has
removed any remaining doubts about the chemotransmitter hypothesis.

Now we are entering an equally exciting phase when attempts
are being made to unravel the mechanisms that control the synthesis
and release of hypothalamic hormones. There is increasing evidence
to implicate neurotransmitter substances, particularly the catecho-
lamines and indoleamines, in these processes. Some experiments
which are included in the present thesis and others which were left
out due to limitations of time and space, were carried out to deter-
mine the effects of catecholamines and other central acting agents on
the secretion of prolactin and growth hormone. Blood and pituitary
levels of these hormones were measured by radioimmunoassays
which have tremendously increased the accuracy and hastened the
pace of endocrine research. It is perhaps one of the most important
research tools ever devised. Some day when the history of radio-

immunoassays will be written, an interesting footnote will perhaps

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describe that Berson pursued hisiidea relentlessly amid skepticism
which was so widespread that one of his earlier papers was rejected
for publication by a reputable journal.

Man has always been intrigued by the physiologic changes that
accompany senescence and has constantly dreamed of conquering it.
From the ancient search for the mythical fountain of youth to the
construction of health spas in EurOpe, we find testimony to man's
desire to remain young longer if not forever. Youth stands not only
for good health and freedom from diseases but also for an active
reproductive life. With the recent growth of neuroendocrinology
there are indications that changes in the central nervous system
might be a factor in the decline of reproductive functions with aging.
This thesis presents evidence that a deficiency in hypothalamic
catecholamines might be responsible for loss of reproductive cycling
in old rats.

Experimental pituitary tumors are important clinically and
also as a laboratory model to study pituitary function. Some of the
experiments in this thesis were devoted to determine the relationship
between tumor size and its secretory activity, and to investigate
the effects of ergot derivatives on the growth of these tumors. It
will come as no surprise if these drugs or other drugs discovered

by the use of these tumors prove to be effective in control of human

pituitary tumor 5 .

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Many Years have gone by since 1889 when Schinzinger suggested
to the Eighteenth Congress of the German Surgical Association that
there might be a. relationship between the ovaries and human breast
cancer (Hayward, 1970). More than 75 years have passed since

Beatson in England gave a practical demonstration of this relationship
by successfully treating breast cancer by ovariectomy and cautioned
his contemporaries that "the parasitic theory of cancer is an unsatis-
factory one in many ways and that in directing all our energies to
working it out we are losing time and searching for what will never

be found simply because it does not exist'l (Hayward, 1970).

Beatson's advice seems as relevant today as it did in the last century.
Acceptable proof of viral involvement in human breast cancer has

not yet been fortthming. More than 50 years have passed since the
first virus (Shope virus) was implicated in animal cancer. Today
virology is an extremely sophisticated and highly resourceful science.
Therefore it does not appear very tenable that discovery of a human
breast cancer virus is being held up due to lack of suitable techniques.
On the other hand there is overwhelming evidence of endocrine
involvement in breast cancer. According to one estimate about 40
percent of human breast cancer cases are susceptible to endocrine
therapy (Pearson _e__t__a_l_. , 1972b). The importance of endocrine
involvement in other cancers was highlighted by the award of a Nobel

Prize to Huggins for his contributions to the endocrine therapy of

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prostate cancer. Yet it is discouraging to see so few endocrinolo-
gists involved in research on the endocrine aspects of cancer. Even
if viruses are implicated as the primary causative agents of breast
cancer, endocrine involvement will still remain an important and
irrefutable factor in the causation and treatment of this dreadful
disease. Lack of glamorous results and the painfully slow nature of
the research on endocrine aspects of breast cancer should not be a
determent, particularly in view of the fact that we have an excellent
laboratory model for breast cancer available. Carcinogen-induced
breast cancers in the rat bear a striking resemblance to human
breast cancer in several aspects, particularly in their dependency
on hormones for development and growth. These tumors are parti-
cularly useful in view of the difficulties inherent in cancer research
in humans. Recently some very interesting reports have been
published to directly implicate the hypothalamus in spontaneous and
carcinogen-induced mammary tumorigenesis in rats (Meites, 1972a,
b). Some of the experiments in this thesis suggest that mammary
tumor growth can be altered by altering catecholamine activity in
the hypothalamus. Other experiments were conducted to determine
the effects of hormones and several central acting drugs, including
ergot derivatives, on the growth of carcinogen-induced mammary
tumors. Several of these drugs, particularly ergocryptine and

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REVIEW OF LITERATURE

The Beginnings of Neuroendocrinology

Beginnings

 

The earliest indications that the nervous system might be
involved in the control of endocrine functions came even before the
birth of endocrinology. Thus, it was reported in the 17th century
that certain birds will lay more eggs if an egg is removed daily from
their nests (Green, 1966). It was known in the 18th century that
rabbit ovaries undergo reflex changes following coitus (Green, 1966).
Several more examples of nervous influences on endocrine functions
are as follows: a female pigeon can lay eggs after seeing her image
in a mirror; daylight affects the breeding season in sheep (Greep,
1963); suckling can induce milk secretion in virgin girls, rats, mice
and cattle (Meites gal. , 1963); an unhappy love affair can alter the

menstrual rhythm (Donovan, 1970).

Neuro secretion

The concept of neurosecretion was born more than 50 years
ago when Spiedal (1919) reported the presence of neurosecretory cells
in the spinal cord of elasmobranch fishes. In the years to come

similar glandular nerve cells were discovered in other fishes,
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amphibia and a number of invertebrates, mainly by the husband and
wife team of Ernst and Berta Scharrer (1954a, 6) and later by
Hanstrom (1953, 1954). Now it is known that several endocrine
functions in these species are controlled by neurosecretions. For
example, neurons in crabs produced a gonad-inhibiting hormone,
and the brain cells in certain insects produce a hormone which con-
trols moulting. For more information in this area the reader is
referred to an excellent book written by the Scharrers (1963).
Hypothalamic Origin of the Posterior
Pituitary Hormones

The clearest demonstration of secretory neurons in the
vertebrate hypothalamus came from the work of Palay (19.45) and
Bargman and Ernst Scharrer (Bargman and Scharrer, 1951; Bargman,
1960) in the late 1940's. They demonstrated that the posterior
pituitary homrones were actually synthesized in the paraventricular
and supraoptic nuclei of the hypothalamus from which the hormone
granules travel down the axons in the hypothalamo-hypophyseal tract
to the pars nervosa of the pituitary for storage and release. This
was soon followed by the synthesis of oxytocin by de Vigneaud _e_t__a_l_.

(1953) for which the senior researcher was rewarded a Nobel prize.

 

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19.39313? Links Between the Hypothalamus
Edthe Anterior Pituitary
While important strides were being made in identification,
isolation and synthesis of neurosecretory material of the posterior
pituitary, a great body of evidence was being gathered to implicate
the hypothalamus in the control of the anterior pituitary function.
Two Rumanian anatomists, Ranier and POpa, had made a great
contribution to the understanding of hypothalamo-hypophyseal relation-
ship by discovering the hypophyseal portal system (POpa and Fielding,
1930a, b, 1933). However, they believed the blood flowed from the
pituitary to the hypothalamus. This error was corrected later by
Wislocki and King (Wislocki and King, 1936; Wislocki, 1937, 1938)
and others (Housseygtil. , 1935; Green, 1947). Dr. Roy Greep
celebrated this event by composing a doggerel.
Hickory dickery dock
The blood ran up the stalk
Then along came Wislocki and King
And the blood ran down again.

The downward flow of blood in the hypophyseal portal vessels
has not been accepted by all. Some very respected anatomists
(Torok, 1965, 1962, 1964; Szentagothai gt_a_l_. , 1957) think that at
least a small amount of blood flows in the opposite direction, from

the pituitary to the hypothalamus. However, the evidence for the

downward flow of blood is overwhelming and many have actually

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seen the blood flow down the stalk to the pars distalis (Hous say _e_t a_l.,
1935’. Green, 1947; Green and Harris, 1949; Barnet and Greep, 1951).
The hypOphyseal portal vessels themselves seem to have a universal
distribution. Green (1951) found them to be common features in

more than 75 vertebrate Species. They originate in the median
eminence and stalk and end in the sinusoid bed of the pars distalis.
Daniel and Prichard (1956) have also described some short vessels

which are supposed to carry blood from the neural lobe to the anterior

pituitary.
The Chemotransmitter Hypothe sis

While evidence for an anatomical link between the hypothalamus
and the anterior pituitary was accumulating, experiments were under-
taken to search for proof of physiological control of the anterior
pituitary by the hypothalamus. Harris (1937) reported that stimula-
tion of the hypothalamus resulted in ovulation in rabbits. The same
year Hinsey (1937) suggested the possibility of hypothalamic neuro-
humors that might reach the anterior pituitary via the portal vessels
and influence its function since there was no evidence of a neural
link between the two organs. Markee 233.1.- (1946) also induced
ovulation in rabbits by stimulation of the hypothalamus. In the late
1940's Harris and Green reviewed all the anatomical and physiological

evidence for hypothalamic control of the anterior pituitary and

 

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suggested the possibility of neurohumoral control of the latter via
the portal vessels (Green and Harris, 1947; Harris, 1948; Harris,
1955). This concept later came to be known as the Chemotransmitter
hypothesis. It proved to be a turning point in endocrinology and
heralded the birth of the science of ne uroendocrin010gy. After this
there was such an avalanche of information and scientific reports
that Greep, a pioneer endocrinologist, complained after the manner 1
of Alexander Graham Bell, "what has Harris wrought"!
Evidence for Hypothalamic Control
of Anterior Pituitary Function

After the publication of the Chemotransmitter hypothesis, some
very elegant and ingenious experiments were done to prove that the
hypothalamus indeed exercised control over anterior pituitary func-
tion. These experiments involved electrical stimulation or lesioning
of the hypothalamus, pituitary stalk section or transplantation of the
pituitary and other techniques. The results of the pituitary stalk
section experiments were contradictory. This problem was solved
by Harris (1950) who prevented regeneration of the portal vessels by
placing a wax barrier between the cut ends of the stalk and produced
permanent disruption of the pituitary function. Nikitovitch-Winer
and Everett (1958) and Evans and Nikitovitch-Winer (1959) demon—
strated that auto-transplantation of the pituitary underneath the

kidney capsule severly impaired gonadotropin secretion. When the

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10

transplanted pituitary was treated with median eminence extracts

or was placed back beneath the median eminence its function was
restored to a great extent. Harris and Jacobson (1952) also reported
that transplantation of the pituitary in the median eminence of
hypophysectomized rats restored normal reproductive functions.
Halasz Stil- (1962) devised a special knife to isolate parts of the
hypothalamus and proposed the ”hypOphysiotropic area'I in the medial
basal hypothalamus as the probable site for production of the sub-
stances involved in the control of the anterior pituitary. By now
there was overwhelming evidence that the "master gland", the
"conductor of endocrine orchestra”, was in fact under the dominance
of the hypothalamus. The next logical step was identification and

isolation of the Specific hypothalamic substances involved in the

control of the pituitary functions.
Hypothalamic Releasing and Inhibiting Factors

The early search for the hypothalamic substances centered
on various neurotransmitters. Markee _e_t_a_1. (1948) and McDermott
$331. (1951) among others, suggested that epinephrine, acetylcholine,
histamine etc. , might be involved in the release of gonadotropins,
ACTH and other pituitary hormones. It was revealed later, but not
before a huge amount of literature had accumulated on the neuro-

transmitters, that actually a special class of polypeptides are the

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hypothalamic substances involved in the anterior pituitary control.
Saffran and Schally (1955) were among the first to report that crude
acidic hypothalamic extracts caused release of ACTH 1211.532 and
331%. They called the active principal corticotropin releasing
factor (CRF). This was also reported independently by Guillemin
333.1. (1957). McCann eta}. (1960) demonstrated the presence of
LRF (luteinizing hormone releasing factor) in acid extracts of rat
hypothalami. Talwalker, Ratner and Meites (1963) and Pasteels
(1963) provided evidence for PIF (prolactin inhibiting factor). The
existance of GHRF was first clearly demonstrated by Deuben and
Meites (1963). In the following years evidence was forthcoming for
the existence of TRF (Shibusawa _e_tal. , 1959; Schreber, 1963:
Guilemin _e_t_a_l_. , 1962), FSH—RF (Igarashi and McCann, 1964;
Mittler and Meites, 1964), MIF and MRF (Kastin e_t31. , 1965;
Taleisnik and Orias, 1964), GIF (Krulich and McCann, 1968) and
PRF (in birds) (Kragt and Meites, 1965; Meites and Nicoll, 1966).
During these years, when these factors were being identified,
Guillemin's and Schally's laboratories mounted a massive effort to
elucidate the structure and synthesis of TRH (Boler still. , 1969;
Burges and Guillemin, 1970; Schallygtgl. , 1973). They succeeded.
TRH is a tripeptide. The same laboratories have also reported the
synthesis of other releasing factors such as LRF, which is a

decapeptide (Schally _e_t a1. , 1973).

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12

Control of Prolactin Secretion

Inhibition of Prolactin Secretion
by the Hypothalamus

There is considerable evidence that the mammalian hypo-
thalamus inhibits the release and synthesis of prolactin. Everett
(19 54) demonstrated that autotransplantation of the pituitary under
the kidney capsule prolongs the life-span of corpora lutea indicating
continued prolactin secretion. These results were confirmed by
Nikitovitch-Winer and Everett (19 58). Destruction of the "final
common pathway" to the anterior pituitary by lesioning the median
eminence also produces several fold increases in serum prolactin
concentrations (Chen £2.31}.- , 1970). Welsch 9.121- (1971) reported
that bilateral lesions placed in the median eminence of ovariectomized
rats produced a ten-fold rise in serum prolactin within 30 minutes
and the concentration remained high for up to six months after the
Operations. Lesions in the hypophysiotropic area, which includes
the arcuate nucleus, the ventral part of the anterior periventricular
nucleus and the medial parvicellular region of the retrochiasmatic
area (Halasz, 1962), also increase prolactin secretion in several
mammalian species (Meites _e_t_a_l_. , 1972). Elevations in prolactin
release have also been reported after pituitary stalk section and

after incubation of the anterior pituitary in vitro (Meites 31311. , 1972).

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13

All these studies indicate that procedures which decrease hypo-
thalamic influence on the pituitary lead to increased prolactin secre-
tion. On the other hand, the same procedures decrease secretion of

the other anterior pituitary hormones.
Prolactin-Inhibiting Factor LPIEJ

Since prolactin release is increased after removal of hypo-
thalamic influence, it was logical to look for a hypothalamic factor
which inhibits prolactin release. Thus it was demonstrated that
hypothalamic extracts could inhibit prolactin release from rat
pituitaries EXILE? (Meites _e_tgl. , 1961; Talwalker _e_t.§_1_. , 1963;
Pasteels, 1961). Similar prolactin-inhibiting activity has been shown
to be present in hypothalamic extracts of several mammalian species
by 3.13.219. and 3.1119519. experiments (Schally _e_t 3.1: , 1965; Grosvener
3211, , 1964). The hypothalamic factor responsible for ‘inhibiting
prolactin release has been named prolactin inhibiting factor (PIF).

In contrast to the mammals, the avian hypothalamus has been shown
to contain a prolactin releasing factor (PRF) (Kragt and Meites,
1965; Nicoll, 1965). The presence of a PRF in the mammalian

hypothalamus also is possible (Meites _e_tal. , 1972).

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14

Control of PIF Activity in the Hypothalamus

PIF activity in the hypothalamus can be altered by several
means, including the use of central acting drugs, hormones and by
physiological stimuli. Thus ergot drugs (Wuttke _e_tgl. , 1971),1-dopa,
monoamine oxidase inhibitors (Lu and Meites, 1971) and dopamine
(Kamberi _e_t_ 9;]: , 1970) increase hypothalamix PIF activity and
decrease prolactin release. On the other hand, reserpine, (Ratner
__£§._1. , 1965), perphenazine (Danon £31. , 1963) and haIOperidol
(Dickerman, S._e_1_:_§_l_,,1972) decrease hypothalamic PIF and increase
prolactin secretion. PIF is also decreased by estrogen (Ratner
and Meites, 1964), testosterone, progesterone, cortisol (Sar and
Meites, 19 68) and EnOvid (norethynodrel-mestranol) (Minaguchi and
Meites, 1967), whereas prolactin itself increases hypothalamic content of
PIF and thus inhibits its own secretion (Voogt and Meites, 1971).

The suckling stimulus has been shown to increase prolactin release
by decreasing PIF. Stresses of several kinds including those of
ether and sodium pentobarbital anesthesias and surgery have been

shown to increase serum prolactin in several species (Wuttke and

Meites, 1970; Frantz 3331. , 1972).

 

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Effects of Biogenic Amines
on Prolactin Secretion

There is considerable evidence that the autonomic nervous
system exercises substantial influence on the endocrine functions.
Some neurotransmitters of sympathetic (catecholamines) and para-
sympathetic (acetylcholine, indoleamines, imidazoleamines) origin
have been shown to profoundly influence the release of prolactin and
other pituitary hormones.

Biosynthesis of catecholamines begins with neuronal uptake
of tyrosine from the blood circulation (Figure 1). After hydroxyla-
tion tyrosine is converted to the catechol amino acid, l-dopa, which
is the immediate precursor of catecholamines. It is decarboxylated
to dopamine which is transformed to norepinephrine and ultimately
to epinephrine (Anton-Tay and Wurtman, 1971). The hypothalamus
is rich in norepinephrine and dOpamine (Brodie _e_t__a_l. , 19 59), but
the presence of epinephrine in the hypothalamus is controversial.
Catecholamines are concentrated in the synaptic vessicles from
which they are released upon nerve stimulation and they then go
across the synaptic cleft and react with the receptors on the post-
synaptic neuron. This results in generation of action potential and
a nerve impulse. The action of norepinephrine ends after its

reuptake into the nerve terminals but catecholamines in the neuron

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17

are degraded by the monoamine oxidase enzyme. The other major
catecholamine degradative enzyme is catechol-o-methyltransferase
(COMT) which is active within the synaptic cleft (Anton-Tay and
Wurtman, 1971).

Catecholamine activity in the brain can be altered in several
ways. It can be increased by administration of l-dOpa or by
inhibiting degradation of catecholamines by monoamine-oxidase
inhibitors (pargyline, iproniazid) or inhibitors of COMT (pyrogallol).
There is no evidence that catecholamines (dOpamine, epinephrine,
norepinephrine) themselves can pass through the blood-brain barrier.
(Innes and Nickerson, 1971). Catecholamine activity in the brain
can be decreased by several central acting drugs. Thus, reserpine
depletes catecholamines, chlorpromazine blocks receptor sites on
post-synaptic membrane, methy1d0pa competes with l-dOpa for
dopa-carboxylase to produce methyleted catecholamines which are
weaker neuro-transmitters, and alpha-methyl-meta tyrosine and
alpha-methyl-paratyrosine (methylated tyrosine analogs) attack
tyrosine hydroxylase which is the rate limiting enzyme in catecho-

lamine biosynthesis (Wurtman, 1970; Wurtman £211.- , 1969; Anton-Tay
and Wurtman, 1971; Coppola, 1968). There seems to be a reciprocal
relationship between hypothalamic catecholamine activity and pro-
lactin secretion. A decrease in catecholamine concentration results

in increased prolactin secretion while an increase in catecholamines

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18

produces just the opposite effect (COppola, 1971). Catecholamines
affect secretion of other pituitary hormones differently. Thus
increased catecholamine concentration in the hypothalamus leads to
increased secretion of LH and FSH (COppola, 1971).

The hypothalamus is also rich in serotonin (Brodie _e_t__a_l. ,
1959) but unlike catecholamines it is present throughout the sero-
tonergic neurons (Wurtman, 1966; Anden _e_tal. , 1965). Brain
serotonin (5-hydroxy‘tryptamine) is synthesized from tryptOphan, but
serotonin itself cannot pass through the blood-brain barrier (Innes
and Nickerson, 1971). Injections of the serotonin precursors,
tryptophan and 5-hydroxytrypt0phan, have been shown to increase
serum prolactin levels. Kamberi ital. (1970) have reported that a
single injection of serotonin or melatonin into the third ventricle also

increased prolactin secretion and decreased secretion of LH and FSH.

Effects of Ergot Derivatives
on Prolactin Secretion

The ergot derivatives, ergocryptine (Figure 2), ergocornine,
lysergic acid diethylamide (LSD) are potent inhibitors of prolactin
secretion (Nagasawa and Meites, 1970b; Wuttke eta}. , 1971; Quadri
and Meites, 1971a; Quadri, Karande, and Meites, unpublished).
Ergocornine has been shown to decrease prolactin secretion both by

a direct action on the pituitary gland and also by increasing PIF

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activity in the hypothalamus (Lu gt 3}. , 1971; Wuttke g_t_ §_l_. , 1971).
Several ergot derivatives have been shown to inhibit growth of pitui-
tary tumors (Quadri _e_t__a_1_. , 1972) and Spontaneous and carcinogen-
induced mammary tumors which depend on prolactin for growth
(Quadri and Meites, 1971b; Cassell £331. , 1971; Quadri _e_t_a_l_. ,

1973a).

Control of Growth Hormone Secretion

 

Stimulation of Growth Hormone Secretion
by the Hypothalamus

Early studies on the hypothalamic control of growth hormone
(GH) secretion were confusing and often contradictory. However,
most of the evidence indicated that the hypothalamus exercised a
stimulatory influence on GH release from the pituitary. Thus,
lesions in the hypothalamus were reported to inhibit body growth
(Hinton and Stevenson, 1962; Bagdanove and Lipner, 1952; Cahane
and Cahane, 1938). Reichlin (1961) found that extensive lesions in
the ventral hypothalamus decreased GH content in the pituitary by
more than 80%. This was later confirmed by Frohman and Bernardis
(19 68) who reported that destruction of certain areas in the hypo-
thalamus decreased pituitary content of GH and increased plasma
GH levels. On the other hand stimulation of the hypothalamus caused

several-fold increases in plasma GH levels in rats (Frohman and

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Bernardis, 1968; Frohman _e_t_a_1. , 1970). They concluded that
ventromedial nucleus of the hypothalamus was the most likely site

for CH control. Abrams eta—l. (1966) reported M. E. lesions and
pituitary stalk section inhibited GH release in monkeys, but electrical

stimulation of the hypothalamus increased body growth (O'Brien 93 11.,

1964).

Growth Hormone Releasing Factor QH-RF)

 

Deuben and Meites (1964, 1965) conclusively demonstrated
GH-RF activity in the hypothalamus. They reported that addition of
rat hypothalamic extracts to pituitary cultures resulted in enhanced
GH release. Pecile 9.1.2.1- (1965) reported that hypothalamic extracts
caused depletion of GH in the anterior pituitary. Several subsequent
reports confirmed the presence of GH releasing activity in the hypo-
thalami of several mammalian species and birds (Schally 53131. , 1965,
1966; Dhariwal eta}. , 1966). Dickerman ital. (1969) found a dose-
response relationship between hypothalamic tissue and GH release
Em. Wilber and Porter (1970) reported that addition of pituitary
portal blood increased GH release from pituitary cultures.

Many attempts have been made to purify GH-RF (Ishida gt a}. ,
1965; Krulich _e_t_1_. , 1965; Dhariwal _e_t_a_1_. , 1966; Schally 2.1.2.1- ,

1965, 1966, 1967). Several good reviews have been published on the

subject (Schally and Arimura, 1972; Schally g 31. , 1968a, b; 1970).

   

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So far there is no convincing evidence that any laboratory has
succeeded in elucidation of the structure and synthesis of GH-R F.
Some investigators have reported that it is a decapolypeptide (Schally
and Arimura, 1972).

There have been some reports that the hypothalamus might also
contain a CH inhibiting factor (GIF) (Krulich 2.2.3.1: , 1968; Dhariwal
gt_a_l., 1969). Schally fatal. (1969) found no evidence for GIF in
porcine hypothalamus. Recently some investigators have reported
isolation and synthesis of a tetradepapeptide from ovine hypothalamus
which inhibits secretion in mg of rat or human growth hormone and
is also active law in rats (Brazeau £13511. , 1973). They have called
it somatotropin-release inhibiting factor (SRIF) or somatostatin
which further complicates the already confusing and inconsistent
terminology for the hypothalamic hormones.

Nueroendocrine Changes Associated
With Aging in Rats

Lhanges in Reproductive Cycles

The most visible change in reproductive activity in aging woman
is manifested by menopause which is characterized by cessation of
CYcling, lack of ovulation and emotional symptoms commonly
referred to as "hot flashes". The ovary produces less estrogen,

FSH levels go up and secondary sexual characters begin to decline.

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The loss of reproductive functions is a gradual process, it usually
begins after the age of 45 years and takes 1-5 years to complete.
The most visible change accompanying cessation of reproduction in
aging rats is the loss of regular estrous cycles. Female rats
exhibit irregular cycles, show long periods of diestrous or go into

a state of constant estrus characterized by constant vaginal cornifica-

tion (Aschheim, 1965).
Changes in Neuroendocrine Organs

The ovaries in aging rats are capable of some functions
(Clemens 3121.1.- , 1969). Frequently they contain large follicles but
no fresh corpora lutea, indicating failure of ovulation and hence a
constant vaginal estrus results. However, it has been demonstrated
that the ovaries of old rats and mice can be reactivated by pituitary
grafts or hypophyseal extracts (Zondek, 1929; Romeris, 1931;
Weisner, 1932; Engelhart and Hauser, 1937). Aschleim (1965)
induced ovulation in old rats by LH administration. These results
indicate that the lack of ovarian activity may actually be due to a
failure at the higher levels of the pituitary or hypothalamus, both
of which are involved in the control of ovarian function. Clemens
and Meites (1971) found that old constant estrous rats of the Sprague-
Dawley strain contained significantly higher levels of FSH and

prolactin and lower levels of LH than 3-month old cycling rats. In

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a subsequent experiment (Riegle, Shaar, Euker, Meites, unpublished)
it was found that in old constant estrous rats of the Long-Evans strain
serum LH was about half and serum prolactin about 5 times as much
as in young female rats of the same strain. Frolkis (1966) reported that
aging causes a decline in neurotransmitter substances in the hypo-
thalamus. Clemens and Meites (1971) found that the hypothalami of
old constant estrous rats contained significantly higher levels of
FHS-RF and possibly low levels of LRF, thus accounting for high
FSH and low LH levels in the blood. There was no significant change
in the PIF levels, therefore it was concluded that the high serum
prolactin levels were due to a direct action of estrogen on the
pituitary.
Reinitiation of Estrous Cycles and Induction
of Ovulation in Old Rats

Aschheim (1965) reported that ovulation could be restored in
old constant estrous rats by LH administration. Clemens £331.
(1969) induced ovulation by injections of progesterone for 3 days or
epinephrine for 10 days. Epinephrine also restored some normal
cycling. They were also able to induce ovulation by electrical
stimulation of the preoptic area in the hypothalamus. Quadri 5131.
(1973d) reported that treatment with epinephrine, 1-d0pa or iproni-

azid reinitiated estrous cycles in many old constant estrous rats.

 

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These studies have given rise to the concept that failure of ovulation
and regular cycling in old constant estrous rats is not due to ovarian
failure, but perhaps is due to a catecholamine deficiency in the hypo-
thalamus. This concept is also supported by the reports that the
hypothalamus in old rats is deficient in neurotransmitter substances
(Frolkis, 1965) and that transplantation of ovaries from young to old
rats does not reinitiate regular cycling, indicating that reproductive

failure does not originate at the ovarian level.
Mammosomatotropic Pituitary Tumors
Induction of Pituitary Tumors

An important year in the history of pituitary tumors is 1936.
In that year at least seven laboratories around the world reported
induction of experimental pituitary tumors in mice and rats after
chronic estrogen treatment. Cramer and Horriing (1936) were the
first to report in Lancet the induction of adenomata of the anterior
lobe of the pituitary by prolonged treatment with ”oestrin". The
tumors, they reported, were of the "chromophobe type”. They con-
firmed their own results in a subsequent publication (Cramer _e_t_a_l_. ,
1936) in which they stated that prolonged treatment with estrogen

. . results functionally in a condition closely resembl-
ing that following hypophysectomy, morphologically it

produces a hyperplasia of the anterior lobe of the pituitary
in which, however, the chromophil cells are greatly

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26

diminished, so that the enlarged anterior lobe consists

mainly of chromophobe cells. On the assumption that

the chromophil cells are responsible for the production

of the Specific hormones of the anterior lobe, the

general condition of hypopituitarism and the extensive

changes in the other endocrine organs produced by oestrin

find their explanation in this disappearance of the

chromOphil cells of the anterior lobe.

Pituitary tumors by estrogen treatment were also induced by Burrows
(1936), Gardner ital. (1936), Martins (1936), Mchen 5131. (1936),
Oberling_e_t_§_1_. (1936) and Zondek (1936). Zondek (1936) reported
production of pituitary tumors after a 28-week treatment with
”follicular hormone”. In the next 20 years more than 20 reports
confirmed these findings.

Gorbman (1949) was the first to report that ”following doses of
radioactive iodine sufficient to cause destruction of most or all of the
thyroid, tumorous enlargements were found in the pituitary glands of
mice". This was confirmed by Furth 53.3.3.1.- (1931, 1953) who
reported that these tumors in rats secreted TSH. They also success-
fully transplanted them into other rats and found a quantitative
relationship between thyroid function and growth of these tumors.
Upton and Furth (1953) were the first to report that pituitary tumors
also can be induced by total-body ionizing irradiation. Furth and
co-workers have done extensive work on experimental pituitary

tumors (Clifton and Furth, 1957; Cohen and Kim, 1963; Furth _e_tgl. ,

1956; Kim 2231. , 1963; Meyer and Clifton, 1956). They have induced

 

 

a - ‘ r
177365 011333303.

:5. annexes of it. a

.r ya," ~ ’

5, ..1ese are 1':

V v
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ferrzg to the Fisher ;

 

 

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xtie tissue in
‘Cour, 1950; 1
.- reported that a
if;

27

many types of transplantable pituitary tumors which secrete one or
more hormones of the anterior lobe. Perhaps the most extensively
used among these are the "mammosomatotropic" pituitary tumors
variously designatedas MtT, MStT, MtT. F4, MtT...W5, with F and W
referring to the Fisher and Wistar strains of rats, respectively.

Characteristics of MammosomatotrOpic
Pituitary Tumors

 

 

Most of the estrogen-induced mammosomatotropic tumors

initially grew only in estrogen-primed rats (Furth e_ta_l. , 19 56).
However, after a few passages they grew in untreated rats of the same
inbred strain. Talwalker and Meites (1966) succeeded in transplant-
ing an MtT. F4 tumor, originally induced in highly inbred Fisher
rats, into randomly bred Sprague-Dawley rats. The tranSplants
grew slowly, never attained large size, the latency period was pro-
longed and the nurnber of successful "takes" was small. Subsequently
the tumor could be transplanted readily into other Sprague-Dawley
rats and it secreted large amounts of prolactin, GH and ACTH.
Most of the tissue in mammosomatotrOpic tumors consists of acido-
phils (LaCour, 1950; Clifton and Meyer, 19 56). Farquhar and Furth
(1959) reported that a unique feature of these cells was the presence
of large cytoplasmic granules, some of which were similar to the

granules in acidOphils of normal pituitaries. MammosomatotrOpic

 

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28

tumors produce extensive hyperplasia of the mammary glands of
intact or gonadectomized hosts (Furth and Clifton, 1958),
Talwalker and Meites (1964a) reported that transplants of MtT. F4
induced extensive lobulo-alveolar growth in adreno-ovariectomized
rats. However there was only slight mammary secretion. The host
rats also exhibited rapid body growth as indicated by increase in the
length of the body and bones and enlargement of liver, spleen,
adrenal cortex and other organs of the body (Furth e_t§_l. , 19 69b).
Rats carrying these tumor transplants have also been reported to
develOp mammary tumors (Meites, 1972a).
Hormone Secretion by Mammosomatotropic
Pituitary Tumors

Most of the mammosomatotropic tumors secrete large amounts
of GH, prolactin and varying amounts of ACTH. Some of the earlier
tumors secreted large amounts of ACTH and caused several fold
increases in plasma corticoid levels (Takemoto _e_t__a_l. , 1962; Cohen
and Kim, 1963). However, some tumors secrete large amounts of
GH and prolactin and little or no ACTH as the MtT. W15 tumor (J.
Furth, personal communication). Mizuno it. a}: (1964) reported that
treatment with estradiol, cortisol acetate or thyroxine of the tumor-
bearing rats caused significant increases in plasma prolactin levels.

Although the pituitary tumors synthesize hormones at extremely

333155 SLY '{ETV ST‘

4

Ivv-O~~Q. 9“:- «. ‘0 4'
-«:-¢.u o L.:S¢e ..SC

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29

rapid rates only very small quantities of the hormones are present

in the tumor tissue itself. Thus, Bates _e_tgl. (1956) reported that
prolactin concentration in tumors was about 3% of that in the normal
rat pituitary. On the other hand the plasma level was more than 50
times the level in normal plasma. GH concentration in tumors was
about 14% of that in the normal pituitary but the plasma level was

30 times the level in normal plasma. Similar relationships were
found for ACTH. Talwalker gt al. (1964a) reported that the amount of
prolactin released into the medium by MtT. F4 tissue during 3 days
of culture was 8-31 times greater than that present initially, indicat-
ing active synthesis and release of prolactin by the tumor, leaving
little in the tissue itself. Meites and co-workers confirmed their
own findings in a subsequent paper (Sinha and Meites, 1967). They
reported that transplantable MtT. W15 contained very little prolactin
but upon incubation for 4 hours it released about 25 times more
prolactin into the medium than was initially present in the fresh
tissue. Meites and co-workers (Chen _e_t_§_1_. , 1967) were the first to
report that high concentrations of prolactin in the blood of tumor
bearing rats significantly increased PIF content and reduced pituitary
prolactin concentration and pituitary weight in the hosts. The high
amounts of PIF in the hypothalami seemed to have no detectable effect
on the secretion rate of pituitary tumors which are situated at a

considerable distance from the hypothalamus. However the tumor is

.. . . .
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susceptible to the inhibitory effect of the hypothalamus as was demon-
strated by Sinha and Meites (1967). They found that when the tumor
was incubated together with rat hypothalami extract for 4 hours,
prolactin release was significantly depressed whereas GH release
was significantly increased. This indicated effectiveness of the
direct action of the hypothalamus 123.3332: even though the tumor
appeared to be free of hypothalamic control Elli/.9. (Sinha and Meites,
1967). Prolactin secretion from pituitary tumor transplants .2 E12
can be markedly reduced if the tumor bearing rats are treated with
ergot derivatives which inhibit prolactin release both by a direct
action on the pituitary and also by increasing PIF, the former being
the major mechanism of action (Quadri and Meites, 1973). Daily
treatment with ergot derivatives also induces significant regression

of pituitary tumor growth (Quadri §_t_ _e_t_l. , 1972).

Carcinogenesis

 

The history of cancer is almost as old as the history of medicine
itself. Attossa, the wife of Darius I of Persia, is known to have
suffered from breast cancer in the fourth century B. C. Amputation
of the breast was performed as early as the first century A.D. and
in the seventh century Paul of Aegina stated that

. . . cancer-- is particularly frequent in the breasts of

women, because owing to their laxity, they readily
admit thick humors which occasion it-- for cancers are

fcrmed by black 'I.

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31

formed by black bile, over—heated. ---The veins are

filled and stretched around like the feet of the animal

called cancer (crab) (Willis, 1967).

In this age it would be unwise to comment on women's laxity, but

Paul of Aegina was not too much off the mark about a possible relation
of bile to cancer; it will be described later in this review that a very
potent carcinogen has been prepared from bile acids.

Fundamental advances in description, classification and hist-
ology of tumors were made in the 19th century. In 1875 Novinsky,
a Russian veterinarian, performed the first successful transplanta-
tion of tumors in dogs. This was followed by extensive work on
transplantation by Loeb in the United States and by Jensen in
Denmark. Apolant (1906) was the first to recognize that mammary
tumors in mice develop from hyperplastic nodules or hyperplastic
plaques. This was confirmed later by others (Gardner, 1941). In
1914 Yamagiwa and Ichikawa of Japan induced papillomas in rabbits
by repeated application of tar to the inner surface of the ear. This
was a landmark achievement, since it heralded the beginning of the
era of chemical carcinogenesis and soon "tar cancer was being
investigated in most cancer research institutes of the world”
(Foulds, 1969). The Japanese workers succeeded where others had
failed because, as Foulds explained, they chose the right species

and had the patience to continue treatment for many months. It is

3-331313! rats 32’.

t ‘.
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32

now known that rats and dogs are refractory whereas mice and rab-

bits respond well to tar application.

Definition

 

There is no universally acceptable definition of a tumor. It
can be explained as an abnormal mass of neOplastic tissue charact-
erized by uncoordinated and uncontrolled proliferation of cells which
multiply, invade adjacent areas or metastasize to distant sites, or
do all three, irrelevant to the needs of the body. NeOplastic cells
arise resembling their parental cells but may undergo histochemical
changes during their pursuit of endless self-propagation. Tumor
cells breed true to type and tumors exhibit varying degrees of auton-
omy and some may become completely independent of the stimuli
which evoked them. Similarly, they show varying degrees of ana-
plasia which is characterized by loss of organization, function and
resemblance to parental tissue. Neoplasms have a universal distribu-
tion in the animal kingdom and can originate from almost all tissues
of the body, but the degree of susceptibility of a given tissue varies
among Species and among strains in a single species. For example,
mammary cancer and lukemia are common in rats whereas breast
and lung cancers are common in the mouse; the Japanese have a high
incidence of stomach cancer and low incidence of breast cancer

whereas the opposite is true in the United States.

.:.Crs are C“““‘C'

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‘Iuisn CA:S€S, DeO¢ ~

 

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33

C la 5 sification

Tumors are divided on the basis of their growth behavior into
two broad classes, benign and malignant. The former are charact-
erized by slow local growth and little or no invasiveness whereas
the latter grow faster and show varying degrees of invasiveness into
adjacent tissues and metastasize to remote areas of the body. This
division is not absolute, since all possible gradations between the
two extremes can be observed. Tumors are classified on the basis
of the histogenesis as follows:

(1) Tumors of connective tissue, (a) benign tumors are des-
cribed by adding the suffix "oma" to the name of the tissue of origin
e. g. , fibroma (fibrous), myoma (muscular), lipoma (fatty), myxoma
(mucinous), osteoma (bony), chondroma (cartilaginous). Other
tumors in the class include osteoclasma, synovioma, angioma
(hemangioma, lymphagioma), meningioma etc.; (b) Malignant tumors
are called sarcomas with a prefix indicating the tissue or origin
e.g., fibrosarcoma, myosarcoma, liposarcoma (Foulds, 1969).

(2) Tumors of epithelial tissue, (a) benign tumors are called
papillomas (wart-like) or adenomas (gland-like) e. g. , fibroadenoma
(b) malignant tumors are called epitheliomas or carcinomas e. g. ,
mammary carcinoma, pulmonary carcinoma, vesical carcinoma

(bladder). If the epithelial cells in the carcinoma have a gland-like

| v
rage: at, the: 1328 '_
I

 

3' lurnors of 'm-

‘—;2t:c lukemia, p;

”ties. etc,

 

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34

arrangement, then the tumor is called adenocarcinoma, if the epi-
thelium resembles epidermis, then it is a squamous or epidermoid
carcinoma.

(3) Tumors of hemopoietic tissues include lymphosarcoma,
lymphatic lukemia, myelogenous lukemia, polycythemia, Hodgkin's
disease, etc.

(4) Tumors of nervous tissue are oligodendroglioma, neuri-
lemmoma, etc.

(5) Special tissue tumors include hepatoma, melanoma, etc. ,

(Willis, 1967).

Etiology

It is generally agreed that cancer is really a collective name
for a multitude of diseases. Therefore, it is not surprising to find
a variety of factors involved in the etiology of cancer. The following

is a brief description of some of them.

Heredity

On the basis of research in homozygous twins and in the so-
called cancer families, there are indications that certain types of
cancer , particularly breast and prostate cancer, might be inherited
(Clemmesen, 1951; Jarvik and Falek, 1962). In 1911, J. A. Murray

reported inheritance of susceptibility to mammary tumors in mice.

”o

 

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35

Later, mainly due to the efforts of Little and his colleagues, several
homozygous strains of mice were established, each showing greater
susceptibility to a particular kind of tumor (Little and Tyzzer, 1915;
Little and Strong, 1924; Little, 19 36). Now we have the C3H strain
which exhibits a high incidence of mammary tumors and hepatomas,
the BALB/c strain with high incidence of mammary tumors. Hered-
ity is not the only factor influencing tumor susceptibility in all homozy-
gous strains of mice. The cross-breeding experiments of Bittner
(1936, 1937, 1952) demonstrated that susceptibility of certain tumors
was due to a virus transmitted in milk. Similar experiments of
Foulds (1949) and Muhlbock (1950) indicated that high incidence of
spontaneous mammary tumors in the inbred descendents of C57BI

and R111 strains of mice was due to a virus transmitted in Sperms.
Virus

In 1908 Ellerman and Bang were the first to show that a
lukemia in the fowl could be transmitted by a cell-free extract. In
1911 Rous established that fowl sarcoma was caused by a virus and
in 1933 Shope and Hurst demonstrated that skin papillomas in cotton-
tail rabbits could be transmitted by a cell-free extract containing a
virus. In a very interesting study Rous and Friedwald investigated
the combined effects of Shope virus and carcinogenic hydrocarbons

from tar (Rous and Friedwald, 1944; Friedwald and Rous, 1950).

1.1- that prior tr1
*- and at ow: r.

~21: that mamma 3V

sensed hr 3 virus 1

p

1.3. 3.355 aroused rm

v A“

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36

They found that prior treatment with the carcinogens accelerated the
development and growth of virus-induced tumors. In 1936 Bittner had
reported that mammary tumors in certain homozygous strains of mice
were caused by a virus transmitted in milk. After a lapse of 15
years, Gross aroused new interest in mice viruses by demonstrating
cell-free transmission of mouse lukemia (Gross, 1951, 1961). The
virus had to be inoculated immediately after birth to be effective.
Subsequently viruses were found to be involved in lukemias in several
inbred strains of mice (Gross, 1961). In 1957 Stewart discovered

the polyoma virus which produces tumors in about twenty tissues in
newborn mice (Stewart and Eddy, 19 59). This virus was not Species
specific and produced tumors in both rats and mice. SV4O virus of
monkey and adenovirus of man also have been shown to produce

tumors in laboratory animals.

Parasites

Some metazoan parasites are carcinogenic, i. e. , Cysticercus
fasiolaris and Cysticercus crassiollis which produce cancer of the
liver and peritoneum in rats, and Schistosoma hematobium which
produces cancer of the bladder in men (Dunning and Curtis, 1946;

Mustacchi and Shimkin, 1958).

. . M-
dwnuU“

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31.2: and 1:.tra'; 1.

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37

Radiation

Solar and ultraviolet radiation can cause skin cancer in man,
mice and rats (Doniach and Mottram, 1940). Roentgen rays or x-
rays have been reported to produce lukemia and cancer of the
thyroid in man and skin cancer in man and rabbits (Heuper, 1954).
Ionized radiation produced lukemias in man in Hiroshima and
Nagasaki. Several radioactive substances are carcinogenic in man
and animals. Parenteral administration of radium, uranium, meso-
thorium and Thorostat or exposure to pure gamma rays have resulted
in production of cancers of soft tissues and bones (Barnes and

Schoental, 19 58).

Biological Substance s

Penile smegma can produce cancer of the penis and cervix
in man and horses and cancer of the skin in mice (Klienberg e_t§._l. ,
1941; Pratt-Thomas, 1957). Human liver extracts are carcinogenic
in laboratory animals (Klienberg _e_tgl. , 1941). Bagg and HagOpian
(19 39) induced mammary tumors in mice by ligation of the milk ducts,
suggesting that local irritation due to retained milk metabolites may
have produced the carcinogenic effect. Some very potent carcino-

genic hydrocarbons have been extracted from bile acids (see below).

 

.-
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38

Hormones

The carcinogenic potential of steroids has been the subject of
much research since the turn of the century. They bear a striking
resemblance in chemical structure to carcinogenic hydrocarbons
(Figure 2). In 1916 LathrOp and Loeb were the first to report that
ovariectomy performed in early age resulted in a decreased incidence
of mammary tumors in mice, but ovariectomy performed at the age
of six months had no effect. Carcinogenic effects of steroids were
also reported by W. S. Murray (1928) who induced mammary tumors
in castrated male mice by transplantation of ovaries. A clearer
demonstration of the carcinogenic effect of steroids was provided
by Lacassagne in 1932-33, who produced mammary cancers in male
and female mice by repeated injections of ”folliculin'l benzoate
(estrone benzoate). Estrogens have also been reported to have
carcinogenic effects on other tissues of the body. Repeated estrogen
administration leads to lukemia and cancer of the cervix, uterus
and pituitary (Gardner, 1953). The effects of steroids on mammary
tumors in rats will be discussed in other sections of this review.

It is not known how steroids exert their carcinogenic effects; perhaps
they are converted to carcinogenic hydrocarbons or produce tumor-
igenic effects after excessive and sustained physiologic actions on

their target tissue. Pituitary hormones have also been shown to

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39

have carcinogenic actions under certain conditions. It has been
reported that tumors of the testis and ovary were produced when
they were transplanted to the spleens of castrated mice and rats
(Twombly fig}. , 1949; Biskind and Biskind, 1949; Furth, 1947).
It was interpreted that gonadal hormones from the grafts were
inactivated by liver, resulting in increased production of pituitary

gonadotropins which in turn caused gonadal tumorigenesis.

Hydrocarbons

It was mentioned earlier that in 1914 Yamagiwa and Ichikawa

were the first to induce experimental tumors in rabbit skin by

repeated application of tar. A year later they also induced skin

carcinoma and demonstrated metastesis to lymph nodes. Since tar

is a mixture of several substances the next logical step was purifica-

tion of the active ingredients. The first carcinogen isolated from

tar was a polycyclic hydrocarbon called 1:2:5z6-dibenzanthracene
(dibenz (a, h) anthracene, DBA) (Kennaway and Heiger, 1930;
Kennaway, 1952). This compound is really a derivative of 1:2-
benzanthracene with an additional benzene ring. Further modifica-
tions and substitutions in the benzathracene nucleus resulted in
PrOduction of several derivatives like 5-methy1-l:2 benzanthracene

With an additional benzene ring. Further modifications and substitu-

ti o . .
ons 1n the benzanthracene nucleus resulted in production of several

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40

derivatives like 5-methy1-1:2 benzanthracene and 10-methy1-1:2
benzanthracene,’ etc. , (Daudel and Daudel, 1966). Most potent of
all such derivatives was a compound called 9:10-dimethy1-122-
benzanthracene (later named 7: lZ-dimethylbenz (a) anthracene,
DMBA) which is now widely used for mammary tumor induction in
rats. Another very strong carcinogen isolated from tar was 3:4
benzpyrene (benz (a) pyrene, BP) which is also a derivative of
benzanthracene with one additional benzene ring (Figure 3). Of
particular interest was the synthesis of 20-methylcholanthrene
(3-methy1cholanthrene, 3MC) from cholic acid and deoxycholic acid
of bile. This indicated that some carcinogens might arise from
normal constituents of the body. This hydrocarbon is also a
benzanthracene derivative. In general it was found that carcinogenic
activity appeared first in carcinogens containing four fused benzene
rings as in 1:2-benzanthracene and 3:4 benzanthracene. Five fused
benzene rings were more potent carcinogens as in the case of 3:4
benzpyrene. Hydrocarbons with six benzene rings are also carcino-
gens but not necessarily very potent as in the case of 3:4:9:10-
dibenzpyrene. Methylation of hydrocarbons increased or decreased
--the carcinogenic index, for example methylation in the 10 position
increased carcinogenic potency but methylation in the 5 position
decreased it. Methylation in two appropriate positions made the

carcinogen very potent as in the case of 9:10-dimethy1-];:'2

 

 

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41

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42

benzanthracene, DMBA (Daudel and Daudel, 1966).

Some hydrocarbons not derived from benzanthracene but
closely resembling it also are carcinogenic e. g. , 1:2:5z6-
dibenzfluorene, 1:2:5z6-dibenzacridine, 1:2:5z6-dibenzcarbazole and
aromatic amines like 2-naphthy1ene and Z-acetylaminofluorene.

Hydrocarbons induce tumors in several tissues of the body
including skin, lungs, viscera, mammary gland, nervous system
and various glands. Most of the hydrocarbons administered are
excreted in urine and bile and only a small quantity is retained in
the body (Haddow, 19 58). It is generally accepted that the cancerous
state produced by hydrocarbons is preceded by a proliferative state
marked by increased mitosis (Glucksman, 1945). Several factors
influence the carcinogenic capacity of hydrocarbons. Some of these
are:

(1) Route of administration. Skin painting of benzacridines
induces epitheliomas but subcutaneous injection produces sarcomas.
Intranasal administration of DMBA produces mammary and lung
cancer but intraperitoneal administration induces only lung cancer.
Single intragastric feeding or intravenous injection of DMBA produces
mammary tumors in rats while feeding of the same carcinogen
results in induction of gastric tumors in mice. Implantation of DMBA
pellets in the pituitary gland results in production of pituitary

tumors (Cheetham, l9 63).

 

 

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43

(2) Role of solvent: Methylcholanthrene is a potent carcinogen
if benzene or lard is used as a vehicle but completely inactive if
lanolin is the solvent.

(3) Dose: The dose of a carcinogen affects the latent period
and incidence of tumors. There is an inverse relationship between
the latent period of tumors in mice and dose of benzpyrene; and a

direct relationship between dose and incidence of tumors.

Miscellaneous Carcinogens

Several unrelated substances like derivatives of iron, nickel, '
plastics (cellophane), silver foils, chloroform, HCl, and even
distilled water(?) are carcinogenic in certain situations (Foulds,
1969; Daudel and Daudel, 1966; Willis, 1967). Urethane (ethyl
carbamate) produces tumors of lung and skin, and goitrogens like
thiouracil and thiourea produce thyroid tumors due to excessive

stimulation of the thyroid by thyrotropin.

Mechanism of Carcinogenesis

Little is known about the mechanism of carcinogenesis. There
seems to be general agreement that perhaps there is no single
mechanism of carcinogenesis. This is not very surprising because
literally hundreds of carcinogens are metabolized and excreted by

different methods and therefore affect the tissues differently. The

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44

problem becomes more complicated when one considers the multitude
of tissues which are susceptible to tumorigenesis and the hundreds
of cellular reactions influenced by carcinogens. This finally leads
us to the definition of cancer as not a single disease but as consist-
ing of a variety of diseases, and therefore it is not unexpected that
scores of hypotheses have been advanced to explain the origin of
neoplasms (see review by Daudel and Daudel, 1966). One of the
earliest was the hypothesis of unicentric origin proposed by Cohnheim
and later vigorously supported by Ribbert. It envisioned that neo-
plasms originated from a single cellor groups of cells (embryonic
cell rests) which failed to develop during the embryonic period but
started to grow at a rapid rate during the later part of life. This
theory is now generally discarded in view of the findings that tumors
arise from several foci extending over large tissue areas, i. e. the
multifocal or multicentric theory (Muir, 1941). The mutational
hypothesis assumes that neOplastic cells arise due to genetic muta-
tions induced by chemical carcinogens or due to incorporation of
viral nucleic acids into cellular nuclear acids. The chronic irrita-
tion hypothesis proposes that persistant stimulation of tissues by
irritation precipitates into neoplasia (Willis, 1967). The immuno-
logical hypothesis postulates that cancerous cells lose some "tissue
specific" proteins, become antigenically inert and therefore become

evasive (Haddow, 1965). This hypothesis also has been severely

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45

criticized. Some of the recent hypotheses are less vague and, more
importantly, are backed by experimental data. Hishizawa _e_t_ El-
(1964) found that methylcholanthrene increased incorporation of
radioactive precursors into RNA, and Gelbibn and coworkers (1961)
found that the same carcinogen also increased incorporation of RNA-
bourid amino (acids into proteins. Chang and Bond (1964) reported
that DMBA andimethylcholanthrene either accelerated or inhibited
fixation of amino acids on sRNA depending on the nature of the amino
acids involved. Proponents of the deletion theory found formation

of a protein-carcinogenic hydrocarbon complex which resulted in
inhibition of protein (enzyme) synthesis leading to uncontrolled
cellular proliferation (Daudel and Daudel, 19 66). Pitot and
Heidelberger (1963) tried to explain carcinogenesis on the basis of
Jacob and Monod's theories of regulation of synthesis in cells. This
involved combination of carcinogens with enzymes resulting in
inhibition of some enzymes and stimulation of others with the net
result being loss of control of growth rate. None of these hypotheses

are applicable to all carcinogens.

Chemothe rapy of Canc er

This review would be incomplete without a brief mention of
chemotherapy of cancer. Potassium arsenite has been in use for

treatment of lukemia since 1865 when it was introduced by Lassauer

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46

(Ackerman and DelRegato, 1962). Nitrogen mustard was introduced
as a chemotherapeutic agent during the Second World War. In 1889,
Schinzinger (Hayward, 1970) was the first to suggest and in 1895
Beatson was the first to perform an ovariectomy for treatment of
breast cancer. Beatson apologized for performing such a drastic
operation by saying that he had "been activated --- primarily by the
interests of those who place themselves under our care and second-
arily, the progress and the advancement of the healing art" (Hayward,
1970). After the successful treatment of breast cancer with ovari-
ectomy, Beatson very bravely rejected the then prevalent theory of
the bacterial basis of cancer and admonished his contemporaries that
the ”---parasitic theory of cancer is unsatisfactory --- we are losing
time and searching for what will never be found, simply because it
does not exist. ” In 1905 DeCourmelles treated breast cancer by
irradiation of the ovaries. Androgens and synthetic estrogens like
triphenylchlorethylene, triphenylmethylethylene and the most effec-
tive estrogen of all, diethylstilbestrol, were introduced for treatment
of breast cancer in the 1940's. Most of the credit for revival of
hormonal treatment for cancer is generally given to Huggins, who

in the 1940's and the 1950's, also introduced adrenalectomy for
treatment of prostate and breast cancer. In 1953 Luft and Olivecrona
introduced hypophysectomy to control breast cancer. Cortisteroids

have been used for treatment of lukemia, and progesterones for

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47

treatment of cancer of the uterus. Folic acid antagonists have been
used for acute lukemia, and antibiotics and bacterial products includ-
ing actinomycin D and mitomycin C have been shown to have anti-
tumor effects. The Cancer Chemotherapy National Service Center
has tested almost 100, 000 chemicals in search of suitable chemo-

therapeutic agents for cancer.
Surgery and Radiotherapy

Total extirpation of malignant tumors before onset of metastasis
is a very successful technique. Irradiation results in immediate
arrest of mitosis in malignant tumors and ultimately in complete
destruction of neoplastic cells. Unfortunately not all tumor cells
respond equally well to radiation therapy. Despite this and other
drawbacks, radiation and surgery are still considered to be the best

treatments for cance r.

Spontaneous Mammary Tumors in the Rat

Incidence

In man cancer is the second leading cause of death, next to
heart diseases. It is estimated that one of every six deaths in the
U.S. is due to cancer. In 1973 it is estimated that a total of

350, 000 Americans (14, 400 in Michigan) will die of cancer, at the

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48

rate of 960 persons a day or more than one every two minutes.

About 53 million Americans now living or two of every three families
or one of every four persons will eventually develop cancer. Of
every six persons who get cancer, four die (Cancer, Fact and
Figures, 1973, American Cancer Society, Inc. ). In a community of
200, 000 persons, 50, 000 will develop cancer of whom 30, 000 will die
if the present trends of diagnosis and treatments continue. The
population of metropolitan Lansing including suburbs was 268, 000 in
January 1970. Cancer is a common occurrence in laboratory rats.
Curtis $31. (19 53) estimated that more than 60% of all rats develop
tumors of some tissue during their normal life span.

In the U. S.A. breast cancer is the most common type of cancer
among women, and one in every 14 get it. Of every four women
developing cancer one gets cancer of the breast, and of every four
women who die from cancer one dies of breast cancer. It is esti-
mated that in 1973 there will be approximately 74, 000 new cases of
breast cancer and 33, 000 will die from it. Spontaneous mammary
cancer is also the most common type of cancer in female rats.
Ratcliff (1940) observed two colonies of rats at Wistar Institute
over a period of 5 years and concluded that the mammary gland was
the most common focus of neoplastic growth in females. Bullock
and Curtis (1930) estimated that up to 90% of all tumors in female

rats could be of mammary origin.

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49

Some Factors Affecting Incidence
of Breast Cancer

 

 

Sex

Breast cancer occurs only occasionally in men (Cancer of
Breast, American Cancer Society, Inc. ). Similarly, it is rare in
male rats. More than 5070 of Sprague-Dawley females deve10p
mammary tumors by the time they are two years old (Meites g g}. ,
1972), whereas only 1-6% of male rats develop tumors of mammary

origin (Noble and Cutts, 1959).
Age

Breast cancer is extremely rare in children (Cancer of the
Breast, American Cancer Society, Inc. ), although more school'
children die of cancer, particularly lukemia, than from any other
disease (Cancer, Fact and Figures, American Cancer Society, Inc. ).
The frequency of cancer of all kinds increases with age, and it is
estimated that more than 50% of all persons over the age of 65 die
of cancer. The incidence of breast cancer is highest among women
past the age of 44 years. Following a similar course, mammary
tumors begin to appear with increasing frequency in female rats
33 they get old. Some investigators (Huggins _e_t_al. , 1965a; Glenn

SELL . 1959a) have stated that mammary tumors seldom, if ever,

:2: ":cre the age
:axxrzence it is n

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50

appear before the age of 18 months in Sprague-Dawley rats. From
our experience it is not uncommon to find mammary tumors develop-

ing in female rats when they are just a year old or even younger.

Heredity

Breast cancer is extremely rare in monkeys, but is a common
occurrence in several other species including mice, rats and human
beings. The incidence of breast cancer varies from strain to strain
within each Species. It appears in about 50% of the females of the
Sprague-Dawley strain but strikes in about 70% of the Wistar strain
(Ratcliff, 1940), and only in about 20770 in the Long-Evans strain
(unpublished data). In women the frequency of breast cancer fluctu-
ates along racial and geographical lines. Unlike the disturbingly
large percentage of women who suffer from breast cancer in the U. S. ,
the proportion of women who are similarly affected in Japan is very
low. The situation is reversed for stomach cancer in these countries
(Willis, 1967). In the U. S. the incidence of cervical cancer is higher
in blacks than in whites, but blacks have lower rates of breast and
uterine cancers (Cancer, Facts and Figures, 1973, American Cancer
Society, Inc. ). Genetic factors seem to influence the incidence of
cancer still farther down at the family level. In the 1950's Dight
Institute for Human Genetics in Minnesota conducted an extensive

study to determine if there was a greater predisposition to cancer

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51

among relatives of breast cancer patients (Anderson gt a}. , 19 58).
Their findings were affirmative. Now it is generally accepted that
daughters and sisters of women with breast cancer may have a
"somewhat greater risk of developing the disease than women without
such history" (Cancer of the Breast, American Cancer Society,

Inc. ).

Importance of Spontaneous Mammary Tumors
of the Rat

 

 

There are close similarities between rat mammary tumors
and certain cancers of the human breast. In 1934 Jacob Heiman
observed a mammary fibroadenoma of a rat "suddenly --- change
into an actively growing sarcomata. " This aroused considerable
interest because there were examples of similar transformations
occurring in human beings where slow-growing, non-metastasizing
fibroadenomas had suddenly changed into sarcomas or had shown
prompt recurrence after incomplete removal (Heiman, 1934). In
1896 Beatson was the first to show hormone-dependency of certain
cancers of the human breast. Now we know that several human
breast cancers regress following ovariectomy, adrenalectomy and
hyp0physectomy (Huggins £22}.- , 1956a). Spontaneous mammary
tumors of the rat show similar hormone responsiveness. Because

of these and other characteristics, rat mammary tumors are

2mm experimer.‘

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52

"important experimental models'' (Huggins 31: 3.}, , 19 56a) for research
in mammary carcinogenesis and serve as "important analytical tools"
for testing anticancer agents (Glenn _e_t_ a}. , 19 59a). The only draw-
back is their late appearance, but this has been overcome by the use
of tumor transplants. As a matter of fact, most of the research on
spontaneous tumors of the rat has actually been done on transplants

of these tumors.

Histological and Growth Characteristics

 

Spontaneous mammary tumors can be defined as the tumors
which develop in the absence of any known extrinsic stimulus. In
the rat they usually appear as a. single tumor per rat (Meites g g}, ,
1972) and only rarely are two or three tumors found in the same rat
(unpublished observations). The majority of these tumors are
fibroadenomas, less frequently adenomas and rarely adenolipomas
or fibromas (Bullock and Curtis, 1930). An extremely small per-
centage may be malignant, and if so, they are mostly fibrosarcomas,
sarcomas and only rarely adenosarcomas (Noble and Cutts, 19 59).
In a study involving approximately 16, 000 rats of the Fischer and
Copenhagen strains, Dunning and Curtis (1956) observed only two
malignant mammary tumors whereas Davis 3133.}. (1956) found 10%
of the mammary tumors to be adenocarcinoma in Sprague-Dawley

rats. Ratcliff (1940) claimed that up to 30% of the tumors in Wistar

a: rats were ma..
mines in 1000 r:

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53

strain rats were malignant, and Dao (1964) encountered 50 adeno-
carcinomas in 1000 rats but failed to mention the proportion of malig-
nant tumors in relation to benign tumors. On the basis of these
reports it appears that some strains of rats are more prone to
develop malignant mammary tumors than others.

It has been shown that the growth characteristics and response
of spontaneous mammary tumors to alterations in hormonal environ-
ment are influenced by their histology (Millar and Noble, 1952,
1954a, b, c). The histology of these tumors has been extensively
reviewed (Bullock and Curtis, 1930; Curtis _e_t_a_1_. , 1933; Heiman,
1934; Wright flil- , 1940). Mammary fibroadenomas appear as
large, firm, encapsulated growths, some lobulated, usually whitish
or pinkish in color and easily separable from skin and fascia
(Heiman, 1936). Microsc0pically they show connective tissue strands
scattered among epithelial cells and duct formations. The gland
cells are cuboidal in appearance with large nuclei. Interspaced among
the stroma fibers are spindle-shaped connective tissue cells and
basophils of connective tissue. Vascularization iS'usually poor.
Robinson and Grauer (1952) found that the ducts and acini were lined
by one or two layers of epithelial cells, some with mitotic figures.
Some of the supportive tissue was hyalinized and showed proliferative
activity. Seilbie (1949) also reported finding bony or cartilagenous

hyalinization of connective tissue in tumor transplants. The

 

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54

proportion of epithelial tissue to fibrous connective tissue varies
between the extremes of adenoma and fibroma (Millar and

Noble, 1954a). Folly and Greenbaum (1947) had reported that
normal mammary glands contained high concentrations of alkaline
phosphatase in the epithelial cells. Huggins 3331. (19 56) reported
that in mammary tumors alkaline phOSphatase content was high in
both the epithelial cells and the blood capillaries, and that there was
a positive correlation between tumor growth and increase in alkaline
phosphatase concentrations.

Heiman (1934) reported that the alveoli in mammary tumors of
pregnant rats contained a pale homogenous secretion. This was
confirmed by Emge and Murray (1938) who reported that epithelium
or adenomatous tissue of tumors underwent hyperplasia during
pregnancy and contained milk after parturition. Robinson and Grauer
also found lactating adenomas in rats and women during pregnancy

(Robinson and Grauer, 19 52; Grauer and Robinson, 1932).

Transformation

A unique feature of mammary tumors in rats and human beings
is their ability to undergo transformations. Heiman (1934) was the
first to report transformation of a benign rat mammary fibroadenoma
into a malignant sarcoma during transplantations. The transformed

sarcoma consisted of polyhydral or spindle—shaped sarcoma cells

72 5122;. amounts OI

 

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with small amounts of interstitial tissue, a large number of mitotic
figures and was carried through 207 successive transplantations
without further transformations. Subsequent investigations reported
that transformations of fibroadenomas into fibrosarcomas were not
infrequent during transplantations‘(Millar and Noble, 19 54a). The
alternative course a tumor can take is to remain a fibroadenoma
during successive transplantations with only minor alterations in

the proportion of fibrous and epithelial tissues or to lose parenchyma
and become a fibroma' composed of white fibrous tissue, or take the
other alternative and become an adenoma (Millar and Noble, 1954a).
Selbie (19 42) was the only investigator to report transformation of a
fibroadenoma into a carcinoma. Transformed fibrosarcomas or
sarcomas have all the characteristics of malignant growths except
the ability to metastasize (Millar and Noble, 19 54a). Fibrosarcomas
also grow faster and have more deleterious effects on the health of
rats, causing loss of weight and death (Millar and Noble, 19 54c).
Millar and Noble (1954a) also estimated that fibrosarcomatous
transformations occur in 15% of all cases. Hormones, particularly
androgenic steroids, have been repeatedly shown to bring about
frequent transformations of rat mammary tumors. The important
feature of transformed tumors is that they frequently lose their

hormone-reSponsiveness, which created considerable confusion in

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earlier days. This topic will be discussed in the latter part of this

review.
Transplantation

As was mentioned before, most of the early investigative work
on spontaneous mammary tumors of rats was actually carried out on
the transplants of these tumors in rats of the same strain. Loeb
(1902) was the first to demonstrate successful autotransplantation of
a rat mammary adenoma into the other side of the abdominal wall.

In 1916 Loeb and Fleisher carried out the first homeotransplantation
of a mammary adenoma in rats. Most of the early workers reported
only a.40-75% incidence of successful takes (Heiman and Krehbiel,
1936; Mohs, 1940a,b; Millar and Noble, 1954a), but in the 1950's
Huggins gt _a_l_. , refined the transplantation techniques and raised the
number of successful takes to almost 100% (Huggins 9321., 19 56a, b).
They transplanted several "seeds" in each rat and noted that not all
transplants grew in every rat and they had neither the same size or
growth rate. They found that for a tran8plant to be able to grow, it
was essential that it contained epithelial cells. Glenn _e_t_al. (1959a)
further standardized transplantation techniques by discarding rats

in which tumor tranSplants did not grow during the first 35 days or
in which the tumors grew too fast. They reported that transplants

grew slowly, if at all, during the initial three weeks (latent period),

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57

after which the growth was rapid. Huggins _e_tal. (1956a) reported
that during the growth period the increase in mean tumor weight
was progressive and logarithmic. They found that morphologically
the transplants consisted of spheroids encircled in a network of
capillaries and each Spheroid consisted of a duct system surrounded
by fibroma cells.
Neuroendocrine Control of Deve10pment and Growth
of Spontaneous Mammary Tumors in the Rat

Soon after Beatson's demonstration at the end of the last cen-
tury that the ovaries were involved in human breast cancer, Loeb
presented evidence of hormonal influences in the development and
growth of mammary tumors in rats. In the early 1900's he reported
that mammary tumor transplants did not grow in males, and in
females they grew faster during pregnancy (Loeb, 1902; Loeb, 1916;
Loeb and Fleisher, 1916). Further proof of the importance of
gonadal hormones in mammary tumors came from the work of
Heiman, Emge and Mohs in the 19 30's and 1940's and of Millar,
Noble, Huggins and Glenn in the 1950's. Meites and co-workers,
in the late 1960's and early 1970's, provided convincing evidence
of the pituitary involvement and were the first to implicate the
hypothalamus in the development and growth of Spontaneous mammary

tumors in rats.

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Role of Ovaries

Estrogens. Heiman and Krehbiel (1936) were the first to

 

discover that ovariectomy retarded growth and reduced the incidence
of "takes” of mammary fibroadenomas in rats. They further demon-
strated that the inhibition of tumor growth could be prevented by
estrogen administration. This was confirmed by Mohs (1940a, b)

who found that transplantability and rate of growth of a pure mammary
fibroma also was enhanced by the presence of physiologic levels of
estrogen in the hosts and that the tumor stimulating effect of estro-
gens was greater in male castrates than in female castrates.

Millar and Noble (1954a, b, c) also found that estrogen was essential
for optimum tumor growth.

In 1940 Heiman reported that the main stimulating effect of
estrogens was on the epithelial components of spontaneous adeno-
fibromas, occasionally resulting in their transformation into adenoma
or cystadenoma. This was in contrast to the inhibitory effect of
androgens on the epithelial. elements of the tumors. Heiman further
found that estrogen in large doses was able to overcome the inhibit-
ory effect of androgens and exert a stimulating effect on the epi-
thelium. The connective tissue elements of the tumor were not
directly affected by~ estrogens and only moderately inhibited by

androgens. Most of Heiman's original findings have been confirmed

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except the transforming effect of estrogen on mammary fibro-
adenomas.

In 1952 Millar and Noble published a brief abstract reporting
for the first time a biphasic effect of estrogen on mammary tumor
growth. They elaborated their findings in a subsequent report
(Millar and Noble, 19 54b). They found that small doses of diethyl-
stilbestrol, DES, (1-10 pg daily) increased tumor growth rate and
epithelial development in the male rats equal to that in the female
rats, but high levels (50-200 pg daily) prevented tumor growth if
treatments were commenced after tumor growth initiation. It was
concluded, on the basis of paired-feeding experiments, that the
inhibitory effect of DES was not a non-Specific reflection of body
growth depression. They speculated that mammary fibroadenomas
were responding like normal immature mammary glands, which had
been shown to be inhibited by large doses of estrogen. The results
of these experiments confirmed earlier reports of similar inhibi-
tion by large doses of estrogen on malignant neoplasms in rats and
mice (Eisen, 1941; Nathanson and Salter, 1939) and in humans
(Nathanson and Kelly, 19 52).

The biphasic effects of estrOgen on tumor growth were con-
firmed by a careful study conducted by Huggins £35.11.- , in 19 56(b).
They reported that phenolic estrogens exerted biphasic effects on

the growth of mammary fibroadenoma transplants in

   
   
   

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60

ovariectomized rats but exerted only a monophasic effect on the
breasts of the hosts causing ductal deveIOpment. With progressive
increase in dosage there was at first an augmentation of tumor size
until a peak was reached with Optimal amounts beyond which there
was a profound decrease in the size of the tumor. The following were
considered to be the Optimum doses for various estrogens, 0. 1 pg
estradiol 1?, 1 pg estrone, 10—20 pg estriol, 50 pg estradiol 3, 16.
The following were the inhibitory doses, 20 pg estradiol 17, 50 pg
estrone, 100 pg estriol and a 10 mg DES pellet implanted subcutane-
ously. The biphasic effects of estrogens were also confirmed by
Glenngtal. (1959a, b).

Some of the earlier reports on the effects of estrogens on
mammary tumors were contradictory (Emge _e_t_.a_l_. , 1938a, b; Mohs,
1940a, b). These contradictions, we now realize, arose for several
reasons including lack of standardized methods of transplantation
resulting in non-uniform transplants or use of transplants that
might have lost their hormone-responsiveness (Huggins e_t_a_l. ,
1956a, b; Millar and Noble, 1954a, b, c; Mohs, 19403, b).

Progesterone. Early reports on the effects of progesterone
on spontaneous mammary tumors were also confusing. Heiman
(1943) reported that progesterone inhibited growth of the adenomatous
portion of mammary fibroadenomas which resulted in shrinkage of

the tumors followed by fibrosis. He also found that progesterone

“fixed the numbe r

 

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61

reduced the number of "takes" and overcame the stimulatory effect
of estrogen on tumor growth. In a subsequent report he confirmed
his earlier findings about the inhibitory effects of progesterone on
number of takes and on epithelial elements of the tumor, but found
that it had no effect on fibromas and that its inhibitory actions were
reinforced when combined with testosterone. However, pregnancy
or simultaneous injections of estrogen were able to offset the
inhibitory actions of progesterone and testosterone (Heiman, 1943).
Heiman's findings were not confirmed by Millar and Noble (1954b)
who found that progesterone had no effect on growth or morphology
of fibroadenomas in female rats. They explained their results as
another example of varied susceptibility of mammary tumors to
hormones. Huggins £1511. (1956b) reported that small doses of
progesterone (1 mg) had no effect but larger doses (204 mg) stimu-
lated mammary tumor growth. Progesterone (1-4 mg) also stimu-
lated tumor growth in ovariectomized rats when injected with either
small or large doses of estrogens. It appeared that progesterone
was partly able to overcome the inhibitory effects of Large doses of
estrogens. Both the mammary fibroadenomas and the normal
mammary glands of the hosts showed characteristic gestational and
proliferative changes seen during pregnancy. Glenn _e_t_a_l_. (1959a)
confirmed most of the findings of Huggins _e_t _a_._l_. , and added that

progesterone treatment beginning at the time of tumor transplant

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enhanced tumor growth but had no effect on the growth of established
tumors, and prior administration of progesterone did not affect
number of ”takes".

Pregnancy. In general it appears that the majority of the
investigators found pregnancy to have a stimulating effect on tumor
transplants but great variation has been reported (Noble and Cutts,
1959). Loeb (1902) was the first to report the stimulating effects of
pregnancy on fibroadenoma transplants. This was confirmed by
several investigators (Robinson and Grauer, 1932; Heiman, 1934),
but Emge _e_t §._I_. , reported that pregnancy had no stimulating effect
on the growth of transplantable fibroadenoma and sarcoma (Emge and
Wolf, 1934;1Emge 9311. , l938a,b). Millar and Noble (1952) on the
contrary, confirmed the earlier findings of the stimulating effect

of pregnancy on benign fibroadenomas.
Role of Androgens

Loeb and Fleischer (1916) were the first to report that mam—
mary fibroadenoma transplants did not grow well in male rats. This
has been confirmed by several investigators (Robinson and Grauer,
1952; Heiman, 1934; Oberling _e_t_al. , 1937). Millar and Noble (1954b)
found that castration of males increased the growth of tumor trans-
plants, whereas castration of females decreased it. Heiman

(1940a, b) reported that administration of exogenous testosterone

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63

decreased the percentage of successful takes of fibroadenomas but
increased the number of takes if the tumors were fibromas or
sarcomas. He also found that testosterone exerted its inhibitory
effect mainly on the epithelial elements of fibroadenomas. This was
confirmed by Mohs who found that fibroadenomas often became pure
fibromas in males but retained their original histological character
in females or estrogen injected male castrates (Mohs, 1940a, b;
1941). Murphy (2 a_l. (19 38) reported that estrogens failed to prevent
the fibromatous transformation of adenofibromas in intact males.
Mohs found that adenofibromas varied in the degree to which they
responded to the fibroma-producing influence of androgens,,and only
when the epithelium was part of the tumors did they become sensi-
tive to the inhibitory effects of androgens. He also found that the
loss of epithelium due to testosterone treatment had no effect on the
tumor growth rate. This was in contradiction to Heiman's findings.
He had reported inhibitory effects of testosterone on both the epithe-
lial elements and growth rate. Later Millar and Noble (19 54b) con-
firmed Heiman's findings. Testosterone has been found to cause
regression of breast carcinomas in women also (Huggins, 1954).
Perhaps the most important series of experiments on the
effects of several androgens were carried out by Huggins and co-
workers (19 57). They found a quantitative relationship between the

dosage of steroids and the extent of inhibition of the tumors. They

   
   
 

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64

also correlated molecular structure of steroids to their inhibitory
effects on tumor growth. Androstan-3-one did not retard tumor
growth but androstan- 17 -01 was moderately effective, and dihydro-
testerone and 2 -methy1-androstan 17 -ol-3-one were powerful
inhibitors; 4-androstene-3, l7-dione was a weak inhibitor. The
growth inhibiting effects of most of the androgens could be increased
by increasing their dosage, but etiocholan-17 -ol-3-one and epi—~
testosterone hadno effect on tumor growth. They concluded that
androgenic inhibitors of tumor growth exert their effects by abolish-
ing the actions of estrogen and progesterone at the tumor cell level
and by inhibiting production of estrogens and progesterones from

the ovaries. Glenn £131. (1959b) confirmed most of the findings of
Huggins _e_t 9:1. , and reported that androgens exerted their antitumor
effects without producing any androgenic effects. They found that
the antitumor effects of 17-methyltestosterone, ll -hydroxy-l7-
methyltestosterone and 9, ll-epoxy- l7-methyltestosterone were the
same, but the androgenic activity of the last two compounds was
considerably less compared to that of the first compound. Similarly
Z-methyl—19-nortestosterone had less masculinizing effect in female
rats compared to 19-nortestosterone, but the two compounds had the

same antitumor activity.

  
 
   
 

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65

Role of the Adrenals

Millar and Noble (19 54b) reported that cortisone did not alter
the growth rate and morphology of tranSplanted fibroadenomas in
female rats. Removal of adrenal glands has been shown to result
in regression of some human breast cancers (Huggins gtgl. , 1956b).
Huggins and coworkers found that ovariectomy decreased mean
tumor weight of established fibroadenoma transplants from 540 mg to
180 mg, but removal of adrenals in addition to ovaries resulted in
greater reduction in mean tumor weight, to 104 mg per tumor.
Glenngtgl. (1959a) used tranSplants of the same fibroadenoma used
by Huggins gt gt. , and found that hydrocortisone (2 mg/day) prevented
body growth but had no effect on the tumor growth. However, if
hydrocortisone was injected 5 days prior to tumor transplantation, it

resulted in greater final tumor size 65 days later.
Role of the Pituitary

Grauer and Robinson (1932) were the first to carry out a
detailed investigation of the effect of lactation on Spontaneous mam-
mary tumors in the rat. They found that lactation in the tranSplanted
adenomas was concurrent with lactation in the breast during the
suckling period after which involution occurred simultaneously in

both the breast and the adenoma. Lactation in the adenoma did not

      

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66

depend on its proximity to the breast tissue since active secretion
occurred in the tumors transplanted within the peritoneal cavity.
They concluded that perhaps the adenoma was being influenced by a
”secretory hormone". In 1936 Heiman and Krehbiel reported that
estrogen (Theelin) in combination with growth hormone (Antuitrin S)
or gonadotropic hormone (Antuitrin G) of the anterior pituitary
increased the number of takes in normal females, although none of
these hormones were effective when used alone. Estrogen plus
gonadotropic hormone also increased the number of takes in male or
female castrates. They concluded that estrogen must be accompanied
by pituitary hormones for stimulation of tumor growth. But Mohs
found that estrogen alone was able to increase the number of takes
in castrates of both sexes. He speculated that the effect of estrogen
upon mammary adenofibroma was indirect through the anterior pitu-
itary, since C. W. Turner and coworkers had shown that estrogens
exerted their effect on mammary glands partly by liberating "mam-
magenic hormone" from the anterior pituitary (Mohs, 1940a, b).
Millar and Noble (1952, 1954b) reported that crude beef and sheep
pituitary extracts stimulated growth of fibroadenoma in intact female
rats. Beef pituitary extract was also effective in males and cas-
trated females. They Speculated that the most likely hormone
component in the pituitary extracts to stimulate tumor growth was

prolactin, since both the extracts had low GH and gonadotrOpic

."I

 

 

 

67

hormone activities and high prolactin activity. They further reported
that biologically pure GH failed to alter the growth or morphology

of the tumors in female rats. They found no synergism between
estrogen and the pituitary hormones to stimulate tumor growth as
was reported by Heiman and Krehbiel in 19 36. In a subsequent

study, Millar and Noble (1954c) found that the same pituitary extract
which stimulated the growth of fibroadenoma failed to have any effect
on the growth of fibrosarcoma, thus emphasizing the relationship
between histological characteristics of mammary tumors and their
hormone-responsiveness.

In the early 19 50's several experiments were conducted to
determine the effects of hyp0physectomy on spontaneous mammary
tumors in rats'and human beings. In 1951 Moon gt 3.1. , reported that
rats failed to develop Spontaneous mammary tumors if they were
hypophysectomized. In 1952-53 Olivecrona and Perrault gtgt. ,
demonstrated that hyp0physectomy caused regression of certain
human breast cancers.‘ Huggins gtgl. (19 56a, b) were the first to
Show that hyp0physectomy retarded growth of mammary fibro-
adenomas in female rats. However, after a period of 70 days, the
tumor growth resumed and no explanation for this phenomenon was
offered. In another experiment estrone plus progesterone induced
tumor growth in the hyp0physectomized rats, and this growth was

accelerated by additional administration of GH or lactogenic

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68

hormone. None of the hormones individually stimulated tumor growth.
In 1971(b) Quadri and Meites reported that two ergot derivatives,
ergocornine and ergocryptin, induced marked regression of spontane-
ous mammary tumors in female rats. It was concluded that the

drugs inhibited tumor growth by depressing prolactin secretion from
the pituitary gland. After termination of the treatment prompt
resumption of tumor growth was observed.

In the late 1960's and early 1970's Meites and coworkers carried
out several experiments to demonstrate that elevations of prolactin
levels lead to increased incidence and to a decreased latency period
for appearance of spontaneous mammary tumors in the rat. These
experiments will be described in the following sections of this review
since, in the strict sense of the word, these tumors were not spontane-
ous but were induced by increasing serum prolactin levels by several

procedures.

Role of the Hypothalamus

Meites and coworkers were the first to directly implicate the
hypothalamus in mammary tumorigenesis in the rat. This topic will

be considered in detail in the latter part of this review.

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69

_Igduced Mammary Cancers in the Rat

Ignpo rtance

As mentioned earlier, spontaneous mammary tumors in the
rat are generally benign, have a low incidence, develop late in life
and Show considerable variation in their cytology, growth charac-
teristics and hormone responsiveness. Because of these and for
other reasons they are not very suitable for experimental work.

By contrast, induced mammary tumors of the rat have proved to be

hydrocarbons. Huggins _e_tgl. (1965) believe that the rat mammary
gland is surpassed in susceptibility to cancer only by the chicken

cells inoculated with Rous sarcoma virus. Malignant mammary

. they closely resemble hormone-responsive human

breast cancers and have already proved of value .

 

 

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70

Induction

It took more than half a century after Yamagawa and Ichikawa
induced tumors in 1914 to synthesize suitable carcinogenic agents
and perfect a method for induction of mammary cancers in the rat.
Lacassagne, Geschickter, Maisin, Coolen, Dunning, Wieland, Dane,
Bachman, Chemerda, Shay, Geyer and Huggins were just a few of
the many scientists who devoted their energies and thoughts to this
area. Today the most common method used to induce mammary
cancers in the rat is through the use of aromatic hydrocarbons,
particularly 7, lZ-dimethylbenz (a) anthracene. Several other hydro-

carbons, irridation and estrogens have been extensively used in the

past.

Irradiation

In 1954 Hamilton, Durbin and Parrot were the first to induce
mammary cancer in rats by irradiation. They gave a single injection
of Astatine211 (EKA-iodine) to 55 day old Sprague-Dawley rats and
induced mammary cancer in about 40% of the rats. Induction of
mammary cancers in rats also was produced by total body irradiation
with x-rays (Maisingtgl. , 1956) or gamma rays (Cronkite gtgt. ,
1960). Malignant tumors can be induced by radiation at an early age

but the latent periods for the appearance of cancers are long, often

~2:2: for a per:
allsercentage o:

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Lacassagne (1

 

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71

extending for a period of several months after which only a relatively
small percentage of treated rats develop tumors. Because of these
drawbacks, this method of inducing mammary tumors in the rat is

not in vogue.

Estrogens

Lacassagne (1932) in France was the first to induce mammary
cancers in 3 male mice by repeated weekly injections of 30 pg
estrone benzoate for about 6 months. Later he found that estrone
treatment of male mice from birth or at an early age caused them
to develop cancers with the same frequency as their sisters
(Lacassagne, 1933). Bonser _e_tgt. (1937) also reported occurrence
of mammary cancers in male or female mice after treatment with
estrogens. The carcinogenic effects of estrogenic substances in
mice have been extensively reviewed (Gardner, 1953, 1941).

Mchen (1938) was the first to report induction of mammary
cancer in rats after treatment with estrone. Estrone was admin-
istered in corn oil by daily vaginal applications from the age of 62
days. Most of the mammary tumors induced were adenofibromas.
The same year Astwood and Geschickter (1938) also reported induc-
tion of breast carcinoma in a castrated female injected with 200
gamma of estrone. For the next five or six years Geschickter

carried out several investigations of the carcinogenic properties

:estrcger. in rats.

2:216 and female
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72

of estrogen in rats. He reported induction of mammary cancer in

26 male and female castrates and noncastrates after injecting

estrone for several months (Geschickter, 1939a). He found that the
time required for appearance of mammary cancers was considerably
reduced if he implanted estrone pellets instead of administering it

by injections. He further concluded (Geschickter, 1939a, b) that

the latent period varied with the dose of estrone; injections of 30 pg
daily required 600-700 days to induce mammary tumors, whereas

a dose of 200 pg daily required only 150-200 days and pellets of

3-10 mg induced tumors in 50-200 days. In other words, the time
required for induction of cancers was inversely proportional to the
size of the daily dose, but the total dose was relatively constant.
Generally a dose of 30-40 mg was needed, but with pellets 3-10

mg was sufficient. He observed a similar relationship between latent
period and dose of estradiol and DES. He also reported an inverse
relationship between the age of the rat at the beginning of the experi-
ment and the latent period. Younger rats took more time to deve10p
tumors, i. e. , one month. Old rats needed almost 300 days compared
to only 90 days for 20 month old rats. Geschickter and Byrnes (1942)
treated 555 rats with estrogens of several kinds and 202 rats
developed mammary cancers. They concluded that the latent period
was dependent on. dosage, estrogenic potency, duration of estrogenic

activity, method of administration and age of the rats. They found

22 52211122180115 a'

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ratttogens we 1‘ e E:

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73

that simultaneous administration of testosterone or progesterone did
not prevent the appearance of cancers. Most of the tumors induced
by estrogens were adeno‘carcinomas, except that estrogen pellets
also tended to produce a number of fibroadenomas.

In the early 1940's several investigators confirmed most of
Geschickter's findings. Noble gtgt. (1940) induced mammary
tumors by implantation of estrone pellets. Mark and Biskind (1941)
also used a similar technique to induce mammary adenocarcinoma
in 233 days in rats, and Nelson (1944) induced mammary carcinomas
in 68 rats by injections or implantations of several estrogens includ-
ing DES. The minimum time of cancer appearance was 300 days
and the majority of the tumors induced were duct carcinoma or
adenocarcinomas while a few were mixed duct and adenocarcinomas.
Metastases to lymph nodes and lungs were observed in 50% of the
animals. Noble and Collip (1941) succeeded in inducing adeno-
carcinomas in 28 of the 49 rats implanted with estrone pellets, the
first tumor appearing in more than 200 days.

The effect of number of estrogen pellets implanted on tumor
induction was studied by Dunning and Curtis (1952). They induced
mammary tumors in four inbred strains of rats by implantation of
estrone pellets weighing 8-12 mg subcutaneously in the scapular
region. They found that absorption of the hormone was more rapid

from two pellets than from one, but the survival period was reduced

ii

I
me half in the I

need tumors in

Although the
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74

by one half in the rats implanted with two pellets. Two pellets also
induced tumors in more rats and reduced the latent period consider-
ably.

Although the mammary gland is the main target of carcinogenic
effects of estrogens, occasionally other organs of the reproductive
system and endocrine glands, particularly the pituitary gland,
undergo neoplastic transformations. Furth and coworkers have made
extensive studies of tumors of the pituitary gland. This topic has
been considered in detail earlier.

Effect of Strain on Induction of Tumors by Estrogens. In the
late 1940's and early 1950's Dunning gtgi. (1947, 1948, 1951),
conducted a series of experiments to determine the effect of strain
differences on induction of mammary cancers by estrogens in rats.
The tumors were induced by implantation of DES pellets weighing
15-25 mg in the scapular region. Different strains of rats showed
considerable variation in the absorption rate of DES. About 80-85%
of the AxC strain males and females developed mammary tumors but
only 17-22% of the Fisher rats and none of the COpenhagen strain
developed mammary cancers. The majority of the induced cancers
were adenocarcinomas with a small number of adenocarcinomas
with squamous cell cancers or solid carcinomas. Pellets that
contained cholesterol plus estrogens were more effective than those

containing estrogens alone, indicating that slow continuous absorption

:estrogens from L

 

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75

of'estrogens from cholesterol pellets was more effective for the
induction of mammary cancer than pellets of larger dose of estro-
gens alone.

Dunning e_tgt. (1947, 1948, 1951) and Dunning and Curtis (1952)
also reported that the difference in susceptibility to carciOgenic
action of estrogens was transmitted equally by both parent strains
to their reciprocal F1 hybrid progeny. Their experiments were
important since they explained the inconsistent results obtained by
early investigators, such as the low frequency of estrogen-induced
mammary tumors reported by Mchen (1938), Eisen (1942) and
Nelson (1944). . It appears that these discrepant results were due
to differences in strains and age of rats, in the types and doses of
estrogens used, and routes of administration. Strain variability to
mammary cancer induction by estrogen was confirmed by MacKenzie
(1955) in an experiment involving three strains of rats and three
kinds of estrogen. They further found that there was a difference in
susceptibility to tumor induction by estrogen between inbred and
random bred rats.

Influence of Diet on Tumor Induction by Estrogen. Dunning
and coworkers (1949, 1950, 19 54) conducted a series of experiments
to study the effect of diet on neoplastic transformation of the mam-
mary gland induced by estrogens. This was important because excess

body weight in man was correlated with a higher incidence of cancer

|.
fazehaum, 194C

as: was shown to

atestticted caior.

 

 

 

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76

(Tannebaum, 1940). Increases in caloric intake of fat content of diet
also was Shown to be related with high incidence of cancers, whereas
a restricted caloric intake had prevented or delayed the onset of both
induced and spontaneously occurring ne0plasms in mice (Dunning
_£__1_. , 1949). Dunning and collaborators (1949) found that in the rat
caloric reduction resulted in prolongation of the latent period for
induction of mammary cancers by estrogens but had no appreciable
effect on tumor incidence. An increase in the fat content of the diet
had just the opposite effect; it decreased latent period and enhanced
the growth rate of tumors. In a subsequent study they studied the
effect of amino acids in the diet on tumor induction by estrogen
(Dunning gtgt. , 1950). They found that addition of 1% tryptophan
appeared to enhance the development of DES-induced mammary
cancers while addition of 4% tryptophan inhibited the formation of
these tumors. In another experiment (Dunning and Curtis, 1954)
they found that if they kept DES-treated AxC line female rats on a
tryptOphan-deficient diet, there was a slight inhibition of tumor
incidence. These results are questionable since a deficient diet
also resulted in decreased survival time.

Mechanism of Carcinogenic Action of Estrogens. It is not
clear if estrogen brings about neoplastic transformation in the
mammary gland by a direct action on the mammary gland or indirectly

via stimulation of prolactin release from the pituitary gland or by

mechanisms.

 

2:}; for 400 days

1:12:13; one rat 6....

 

.xE-‘tCCess‘. 1

77

both mechanisms. Huggins gtgt. (1966) injected 50 pg estradiol
daily for 400 days to 15 ovariectomized and hypophysectomized rats
and only one rat develOped mammary carcinoma. Jensen and
Jacobson (19 60) and others have demonstrated the presence of
receptor molecules for estrogen in the mammary gland, uterus,
vagina and pituitary gland. The nature of this receptor, its inter-
action with estrogen and the mechanism by which estrogen brings
about carcinogenous transformation of the mammary gland await
elucidation.

Although it is established that chronic treatment with estrogens
elicits mammary cancers in the rat, this method of tumor induction
failed to gain popularity with investigators. There were several
reasons for this and some of these have been discussed. They
include long latent periods, low incidence of tumors, need for pro-
longed treatment, etc. Also, estrogen treatment has not always
been successful in inducing tumors. For example, Maisin _e_t_a_t.
(1956) reported that pellets of DES produced no mammary tumors
in rats in 300 days. Thus it appears that this method of inducing

mammary tumors in rats is not very efficient.
Hydrocarbons

As has been mentioned earlier in this review, hydrocarbons

isolated from coal tar proved very useful for inducing tumors of

 

seal tissue 5 in ;

maiar interest

 

triers or their (in:
2. Ihese compG
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78

several tissues in several Species of laboratory animals. Of
particular interest in this review are the polycyclic aromatic hydro-
carbons or their derivatives used to induce mammary tumors in the
rat. These compounds include 3-methy1cholanthrene, 7, 12-
dimethylbenz (a) anthracene and aromatic amines.

Aromatic Amines. Unlike 3-methylcholanthrene and 7, 12-

 

dimethylbenz (a) anthracene, aromatic amines are not derived from
modifications of benzanthracene nucleus but closely resemble it
(Figure 3). Two aromatic amines, 2-aminof1uorene (AF) and 2-
acetylaminofluorene (AAF) have been most extensively used in the
past to induce mammary tumors in the rat.

Wilson gtg._l_. (1941) were the first to discover that feeding of
a hydrocarbon, AAF, for many months to rats produced mammary
adenocarcinomas and tumors of various other tissues including the
liver, bladder, etc. Bielschowsky (1946) confirmed these findings
and evoked mammary cancers by feeding AAF in'70% of Wistar rats
and in 4% of Long-Evans rats. Several other investigators, includ-
ing Dunning gtgt. (19 47), confirmed the variations in susceptibility
to aromatic amines depending upon the strain of the rats. Fisher
rats were found to be the most susceptible strain to the carcinogenic
effect of these compounds. Bielschowsky (1947) found that feeding
Of AAF at an early age increased the incidence of mammary cancer.

This was confirmed by Engel and Copeland (1948). Wilson _e_t_gl.

.342: reported tha
estate were a’

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79

(1941) reported that the tumor incidence and latent period for tumor
appearance were also influenced by dose of AAF and duration of treat-
ment. Most of the tumors induced by AAF were adenocarcinomas

and a few were fibroadenomas (Bielschowsky, 19 47). Several
investigators reported that composition of the diet had an important
influence on the incidence of mammary tumors. A low caloric intake,
low fat or high protein content, generally reduced the tumor inci-
dence (Noble and Cutts, 1959).

The use of aromatic amines to induce mammary cancers never
became popular because of the several month-long latent periods
required to induce mammary tumors, low incidence of tumors induced,
appearance of tumors of various other tissues which almost always
arose and resulted in early deaths of the rats, and more importantly
the discoveries of more potent carcinOgens like 3-
methylcholanthrene and 7, lZ-dimenthylbenz (a) anthracene which
selectively evoked mammary cancers in shorter lengths of time.

3-Methy1cholanthrene. The first carcinogenic polycyclic
hydrocarbon isolated from tar was 1: 2*: 5:6-dibenzanthracene (dibenz
(a, h) anthracene, DBA) (Kennaway and Heiger, 1930). Burrows
3331. (1932) were the first to demonstrate the carcinogenic activity
of this compound. Cook gtgl. (1933) were the first to identify 3:4

benzpyrene1BP) as the active carcinogenic agent in coal tar.

are: and Dane (

 

It: deoxycholic at
The first mar
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red the skin of :

 

80

Wieland and Dane (1936) synthesized 3-methylcholanthrene (MC)
from deoxycholic acid.

The first mammary cancers were induced accidentally by the
French investigators, Maisin and Coolen (1936). They repeatedly
painted the skin of mice with MC and BP to induce skin cancer.

They discovered that in addition to Skin cancer, mammary tumors
had developed in 18% of the mice in seven months. The same year
Dunning gtgt. (1936) demonstrated that subcutaneous injections of

a 1% solution of DBA in wax or paraffin into mammary tissue of

rats and mice resulted in production of malignant mammary tumors.
The latent period for appearance of the tumors depended upon the
dose of the carcinogen. Dunninggtgl. (1940) were the first to
report that subcutaneous injection of MC also resulted in production
of mammary tumors in 60% of treated rats. But they found that
stronger solutions of MC (1%) in wax or paraffin produced significantly
smaller numbers of tumors than the weaker (0. 5%) solutions. This,
they explained, was due to the toxicity of the stronger solution. The
majority of the tumors produced by subcutaneous injections of MC,
BP or DBA were sarcomas and only rarely adenocarcinomas. This
has been confirmed by others (Dao, 1964). Dao (1964) found that
most of the hydrocarbons injected by the subcutaneous route did not
enter epithelial cells but concentrated locally at the injection site

resulting in sarcoma production. The latent period for tumor

entrance was lo
seeing one yea:

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81

appearance was long, extending over several months and often
exceeding one year. Ge schickter (1939) reported that implantation

of DBA or BP pellets into mammary tissue also resulted in production
of sarcomas after a latent period of several months. Shear (1936)
also reported that intramuscular injections of hydrocarbons also
evoked mainly sarcomas, and Strong and Smith (1939) found that a
single injection of MC into the breast of mice produced both sarcomas
and adenocarcinomas of the mammary gland.

In the years succeeding these earlier reports, several routes
of administration were tried to evoke mammary cancers in greater
number of rats and in shorter latent periods. Proshaka gtgl. (1959)
induced mammary tumors by repeated instillation of MC in the mouth,
and Orr (1943) succeeded in eliciting mammary tumors by repeated
instillation of MC in the nares. Shay gt gt. (1949) were the first to
induce mammary tumors in male and female Wistar rats by daily
gastric instillation of 2 mg MC in oil for up to 10 months. About
80-85% of the animals developed mammary tumors. The range
of latent period was 129-383 days. They confirmed induction of
mammary tumors by this route in several subsequent publications
(Shaygtgl. , 1951, 1952, 1956, 19 59). This route of administration
of hydrocarbons proved very useful and was employed successfully
for several years to induce tumors by several hydrocarbons.

Shay's technique of inducing mammary tumors in rats by

 

 

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82

gastric instillation of MC was improved and perfected by Huggins
and coworkers (19 59a). They found that dose was important in
determining the length of latent period. For example, a single
instillation of 20 mg, 50 mg or 75 mg MC induced mammary tumors
in 74-167, 44-83 and 43-69 days respectively. Multiple feedings by
gastric tube of 2 mg MC daily for 6 days a week took up to 9 months
to induce tumors, whereas multiple feedings of 15 mg elicited mam-
mary tumors in only 34-69 days. However, they found that excessive
dosage was toxic, for example, 15 mg MC killed a Significant num-
ber of rats. They concluded that the optimal dose was 10 mg daily,
6 days a week, for 7 weeks. With this procedure the earliest
mammary tumors appeared in 20 days, and within 60 days 100% of
the rats had developed tumors. This was a significant achievement.
They further found that this dose of MC had no effect on body weight,
although it decreased the weights of the ovaries, uterus and pituitary
gland, and reduced the concentration of alkaline phosphatase enzyme
in the mammary gland.

Ina subsequent experiment they further improved the technique
of tumor induction (Huggins gtgt. , 1961a). They gave a single
gastric instillation of 100 mg MC in sesame oil into female Sprague-
Dawley rats of 50-65 days. This procedure resulted in production
of mammary tumors in every rat. The earliest cancer was detected

by palpation in 31 days and the last by day 130. By histological

 

narration the ea
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reared from a;
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83

examination the earliest mammary tumor was detected in 14 days.
In the same publication they also reported that the tumor incidence
increased from age 23 to 50 days when 100% of the rats were
instilled with MC at an age of 75 days, and only a small number of
rats developed tumors if MC was fed at the age of 100-365 days.
They found that 90% of MC given orally was excreted in feces; there-
fore it appeared that only a trace amount of MC was needed to induce
tumors. This speculation was substantiated by their findings that
an MC pellet implanted in the spleen could be recovered after tumor
appearance with loss of only insignificant amounts of carcinogen.
LlZ-dimethyl (a) Benzanthracene. Bachmann and Chemerda
(1938) synthesized 3 new polycyclic hydrocarbons: 9, lO-dimethyl-
l-2-benzanthracene (DMBA), 9, 10-diethy1-l, Z-benzanthracene and
5, 9, 10-triethy1 l, Z-benzanthracene (7, 8, 12, TMBA) in the Chemistry
Laboratory of the University of Michigan. This was an important
achievement since DMBA is now used almost exclusively for induc-
tion of experimental mammary cancers in laboratory animals, and
TMBA is a very efficient inducer of experimental leukemia in rats.
The same year Bachmann collaborated with the husband and wife
team of the Kennaways of Research Institute at Royal Cancer Hospital,
London, to test the carcinogenic prOperties of the newly synthesized
hydrocarbons. The Kennaways had previously contributed exten-

sively to the synthesis of aromatic hydrocarbons. Bachmann and the

Eire-rays (3a c hrr.

 

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Kennaways (Bachmannifl, , 1938) applied DMBA and TMBA in O. 3%
benzene to the skin of mice and induced epitheliomas and papillomas
in 35 days. DMBA proved to be a very potent carcinogen. It induced
tumors twice as quickly as did MC. DMBA also had two unusual
features: (1) it produced a ”multiplicity of papillomas to an extent
greater than that produced hitherto by other compounds and (2) it
produced epilation over an eSpecially large area". TMBA also
caused rapid production of multiple papillomas and considerable
epilation, but the survival rate in the mice was considerably
decreased indicating that introduction of a methyl group into position
5 of the molecule of 9:10 dimethyl-l, Z-benzanthracene had an
adverse effect on carcinogenic activity.

Geyer 5:331. (1951) were the first to induce mammary tumors
in rats by 12 weekly intravenous injections of a total of 4. 3 mg
DMBA/100 g body weight. The incidence of tumors in females was
about 30%. Similar injections of DBA, MC and other hydrocarbons
failed to induce mammary tumors. In a subsequent experiment
(Geyer £1211, , 1953) they raised the total dose of DMBA to 5. 2
mg/100 g body weight and injected it intravenously as an emulsion
in corn oil. The injection protocol consisted of 3 injections on
alternate days followed three weeks later by a similar set of
injections. This procedure raised the tumor incidence to approx-

imately 90% in 19 weeks. This contrasted with an incidence of 80%

\

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85

in 47 weeks if the total dose of DMBA injected was 1. 25 mg/100 g
body weight. The rats which received the higher dose had twice

as many tumors per rat as the rats which received the lower dose.
They further reported that simultaneous injections of O. 6 mg
estradiol decreased the latent period to 16 weeks and increased the
tumor incidence to 10070 and yielded mammary adenocarcinomas
exclusively, whereas DMBA alone produced some adenofibromas.

A similar dose of DES also decreased the latent period and increased
the tumor incidence but the effect was not quite so great as that of
estradiol. In all these experiments the mammary gland was the main
target of neoplastic transformation and extremely few tumors were
induced in other tissues of the body. These experiments were very
significant. They had succeeded in inducing mammary tumors
selectively and invariably with a considerable shortening of the latent
periods. The present method of inducing mammary tumors by DMBA
is essentially a modification of this procedure.

Several other investigators also were attempting to induce
mammary tumors by DMBA using all possible routes of administra-
tion and procedures. Noble and Walters (1954) succeeded in eliciting
mammary tumors in rats by injecting 5 mg of DMBA in sesame oil
intramuscularly into the thigh. The tumor incidence was low and
only 4 out of 21 female rats developed breast adenocarcinomas in

4-5 months. In a subsequent experiment Noble (19 58) injected 5

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86

mg DMBA with 10 mg cholesterol in O. 5 m1 sesame oil intramus-
cularly into the thigh. This resulted in develOpment of hormone-
independent adenofibromas instead of the usual adenocarcinomas.
However if the injections were given near the breast adenocarcinomas
were produced.

Orr (1955-1957) was the first to induce mammary tumors in
rats by painting the skin with DMBA solution in oil. The incidence
of tumors was more than 70% and the latent period was 12 or 27
weeks depending on the strength of the DMBA solution. Histologic-
ally most of the tumors were adenocarcinomas. Noble and Cutts
(19 59) speculated that skin painting with DMBA must have been
followed by general absorption of the carcinogen so that the breast
was influenced by a systemic rather than a local action.

In 1958 Scholler and Carnes used Geyer's intravenous technique
and induced mammary tumors in about 9070 Wistar rats within 14
weeks, the median time of tumor appearance was 59 days. No
significant difference in histological and growth characteristics
were noted among tumors produced by DMBA alone and those pro-
duced by DMBA plus estradiol. When Huggins and coworkers (1959a)
entered the field of carcinogen-induced mammary tumorigenesis
in rats, many of the important criteria and procedures for induction
of mammary cancers in rats by hydrocarbons already had been

established. A dose-response relationship between dose of DMBA

zit'rnor incidenc‘
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and tumor incidence had been established, tumor incidence had been
raised to almost 100% and the latency period was reduced to a few
weeks. Still none of the procedures used to induce tumors by DMBA
were completely reproducible. Huggins and collaborators (1959a)
greatly improved the procedures previously used and finally suc-
ceeded in devising a procedure which induced mammary tumors in
rats selectively and invariably in less than 60 days. This procedure
is now the standard procedure for inducing mammary cancers in the
rat.

Huggins £132.}.- , published their first paper on MC and DMBA
induced mammary tumors in 1959. They used Shay and coworkers'
(1949) technique of oral administration and instilled 1 mg DMBA in
sesame oil by stomach tube, 6 days each week for 100 days into 50
day old female rats of the Sprague-Dawley strain. This resulted in
development of mammary cancers in each of 9 rats treated during a
period of 47-100 days with a mean of 78 days. Huggins 3’53}. (1961a)
reported induction of mammary tumors after a single feeding of
DMBA. The lowest dose of DMBA which evoked cancers was 1 mg.
There was a progressive rise in the incidence of cancers with
increasing dosage until the optimal dose was reached. The optimal
dose was 20 mg because (1) every rat survived (2) mammary cancers
developed in every rat without exception (3) the first cancer was

visible in 28 days and all the rats had tumors of the breast before

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60 days (4) the average number of active centers in each rat was 6. 8
with a range of 3 to 21. A single feeding by gastric intubation of

15 mg DMBA also induced mammary cancers in all rats but there
were fewer active centers per rat. This method had several advan-
tages over the multiple feeding method. It was simple and saved
labor and materials and induced tumors in all rats within a short
period. This method was later successfully used by several
investigators.

Huggins g_t_a_l_. (1961b) modified Geyer and coworkers' (1951)
intravenous technique and induced mammary cancers in every rat
by injections of 2. 5-5 mg of emulsified DMBA. Two years later
Huggins and Fukunishi (1963) also succeeded in evoking mammary
cancers by intraperitoneal injection of 5 mg of emulsified DMBA in
50 day old Sprague-Dawley rats. This resulted in 1007/0 tumor
incidence in 60 days. They concluded that ”quantitatively the intra-
peritoneal injection resembled the intravenous injection in its effici- '
ency in evoking mammary cancer. " A single feeding of the same
amount of DMBA was less efficient since it elicited cancers in only
50% of the rats (Huggins eta}. , 1961a, b). Surprisingly the intra-
peritoneal injection of DMBA produced no peritoneal tumors but
implantation of DMBA pellets in the peritoneal cavity produced
sarcomas of the peritoneum, indicating that not all cells are sus-

ceptible to malignancy after brief contact with hydrocarbons.

 

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Peritoneal cells apparently need longer contact with carcinogens.

The lipid emulsion (1 gm DMBA/l gm emulsion) used in the
intraperitoneal and intravenous injections of DMBA was prepared
by Dr. P. E. Schurr of the Upjohn Company, Kalamazoo, Michigan
(Huggins and Fukunishi, 1963). The hydrocarbon was dissolved in
the oil phase of Upjohn Lipomul IV and emulsified in the aqueous
phase; particles in the emulsion were submicroscopic. The previ-
ously used concentrated oil solutions were unsatisfactory because as
Huggins explained, they elicited large amounts of fibrous tissue
which covered the abdominal viscera and caused death in many
animals. The emulsion had the advantage of a high concentration of
the hydrocarbon, ease of preparation, sterility and stability for
long periods. The vehicle itself was well tolerated by the animals
and was not carcinogenic. At present the standard method of induc-
ing mammary cancers in rats is to inject 1 m1 of this lipid emulsion
containing 5 mg of DMBA into the tail vein. It is less hazardous to
handle and more convenient and economical (Huggins, 1962).

In 1962 Huggins and Yang summarized the conditions which
influence production of mammary cancers by hydrocarbons:

Nature of Hydrocarbons. Aromatic amines, particularly AAF

 

and AF are weak carcinogens as far as production of mammary
tumors in rats is concerned. MC is a powerful hydrocarbon for

inducing cancers of the breast in the rat, but DMBA is considered

90

to be the most potent carcinogen for this purpose. TMBA is the
hydrocarbon of choice for inducing leukemia in rats. The leukemo-
genic action of hydrocarbons was discovered by Morton and Mider
(1940), and Huggins and Sugiyama (1966) were the first to induce
leukemia in rats by intravenous injection of hydrocarbons. Huggins
_e_til. (1970) induced leukemia in Long Evans rats by giving a series
of pulses of TMBA. The most common form of leukemia induced by
this method is a diffuse hepatic form of stem-cell leukemia often
associated with erythroblastosis. DMBA rarely induces leukemia;
however, by rearrangement of experimental conditions, involving
multiple injections, DMBA has been shown to induce leukosis and
leukemia in a large percentage of rats (Huggins, and Sugiyama,
1966).

Dosage of Hydrocarbons. The dose of hydrocarbons has impor-
tant influence upon their carcinogenic properties. It has been men-
tioned earlier that both the latent period and tumor incidence are
influenced by the dose of DMBA administered by gastric instillation
(Huggins _e_tal. , 1961a, b). Huggins and coworkers (1964) conducted
an extensive study to determine the stoichiometric relationship
between dose of DMBA given intravenously, the frequency of its
administration and the yield of mammary cancers. Mammary tumors
were induced by injection of 2-6 mg DMBA to 50 day old Sprague-

Dawley rats. They found that the large dose produced more tumors

 

‘

:1: the smaller c
:tfpie injection .-

;'e:as a single 1

 

 

91

than the smaller doses. When the large dose of 6 mg was given in
multiple injections more tumors were produced than when it was
given as a single injection. They hypothesized that only a small
number of mammary cells at a time are susceptible to cancerous
transformation, therefore increasing the number of exposures
increases the ''chances of a hit by hydrocarbon. "

On the other hand, induction of mammary cancers was inhibited
when hydrocarbons were fed repeatedly during a period of time that
overlapped multiple injections of optimal amounts of DMBA which
otherwise produced a large number of tumors. This protective
action of hydrocarbons had been described earlier by other investi-
gators as well.

§pecies of Animals. Target tissues for carcinogenic action of
hydrocarbons vary from species to species. Subcutaneous injections
of DMBA into newborn mice results in production of lymphomas and
pulmonary tumors and no breast cancers, whereas it produces
mammary tumors in newborn rats and few lymphomas. The LD50
of DMBA also varies from species to species; it is 60 mg/kg for
rats (Huggins _e_t_a_L , 1972), 130 mg/kg for guinea pigs and for
Carworth No. l (C F1) mice it is 10 times that of Sprague-Dawley
rats (Huggins gal. , 1965).

Strains. Many investigators have demonstrated that various

strains of rats differ in their susceptibility to the carcinogenic effects

:fi'rlroca rbons.
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92

of hydrocarbons. Dunninggtal. (1936, 1940) studied five strains

of rats and concluded that the Long-Evans strain was least susceptible
to the carcinogenic effect of MC. Bielschowsky (19 46) reported that
feeding of AAF produced higher incidence of mammary tumors in
Wistar rats than in piebald rats. Syndor _e_t_gl. (1962) reported that
a single intravenous injection of 5 mg DMBA produced multiple can-
cers of the mammary gland in all 50 day old Sprague-Dawley rats
whereas only 16% of Long-Evans rats developed similar tumors.
The Long-Evans rats also had longer latent periods,fewer tumors
per rat and occasionally showed regression of the cancers. The
results were not significantly modified when newborn rats of one
strain were foster-nursed by mothers of the other strain. In cross-
breeding experiments both types of F1 hybrids had only 75% incidence
of mammary cancers, but in the first backcross hybrids which
derived three-fourths of their genetic material from either the
Sprague-Dawley or Long-Evans strain the incidence of mammary
cancers approached that of a genetically undiluted strain. This
indicated that susceptibility to hydrocarbon-induced mammary can-
cers was inherited and it‘was speculated that this was due to varying
susceptibility of the mammary gland to hydrocarbons due to dif-
ferences in endocrine constitution particularly in the pituitary func-
tion. The differences between these two strains vanished when

repeated injections of DMBA produced only 9% mammary tumors in

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this strain, but if four intravenous injections were given then the
incidence rose to 94%; in the Sprague-Dawley rats a single injection
evoked cancers in 100% of the rats.

Age. Huggins and collaborators (1961) demonstrated that the
carcinogenic effect of hydrocarbons was influenced by the age of the
rat. They reported that 100% of the rats developed mammary cancers
when treated with DMBA 50-65 days of age but seldom any tumors
developed when the rats were 100 days old. Similarly tumor inci-
dence was low before the age of 50 days; only 50% developed tumors
when rats were treated with DMBA soon after birth (Huggins and
Fukunishi, 1963). Age also influenced tolerance to hydrocarbons.
Huggins and Sugiyama (1966) concluded that adolescent rats were
more tolerant of DMBA than older rats.

Route of Administration of Hydrocarbons. This has been dis-
cussed earlier.

Condition of Animals. Animals should have had no previous
contact with cancer-protective substances including hydrocarbons.
This has been discussed earlier.

Animals should be free from disease, otherwise they will die
before developing cancer.

Hormonal Status of Animals. This will be considered in detail

in the latter part of this review.

till?

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94

Growth and HistologLical Characteristics of Hydrocarbon-Induced

Mammary Cancers in the Rat. Huggins and Yang (1962) treated 38

 

Sprague-Dawley rats with DMBA at the age of 50 days and observed
them for 180 days. During this period the following kinds of tumors
were found: mammary adenocarcinomas in 100% of rats, mammary
fibroadenomas in 89%, ear-duct tumors in 2 rats and leukemia in l
rat. Tumors of other tissues were less common. In another study
(Huggins g3}, , 19 593., b) they found that most of the mammary
tumors evoked by hydrocarbons were adenocarcinomas; sarcomas,
benign fibroadenomas and adenomas occurred rarely. The first
tumors to appear after treatment with hydrocarbons were mammary
carcinomas, usually within 60 days; benign tumors and tumors of
the other tissues appear later (Huggins and Yang, 1962). Mammary
cancers could be detected by palpation at 20 days and by histological
examination at 11 days after exposure to hydrocarbons (Huggins,
1967).

These studies demonstrate that after a brief single exposure
to hydrocarbons not all cells in the body become malignant. They
also indicate that not all cells in the mammary gland become malig-
nant, some ”don't go all the way to a malignant state but become
benign tumors" (Huggins and Yang, 1962).

All mammary cancers evoked by hydrocarbons are similar in

cytologic appearances; all are papillary adenocarcinomas with

 

my. mitosis
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imary fat.
ithe host by 2

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95

abundant mitosis. Some have islands of squamous carcinomas.
Many of the mammary cancers invade adjacent muscles, skin and
mammary fat. They rarely show metastases to distant sites but
kill the host by attaining great size and infiltrating adjacent tissues,
resulting in ulceration, hemorrhage and death (Huggins 2221' , 1959;
Shaygtgl. , 1949; Bielschowsky, 1947). Some investigators have
reported occasional metastasis from mammary cancers to lungs,
liver and lymph nodes (Shay _e_t_ 11.. , 1952). Dao (1964) was also able
to induce metastasis in mammary cancer cells. He inoculated cell
suspension of DMBA-induced mammary tumor cells into the portal
veins which resulted in development of extensive tumor foci in the
liver. The tumor foci later transformed into metastatic adeno-
carcinomas and caused atrophy of the liver. These cancers retained
their hormone dependency; they regressed after ovariectomy and
metastasis disappeared.

Young and Cowan (1963) reported that a number of mammary
cancers underwent spontaneous regression. This phenomenon has
been observed by other investigators also, but there is no adequate
explanation for it at present.

Hydrocarbon-induced mammary tumors have been successfully
transplanted to other rats in the same strain but the number of
successful takes is small (30%) compared to the number of takes

in case of benign fibroadenomas (100%) (Huggins gig}, . 1959a, b)-

 

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96

Dorfrnan and coworkers (Abe SEE}: , 1962; Harda 2311. , 1965) have
done extensive work with transplants of DMBA-induced mammary
cancer. This will be discussed in detail later.

Not much information is available about the metabolism of
hydrocarbon induced mammary cancer cells. Their respiration
values are similar to these of the normal lactating mammary glands
(Rees and Huggins, 1960). They have a high rate of aerobic and
anaerobic glycolysis which is a distinctive feature of all cancer
cells (Huggins, 1965). They contain high quantities of certain
enzymes, particularly lactic and malic dehydrogenases (Rees and
Huggins, 19 60). After ovariectomy or hyp0physectomy the tumors
decrease in size and the cells are destroyed in some tumors, in
others the many layers of cells in the acini are replaced by a single
layer of flat cells and the epithelial cells are flattened (Huggins,
1965).

Metabolism of Hydrocarbons. The metabolism of hydrocarbons
has not been extensively investigated. The following has been
excerpted from Dao (1967). The clearance of hydrocarbons from the
body is related to the size of the molecules; big molecules like
DMBA and MC are retained up to 72 hours, while small molecules
like BP, phenanthrene and anthracene are completely cleared within
48 hours. Cleared hydrocarbons appear in bile as oxygenated

metabolites. The adipose tissue and the mammary gland are slow in

 

 

97

 

Mammary

Mammary gland from a pregnant rat.
development is not complete, the alveoli are
tightly closed and not fully developed. Abundance

of fat cells.

Figure 4.

 

98

 

 

 

 

Figure 5. Mammary gland from a lactating rat. -Note
the fully developed alveoli. Each alveolus
is lined by a single layer of cells and the

‘ ' luminus is filled with milk and fat globules.

 

 

 

"1

(I13

 

 

99

 

 

 

Figure 6. A mammary adenocarcinoma, showing uncon-
trolled growth of epithelial cells which have
invaded ductal lumina and have formed many
layers around the alveoli. The lumina are
almost extinguished.

100

 

 

 

Figure 7.. A mammary adenocarcinoma inan early stage.

 

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101

clearing carcinogens. The fat pad of the mammary gland acts as a
trap and retains hydrocarbons within adipose cells from which they
are released slowly.

Localization of hydrocarbons in the target tissue depends on
effective transport of carcinogen to the target tissue, and the trans-
port of carcinogen in turn depends on the nature of the vehicle.
Aqueous suspensions of MC have failed to induce tumors. Dao (1967)
found that the level of MC in the adipose tissue after feeding 100 mg
in aqueous suSpension was only 6%: of that found after feeding 30 mg
in sesame oil. Localization of hydrocarbons is also affected by the
endocrine status of the animal. Hydrocarbon levels were low in the
tissues during pregnancy and lactation but high in castrates where
more fat is present.

Mechanism of Carcinogenic Action of Phdrocarbons. This
topic has been reviewed by Yang eta}. (1961) and by Dao (1964).

Not much is known about the relationship between structure of hydro-
carbons and their carcinogenic prOperties. It was mentioned earlier
that addition of methyl or amino groups or a benzene ring to the
basic benzanthracene nucleus generally results in increased carcino-
genic potency. Huggins and Yang (1962) indicate that the aromatic
hydrocarbons have to be strong electron donors or acceptors to be
able to induce cancers. Yang _e_t_al. (1961) found that there was an

increase in carcinogenesis as hydrocarbons became sterically

 

 

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102

similar to steroids. Huggins and Yang (1962) found strong similarities
between aromatic hydrocarbons, steroids, and the base pairs of
nucleic acids. They state that three critical molecular factors
determine mammary carcinogenecity of polynuclear hydrocarbons
(1) the electron transfer factor (2) the geometric factor - the
configuration must resemble that of the steroids or the purine-
pyrimidine base pairs of nucleic acids (3) molecular thickness - it
must not exceed the thickness of the base pairs. In brief the mam-
mary carcinogens must resemble the base pairs of nucleic acids in
geometrical configuration, and be able to form molecular complexes.

The mechanism of carcinogenesis by hydrocarbons is not
understood. It is generally agreed that only a brief contact between
hydrocarbons and the target tissues is needed to initiate the process
of carcinogenesis. Dao (1967) removed a mammary gland from a
rat 10 minutes after administration of DMBA and transplanted it
into another rat which had no previous exposure to DMBA. After
an appropriate latent period, the transplant was transformed into a
tumor. They also found that if mammary glands were exposed If}.
lit—1:2. to DMBA solution for 20 minutes, then washed and reimplanted
into the interscapular area in the same rat, 95% of such grafts
developed into tumors.

Dao (19 67) believes that hydrocarbons must alter the informa-

tional molecules so that the new prOperties can become inheritable.

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103

Huggins and Yang (1962) also speculate that hydrocarbons cause
selective changes in nucleic acids of the tumor cells. Several
hydrocarbons have been reported to bind to proteins, or nucleic
acids or to both. This topic has been reviewed by Daudel and
Daudel (1966).

Neuroendocrine Control of Development and Growth
of Carcinogen-Induced Mammary Tumors in Rats

 

Most of the early information on the involvement of endocrine
factors in mammary carcinogenesis came from studies on spontane-
ous mammary cancers in humans, mice and rats. Some of the
most important contributions in this area were made by such
pioneers as Beatson (1896), Loeb (1902), Lacassagne (1932) and
later by Heiman (1934), Mohs (1940a, b), Emge and Wolf (1934),
Dunning and Curtis (1946), Miller and Noble (1954a, b, c), Huggins
(1963), Glenn e_tgl. (1959a, b), and numerous other investigators
mentioned earlier in this review. These great scientists not only
laid the foundations for endocrine research in mammary carcino-
genesis but pointed out the significance and relevancy of mammary
cancer research in laboratory animals. The present widespread
use of animal models for cancer research and the extensive use of
endocrine methods for treatment of human cancer stand as a monu-

ment to the foresight and hard work of these men and women.

 

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The second phase in mammary cancer research, especially in
the rat, involved attempts to induce mammary cancers by aromatic
hydrocarbons. The first efforts in this area were made in the
beginning of this century, reached a peak in the late 30's and 40's
and ended in the late 50's when a rapid and easily reproducible
method for induction of mammary cancers in the rat was perfected.
Some of the more prominent researchers in this area included
Yamagawa and Ichikawa (1918), Kenway and Heiger (1930), Wieland
and Dane (1936), Bachmann and Chemerda (1938), Maisin and
Coolen (1936), Bielschowsky (1946), Shay (1951), Scholler (1958),
Geyer gta_l.. (1951) and Huggins (1967) and their colleagues. This
was an achievement of great significance. It provided investigators
with a laboratory cancer model which could be developed in rats
economically, invariably and at an early age. Most importantly it
resembled certain human breast cancers not only histologically but
in its dependency on hormones for development and growth. Some
of the scientists who contributed extensively to our knowledge of the
endocrine factors involved in hydrocarbon-induced mammary carcino-
genesis in the rat during the 60's and 70's include Huggins (1963),
Meites (1972a, b), Dao (1964), Pearsongtgl” 1972a, b), Dorfrnan
(1965) and their coworkers. Meites and coworkers (1972) went a
step further in a series of publications and directly implicated the

hypothalamus in spontaneous and DMBA—induced mammary

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105

tumorigenesis in the rat. What follows is a brief review of the
neuroendocrine factors involved in carcinogen-induced mammary

cancers in rats.

Influence of Sex

 

The earliest indications that sex hormones might play a role
in the development and growth of hydrocarbon-induced mammary
cancers came from the observations that male mice and rats were
less susceptible to the carcinogenic effects of hydrocarbons on the
mammary gland than females of the same species. Englebreth and
Holm (1941) reported that skin painting of mice with hydrocarbons
resulted in develOpment of mammary cancers in females, whereas
in the males the incidence of cancers was very low and the latency
period was very long. They suSpected that a sex hormonal factor
was involved in hydrocarbon-induced mammary tumorigenesis in
mice. A few years later Bielschowsky (1944) reported that feeding
of AAF evoked mammary tumors in only 7% of male rats compared
to a much higher incidence in the female rats. Several other
investigators including Cantero £11. (1948) and Stasney _e_t_il.
(1947) also reported that male rats were either less susceptible or
completely refractory to neoplastic transformation when exposed
to aromatic amines AF and AAF.

An influence of sex also was found in mammary turmorigenesis

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induced by polycyclic aromatic hydrocarbons. Shay _e_til. (1952,
1956, 1959) were the first to report that female rats were more
susceptible to develOpment of mammary tumors after gastric instil-
lation of MC than either male rats or Spayed female rats. Male
rats not only had a smaller incidence of mammary tumors but the
latency period in the males for mammary tumor appearance was
twice as long as that in the females. They speculated that estrogen
either sensitizes the breast tissue to the carcinogenic effect of MC
or works synergistically with it, in contrast to testosterone or
progesterone which interfere with the tumorigenic action of the
carcinogen and prevent neoplastic transformation of the mammary
tissue. They also reported that sex hormones affected the type of
mammary tumors produced under the influence of carcinogens.
Glandular tumors were more common in females or female hormone-
treated rats while spindle-cell tumors and collagenous tumors were
more common in males. A few years later Huggins _e_t_gl. (l959a,b)
and Dao and Sunderland (1959) published two very important papers
which besides dealing with several aspects of the endocrine control
of carcinogen-induced mammary tumorigenesis in the rat, also
confirmed the earlier findings that oral administration of MC or
DMBA to rats resulted in develOpment of mammary cancers in intact
females but not in intact males. Kim and Furth (1960) also reported

that MC failed to induce mammary cancers in male rats.

 

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107

Role of the Ovaries

After demonstrating the influence of sex in carcinogen-induced
mammary tumorigenesis, the next logical step was to investigate
more specifically the role of the ovaries in this process. Biel-
schowsky (1944, 1947) was among the first to report that ovariectomy
decreased the incidence of AAF-induced mammary tumors in the
rat but had no effect on the growth of established mammary tumors.
Huggins _e_ta_l_. (19 59a) and Dao and Sunderland (19 59) reported that
castration of female rats about a week before administration of
carcinogen decreased the incidence of MC-induced mammary tumors
but did not completely prevent tumor induction. In a subsequent
experiment Dao (1962) found that if carcinogens were given to female
rats 30 days or more after castration, no mammary tumors were
induced. Huggins _e_t_al. (1959a) found that the tumors induced with
oral feeding of MC in ovariectomized rats differed morphologically
from the tumors produced in the intact rats, particularly in the
arrangement of epithelial cells and in secretion of the tumor acini.
In the ovariectomized rats the lumina of tumor acini were devoid of
alkaline phosphatase and some of the acini were lined with a single
layer of flat cells while other glands had many layers of epithelial
cells. Huggins 3331' (19 59a) also found that ovariectomy decreased

the size of most of the established mammary tumors; however, the

3335 (00.1.11

 

 

 
   
 

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108

tumors continued to grow in a very small percentage of ovariecto-
mized rats and in others the decrease in tumor size was temporary
and growth was soon resumed. Presumably the latter were hormone-
independent (autonomous). The mammary tumors which diminished
in size after ovariectomy showed a characteristic cytologic appear-
ance of atrophy of epithelial cells which was very similar to that

seen after hyp0physectomy or administration of dihydrotestosterone.
Many layers of epithelial cells in the tumor acini were replaced by

a single layer of flat cells and the acini no longer contained alkaline
phosphatase and carbohydrate.

The inhibitory effect of ovariectomy, performed before or
after carcinogen treatment, on mammary tumorigenesis has been
confirmed by several investigators (Meites gig}. , 1972a, b;
Pearson_£__l. , 1972a, b; Dao, 1964).Dao and Gr‘iener (1961) also
reported that MC can successfully induce mammary tumors in male
rats if they are grafted with ovaries. There was a greater increase
in tumor incidence if the ovaries were grafted in castrated males
rather than in intact males.

It has been mentioned earlier that ovariectomy produced
remission in a substantial number of human breast cancer patients.
Nissen-Meyer (1968) and Cole (1968) reported that castration per-
formed in human females at the time of mastectomy was very effec-

tive in controlling metastasis.

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109

Role of Estrogens

Huggins e_tai. (19 59a) reported that the inhibitory effect of
ovariectomy on the development of MC-induced mammary tumors
was overcome by daily treatment with 0. l or 1 pg estradiol-173.
Both these doses restored mammary tumor incidence to 100 percent,
but the mean latency period was prolonged. It was 63-72 days
compared with 50 days in the intact controls. A dose of 20 pg
estradiol 17B depressed the incidence of mammary cancers to about
33 percent and lengthened the time of tumor appearance. Dao and
Griener (1961) and later Sterenthal SEE}: (19 63) also demonstrated
that administration of estrogens overcomes the inhibitory effect of
ovariectomy on the deve10pment and growth of carcinogen-induced
mammary tumors. Sterenthal _e_t_al. (1963) and Pearson _e_t-341.
(1972a) demonstrated very clearly the dramatic effects of estradiol
benzoate on mammary tumor growth. Ovariectomy and adrenalectomy
caused a rapid decrease in the tumor size but after 11 to 19 days of
treatment with daily injections of 1-5 pg estradiol benzoate, tumor
growth was reactivated and a several fold increase in tumor size
occurred. When estradiol benzoate was withdrawn there was a rapid
decrease in tumor size within 5 to 15 days. Unlike low doses of
estradiol benzoate, a high dose of 500 lug estradiol benzoate daily

induced a significant regression of the mammary tumor area.

 

 

 

 

 

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110

Young (1961) found that administration of estradiol 178 with proges-
terone and bovine growth hormone also restored MC-induced mam-
mary tumors in hyp0physectomized rats. Dao (1962b) stated that
ovarian hormones should be present at the time of carcinogen
administration for a successful neoplastic transformation of the
mammary gland. He found that grafting of ovaries to male castrates
raised the incidence of MC-induced mammary tumors to about 60
percent but no change occurred if the ovaries were grafted 25 days
after MC administration. He later reported (Dao, 1967) that there
was no significant difference in mammary tumor incidence if ovarian
grafts were given immediately or 30 days after feeding DMBA,indicat-
ing that "latent tumor cells" survive through a prolonged period of
ovarian hormone deficiency.

In 1964 Meites and colleagues published their first paper on
carcinogen-induced mammary tumors in the rat (Talwalker _e_t_gl. .
1964b). Earlier they had experimented with skin tumors induced
by DMBA and croton oil (Meites, 19 58). Talwalker gt 3.1. (1964)
reported that bilateral ovariectomy 7 days before feeding 20 mg
DMBA in corn oil by gastric instillation completely suppressed
development of mammary cancers in Sprague-Dawley rats during a
40 week observation period. Daily injections of 1 pg or 10 pg
estradiol 7 days before and 7 days after (total of 14 days) DMBA

administration induced mammary tumors in 33% and 23% of the

 

 

 

 

 

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111

treated rats respectively compared to a 100% tumor incidence in

the intact controls and 0% tumor incidence in the ovariectomized
controls. The average number of tumors per rat in the rats which
received 1 and 10 pg estradiol were 1. 5 and l. 0 respectively com-
pared to 3.9 tumors per rat in the intact controls. The ovariec-
tomized estradiol treated rats also had longer latency periods of 104
and 89 days compared to 68 days in the intact controls. In a sub-
sequent experiment Nagasawa and Meites (Meites, 1972b) attempted
to find the optimum amount of estrogen needed for carcinogen-
induced mammary tumorigenesis in rats. Sprague-Dawley rats
were ovariectomized at 45 days of age; 17 days later they were
given a single dose of DMBA and beginning on the next day they were
injected on alternate days with different doses of estradiol benzoate
(EB) for a total of 150 days. No mammary tumors appeared in the
ovariectomized rats. Of the ovariectomized rats treated with EB,
the greatest incidence of mammary tumors occurred in the rats
treated with 2 pg EB. In this group 17 out of 21 rats developed mama
mary cancers compared to 12 out of 21 in the 0. 2 pg EB group and
10 out of 19 in the 20 pg EB group. The'mean latency period (73
days) was also shortest in the 2 pg EB group compared to the latency
periods in 0. 2 pg (98 days) and 20 pg (119 days) groups. On the

basis of these data they concluded that 2 pg EB administered on

 

 

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112

alternate days was optimal for DMBA-induced mammary carcino-
genesis.

The above mentioned effects of ovariectomy and small doses
of estrogen on carcinogen-induced mammary tumors in rats have
now been confirmed repeatedly by several investigators. Pearson
53311. (19 54b) have reported that estrogen treatment reactivates
tumor growth in humans also after oophorectomy-induced remis-
sion. The mechanisms by which estrogen stimulates breast cancer
in humans or carcinogen-induced mammary cancers in rats are not
clear. Yang e_tal. (1961) Speculated that because of structural
similarities steroids may act at the same sites in the mammary

tissue as hydrocarbons.
Biphasic Effects of Estrogens

It has been mentioned earlier that estrogens have a biphasic
effect on spontaneous mammary tumors in the rat (Miller and Noble,
1952, 1954a, b, c; Huggins ital. , 1956b) i. e. small doses of estro-
gens stimulate growth while large doses have an inhibitory effect.
Huggins eta}, (1959a) were the first to report that estrogens had
a similar effect on the growth of carcinogen-induced mammary
tumors in rats. It has been mentioned before that daily injections
of 0. l or 1 pg estradiol (3 restored MC-induced mammary tumors

in ovariectomized rats while a dose of 20 pg estradiol depressed

 

 

 

 

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113

the incidence of mammary cancers and lengthened the tumor appear-
ance. The biphasic effect of estrogen on carcinogen-induced mam-
mary tumor growth was later confirmed by Pearson and coworkers
(Pearson 3153.1. , 1972a; Sterenthal e_t §_1_. , 1963). They reported
that a dose of 1-5 pg estradiol accelerated mammary tumor growth
whereas a dose of 500 pg daily induced a marked regression of
mammary tumor size. Nagasawa and Meites (Meites, 1972b) also
found that a dose of 20 pg estradiol benzoate injected on alternate
days to ovariectomized rats had an inhibitory effect on the develop-
ment of DMBA-induced mammary cancers: the incidence of tumors
was low, the mean latency period was long and a high percentage

of cancers underwent spontaneous regression. The inhibitory effect
of large doses of estrogen on carcinogen-induced mammary cancers
in rats has since been confirmed by several investigators. Large
doses of estrogen have also been found to cause remission of breast
cancer in about 20% of human patients (Hayward, 19 70).

The mechanism of inhibition of mammary tumor growth by
large doses of estrogen has not been entirely elucidated. Meites
(1972a, b) and Pearson 9.211: (1972a) hypothesized that estrogens
interfere with the peripheral action of prolactin on mammary tissue.
This matter will be discussed in more detail later.

Some investigators have tried to compare the inhibitory actions

of several estrogens on mammary tumor growth. Lemon (1972)

9;:05p

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114

tested the effectiveness of estriol, l6 epi-estriol, estradiol 175 ,
l6 ketoestradiol 17(5, 17 alpha estradiol 17p, estrone, dehydro-
epiandrosterone on the basis of survival of rats carrying DMBA-
induced mammary adenomas. He concluded that estriol and 16
epi-estriol were the most effective inhibitors of tumor growth.
These results need confirmation.

Recently Caldwell _e_tgl. (1971) reported the antitumor effects
of estrogen antibodies on a transplantable mammary adenocarcinoma
(MT/W9A) in female Wistar-Furth rats. They found that the time
between implantation and the first evidence of tumor growth was
prolonged from a mean of 36 days in the control group to 50 days
in the group that was actively immunized against a bovine serum
albumin-estrogen conjugate. The immunized animals had a longer
survival time of about 100 days compared with about 60 days for the
control group. They also found that the animals which had the highest
titer of antibodies survived for the longest time, while few immunized
aminals in which no antibodies were detected had survival times

resembling those in the control group.

Role of Progesterone

Cantarow 2321' (1948) reported that simultaneous administra-
tion of progesterone increased the incidence of mammary tumors

induced by feeding AAF. But Shay _e_t_gl. (1952, 1956, 19 59)

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115

reported that subcutaneous implantation of progesterone pellets into
rats fed MC decreased the incidence of mammary tumors. Huggins
3511. (19 59b) confirmed the findings of Cantarow and coworkers
(1948). They found that concurrent daily intramuscular injections

of 4 mg progesterone markedly accelerated the development of mam-
mary cancers in rats fed MC. The incidence of tumors was 100%
and the mean latency period for the appearance of mammary tumors
was shortened to 40 days compared to 50 days in the intact rats.
They also reported an unexpected finding that the deve10pment of
MC-induced mammary tumors was not delayed substantially if 4 mg
progesterone was injected daily into ovariectomized rats. The

. tumor incidence was 100% and the mean latency period 58 days com-
pared to 50 days in untreated controls and 73 days in ovariectomized
rats which had a considerably lower tumor incidence. Huggins 333.1.
(1959b) concluded from these results that progesterone enhanced
mammary growth incited by estrogens in intact MC-fed rats, while
MC substituted for estrogens to promote mammary growth in the
ovariectomized rats. In a subsequentpaper Huggins and Morii
(1961) reported that daily injections of 4 mg progesterone for 50
days beginning 15 days after a single intragastric instillation of MC
decreased the tumor incidence by 20% and increased the mean
latency period to 61 days compared to 58 days in the untreated

controls. However, the number of active tumor centers increased

e T

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116

in the progesterone-treated as compared to that in the untreated
controls. No statistical evaluation of these results was given. A
year later Huggins and Yang (1962) reported that administration of
4 mg progesterone to intact rats for 30 days beginning 15 days after
a single feeding of 20 mg DMBA accelerated the appearance of
cancers, increased their number and augmented their growth rate.
Huggins 333.1. (1962) confirmed these results in a subsequent paper.
Mammary tumors were induced by a single feeding of DMBA at 50
days of age and the rats were injected with 4 mg progesterone from
age 65 to 95 days. Again progesterone was found to profoundly
accelerate mammary tumor development and growth. The incidence
of cancer was 100%, the time of appearance of tumors was short and
the number of active tumor centers was large. It was concluded that
progesterone mimicked the tumor-stimulating action of pregnancy.
Gruenstein _e_t_ai. (1964) also reported that simultaneous administra-
tion of 4 mg progesterone and MC accelerated mammary tumor
development and increased their incidence in female rats. Jabara
(1967) found that administration of progesterone beginning two days
before DMBA treatment also accelerated mammary tumor develop-
ment in rats.

From the above studies it can be concluded that concurrent
administration of progesterone and a carcinogen stimulates neo-

plastic transformation of the mammary gland in the rat. In these

Tn

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117

conditions progesterone can be considered as a co-carcinogen.
Progesterone also appears to be a strong stimulator of growth of
established mammary tumors, since it accelerated appearance of
palpable mammary tumors when injected 15 days after carcinogen
treatment.

It appears that progesterone also can inhibit carcinogen-
induced mammary tumorigenesis under certain conditions. Welsch
_e_t_al. (1968) reported that daily subcutaneous injections of 4 mg
progesterone for 40 days beginning 25 days before administration
of DMBA at the age of 55 days, reduced mammary tumor incidence
to 79% compared to 100% in the untreated controls. The average
number of tumors per rat also decreased to 3. 3 compared to 12 in
the controls, and the average total weight of tumors per rat declined
to 5.9 g compared to 16 g in the controls. However, the mean
latency period remained unchanged. The mammary glands which
did not undergo neoplastic treatment showed enhanced growth.
Welsch eta}, (1968) concluded that if progesterone treatment
precedes carcinogen administration by at least 25 days, then it
results in enhanced development of the mammary gland which in
turn becomes refractory to carcinogen-induced neoplastic trans-

formation.

Lin!

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118

Influence of Pre gnancy

Cantarow e_t 31. (19 48) found that pregnancy increased the
incidence of AAF—induced mammary tumors in rats but Scholler
(19 58) and Scholler and Carnes (19 58) reported that pregnancy had
no effect on the incidence and growth of DMBA—induced mammary
tumors in Wistar rats. Dao and Sunderland (19 59) reported that both
pregnancy and pseudopregnancy stimulated carcinogen—induced
mammary tumor growth in rats. When pregnancy was induced after
repeated administration of MC, then mean latent period for the
appearance of mammary tumors shortened and the number of mam-
mary tumors per rat increased significantly. After parturition there
was a dramatic regression of all mammary tumors. If these rats
became pregnant again, the tumors also resumed rapid growth.
Pseudopregnancy had a similar stimulating effect on carcinogen-
induced mammary tumor growth. Dao and Sunderland (1959) con-
cluded that progesterone was perhaps responsible for the acceleration
of development and growth of mammary tumors during pregnancy
and pseudopregnancy. These findings were confirmed by Huggins
31331. (1962). They reported that when pregnancy was induced 15
days after a single feeding of DMBA at the age of 50 days the mean
time of appearance of mammary cancers was shortened from 41

days in non-pregnant control rats to 30 days in the pregnant rats and

 

 

 

 

 

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119

the mean number of active centers increased from 2. 7 in virgin rats
to 3. 8 in the pregnant rats. They compared the tumor stimulating
effect of pregnancy to the similar acceleration of mammary tumor
growth by progesterone. However, a year earlier Huggins and Morii
(1961) had reported exactly the Opposite results. They had found

that the incidence of mammary cancers lessened considerably if the
rats were bred 15 days after a single feeding of 100 mg MC. They
also found that pregnancy "destroyed many young cancers that were
obtaining a foothold, and rats in this class never suffered a recrudes-
cence of their mammary cancers. " Dao e_tal. (1960) found that if

the pregnancy occurred prior to administration of a chemical car-
cinogen, it prevented induction of mammary cancer. They also found
that if the rats became pregnant within 10 days after a single dose of
MC then mammary tumor incidence was markedly reduced from 60 %
in the control rats to 9% in the pregnant rats. They hypothesized

that hyperfunctioning mammary glands are refractory to carcinogenic
stimulation because "mammary epithelial cells under strong hor-
monal stimulation are protected from injury by carcinogens" (Dao,
1964). They also found that DMBA treatment of pregnant rats

failed to inhibit RNA synthesis indicating a lack of interaction between
the carcinogen and hyperfunctioning epithelial cells. They con-

cluded that the time interval between pregnancy and carcinogen

17m

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120

treatment was critical in inhibiting or stimulating carcinogen-induced
mammary tumorigenesis (Dao _etal. , 1960; Dao, 1964).

From these studies it appears that pregnancy induced before
or immediately after carcinogen treatment inhibits mammary tumor-
igenesis whereas it greatly stimulates growth of established mam-
mary tumors (Huggins and Yang, 1962). MacMohan EEEl- (1970)
reported that early pregnancies reduced the risk of breast cancer

in women.
Combined Effect of Estrogen and Progesterone

Huggins and Morii (1961) reported that combined daily adminis-
tration of 4 mg progesterone with 1 or 10 pg estradiol-17f} for 50 days
beginning 15 days after a single feeding of 100 mg MC at the age of
50 days, produced a marked decrease in the incidence of mammary
tumors. Only 20 to 30% rats in progesterone and estrogen treated
groups developed mammary tumors compared to a 100% incidence
in the controls. The time of appearance of mammary tumors in the
progesterone and estrogen treated groups also increased to 42-133
days compared to 46-84 days in the untreated controls and the num-
ber of active centers decreased to l. 0 compared to 1:6 in the con-
trols. The combined treatment with progesterone and estrogen

produced a "characteristic gestational type of hyperplasia of the

mammary glands which resembles that found in rats on day 15 of

 

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121

pregnancy. " On the other hand when 4 mg progesterone was admin-
istered with a lower dose of 0. 01 pg estradiol 17‘}, then the mean
latency period decreased to 51. 9 days compared to 58. 0 days in the
untreated controls and the number of active centers increased to 3. 2
compared to 1. 6 in the controls. Unfortunately the investigators
failed to give the statistical significance of any of these results.
Still these were important findings. They confirmed some of the
above findings in a subsequent publication (Huggins £1311. , 1962).
Concurrent administration of 4 mg progesterone and 20 pg estradiol
175 for 30 days beginning 15 days after a single dose of 20 mg of
DMBA at the age of 50 days resulted in a decrease of more than 50%
in the incidence of tumors and more than tripled the mean latency
period and reduced to less than a third the number of mean active
centers. Treatment with progesterone alone accelerated tumor
growth whereas combined treatment with a similar dose of estradiol
17(3 and progesterone inhibited tumorigenesis. These were impor-
tant findings. They suggest that certain dose levels of estradiol
175 may convert progesterone from an accelerator to a suppressor
of mammary cancers in the rat.

Huggins _e_tgl. (1962) carried these experiments further to
determine the dose of estradiol 1715 which would have the most sup-
pressive effect when injected with 4 mg progesterone. They found

that 0- 1'1 IJ-g estradiol 179 did not abolish the cancer stimulating

 

 

 

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122

effect of progesterone, while a dose of 10 pg delayed somewhat the
onset of tumorigenesis, but doses of 20-50 pg abolished mammary
cancers in about 60% of the rats and postponed the development of
cancers in the remainder by several months, and greatly reduced

the number of active centers. They also found that treatment with

20 pg estradiol 176 and 4 mg progesterone for 30 days was more
effective than administration for 10 or 20 days. The mammary glands
of the rats treated with both estradiol 17t3 and progesterone were
extremely hyperplastic although without lactation. Another important
finding in these experiments was that benign mammary tumors and
ear duct tumors were not affected by the combined administration

of estrogen and progesterone. These were significant findings
showing the dual effects of progesterone, stimulatory to tumor
growth with low doses of estrogen and inhibitory with high doses of
estrogen. The discovery of the suppressive effect of estrogen and
progesterone was important clinically since it was later successfully
used to treat human breast cancer (Landau e_t_ a_1_. , 1962).

Other investigators also experimented with combinations of
estrogen and progesterone. Gruenstein _e_t_al. (1964) reported that
Enovid which consists of 98. 5% norethynodrel and 1. 5% mestranol
had no significant effect on rat mammary tumor development when
injected simultaneously with MC. Enovid is an oral contraceptive

and believed to have mainly estrogenic effects. Welsch and Meites

I

 

 

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123

(1969) reported that daily injections of 10 or 100 pg Enovid into
immature (30 day old) rats for 25 days before and 15 days after
DMBA treatment resulted in a significant reduction in number of
tumors per rat. There was also lengthening of the mean latency
period and a decrease in the total weight of the tumors per rat.

They concluded that Enovid stimulated the mammary gland growth
and thus exerted a protective action against tumorigene sis by DMBA.
They believed that Enovid exerted its stimulatory action on the
mammary gland by increasing prolactin secretion both via the
hypothalamus and the pituitary and by a direct action on the mammary
gland. Enovid had a different action when injected for 25 days into
mature rats bearing DMBA-induced tumors. It significantly stimu-

lated the tumor growth.

Role of Androgens

 

Farrow and Adair (1942) reported that orchidectomy caused
regression of mammary cancers in men. Since then several investi-
gators have reported the usefulness of this surgical procedure in
treatment of cancer of the male breast. The situation appears to be
different in rats. Millar and Noble (19 54b) found that castration of
males increased the growth of Spontaneous mammary tumor trans-
plants and administration of androgens of several kinds has been

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growth of spontaneous mammary tumor transplants in female rats
(Heiman, 1940a, b; Huggins and Mainzer, 1957; Glenn gtua_l. ,
1959b). Testosterone has been found to cause regression of breast
carcinomas in women also (Huggins, 19 54).

Male rats are relatively refractory to induction of mammary
cancer by polynuclear hydrocarbons. Shay §_t_a_l. (1949) reported a
low mammary tumor incidence of 11% in male Wistar rats after
gastric instillation of MC. Kim and Furth (1960a, b) were unable to
induce mammary tumors in male rats after treatment with MC.

Dao (1962a, b) and Dao and Griener (1961) also reported the inability
of polynuclear hydrocarbons to induce mammary cancer in male
rats. Huggins and Grand (1966) made a very determined effort to
induce mammary cancers in male Sprague-Dawley rats by intra—
venous injections of DMBA, 35 mg/kg or 6 mg, whichever amount
was smaller. They reported that a single dose of DMBA elicited
leukemia, mammary cancer and periauricular cancer in 32% of the
rats, whereas with 6 doses of DMBA the incidence of multiple
classes of neoplasms rose to 100% but only 14% of the rats developed
mammary cancer exclusively. The mortality rate was very high in
these experiments. The most important findings of these experi-
ments was that bilateral orchiectomy had no effect on growth of
established mammary tumors which showed a progressive increase

in size during a 4 week postOperative period. Similarly,

 

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hyp0physectomy had no significant effect on the growth of mammary
tumors which continued to increase in size during a 28-52 days
observation period and active mammary cancers were observed at
necropsy. Thus mammary cancers in male rats differ profoundly
from breast cancers in human male and from hydrocarbon-induced
mammary cancers in female rats. Huggins and Grand (1966) con—
cluded that "aromatic-induced cancers of the breast of male rats
are, by definition, hormone-independent".

Shay and collaborators (1952, 1956, 1959) reported that sub—
cutaneous implantation of testosterone pellets decreased development
of mammary cancers in female rats after repeated gastric instilla-
tion of MC. Huggins 31331. (19 59b) found that daily injections of 1 mg
dihydrotestosterone had an inhibitory effect on deve10pment of mam-
mary cancers in female rats treated concurrently with 1 mg DMBA.
DMBA was given by stomach tube 6 days each week for 100 days to
50 day old female rats and the androgen was injected intramuscularly
for 84 days. This decreased the incidence of mammary tumors by
about 45% and prolonged the mean time for appearance of mammary
tumors to 177. 2 days compared to 78. 9 days in the control rats. In
another experiment dihydrotestosterone, 1 mg or 2 mg, was injected
into 17 rats bearing DMBA-induced tumors. A considerable regres-
sion 9f miil’l'lrnary cancer occurred in 14 animals while the tumors

continued to grow in 3 animals. The tumors which underwent

 

 

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regression showed a cytological picture similar to that observed
after ovariectomy or hyp0physectomy. There was atrophy of
epithelial cells and many layers of the acini were replaced by a
single layer of flat cells and acini lost their alkaline phOSphatase
and carbohydrate contents.

Dorfrnan and colleagues reported extensive work on the
influence of androgens on carcinogen-induced tumors and on trans-
plants of Spontaneous mammary tumors in rats (Dorfrnan, 1965;
Harda 232.1. , 1965; Rooks 3331. , 1963, 1966; Abe 3331. , 1962).
Huggins and Mainzer (19 57) had found earlier that 2a-methy1-17fi-
hydroxy 5 a-androstan—3-one (drostanolone) was an effective inhibitor
of a transplantable spontaneous mammary fibroadenoma in rats.
Abe £31. (1962) confirmed the suppressive actions of drostanolone
and also of testosterone on the growth of a transplantable mammary
fibroadenoma in rats on the basis of inhibition of glycine-2--Cl4
incorporation into the tumor proteins. They found that drostanolone
was a much more potent inhibitor of tumor growth than testosterone
propionate. In a subsequent publication Rooks 22.3.1.0 (1963) tested
several steroids for their antitumor effects by using the Glycine-2-
C14 uptake bioassay method. They found 2-hydroxymethy1ene-17a-
methyl- 17(3- hydroxy- Sa-androstan- 3- one , 2a-hydroxymethy1-17fl-

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were effective inhibitors of tumor growth but none was as effective
as drostanolone.

Thus far drostanolone had been tested for its effect on trans-
plants of spontaneous mammary fibroadenomas only, and it was not
known if this androgen was as effective an inhibitor of carcinogen-
induced mammary tumors which had by that time begun to replace
spontaneous mammary tumors as laboratory models for research on
endocrine aspects of mammary carcinogenesis. In another paper
(Harda -e_t§_._1_. , 1965), from Dr. Dorfman's laboratory, this need was
fulfilled. Mammary tumors were induced in female Sprague-Dawley
rats by repeated administration by gavage of MC or by a single
administrationofDMBA. They reported on the basis of Glycine-Z-C14
uptake bioassay, that drostanolone was again the most effective
inhibitor of these tumors. A corresponding l7a-methy1 derivative
also possessed antitumor properties. In a subsequent paper Rooks
3311, (1966) tested 12 compounds of the androstane series, many of
which exhibited significant inhibitory activity approaching or equal
to that of drostanolone. Rooks and Dorfman (1966) found that
drostanolone not only inhibited growth of spontaneous and MC and
DMBA-induced mammary tumors, but also significantly increased
the survival time of tumor-bearing rats. Drostanolone has been

reported to be of value in treatment of human breast cancer and to

 

   

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provide relief from pain (Blackburn, 1962; Goldenburg and Hayes,

1962; Thomas 2321- , 1962).
Role of the Adrenal Glands

Huggins and coworkers introduced adrenalectomy for treatment
of human breast and prostatic cancers (Huggins and Scott, 1945;
Huggins and Bergenstal, 1951). They published several papers
explaining the theoretical and therapeutic importance of this pro-
cedure (Huggins and Dao, 1952, 1953: Huggins and Bergenstal, 19 52).
During a five year period Dao and Huggins (19 57) performed adrenal-
ectomy in a total of 52 patients with metastic mammary cancer.
They reported only 2 postoperative deaths, remissions of various
durations in 12 of 32 patients who had bilateral adrenalectomy alone
and in 8 of 18 patients who also underwent 00phorectomy. They
concluded that breast cancer patients are most likely to be helped
if selected on the basis of age, rate of neoplastic growth, titer of
estrogenic substances in the urine and microscopic appearance of
the tumor tissue. Huggins (19 56) explained that the use of adrenal-
ectomy for treatment of mammary cancer was based on the knowledge
that the human adrenal gland secretes sufficient quantities of growth-
promoting steroids to maintain hormone dependent neoplasma. The
baSiC theoretical considerations, as Huggins explained, which led to

the in'5r0dlICtion of use of adrenalectomy were: (1) steroids which

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promote growthof secondary sex structures are elaborated by the
adrenal cortex in patients with tumors or hyperplasia of this struc-
ture (2) steroids of this kind are produced in certain women after
me’n0pause (3) orchiectomy in patients with cancer of the prostate
induces a rise in the amount of 17-ketosteroids excreted in the urine
(4) gonadectomy performed at an early age in certain strains of mice
leads to hyperplasia and neoplasms of the adrenal cortex which
secretes steroids that induce growth in the mammary gland (5) in
forced-fed ovariectomized rats, adrenalectomy retards the growth
of Walker carcinoma 256. These and other considerations led
Huggins to revive hormonal treatment for prostatic cancer (for which
he was awarded the Noble Prize) and mammary cancer.

Shay _e_t 31. (1960) reported that adrenalectomy had no effect on
the incidence of MC-induced mammary tumors in rats. Kim (1965)
found that adrenalectomyaccelerated growth in DMBA-induced
mammary tumors in rats. But Gruenstein _e_t_a_l_. (1966) reported
that removal of the adrenals had slight but insignificant effects on
the development of MC-induced mammary cancers in Wistar rats.
Huggins 31331 (195921) also found that adrenalectomy had no major
effect on development of carcinogen-induced mammary cancersin rats.
Daniel and Prichard (1967) did an extensive study to determine the
effeCt Of adrenalectomy on the growth of carcinogen-induced mam-

mary tumors in Sprague-Dawley rats. Tumors were induced by

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repeated gastric instillation of MC for 7 weeks into 34-38 day old
rats. They found a significantly higher incidence of tumors in the
adrenalectomized than in the intact rats. Unfortunately however,
they do not mention the exact schedules of operations performed with
regard to MC-administration, use statistics rarely and their
schedule of MC administration was not the best available. They
found that adrenalectomy had no effect on established mammary
tumors which continued to grow, often extremely rapidly. Ovari-
ectomy exerted an inhibitory effect on the tumors which before were
"stimulated to grow by adrenalectomy". They state that ovariectomy
and adrenalectomy had greater inhibitory effects on tumor growth
than ovariectomy alone, although the difference was not significant
statistically.

Additional work is needed to more precisely determine the
effects of adrenalectomy on the development and growth of DMBA-
induced mammary tumors. It appears that a significant interaction
takes place between carcinogens and the adrenal cortex. Huggins
and Morii (1961) reported that intravenous injections of DMBA caused
massive necrosis in the inner zones of the adrenal cortex, but the
zona glomerulosa and the adrenal medulla were unaffected. They
postulated that because of structural similarity, DMBA enters the
adrenal cortex, gets localized there and causes damage to the cell.

Its full significance has not been elucidated.

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131

Role of the Pituitary Gland
Effect of Anterior Pituitary Extracts

About 45 years ago Evans and Simpson (1929) reported that
treatment of normal female rats for three or four weeks with aqueous
alkaline extracts of the anterior hypophysis resulted in marked
proliferation of the mammary gland. They were particularly fascin-
ated by the effects of more chronic administration of such hypophyseal
extracts. "So striking was the phenomenon", they reported (Evans
and Simpson, 1931), "that in some cases milk exuded from the mam-
mary gland when the latter was incised at autopsy . . . the mammary
development resembled closely that at the height of lactation". They
were in for a bigger surprise when they injected such extracts for
periods of 12 to 18 months. It resulted in the formation of multiple
mammary tumors (Evans and Simpson, 1931). They reported that
"localized enlargements in the mammary line began to appear in
animals of 15 months of age which had been injected for 11 months. "
These enlargements were diagnosed to be adenofibromas. Sixty-two
percent (10 out of 16) of the animals which lived to the age of 16
months and had received daily intraperitoneal injections of the above
mentioned hypophyseal extracts developed one to six lobulated tumors,
some of which reached great dimensions and weighed up to 263

grams". None of the tumors metastasized. Evans and Simpson

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(1931) speculated that "the tumor formation observed in these cases
may be related to the greater frequency of mammary tumors in
women in whom ovarian dysfunction has for years produced menstrual
irregularities". No tumors deve10ped in similarly treated male or
ovariectomized female rats. They also reported that in intact female
rats "striking reactions" also occurred after treatment with "implants
of rat hypophysis or with extracts of human placenta and the urine of
pregnant women". They failed to be more specific about the nature
of the "striking reactions", but it appears safe to assume after read-
ing their paper that they would have included marked mammary
hyperplasia and possibly mammary fibroadenomas.

These were very important observations. For the first time
they directly implicated the pituitary in neoplastic transformation of
the mammary gland and also pointed out the need for ovarian hor-
mones in such a phenomenon.

Induction of Mammary Cancers
in Hypophysectomized Rats

The same group of investigators (Moon, Simpson and Evans,

19 52) were also the first to demonstrate the importance of the
pituitary gland in carcinogen-induced tumors in the rat. They
implanted MC pellets into the right gastrocnemius muscle of 15

adult female rats of the Long-Evans strain. During an observation

 

 

 

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period of 316 days, 8 of the 15 rats deve10ped sarcomas at the site
of the implants. However, of the 15 rats which were hypophysec-
tomized two weeks before MC implantation only one developed a
sarcoma at the site of the implantation. It is unfortunate that the
carcinogen was implanted in the gastrocnemius muscle rather than
in the mammary gland, although it is not certain if that would have
resulted in the production of mammary carcinomas. Nevertheless,
this experiment demonstrated that hypophysectomized rats were
refractory to the carcinogenic effects of MC. Two years later Noble
and Walters (19 54) carried out a similar experiment except that
they injected 5 mg of DMBA intramuscularly and reported production
of adenocarcinomas of the breast near the site of injection in 4 out
of 21 female rats in a period of 4-5 months. Most of the remaining
animals developed discrete sarcomas. In 19 hypophysectomized
animals a similar DMBA-treatment failed to induce any mammary
tumors and only 3 rats developed sarcomas. Huggins £331. (19 59a)
perfected a method by which they could induce mammary adenocar-
cinomas in almost 100% of the female rats treated with DMBA or
MC. The same year Huggins and Briziarelli (19 59) reported that
the p01ycyclic hydrocarbons failed to induce mammary cancers in
hypophysectomized female rats during an eight month period. How-
ever they failed to give the schedules of the surgical operation or

carcinogen treatment. Not many attempts were made to induce

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mammary cancers by carcinogens in hypophysectomized rats because
of the low survival rate of such animals. Dao (1964) reported that
six rats which were hypophysectomized and treated with 30 mg MC
died within four months without developing any tumors. Earlier
Dao and Sunderland (19 59) had also reported that feeding of MC for
15 days failed to elicit mammary tumors in hypophysectomized rats.
Furth (1967) reported that estrogen also failed to induce mammary
cancers in hyp0physectomized rats.
Effect of Hypophysectomy on Established
Mammary Cancers

The Swedish workers, Luft, Olivecrona and Sjogren (1952)
introduced hyp0physectomy for treatment of human mammary cancer.
It was based on observations that this surgical procedure results in
marked atrophy of secondary sex organs in experimental animals.
In a subsequent publication Luft and Olivecrona (1953) described the
many beneficial effects of hyp0physectomy in patients suffering from
breast cancer. These findings were confirmed by Perrault __e_t§._l.
(1954) in France and by Pearson 3131. (1954) in the U. S. A. Pearson
e_tél. (1954) reported that hyp0physectomy produced remission in
50% of treated cases while Atkins gta_l. (1960) reported remission
of breast cancer in 30% of the patients. Lawrence and Tobias (19 56)

have tried to induce remission of breast cancer by abolishing the

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pituitary function by external radiation.

Ehni and Eckles (19 59) reported that the pituitary stalk section
also led to remission of breast cancer in humans. These results
seem to create a paradox. However, in most of the stalk section
studies, cauterization of the pituitary was also performed which
might have affected the pituitary function (Pearson e_t §_l_. , 1969).
Daniel and Prichard (1963) reported that the pituitary stalk section
produced slight regression of experimentally induced mammary
cancers in rats also. However, they also reported extensive infarc-
tion of the pituitary tissue.

Several investigators have reported the effects of hyp0phy-
sectomy on carcinogen-induced mammary tumors in rats. Furth
and Clifton (19 57) reported that it induced regression of almost all
DMBA-induced mammary tumors. Huggins _e_t_gl. (l959a) reported
that hyp0physectomy 25 days after development of MC-induced
palpable mammary cancers in rats resulted in profound decrease
in size of the tumors. The regressed tumors had a characteristic
cytologic appearance of atrophy of epithelial cells. The many layers
of epithelial cells surrounding the tumor acini were replaced by a
single row of flat cells. These tumors resembled in histologic
appearance those which regressed after ovariectomy or dihydro-
testosterone treatment. They concluded, perhaps because of this

histologic resemblance, that the regression of the mammary tumors

136

was due to abolition of ovarian function after hyp0physectomy. They
also reported that hyp0physectomy was more effective than either
ovariectomy or dihydrotestosterone treatment in producing regres-
sion of carcinogen—induced mammary cancers in rats. The same
year Dao and Sunderland (19 59) confirmed the findings of Huggins
3311, They reported a decrease in size of MC-induced mammary
tumors within 2-4 days after hyp0physectomy. Kim and Furth
(1960a, b) also reported complete regression of 51 of 59 MC-induced
mammary tumors in 20 rats following hyp0physectomy. About 54%
of the regressed tumors remained "minute" throughout the observa-
tion period, about 37% could not be found at autopsy and only about
9% of the tumors resumed growth but at a very slow rate. No new
tumors appeared in the hyp0physectomized rats. Sterenthal 2E2}.-
(1963) also reported that DMBA—induced mammary tumors regressed
after hyp0physectomy and that the animals lived for several months
without recurrence of the tumor growth. They reported similar
results a few years later (Pearson _e_tgl. , 1969). In this study the
average area of DMBA-induced mammary tumors was reduced to
less than one third within 7 days after hyp0physectomy. Daniel and
Prichard (1963) compared the effects of hyp0physectomy and the
pituitary stalk section on carcinogen-induced mammary tumors in
rats, Whereas stalk section caused only a minor tumor regression

as mentioned above, hyp0physectomy produced a complete regression.

 

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Effects of Pituitary Grafts

Since pituitary grafts are to a great extent free from direct
hYpothalamic control and secrete large amounts of prolactin it was
1C>gical that the effects of such grafts on mammary tumorigenesis
Were investigated. Several people have reported tumorigenic effects
of pituitary grafts in mice. Loeb and Kirtz (1939) were the first to
report that subcutaneous implantation of several pituitaries increased
the incidence of mammary tumors in several strains of mice.
Muhlbock and Boot/(19.59) and Boot _et_a_i_l_. (1962a, b) reported induc-
tion of mammary cancer in mice without the mammary tumor agent
by isografts of several pituitaries. Liebelt and Liebelt (1961)
reported similar results using a single pituitary isograft. The same
year Bittner and Cole (1961) were successful in inducing mammary
tumors in viral agent-free, ovariectomized mice. During the sub-
sequent years Hagen (1966) and Hagen and Rawlinson (1964) induced
mammary cancers in male mice by isologous pituitary implants.
Stimulation of neoplastic transformation of the mammary gland in
mice by pituitary isografts has been reported by several other investi-
gators including Dux and Muhlbock (1969) and Muhlbock and Boot
(1967).

In contrast to the extensive work done in mice on the tumori-

genic effects of pituitary grafts, very little attention has been paid to

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this area in rats. Dao (1962a) and Dao and Gawlack (1963) reported
that subcutaneously tranSplanted pituitaries induced marked growth
0f the mammary glands but failed to induce mammary tumors.
Welsch 23.9.1- (1970a) reported that grafting of female rats with
Several pituitaries increased the incidence of spontaneous tumors.

A total of 7 pituitaries were implanted in each rat, 5 subcutaneously
in inguinal, abdominal and thoracic regions of the mammary gland
and 2 underneath the kidney capsule. Nine months after grafting,

the tumor incidence was 30% in the rats which were 2 months old at
the time of implants and 61% to 71% if they were 8 months old when
they received the pituitary hemografts. This contrasted with 7% and
8% to 19% respectively in nongrafted controls of the same age. These
results also indicated that older rats are more prone to develop
mammary tumors after receiving pituitary grafts than younger rats.
They also reported that tumorigenic effects of the grafts were more
apparent in nulliparous than in multiparous rats. All rats receiving
the grafts had shorter latency periods for tumor appearance than the
nongrafted controls. All mammary tumors were almost exclusively
adenomas with varying amounts of connective tissue. This contrasts
with adenocarcinomas produced by prolonged estrogen administration
as was discussed earlier in this review. Welsch _e_t_-'11. (1970a) con-
cluded that enhanced tumorigenesis was due to increased prolactin

secretion from the pituitary transplants.

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Dao (1962a) investigated the effects of pituitary transplants on
carcinogen-induced mammary tumorigenesis in rats. He reported
subcutaneous transplantation of single pituitaries in intact or ovari-
ectomized rats prior to MC administration had no effect on mammary
tumor induction. Welsch_e_t_a;l. (1968) investigated the effects of
multiple pituitary transplants on DMBA-induced mammary tumori-
genesis. They transplanted immature 25 day old female rats with 4
pituitaries underneath the kidney capsule 30 days before DMBA treat-
ment at the age of 55 days. This reduced the tumor incidence to 73%
compared to 100% in the controls. The average number of tumors per
rat also decreased to 4. 7 compared to 12 in the controls. However
the mean latency period for tumor appearance was shortened con-
siderably from 68 days in the controls to 54 days in the grafted rats.
Theyconcluded that increased prolactin secretion from the pituitary
grafts prior to carcinogen treatment stimulated mammary growth and
inhibited tumorigenesis. They also hypothesized that the inhibitory
effect of the graft was also "partly indirect i. e. by a luteotropic
action of prolactin on the ovary, resulting in elevated progesterone
Secretion". They explained that the short latency periods in the
pituitary grafted rats were due to the fact that the pituitary grafts
Stimulated growth of the mammary tumors once the neOplastic process
began thus contributing to shortened latency periods.

Dao (1964) reported that single pituitary grafts into

 

 

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hyp0physectomized-ovariectomized rats failed to initiate mammary
tumorigenesis after a single feeding of DMBA. Welsch _e_tgl. (1968)
reported that multiple pituitary grafts into immature (25 day old)
ovariectomized rats 30 days before DMBA treatment also failed to
induce mammary tumors. This, they concluded, suggested a
Synergistic role for pituitary and ovarian hormones in mammary

carcinogenesis.

Effect of Pituitary Tumor Transplants

Furth e_t_al. (19 56) induced pituitary tumors in rats by chronic
eStIOgen treatment. These tumors secrete large amounts of pro-
lactin and growth hormone. They have been described in detail
earlier in this thesis. Furth and co-workers published a series of
Papers describing their effects on spontaneous and carcinogen-
induced tumorigenesis in rats (Kim and Furth, 1960a, b; Kim 3511. ,
1960). In the first of these papers Kim and Furth (1960a) reported
that neither subcarcinogenic single dose of MC or the pituitary tumor
transplant alone induced any mammary carcinomas during a period
Of 158-213 days. Both the treatments induced a single fibroadenoma
each in 8 rats. However when the pituitary tumor was grafted 2
Weeks prior to MC administration,it resulted in production of several
mammary carcinomas. They concluded that "mammotropin"

secreted from the pituitary tumors was involved in initiating

 

 

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carcinogen-induced mammary tumorigenesis. In a subsequent
experiment (Kim and Furth, 1960b) they studied the role of
"mammotropic hormone" (Mammotropin, MtH) of the pituitary tumor
on growth of carcinogen-induced mammary tumors. They found that
implantation of pituitary tumors, 33 days after ovariectomy, reacti-
vated growth of the regressed mammary tumors. Similar resump-
tion of tumor growth followed pituitary tumor implantation in hypo-
PhYsectomized rats. In this group of rats not only tumor growth
resumed but several new tumors appeared. They concluded that
"MtH" can not only stimulate tumor growth but also "can unfold

latent neoplastic potentialities". Kim _etgl. (1960) also studied the
effects of pituitary tumors on growth of transplants of MC-induced
mammary tumors. These transplants regressed following ovari-
eCtomy or hyp0physectomy but resumed growth, often very rapidly,
When the rats were grafted with pituitary transplants. Based on

all the experiments described above, Kim and Furth (1960a,b) and Kim
ital. (1960) concluded that "Mammotropin" from the pituitary tumors
was "the main direct stimulant of the mammary gland and its
tumors". It is interesting that the authors failed to mention the

role of GH in mammary tumorigenesis, although it is also known to
be secreted from these pituitary tumors. Meites and co-workers
(Meites, 1972b) have also studied the role of mammosomatotropic

tumors (MtT.W15 and MtT. W5) in mammary tumorigenesis in inbred

142

Wistar-Furth rats. They reported that some of the rats developed
palpable mammary tumors 3. 5 to 4. 5 months after they were grafted
with pituitary tumors. Histologically the tumors were adenocar-
cinomas. Transplants of these tumors into other rats of the same
strain grew only if the rats were simultaneously transplanted with
the pituitary tumors. The mammary tumor transplants did not
grow in ovariectomized—adrenalectomized rats but grew well if the
doubly-operated rats were also grafted with pituitary tumors. This
experiment indicated that the mammary tumor was primarily
dependent for its growth on the hormones secreted by the pituitary
tumor rather than on the ovarian and adrenal steroids. In a subse-
Cluent passage the mammary tumor transplants grew without any
latency period if the host rats already had a well grown pituitary
tumor transplant. The mammary tumors also showed successful
"takes" in intact rats without pituitary tumor grafts but the latency
Period was very long and the growth was limited. Again, this indi-
cated primary dependency of this tumor on the homrones of the
pituitary tumors MtT. W15 and MtT. W5, namely prolactin, GH and

possibly small amounts of ACTH.

Effect of Exogenous Prolactin

The literature reviewed thus far dealt with the effects on

mammary tumorigenesis of i_n situ pituitary which secretes several

143

hormones, of lack of _ingi_tg pituitary (hyp0physectomy), of pituitary
grafts which secrete prolactin and GH and of pituitary tumors which
secrete both prolactin and GH in large amounts. Although in all
these studies it was assumed that of all the pituitary hormones
prolactin was the main hormone involved in mammary carcinogenesis,
yet it was logical to investigate the effects of prolactin alone on the
development and growth of mammary tumors in rats. Boot and
collaborators (Boot _e_t_al. , 1962; Boot, 1970) reported that chronic
injections of prolactin increased mammary tumor incidence in mice.
Talwalker _e_t_a_l_. (1964b) reported that twice daily injection of 1 mg
Porcine GH and 1 mg ovine prolactin for 7 days before and 7 days
after DMBA administration into ovariectomized rats resulted in a
44% tumor incidence as compared to 0% tumor incidence in the
ovariectomized rats. If the same rats were injected daily with the
increasing doses of GH and prolactin (1 to 3 mg) for 75 days then

the tumor incidence increased to 66%. This was the first study
Which clearly demonstrated that 2 pituitary hormones, prolactin and
CH, can support carcinogen-induced mammary tumorigenesis in the
absence of ovarian steroids. Pearson _e_t_al. (1969) investigated the
role of prolactin on the maintenance and growth of carcinogen-
induced mammary tumors in rats. They reported that daily injections
0f 1. 5 mg ovine prolactin reactivated growth of DMBA-induced

mammary tumors which had regressed after hyp0physectomy. They

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concluded that DMBA-induced rat mammary carcinoma is prolactin-
dependent and that the stimulating effects of estrogens on tumor
growth might be mediated through stimulation of pituitary prolactin
secretion. Nagasawa and Yanai (1970) investigated theeffects of
prolactin or GH alone on DMBA-induced mammary tumors in
adreno-ovariectomized rats. Rats were adreno-ovariectomized 15
days after the first palpable mammary tumors appeared and were
treated with either prolactin or CH beginning the next day. They
reported that twice daily injections of 1. 25 mg and 2. 5 mg of bovine
prolactin increased tumor incidence by 201% and 390% reSpectively
by the end of 20 days compared to an increase of less than 50% in
intact controls. Prolactin treatment also increased average tumor
size and average total tumor weight per rat. Administration of 2. 5
mg prolactin twice daily for 10 days, beginning 20 days after adreno-
Ovariectomy reactivated growth of the regressed tumors to pre-
operative levels. In contrast to prolactin, similar treatment with
l. 5 mg bovine GH failed to prevent tumor regression that ensued
after adreno-ovariectomy although it stimulated normal mammary
gland development and increased body weights. It was concluded
that prolactin is the principal hormone responsible for maintenance
and growth of DMBA-induced mammary tumors in rats. We have
found that daily injections of l. 0 mg ovine prolactin for 3 weeks

into DMBA-induced mammary tumor-bearing rats resulted in several

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fold increases in mean tumor number and in mean total tumor dia-
meter per rat (Quadri, Kledzik and Meites, unpublished data). On
the other hand prolactin antibody has been reported to cause regres-
sion of established carcinogen-induced mammary tumors in intact
rats (Pearsongtal. , 1972a, b; Butler and Pearson, 1970, 1971).
Twice daily treatment with antirat prolactin antibody for 36 days
caused regression of 50% DMBA-induced tumors compared to a
regression of only 13% tumors in the controls during the same period.
Almost all tumors regressed in about 50% of the antiserum~treated
rats. Anitbody treatment also prevented tumor growth, and there

W as only a small increase in tumor area in the antiserum treated

rats compared to a four-fold increase in tumor area of the control
rats. The investigators concluded that prolactin antibody interfered
With growth of mammary tumors by inactivating endogenous prolactin.
Cessation of antiserum treatment resulted in resumption of tumor
growth.

In a recent paper Nagasawa _e_t_al. (1973) attempted to determine
the relation of blood prolactin levels to growth of carcinogen-induced
mammary cancers. They measured serum prolactin by radioimmuno-
assay beginning about 45 days after the appearance of DMBA-induced
mammary tumors and then at monthly intervals for a total of 165
days. They found that although the average number of tumors

increased from 1. 5 to 7. 5 per rat and average tumor diameter

 

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146

increased from 0. 7 to 1. 4 cm, serum prolactin concentrations
fluctuated insignificantly within a constant range of 117 to 91 ng/ml.
They concluded that DMBA-induced mammary tumors grow in the

presence of normal serum prolactin concentrations.
Effect of Central Acting Drugs

Since secretion of prolactin and other anterior pituitary
hormones is controlled by the central nervous system and hence can
be altered by treatment with central acting drugs, it was logical to
investigate the effects of these drugs on mammary tumorigenesis.
Lacassagne and Duplan (1959) reported that treatment with reserpine
Shortened the latency period for appearance of mammary tumors in
C3H mice, but others (Cranston, 1958; Dukor e_tgl. , 1966) reported
that reserpine, chlorpromazine and other central acting drugs had
no effect on growth of established mammary tumors or mammary
tumor grafts in mice. It appears that these tumors in all probability
Were not hormone-responsive or had lost their responsiveness dur-
ing repeated passages as discussed earlier in this review. Pearson
e493}. (1969) reported that injections of 4 mg perphenazine three
times each week to rats which had been fed DMBA two months
previously but had not yet developed palpable mammary tumors,

did not shorten the latency period for appearance of mammary

tumors but markedly increased the number and size of mammary

 

 
 

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tumors. When perphenazine injections were begun immediately

after feeding DMBA then again there was a marked increase in the
incidence, number and size of the mammary tumors induced.
Perphenazine has been shown to increase prolactin secretion in rats
(Ben David, 1968; Pearson gtgl. , 1969; Quadri, Karande and Meites,
unpublished data). Muckter e_t_ a_l. (1969, 1970) reported that a
thalidomide derivative, CG 603, was more effective in inhibiting
DMBA-induced mammary tumors than ovariectomy, estrogen or
andrOgens. Its antitumor activity was equal to that of hyp0physectomy.
When CG 603 was given in combination with drostanolone propionate,
there was enhanced regression of mammary tumor weight and
number and prolongation of the survival time of the tumor-bearing
rats. Gelato _e__t__a_l_. (1972) have reported that CG 603 inhibits pro-
lactin secretion in rats.

Meites and co-workers have done extensive research on the
effects of central acting drugs on the growth and development of
carcinogen—induced mammary tumors in rats. Nagasawa and Meites
(1970b) reported that daily treatment with 7. 5 mg iproniazid for 25
days resulted in complete suppression of DMBA—induced mammary
tumor growth. Quadri gal. (1973a) reported that 3-week treatment
With daily injection of 2.. 5 mg pargyline resulted in 30% loss in aver-
age tumor diameter and a reduction of 26 70 in average tumor number

per rat compared to gains of 40% and 42%, respectively, in the

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control rats. No new tumors appeared during the entire treatment
period. Quadri e_t_a_l_. (1973b) found that injections of 20 mg of
l—dopa three times daily for 21 days completely suppressed growth of
DMBA-induced mammary tumors. At the end of treatment the mean
tumor diameter and mean tumor number in the l-dopa treated rats
had been reduced to 1. 6 cm and 2. 0 tumors per rat respectively
compared to 6. 7 cm and 6. 2 tumors per rat in the control rats.
Five tumors completely disappeared in the l-dopa treated rats and
a total of 16 tumors were in a state of active regression compared
to 55 actively growing tumors in the control rats. L-dopa treatment
also prevented development of new tumors. Recently there have been
unpublished reports that l-dopa also was effective in treatment of
human. breast cancer. L-dopa, iproniazid, and pargyline increase
catecholamine activity in the hypothalamus. The former is the
immediate precursor of catecholamines while the latter two are
monoamine oxidase inhibitors and produce increased accumulation
of catecholamines by inhibiting their degradation. All three have
been shown to increase hypothalamic PIF levels and decrease
pituitary prolactin, secretion, which leads to suppression of mammary
tumor growth (Meites _e_t_gl. , 1972).

Drugs which decrease hypothalamic catecholamine activity
and increase pituitary prolactin secretion had exactly the opposite

effects on mammary tumor growth. Welsch and Meites (1970)

 

149

reported that treatment of rats with 10 or 100 pg reserpine per 100
g body weight beginning 5 or 25 days before DMBA treatment signifi-
cantly reduced the incidence and growth of tumors. The higher

dose of reserpine also prolonged the mean latency period for tumor
appearance to 98 days compared to 70 days in the control rats. On
the other hand, reserpine treatment of tumor-bearing rats resulted
in marked stimulation of tumor growth. Quadri _eta_l. (1973a)
reported that daily injections of 50 pg haloperidol for a total of 28
days increased average tumor diameter and tumor number of
established DMBA-induced tumors by 340% and 240% respectively
compared to respective increases of 40% and 42% in the control rats.
Similarly twice daily treatment of tumor-bearing rats with 20 mg

0f methyldopa increased tumor size to 10. 6 cm and tumor number

t0 10. 6 at the end of a 4 week treatment period (Quadri 932.1. ,

1973b). This compared with a tumor diameter of 6. 7 and a tumor
number of 6. 2 in the controls during the same period. At the end

0f the treatment the methyldopa treated rats had a total of 99 actively
growing tumors compared to 55 such tumors in the control group.
Reserpine depletes hypothalamic catecholamines. Haloperidol is

a butyrophenone that blocks the actions of catecholamines,and
methy1d0pa is a false precursor that decreases catecholamine
Synthesis. All three have been shown to reduce hypothalamic PIF

levels and raise serum prolactin concentrations by several hundred

150

percent (Meites gt _a_l. , 1972). It was concluded that increased
prolactin levels produced the marked acceleration of established
mammary tumor growth. When treatment with these drugs was
terminated there was a prompt reduction in the tumor growth. On
the other hand, cessation of treatment with l-dopa, iproniazid or
pargyline, resulted in resumption of tumor growth which often
exceeded that of the control rats. These reversals of growth pat-
terns were believed to reflect alterations in serum prolactin release.
Another class of drugs reported to be potent inhibitors of
spontaneous and carcinogen-induced mammary tumors consists of
several ergot derivatives. Shelesnyak and co-workers discovered
that ergot drugs were potent suppressors of reproductive phenomena
such as deciduoma formation, pseudopregnancy, early pregnancy
and lactation, all of which depend on adequate prolactin secretion
(Shelesnyak, 1954; 1958; Carlsen _e_t_gl. , 1961; Zeilmaker and Carlsen,
1962). Several ergot derivatives including ergocornine, ergocryp-
tine, LSD (lysergic acid diethylamide) have been found to inhibit
prolactin secretion directly by acting on the pituitary and indirectly
by increasing hypothalamic PIF activity (Wuttke gig}. , 1971; Lu
£1531. , 1971; Quadri and Meites, 1971a; Nasr and Pearson, 1971;
Quadri, Karande and Meites, unpublished data). Nagasawa and
Meites (1970) were the first to report that ergocornine was a potent

inhibitor of growth of DMBA-induced mammary tumors in rats.

151

Heuson _e_t_a_l_. (1970) reported that a 6 week treatment with ergocryp-
tine caused regression of established DMBA-induced tumors in rats
and inhibited development of new tumors. Cassell 21.3.1: (1971)
reported that the inhibitory actions of ergocornine and ergocryptin
on established mammary tumors paralleled that seen after ovariec-
tomy. Nasr and Pearson (1971) also reported that ergocornine
inhibited mammary tumor growth and reduced serum prolactin con-
centrations. Quadri and Meites (1971b) found that ergocornine and
ergocryptine also caused regression of spontaneous mammary tumors
in rats. Quadri _e_t_ai. (1973a) reported that LSD also has antitumor
properties. Daily treatment with 2 pg LSD per 100 gm body weight
prevented increases in both tumor diameter and number of estab—
lished DMBA-induced mammary tumors in rats. Recently Quadri
£511. (1973c) reported that treatment with combinations of ergocor-
nine and ovariectomy or ergocornine and high doses of estrogen
were more effective in causing mammary tumor regression than any
single treatment alone. Clemens and Shaar (1972) reported that
ergocornine treatment also inhibited development of DMBA-induced
mammary tumors in rats.

Ergot derivatives also inhibit deve10pment and growth of
mammary tumors in mice. Yanai and Nagasawa (1970, 1971)
reported that ergocornine and ergocryptine decreased the incidence

of spontaneous mammary tumors in mice and suppressed development

152

of hyperplastic nodules. Welsch (1972) confirmed these findings.
Singh 23.11. (1972) reported that ergocornine inhibited growth of

transplantable D ~mammary tumors in BALB/c mice.

2

The studies described above provided strong indirect evidence
that in rats and probably in mice, prolactin is intimately involved
in development and growth of spontaneous and carcinogen-induced

mammary tumors.

Effects of Lactation

In human beings lactation appears to inhibit development of
breast cancer. MacMahon eta}. (1970) reported a low incidence of
breast cancer in countries where babies are breast fed for long
periods of time. Not much research has been done on the effects
of lactation on carcinogen-induced mammary turmoigenesis in rats.
Bielschowsky (1947) reported that AAF-induced mammary tumors
in rats regressed during lactation and reappeared after the end of
lactation. Foulds (19 49) reported that spontaneous mammary tumors
in certain hybrid mice also regressed during lactation. Scholler
(19 58) and Scholler and Carnes (19 58) reported that lactation had no
effect on the incidence and growth of DMBA-induced mammary
tumors in rats. It has been mentioned earlier that Dao and
Sunderland (1959) found that pseudopregnancy induced by mating

with sterile males accelerated MC-induced mammary tumorigenesis

153

in rats, however, tumor growth was retarded after a period of 5 to

9 days. Similarly, pregnancy also enhanced tumor development and
growth, especially during the latter stages. However, the tumors
regressed rapidly after parturition. They claimed that they did not
find ”a single instance in which a tumor failed to regress after
parturition/1'. When these rats were mated again,they became
pregnant and the tumor growth resumed promptly. Parturition again
caused regression of all tumors and the tumor regression continued
during lactation. However, Dao and Sunderland (19 59) argue that
lactation itself was not important in tumor regression since regres-
sion continued even if lactation was suppressed by limited feeding of
the newborns or when milk removal was prevented by performing
cesarean sections near the end of pregnancies. They concluded that
tumor regression occurs after parturition due to withdrawal of
hormonal stimulation which is available during pregnancy, perhaps
fromthe placenta. McCormick and Moon (1964) were not able to fully
confirm the results obtained by Dao and Sunderland (1959). They
found regression of DMBA-induced tumors not to be a ”100%
phenomenon” during lactation. Some tumors regressed slowly while
others continued to grow rapidly. They suggested that the tumors
which grew during lactation might have been "hormone-independent”.
However, they also found a negative correlation between the number

of regressing tumors and the number of suckling young i. e. with

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larger litters more tumors continued to grow. They suggested that
this was perhaps due to elevated secretions of prolactin and ovarian

hormones , particularly proge sterone .

Effect of GH and Other Pituitary Hormones

Very little work has been done on the effects of gonadotropins
on carcinogen-induced mammary tumorigenesis in rats. Huggins
_e_t_al. (1962) injected rats with 1 unit of equine gonadotrOpin for 30
days beginning 15 days after a single feeding of DMBA at the age of
50 days. Six of the 20 animals (3070) treated in the above manner
were free of mammary cancers compared to a 100% tumor incidence
in controls. All animals had large cystic ovaries and hyperplastic
mammary glands characterized by marked epithelial proliferation.
They concluded that inhibition of mammary tumor development
occurred due to secretion of ovarian hormones.

Next to prolactin, GH has been more often implicated in spon-
taneous and carcinogen-induced tumorigenesis in rats than any other
pituitary hormone. Moon e_t_al. (1950) reported that injections of
0. 4-3. 0 mg of pituitary GH, 6 days a week, for a maximum period
of 485 days into adult female rats of a Long-Evans strain resulted
in production of mammary fibroadenomas and fibromas in more than
50% of the animals. About 20% of the control rats injected with

albumin also developed similar tumors. They stated that the GH

155

preparation was "pure by physiochemical criteria". Several of the
GH treated rats developed tumors of other organs including the
ovaries and uteri. There was also marked deve10pment of mammary
glands. They concluded that these changes occurred due ”to marked
disturbance in the normal function of the pituitary gland" but failed

to speculate on the nature of such disturbances. However, in a
subsequent experiment (Moon _e_t_ a1. , 1951) they found that similar

GH injections into hypophysectomized rats failed to elicit any tumors.
Very few pe0ple have investigated the role of GH in carcinogen-
induced mammary tumorigenesis in rats. Talwalker 223.1: (1964b)
reported that daily injections of 1 mg of GH and 1 mg of prolactin,
twice daily 7 days before and 7 days after DMBA administration
elicited mammary tumors in 44% of ovariectomized rats, while
continuous daily treatment with increasing doses of (3H and prolactin
for 75 days increased mammary tumor incidence to 66%. This study
for the first time clearly indicated the importance of GH and prolactin
in development of mammary tumors but the role of GH alone remained
unsolved. Furth (1967) demonstrated the stimulatory actions of
pituitary tumors in mammary carcinogenesis but these tumors also
secrete both prolactin and GH. Recently Nagasawa and Yanai (1970)
reported that twice daily injections of l. 5 mg of bovine GH for 20
days beginning a day after adreno-ovariectomy of DMBA-induced

tumor-bearing rats failed to prevent regression of the tumors. On

156

the other hand, similar treatment with prolactin stimulated mammary
tumor growth. They concluded that prolactin is the principal

hormone involved in maintenance and growth of carcinogen-induced
mammary tumors whereas GH has only a minimal role. Pearson
EEEE° (1969) also reported that daily treatment with 2 mg of bovine
GH for 7-41 days failed to reactivate growth in DMBA-induced mam-
mary tumors which had regressed following 00phorectomy and
adrenalectomy. There was no evidence of tumor growth in 2 animals
which received 4 mg of bovine GH for an additional period of 43-52
days. They reported that similar treatments with ovine prolactin,

on the other hand, reactivated growth in regressed tumors.

Role of the Hypothalamus

The earliest indications that the central nervous system might
play a role in mammary tumorigenesis came from work in mice.
Cranston (195 8) and Lacassagne and Duplan (195 9) indicated that
reserpine, chlorpromazine and other central acting drugs might
affect spontaneous mammary tumorigenesis in mice. Liebelt (195 9)
reported that hypothalamic lesions induced by gold thioglucose
increased the incidence and decreased the mean latency period for
appearance of spontaneous mammary tumors in RllGCBA mice.
They hypothesized that disturbances in hormonal mechanism were

responsible for stimulation of tumorigenic process. Toh (196 7)

157

reported that hypothalamic lesions, regardless of location, greatly
stimulated mammary tumorigenesis in mice. These results were
later confirmed by Montemurro and Toh (1968). They indicated
that hypothalamic lesions enhanced prolactin secretion from the
pituitary gland which led to stimulation of mammary cancer develop-
ment in mice.

Meites and co-workers were the first to directly implicate
the hypothalamus in spontaneous and carcinogen-induced mammary
tumorigenesis in rats. In the first of a series of papers (Clemens
$11. , 1968) they reported that median eminence (ME) lesions 6
days before DMBA treatment decreased mammary tumor incidence
to 30% compared with a 95% tumor incidence in intact rats. In the
lesioned rats the average number of tumors decreased to l. 1 tumors
per rat and the mean latency period increased to 104 days compared
to 3. 1 tumors per rat and 78 days respectively in the intact controls.
When the ME was lesioned 6 days prior to DMBA administration
followed 8 days later by ovariectomy, then the tumor incidence was
reduced to 0% compared with a tumor incidence of 54% in non-
lesioned ovariectomized rats. They concluded that ME lesions
inhibited tumorigenesis by increasing prolactin secretion which
stimulated mammary growth and rendered the mammary glands
refractory to carcinogenic action of DMBA. When the rats were

lesioned in the ME or both lesioned in the ME and ovariectomized

158

75 days after DMBA treatment, then within 10 days there were
increases of 120% and 80% respectively in the average number of
tumors per rat compared with an increase of 19% and a decrease of
27% respectively in non-lesioned intact and ovariectomized rats.
However, 25 days after lesioning marked tumor regression occurred
in the lesioned-ovariectomized rats and the average number of
tumors regressed by 39% whereas tumor growth continued progres-
sively in lesioned-non-ovariectomized rats. It was concluded that
both prolactin and ovarian hormones are needed for Optimal tumor
growth. Most of these findings are confirmed by Klaiber e_t_ al. (1969).
They reported that lesioning of the ME after appearance of DMBA-
induced mammary tumors increased the average number of tumors to
9.1 tumors per rat compared with 5. 8 tumors per rat in the intact
controls. The total volume of tumors also increased to 34 cm per
rat compared to 17 cm per rat in the controls. Ovariectomy alone
caused marked tumor regression but the inhibitory effect of
ovariectomy on mammary tumor growth was overcome by simul-
taneous placement of ME lesions. There were no significant dif-
ferences in the number and growth of tumors between the intact
control rats and the lesioned-ovariectomized rats 21 days after the
operations. At this point these results differed from those of
Clemens _e_t_a__l_. (1968) who had reported a decrease of 39% in the

average number of tumors per rat in the lesioned-ovariectomized

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rats 25 days after the operations compared with an increase of 33%
in the intact controls. Klaibergtgl. (1969) suggested, on the basis
of their results, that ovarian steroids may promote tumor growth
not only by direct action on the mammary gland but also by feedback
effects on the hypothalamus. Evidence for the latter mechanism was
forthcoming in a paper by Nagasawa and Meites (1970a) which will
be described later,

Stimulation of growth of established mammary tumors after
induction of ME lesions also was confirmed in yet another paper
(Welsch _e_t_ a1. , 1969). But again when the rats were both lesioned
and ovariectomized, there was an initial stimulation of growth of
established tumors by 10 days followed by inhibition of tumor growth
after 25 days, compared to inhibition of tumor growth in non- lesioned
ovariectomized rats at both time intervals. ME lesions placed 10
days after ovariectomy did not prevent tumor regression. It
appeared that after removal of ovarian steroids, prolactin can sus-
tain tumor growth only temporarily and can not reactivate tumors
which have regressed following ovariectomy.

Lesions placed in the preOptic area or amygdaloid complex
induced tumor regression due to, it was hypothesized, decreased
gonadotropin secretion followed by decreased estrogen secretion
which also may have diminished prolactin secretion. ME lesions

also markedly increased the incidence of spontaneous mammary

160

tumors in lO-month old rats (Welsch e_t_ a_l_. , 1970b). During a 6
month period, the lesioned rats had a tumor incidence of 52% com-
pared with 19% in the non-lesioned controls. Serum prolactin levels
increased to 179 ng/ml compared with 50 ng/ml in the controls.
Histologically the tumors were adenomas in the lesioned rats and
fibromas in the controls and no carcinomas appeared in either group.
It was concluded that disruption of the final common pathway from
the hypothalamus to the anterior pituitary increased prolactin
secretion and enhanced mammary tumorigene sis. In another publica-
tion, Nagasawa and Meites (1970a) reported that estrogen implants
in the ME significantly stimulated growth of existing DMBA-induced
mammary tumors in rats. There was a linear increase in the
number of tumors beginning 5 days after estrogen implantation.
Average tumor size and tumor weight also increased almost linearly
with time. Estrogen implants outside the ME or in the cerebral
cortex had no effect on mammary tumor growth. Serum prolactin
concentration was 70 ng/ml in the rats with estrogen implants in

the ME comparedto 15-19 ng/ml in the rats which had estrogen
implants in other parts of the brain. It was concluded that estrogen
implants in the ME accelerated mammary tumor growth by increas-

ing prolactin secretion.

 

161

Prolactin Versus Estrogen in Carcinogen-
Induced Mammary Tumorigenesis

From the literature survey presented above it is apparent that

2 hormones, prolactin and estrogen, are the dominant factors involved
in both spontaneous and carcinogen-induced mammary tumors in
rats. However, the relative importance of each of these hormones
has become the subject of a lively controversy among research
workers led by Meites, Pearson and Dao. Pearson and collaborators
(1969, 1972a, b) tend to think of prolactin as the only hormone of
significance in DMBA-induced mammary tumorigenesis and assign
only a secondary role to estrogen. Meites _e_t_gl. (Meites _e_t_gl. ,
1971, 1972; Meites 1972a, b, c) while contending that both hor-
mones are important, appear to incline towards prolactin as the
dominant of the two. Dao and co-workers (Dao and Sinha, 1972:
Sinha and Dao, 1972; Sinha ga—l. , 1973) do not completely deny
prolactin a role, but argue that estrogen is more important in DMBA-
induced mammary oncogenesis in rats. The following paragraphs
are devoted to present these points of view in more detail. They
are presented here in the order in which they were stated above
since in that sequence they go from one extreme to the other i. e.
”prolactin more important” to ”estrogen more important".

Pearson _e_t_al. (1969. 1972a, b) support their point of view

mostly by the experiments conducted in their own laboratory. In

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some of their early studies (Pearson g _a_l. , 1954a; Pearson and

Ray, 1954) in humans they found that estrogen treatment reactivated
tumor growth after 00phorectomy-induced remission but failed to do
so following hypophysectomy- induced remission, suggesting that the
pituitary was required for estrogenic stimulation of mammary tumor
growth in humans. They obtained similar results in rats (Sterenthal
_§_t___l_. , 1963). Daily administration of 1-5 pg of estradiol benzoate
(EB) into rats reactivated DMBA-induced mammary tumors which
had undergone regression following adreno-oophorectomy but the
same treatment failed to reactivate tumor growth following
hypophysectomy- induced remission. In an attempt to define the
pituitary factor involved they treated rats whose tumors had
regressed following adreno-oophorectomy with OH or prolactin
(Pearson _e_t_a_l. , 1969, 1972a). GH had no effect but daily treatment
with 2 mg prolactin reactivated growth in tumors which very dramatic-
ally reached their original size in an average of 18 days. Prolactin
treatment (1. 5 mg daily), but not estrogen treatment (5 pg daily),
had similar effects in hyp0physectomized rats. They obtained still
more dramatic effects in rats whose mammary tumors had regressed
f9110Wing 00phorectomy, adrenalectomy and hyp0physectomy.
IDrohfictin treatment reactivated tumor growth in all rats whereas

EB tr(Batment failed to do so. They concluded that estrogen exerts

Its effects on tumor growth by increasing pituitary prolactin

 
 
 

163

secretion and that tumor regression after adreno-oophorectomy is
really due to reduction in serum prolactin levels. They substantiated
their hypothesis by an experiment in which adreno-oophorectomy
produced a significant fall in serum prolactin levels and a reduction
in the size of existing tumors. But after a 5-11 day treatment with

5 pg EB daily there was a 2 fold increase in mean tumor area and

a 5-fold increase in serum prolactin levels. Again 5-15 days after
EB was withdrawn tumor area and serum prolactin concentrations
both declined. They concluded that small doses of EB stimulate
prolactin release which in turn accelerates mammary tumor growth.
They have also attempted to explain the paradoxical effect of large
doses of EB which induce regression, rather than stimulation, of
mammary cancers in women and rats. A dose of 20 or 500 pg EB
induced tumor regression and also increased serum prolactin levels.
Since tumor regression could not be explained on the basis of sup-
pression of prolactin secretion, they suggested, like Meites (1972b)
that large doses of EB exert antitumor effects by inhibiting the
peripheral action of prolactin on mammary tumors. To substantiate
this idea they induced partial tumor regression by 00phorectomy,
then injected the rats with 1 mg perphenazine which increased serum
prolactin levels and stimulated tumor growth. When perphenazine
injections were maintained and 500 pg EB was added to the regimen,

it resulted in tumor regression. Thus, they concluded that

164

estrOgen was interfering with the action of prolactin.

Pearson e_t_al. (1969; 1972a, b) also presented evidence to
indicate that prolactin was important in development of carcinogen-
induced mammary tumors. Daily treatment with 4 mg perphenazine
beginning either immediately after DMBA treatment or 2 months
after DMBA administration but before tumor appearance increased
the incidence, number and size of mammary tumors. There was
no effect on mean latency period for appearance of tumors. Per-
phenazine treated animals had consistently high serum prolactin
levels. In another experiment adreno-ovariectomy was performed
6 days after feeding DMBA and perphenazine injections (1 mg daily)
were started 2 days later. After 5 months of treatment, 7 of 15
rats developed mammary tumors whereas none of the 11 control
rats developed any tumors. All perphenazine treated rats had higher
serum prolactin values. These experiments indicated that DMBA
can induce neoplastic transformation of the mammary gland in the
presence of prolactin alone although the tumor incidence was not
100% and adreno-ovariectomy was performed 6 days after DMBA
treatment.

Based on these experiments Pearson and co-workers (1969,
1972a, b) conclude that ”DMBA-induced rat mammary carcinoma is
prolactin-dependent and this hormone may be the only one of signifi-

cance in maintaining the growth of this tumor”. They reject the idea

165

of synergism between estrogen and prolactin and state that there is
no evidence that estrogen potentiates the action of prolactin". The
results presented by Pearson and co-workers strongly support the
idea that prolactin might be the only hormone responsible for
maintenance and growth of DMBA-induced mammary tumors if not
for development of these tumors. However, this concept is not
strongly supported by the results obtained by Meites and co-workers
and by Dao and Sinha (see below) and only a few of their results
have been confirmed by others (Nagasawa and Yanai, 1970). The
rest needs confirmation.

Meites and co-workers (1971, 1972; Meites, 1972a, b, c)
have exercised restraint in arriving at their conclusions. They were
the first (Talwalker Eli}.- , 1964b) to report that treatment of ovari—
ectomized rats with exogenous GH and prolactin can support develop-
ment of DMBA-induced mammary tumors. However, they refused
to exclude the possible participation of adrenal steroids and con-
cluded that "pituitary hormones as well as estrogen participate in
initiating and promoting phases of mammary carcinogenesis and that
the effects of estrogen may be partially mediated through stimulation
of STH (GH) and prolactin secretion by the pituitary". In their
experiments with hypothalamic lesions, they (Clemens _e_t_ a1. , 1968)
found that increases in serum prolactin levels due to ME lesions

can not support tumor growth indefinitely in the absence of ovarian

166

hormones. When rats were simultaneously ovariectomized and
lesioned in the ME, there was a striking increase in number of
tumors during the first 10 days but the stimulatory effects did not
last and marked tumor regression was observed by the end of 25
days. They concluded that "a sustained estrogen secretion was
necessary to permit high serum levels of prolactin and to promote
mammary tumor growth beyond a relatively short period”. They
confirmed these results in a subsequent publication (Welsch _e_t_a_l.. ,
1969). When lesions were made in the ME and ovariectomy was
performed immediately thereafter, initial stimulation up to 10 days
after the operation was followed by regression of mammary tumors
at 25 days. They concluded that, in the absence of ovaries, prolactin
alone can at least initially promote the growth of mammary cancers,
and the subsequent regression could have been due to inadequate
pituitary prolactin secretion, due to removal of ovaries. Although
serum prolactin concentrations were not measured in these experi-
ments, there is good reason to believe that they remained elevated
for long periods of time since in a subsequent experiment (Welsch
J4. , 1970b, 1971) bilateral lesions in the ME of ovariectomized
rats resulted in about a ten-fold rise in serum prolactin by the end
of 30 minutes,and the concentration remained elevated for at least
six months thereafter. Welsch _e_t__a_._l. (1969) also reported that

elevations in serum prolactin levels due to ME lesions were unable

167

to reactivate growth in tumors which had undergone regression follow-
ing ovariectomy. These results conflict with those reported by
Pearsongtgl. (1969, 1972a, b)-

However, on the basis of the above results they did not com-
pletely discount the possibility that prolactin alone can sustain the
process of tumorigenesis initiated by DMBA. They found that
multiple pituitary grafts or chronic prolactin and GH administration
into ovariectomized—adrenalectomized rats given local DMBA
implants in the mammary glands resulted in development of mam-
mary tumors (Quadri and Meites, unpublished). Similarly they
reported that transplants of a mammary tumor which had originated
after grafting of a pituitary tumor, could grow in ovariectomized-
adrenalectomized rats if they were grafted with a pituitary tumor
which secretes large amounts of prolactin and GH. While these
results demonstrate that high serum prolactin concentrations can
sustain both development and growth of mammary tumors in the
absence of ovarian hormones, high serum prolactin levels are not
required to sustain carcinogen-induced mammary tumorigenesis in
intact rats. In a recent publication (Nagasawa 3531. , 1973) they
reported that serum prolactin concentrations measured from 45 days
to 165 days after DMBA treatment showed no significant elevations
although tumor number and size increased markedly during the

same period. On the other hand they have repeatedly demonstrated

168

that elevations in serum prolactin concentrations after initiation of
the process of carcinogen-induced tumorigenesis can significantly
stimulate this process. Thus increase in pituitary prolactin secre-
tion induced by methyldopa or haloperidol can cause several hundred
percent increase in the number and size of mammary tumors
(Quadri _e_t_al. , 1973a, b). By contrast decrease in serum prolactin
levels induced by ergot derivatives, monoamine oxidase inhibitors
and l-dopa can markedly reduce mammary tumor growth in intact
rats (Cassellgtal. , 1971; Quadri and Meites, 1971b; Quadri _e_t_al, ,
1973a, b, c).

Meites and co-workers have also explored a new aspect of
the role of prolactin in carcinogen-induced mammary tumorigenesis.
In a series of papers they reported that elevations in serum prolactin
levels before DMBA—administration stimulates mammary growth
and makes the mammary gland refractory to carcinogen-induced
neoplastic transformationiMeites _e_ta_1. , 1971, 1972; Meites, 1972a,
b, c). In another experiment they found that large doses of estrogen
induce mammary tumor regression not by inhibiting serum prolactin
levels but by interfering with peripheral actions of prolactin of
mammary tumor cells (Meite.s_e_t.a_l_.,1972; Meites, 1973b). In short,
Meites and co-workers have resisted the temptation to assign a
single, blanket role to prolactin in the process of mammary tumori-

genesis. Instead, they have attempted to investigate its effect in

169

different stages of the tumorigenic process, viz, before carcinogen
treatment, after carcinogen treatment, after appearance of mam-
mary tumors, and in different situations such as in the absence of
ovaries, in the absence of ovaries and adrenals, etc. They have
discovered different roles for prolactin in each of these different
situations.

Dao and co-workers (Dao and Sinha, 1972; Sinha and Dao,
1972; Sinha ital. , 1973) give more emphasis to the role of estrogen
in carcinogen-induced mammary tumorigenesis, although they
appear to agree that perhaps both estrogen and prolactin are impor-
tant. They reported (Sinha and Dao, 1972) that local implantation of
a combination of various amounts of DMBA and 50-100 pg of DES
resulted in higher tumor incidence and shorter latency periods com-
pared with implantation of DMBA alone. They concluded that
"estrogen acts in synergism with chemical carcinogen in inducing
mammary cancers in rats” and that action of estrogen is localized
in the mammary gland. It has been demonstrated several years ago
that estrogen enhances the tumorigenic effect of carcinogens,
however, its mechanism of action has not been elucidated (see earlier
parts of this review). It's rather difficult to accept Dao and Sinha's
conclusions that the action of estrogen was localized in the mammary
gland since they failed to measure changes in other hormones and

used lack of changes in the hist010gy and weights of the ovaries,

170

pituitaries, adrenals and uteri as the sole criterion to exclude the
possibility of a systemic effect. It would have been easier to accept
their conclusions had they used hypophysectomized, rather than
intact, rats in their experiment. Dao and Sinha (1972) also reported
that ME lesions placed after ovariectomy prevented further regres-
sion of the tumors while ovariectomy after placement of ME lesions
resulted in complete regression. Subsequent grafting of ovaries

in both situations resulted in tumor growth. They concluded that
"these experiments provide conclusive evidence that mammary
cancers regress or fail to grow in the absence of ovarian hormones
even when there are continued high levels of plasma prolactin”.

These results differ from the results obtained in the ME lesion-
experiments by Meites and co-workers (Clemens eta}. , 1968;
Welsch _1_:_a_1_. , 1969) and completely contradict the results obtained
by Pearson _e_t_ai. (1969, 1972a, b). Dao and Sinha failed to measure
prolactin levels and used extremely small numbers of animals.

For example, in the first experiment only 2 rats were used as
controls and 4 as experimentals, 1 of which had ”hormone-
independent" tumors. They have tried to correct some of these
deficiencies in a subsequent report of the same experiment (Sinha

et 1. , 1973).

GENERAL MATERIALS AND METHODS
Animals

Sprague-Dawley rats were obtained from Spartan Research
Animals, Inc. , Haslet, Michigan or the Holtzman Company, Madison,

Wisconsin. Inbred female rats of Wistar-Furth strain were pur-

Chased from Microbiological Associates, Inc. , Bethesda, Maryland.
All rats were housed in steel cages in air conditioned, temperature

controlled (75 :1: 101?) rooms which were lighted for 14 hours each

day from 5 a. m. to 7 p. m. with fluorescent lights. All rats were

fed a standard laboratory diet of Wayne Lab Blox pellets (Allied
Mills, Chicago, Ill.) and given tap water_aii libitum. The diet given
to old rats was supplemented, twice each week, with oranges and
carrots, Old rats were also given, once every 4 months, for a

period Of 5-7 days, oxytetracycline (Pfizer) in their drinking water

as a Prophylactic measure against infections.

Blood Collection for Radioimmunoassays
by Cardiac Puncture
Before actual blood collection, an ice bath was prepared by
muflng iCe and water in a large tray. A test-tube rack containing
the .
req‘n’l‘ed number of appropriately labeled disposable culture tubes,

each 1
2 x '75 mm (Kimble Owens, Illinois), were placed in the ice
171

172

bath a few minutes before blood collection. In the next step an ether
chamber was made available by placing a small amount of liquid

ether in the chamber, immediately placing the lid on top and allowing
the ether to vaporize so that in a minute or two the chamber contained
a light mixture of air and ether. Rats were lightly anesthetized by
placing them in the ether chamber for 0. 75 to 1. 0 minute. , To avoid
variability care was taken to see that all rats were exposed to the
air-ether mixture for the same length of time. Concentration of
ether vapors in the air-ether mixture was maintained by frequent
additions of liquid ether to the chamber. As a precautionary measure
against fire, smoking or other kinds of fires were not allowed when
the ether was being used.

As soon as a rat was anesthetized, it was taken out of the
chamber and blood was collected by cardiac puncture with a 26 gauge
1/2 inch needle attached to a 1 ml plastic tuberculin syringe. Care
was taken to avoid more than one heart puncture per sampling and
other unnecessary stress to the animal. A single gentle thrust
with the needle was all that was needed for a successful heart punc-
ture. After withdrawing the required volume of blood in the syringe
it was slowly and gently expelled down into the collection tubes by
the walls of the test tube without applying too much pressure on the
plunger of the syringe. ‘ These measures were taken to avoid hemo-

lysis.

173

To obtain serum for LH, FSH, and prolactin assays the blood
was allowed to undergo complete coagulation for 15-20 minutes and
then centrifuged for 25 minutes at 2200 rpm in an International
Centrifuge at room temperature. The serum was withdrawn with a
20 gauge needle attached to a 1 ml tuberculin syringe. Care was
taken to avoid disturbing the clot. This procedure resulted in collec-
tion of clear and cell-free serum. After collection of serum the
needle and the syringe were rinsed several times with distilled water
and used to separate serum from the next tube. All sera were
stored in a freezer at ~200C until assayed.

A slightly different technique was employed to obtain plasma
for CH radioimmunoassay. Blood was collected in a heparinized
tuberculin syringe containing 0. 1 ml of sodium heparin solution
(100 mg/100 ml) and immediately transferred to previously chilled
tubes. After transfer of blood, the tubes were gently shaken and
centrifuged within 15 minutes at 2, 200 rpm for 25 minutes. Plasma
was separated and stored at -200C in the freezer until assayed for

CH.

Rat Prolactin Radioimmunoas say

 

The radioimmunoassay for rat prolactin was developed in
collaboration with Dr. A. Rees Midgley's laboratory, Department of

Pathology, University of Michigan, Ann Arbor, Michigan. Details

174

of this procedure have been described elsewhere (Niswender 2231' ,
1969; Chen, 1969). This radioimmunoassay utilizes a double anti-
body system. Antibody against purified rat prolactin (first antibody)
was made in rabbits. Anti-rat prolactin antibody no. 625 diluted
to 1:4000 was used in the experiments to be described in these pages.
This concentration of antibody gave a consistent binding of 35-50%
with radiolabelled prolactin.

Purified rat prolactin (HIV-8-C and H-lO-lO-B) for radiolabel-

ling was obtained from Dr. S. Ellis (NASA, Ames Research Center,

131 125
or

Moffett Field, California). It was labelled with either I I

(Cambridge Nuclear Corp. , Cambridge, Mass.) in a standard
radioiodination procedure using chloramine-T as an oxidizing agent.
The hormone (2. 5 pg) was iodinated with l millicurrie of the isotope
and the oxidation was allowed to proceed for 2 minutes, after which
the reaction was stOpped by sodium metabisulfite and the mixture
eluted through a 1 x 15 cm Bio-gel p60 column. Eluted fractions
containing the peaks of labelled prolactin were diluted in 0. 1%
BSA-PBS (bovine serum albumin-phosphate buffered saline) or 0. 1%
egg white (EW)-PBS to working concentrations which varied from
30, 000 to 60, 000 cpm (counts per minute) per 100 pl in a Nuclear-
Chicago autogamma counter.

For reference standards either the rat prolactin (HIV-8-C,

H-lO-lO-B) or NIAMD (National Institute of Arthritis and Metabolic

175

Diseases) rat prolactin RP-l provided by Dr. A. Parlow under the
NIH Rat Pituitary Program was used.

The second antibody, against a commercial preparation of
rabbit gamma globulins, was prepared in sheep using standard
immunization procedures. This antibody was used at concentrations
of 1:8 to 1: 12 to obtain maximum precipitate in the assay tubes.

Various steps of the radioimmunoassay were carried out at low
temperatures in an ice bath. The assay reagents were mixed in
glass test tubes (12 x 75 mm) obtained from several commercial
sources. The assay was 6 —day long. On day 1, unlabelled prolactin
(unknown in serum or known standard reference rat prolactin) 1%
BSA-PBS or EW-PBS diluent (total volume 500 pl) and 200 pl of the
first-antibody were mixed in the reaction tube. The reaction was
allowed to proceed for 24 hours in a cold room at 40C. On day 2,
labelled rat prolactin (100 p1) was added, mixed and allowed to
incubate, for 24 hours. On day 3, the second antibody (200 pl) was
added, mixed and allowed to incubate for an additional period of 72
hours. On day 6, 3 m1 of PBS was added and the mixture was
centrifuged at 2000-2, 200 rpm for 25-30 minutes. The supernatant
was carefully decanted and the precipitate counted in an automatic
gamma counter (Nuclear-Chicago Corp. , Des Plaines, Illinois).

Standard curves were drawn on semi-logarithmic paper from

10-12 doses of standard reference prolactin preparation and the

176

values for the unknown were read from the curve and expressed as
ng or pg of prolactin per ml serum. Unknowns were usually done

in duplicate or triplicate. Standard curves were also made in dupli-
cate or triplicate depending on the number of samples assayed and
were counted at the onset, middle and at the end of the assay to
allow for decay of the isotope and other variable conditions of the

assay.

Growth Hormone Radioimmunoassay

 

The growth hormone (GH) radioimmunoassay was deve10ped in
our laboratory by Dr. Elias Dickerman as part of his Ph. D. research
project. Details of this assay have been published elsewhere
(Dickerman e_tgl., 1972; Dickerman and Mack, 1970). This assay is based
onthe same principle as the radioimmunoassays for other hormones
but contains several procedural variations.

The antiserum to rat GH (the first antibody) was obtained from
Dr. A. Parlow, National Institute of Arthritis and Metabolic Diseases,
NIH Rat Pituitary Program. This antiserum was made in monkeys.

It will henceforth be referred to as NIAMD-A-Rat GHS-l. The anti-
serum was diluted to a working concentration of 1:50, 000, 200 pl

of which consistently bound 45-50% of radiolabelled CH. The diluent
used was normal monkey serum diluted to 1:600 in 0. 05 M EDTA-

PBS (ethylenedinitrillo tetraacetic acid disodium, pH 7. 2).

177

For radiolabelling a highly purified rat GH, NIAMD-RGH-I-l
provided by Dr. Parlow was used. Twenty pg of GH was labelled
with 1 millicurrie (me) of carrier free 112'5 of high specific activity
(Cambridge Nuclear, Cambridge, Massachusetts) with Chloramin-T
(87. 5 pg) used as an oxidizing agent. The reaction was stopped by
sodium metabisulfate (Na25205)° The mixture was eluted through
a 0.9 x 20 cm column of Sephadex G- 50 expanded in 0. 01 m barbital
buffer. Fractions containing peak radioactivity were repurified
through a 0. 9 x 50 cm column of Sephadex G-100. The peak contain-
ing radiolabelled GH was diluted in 1% BSA-PBS at 7. 2 pH so that
100 pl of the diluted preparation gave a reading of 19-21, 000 cpm
on the day of iodination. The diluted preparation was usable for up
to 1 to 1-1/2 months.

NIAMD-RGH-RP-l, diluted to concentrations of 1 ng/pl and
1 ng/10 pl, was used as a reference standard. This preparation
was also obtained from Dr. Parlow's NIAMD program.

Antiserum to monkey gamma globulin was prepared by us in
goats using standard immunization procedures. The antiserum was
diluted to a working concentration which varied from 1:7 to 1:26
depending on the quality of the antiserum harvested. PBS was used
as diluent. A volume of 200 pl of the diluted antiserum per assay
tube was used. This volume at the proper dilution gave the maximum

precipitate.

178

The GH radioimmunoassay differs from prolactin radioimmuno-
assay in the following respects: the GH radioimmunoassay is an
equilibrium assay i. e. all the three reagents, 'the first antibody,
the radiolabelled GH and the unknown GH in the plasma are added
on the same day. Unlike the 6-day prolactin radioimmunoassay, the
GH radioimmunoassay is only 5-day long. Although both serum and
plasma can be used to determine (3H, plasma has been most fre—
quently used in our laboratory as it consistently gave higher GH
values, indicating that GH was losing its immunological activity or
was being degraded in serum.

The assay was carried out in 12 x 75 mm disposable culture
tubes. On day 1, 100 p1 of the iodinated CH, 200 p1 of the first
antibody and the unknown plasma OH or reference standard GH (total
volume of 500 pl) were added to the reaction tubes and mixed. The
mixture was incubated at 40C for 72 hours. On day 4, 200 pl of the
second antibody was added and mixed. After a further incubation
period of 24 hours, the assay tubes were centrifuged at 2, 200 rpm
for 20 minutes (International‘Equipment Company, Needham Heights,
Massachusetts) at room temperature. At the end of 20 minutes 3 ml
PBS was added to each tube and the mixture was centrifuged again
for an additional 20 minutes as described above. Here again the
GH radioimmunoassay differed from the prolactin radioimmunoassay.

The extra centrifugation resulted in maximum precipitation of the

179

antigen-antibody complex. The supernatant was decanted and the
precipitate counted in a Nuclear-Chicago counter with automatic
changer. A standard curve was drawn on a semi-logarithmic paper
with 10-15 standard reference points and the unknowns read off the
curve.

Several precautions were taken during the assay to insure
accuracy and reproducibility. The whole assay was carried out in
ice baths and only one rack at a time was taken out of the cold room.
At low standard reference points, the standard reference GH prepara-
tion was diluted to 1 ng/lO pl to achieve maximum accuracy in pipet-
ting. To minimize variations, large volumes of different reagents
were made and kept frozen; 50 % binding was considered as a quality

control.

Transplantation of Pituitary Tumors

 

Donor Rats

 

Inbred female Wistar-Furth rats carrying MtT. W15 pituitary
tumor transplants originally were obtained from Dr. Jacob Furth,
Department of Pathology, Francis Delafield Hospital, New York,
N. Y. This pituitary tumor has been maintained in our laboratory
for the past three years by serial subcutaneous transplantation into

female rats of the same strain.

180

Recipient Rats

Inbred virgin female rats of the Wistar-Furth strain, 50-55
days old, were obtained from Microbiological Associates, Bethesda,
Maryland. They were housed in separate animal cages and were

fed the same standard laboratory diet as the other rats.
Tumor Transplantation

Two donor rats were given light ether anesthesia by placing
them in a glass ether chamber containing a light ether-air mixture.
The area surrounding the tumors was carefully shaved and cleaned
with an antiseptic solution. The tumors were exposed and some
tumor tissue was removed from each rat using a strictly sterile
technique. The tumor tissue was placed in a small beaker containing
0. 85% physiological saline and minced with a pair of small scissors.
Care was taken to see that the tumor tissue was minced finely and
no coarse bits and pieces remained. The recipient rats were given
light ether anesthesia and their backs were shaven and sterilized
at the posterior part of the back. The tumor mince was withdrawn
in a 1 ml sterile glass syringe containing a long 15 gauge needle.
The needle was inserted subcutaneously into the posterior part of

the back and pushed all the way up to the post-cervical area where

0. 2 ml of the tumor mince was deposited. Extreme care was taken

181

to see that all the tumor tissue was deposited in the same place and
not throughout the back. Care was also taken to insure that each
rat received about the same amount of tumor tissue. These two
precautions were undertaken in an attempt to develop transplants of
uniform size and shape. The wound in the back was closed by a
single metal clip. This technique resulted in 100% incidence of
”takes”'and the tumors reached a size of 0. 5 cm or more 5-6 weeks
after transplantation. It was very interesting to see the rapid gain
in body weights and body lengths of the rats carrying these tumor
transplants. Their mammary glands also were more developed,

particularly in the older rats.

DMBA-Induced Mammary Tumors in Rats

Induction

Sprague-Dawley virgin female rats were obtained at 40-45 days
of age. They were first allowed 5-10 days in our animal quarters
to get acclimatized to the new surroundings. At the age of 50-55
days, they were given light ether anesthesia by placing them in an
ether chamber containing a mixture of air and ether, and were then
injected via the tail vein with a single dose of a suspension of 5 mg
of 7, lZ-dimethylbenz (a) anthracene (DMBA) in 1 m1 of a lipid

eumulsion. The lipid emulsion containing DMBA was kindly supplied

182

to us by Dr. P. E. Schurr of the Upjohn Company, Kalamazoo,
Michigan. Beginning 4-5 weeks after injection and once a week
thereafter, the rats were palpated for appearance of mammary
cancers. About 60-80 days after injection, when each rat had
deve10ped one tumor at least 1 cm in diameter, the rats were divided

into the required number of control and experimental groups.

Mea surement

Beginning on the first day of treatment and once a week during
the treatment period of 3—4 weeks, the rats were placed under light
ether anesthesia and the largest diameter of each tumor was mea-
sured with a vernier caliper. Total number of tumors per rat and
body weight also were recorded. All measurements were continued
during the post-treatment period, which usually was of the same

length as the treatment period.

Calculations

Average number of tumors per rat and average total tumor
diameter per rat were calculated for each group. Percent changes
in average number of tumors per rat and average total tumor dia-
meter during the treatment period were calculated by comparing
weeks 1, 2, 3 etc. with week 0. Similar procedures were used to

calculate percent changes in average numbers of tumors per rat and

183

average total tumor diameter during the post—treatment period.
Significance of differences between average tumor number per rat,
average tumor diameter and average body weights between any 2

groups were evaluated by Student's ”t” test.

184

EXPERIMENTAL

Inhibition of Prolactin Secretion in Female Sprague-Dawley Rats
by a Sinile Injection of Lysergic Acid Diethylamide (LSD)

Objective 5

Ergot derivatives such as ergocornine, ergocryptine and C-
Lysergic acid diethylamide (LSD) inhibit growth of spontaneous
(Quadri and Meites, 1971b) and carcinogen-induced (Nagasawa and
Meites, 1970b; Cassell g_t_§_1. , 1971) mammary tumors in Sprague-
Dawley rats. Ergocornine markedly reduces serum prolactin levels
in rats, both by a direct action on the pituitary gland and also via
the hypothalamus by increasing prolactin inhibiting factor (PIF)
activity (Wuttke _e_t_al. , 1971; Lu gal. , 1971). It was of interest
therefore to determine whether LSD could similarly decrease serum

prolactin levels in rats.
Materials and Methods

Female 3- to 4-month-old Sprague-Dawley rats, (Spartan
Research Animals, Haslett, Mich. ), weighing 200-250 g, were used.
At least 2 estrous cycles were followed by taking daily vaginal
smears and only regular 4-day cycling rats were used. On the day

of pro-estrus, a pretreatment blood sample was collected at 11:30

185

A.M. At 12 noon, the rats were injected intraperitoneally with 25,
50 or 100 pg of LSD/100 g of body weight in a volume of O. 2 ml of

0. 85% NaCl. Control rats were injected with a similar volume of

0. 85% NaCl only. Blood samples were collected again at 1:30, 3:30,
and 5:30 P. M. by cardiac puncture under light ether anesthesia.
This method of collecting blood was shown not to alter normal serum
prolactin values (Wuttke and Meites, 1970). Serum was separated
and stored at -200 until assayed for prolactin by radioimmunoassay

(Niswender _e_t_gl. , 19 69).
Results

Table 1 shows that a marked rise occurred in serum prolactin
values in control rats on the afternoon of proestrus, in agreement
with previous reports (Wuttke gta_l. , 1971; Wuttke and Meites, 1970).
By contrast, all doses of LSD injected prevented the normal afternoon
rise and evoked a significant decrease in serum prolactin values.

The 100 pg dose of LSD produced a significantly greater reduction

in serum prolactin concentration than the 25 pg dose (P < . 001).

Conclusions

 

These results show that LSD, like other ergot derivatives
(Wuttke _e_t_al. , 1971; Lu 3:511. , 1971), is a potent inhibitor of

prolactin release in female Sprague-Dawley rats. All three doses of

186

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187

LSD prevented the normal afternoon rise in serum prolactin on the
day of proestrus. Earlier it was reported (Meites, 1962) that LSD,
unlike many other drugs tested, failed to stimulate mammary growth
and initiate lactation in virgin female rats. On the basis of the
present observations, it is probable that LSD inhibits mammary
growth and lactation. Whether LSD can influence release of hormones
other than prolactin remains to be studied.

Inhibition of Prolactin Secretion in Female Sprague-Dawley Rats
by Ergocryptine; Counteraction by Perphenazine

Objectives

 

Ergot derivatives are known to inhibit deciduoma formation
(Shelesnyak, 19 54), pseudo-pregnancy (Carlsen e_t_gl. , 1961) and
lactation (Zeilmaker and Carlsen, 1962), all dependent on adequate
prolactin secretion from the pituitary gland. Recently it was shown
that ergot derivatives also have potent anti-tumor effects in rats;
ergocornine, ergocryptine, and LSD induced regression in rats of
spontaneous (Quadri and Meites, 1971b) and carcinogen-induced
mammary tumors (Nagasawa and Meites, 1970b; Cassell_e_t_§;1. ,
1971, Quadri _e_t_ _a_l. , 1972). It also was demonstrated that several
ergot derivatives cause profound regression of a prolactin
and growth-secreting hormone pituitary tumor (MtT.W15) and

markedly reduce serum prolactin levels in rats carrying these tumors.

188

Although ergocryptine has been demonstrated to inhibit several
of the above processes which are dependent on prolactin, the effects
of ergocryptine on prolactin secretion have received little attention.
The present study was conducted to investigate (l) the effect of
ergocryptine on serum prolactin concentrations, (2) the duration of
this effect, and (3) to see if a central acting drug, perphenazine,

could counter inhibition of prolactin release induced by ergocryptine.

Materials and Method 5

 

Female 3-4 month old Sprague-Dawley rats (Spartan Research
Animals, Haslett, Michigan) were maintained in an air conditioned,
temperature (74 :1: 20F) and light (14 hours light) controlled room.
They were given a standard laboratory diet and water ad libitum.
Only 5 day cycling rats, as judged by daily vaginal smears, were
used. Ergocryptine and perphenazine were injected in a solution
containing 95% physiological saline and 4% ethanol of 70% strength.
Control injections consisted of saline-ethanol vehicle. Injections
were given intraperitoneally and all blood samples (1 ml) were
collected by cardiac puncture under light ether anesthesia. Serum
was separated from blood immediately after collection and stored
at -200C for 2—3 weeks before being assayed for prolactin using a
double antibody radioimmunoassay. Statistical evaluation was done

using Student's _t_ test.

189

Experiment 1

A pre-treatment blood sample was collected at 11:30 A. M.

on the day of proestrus. At 12 noon, 1 pg, 5 pg, 50 pg or 100 pg
ergocryptine per 100 gm body weight was injected. Control rats

received saline-ethanol vehicle alone. All rats were bled again at

1, 3 and 5 P. M. At the end of the last bleeding, the rats were

laparotomized and proestrus was confirmed by observing whether

the uteri were ballooned.

Experiment 2

A blood sample was collected at 8 P. M. on the day of proestrus.

Another blood sample was collected at 8 P. M. on the second day of

diestrus of the same estrous cycle. Half an hour later a single

iInjection of 1 pg. 5 [-18, 50 pg or 100 pg ergocryptine per 100 gm
body weight was given. An hour later a third blood sample was

col163cted. The rats were bled again approximately 24 hours later

at 8 P. M. on the day of proestrus.

ExPe riment 3

A pre-treatment blood sample was collected at l P. M. on the
second day of diestrus. At 1:30 P. M. the rats were injected with

perphenazine, ergocryptine or both as indicated in Table 4. They

190

were bled again at 3:30 P. M., and at 4 P. M. were injected with

ergocryptine. The final blood sample was obtained at 5 P. M.

Results

Effects of a Single Injection of Ergocryptine
on Serum Prolactin Levels

The control rats showed a gradual rise in serum prolactin
concentration as normally seen on the afternoon of proestrus
(Figure 8). By contrast all doses of ergocryptine except the 1 pg
per 100 g body weight, prevented the proestrous rise and reduced
serum prolactin values. These values were significantly lower
(P < . 05 - . 001) as compared to the pre-treatment values in the
control group. The higher doses of ergocryptine produced more
marked effects than the lower doses indicating a dose-response
relationship. The lowest dose of 1 pg ergocryptine per 100 g body
weight produced the least effect and did not block the proestrous rise
in serum prolactin levels (Table 2).

Duration of the Inhibitory Effect of a Single Injection
of Ergocryptine on Serum Prolactin Concentrations

There were no significant differences in serum prolactin
concentrations between the proestrous or the two diestrous values
in the control group (Table 3). On the other hand all doses of

ergocryptine significantly reduced prolactin levels 1 hr after

191

 

Figure 8. Effects of a single injection of ergocryptine on serum
prolactin levels in female Sprague-Dawley rats.

192

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195

treatment on the day of diestrus. The 100 pg dose of ergocryptine
also suppressed the prolactin peak on the day of proestrus, 24 hours
later.
Effect of Ergocryptine on Perphenazine-Induced
Increase in Serum Prolactin

There was no significant change in prolactin concentrations in
the control group (Table 4). A dose of 50 pg perphenazine caused a
10 fold increase in serum prolactin levels which was not affected by
a subsequent injection of vehicle. A dose of 50 pg erocryptine
reduced serum prolactin levels to almost one third of the pre-
treatment value, but the same dose was unable to suppress the
prolactin rise induced by 50 or 10 pg perphenazine. Simultaneous
injections of 50 pg perphenazine and 50 pg ergocryptine produced a
10 fold increase in prolactin concentrations, similar to that produced

by 50 pg of perphenazine alone.

Conclusions

These results show that ergocryptine, like other ergot-
derivatives, such as ergocornine (Wuttke 912.1. , 1971) and LSD
(Quadri and Meites, 1971a), is a potent inhibitor of prolactin release
from the pituitary. Three doses of ergocryptine each blocked the

proestrous rise in serum prolactin and reduced serum prolactin

196

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197

concentration to very low levels. The lowest dose of 1 pg ergocryptine
per 100 gm body weight did not block the proestrous prolactin peak

but reduced serum prolactin. The same dose however, caused a
significant reduction in prolactin levels within 1 hour of injection on
the day of diestrus. The 100 pg dose of ergocryptine injected in
saline-ethanol on the second day of diestrus blocked the proestrous
peak on the next day. The metabolic clearance rate of ergocryptine

is not known; however it appears that a high dose of ergocryptine

given in saline-ethanol can remain in the body for at least 24 hours
after it is administered.

The prolactin inhibitory action of ergocryptine may further be
prolonged by administering it in oil or by controlling its release
from a subcutaneously implanted pellet. This could be of particular
value in mammary tumor work in rats where daily injections of ergot
drugs are given to lower serum prolactin values for long periods.

Ergocryptine was not able to inhibit the increase in prolactin
concentrations produced by perphenazine. Similar results have been
reported for ergocornine (Lu and Meites, unpublished). Perphenazine
and the related central acting drug, chlorpromazine, decrease
catecholamine turnover in the hypothalamus, resulting in release of
prolactin inhibiting factor (PIF) fromthe hypothalamus (Lu e_t_ El. ,
1971). On the other hand, the major action of ergot drugs seems

to be directly on the pituitary gland (Lu SEE}: , 1971). Ergocornine

198

acts directly to decrease the size of a normal pituitary gland that is
grafted under the kidney capsule, and prevents estrogen from
increasing the size of pituitary and secretion of prolactin. In the
present study it appears that the prolactin releasing action of perphen-
azine on the hypothalamus dominated over the prolactin release-
inhibiting action of ergocryptine on the pituitary,

Inhibition of Prolactin Secretion in Rats
by Pyrogallol

 

 

Objectives

 

It was reported earlier that monoamine oxidase inhibitors such
as iproniazid and pargyline prevent degradation of catecholamines
and thereby depress serum prolactin values (Lu and Meites, 1971).
The purpose of this study was to determine whether pyrogallol,
which inhibits the other catecholamine degradative enzyme, catechol-
o-methyl transferase (COMT), also can decrease serum prolactin

leveIS.

Materials and Methods

 

Virgin female 3-4 month old Sprague-Dawley rats, (Spartan
Research Animals, Haslett, Mich.) were used. Two estrous cycles
were followed by collecting daily vaginal smears and only rats

showing regular four day cycles were used.

199

At 11 A. M. on the day of diestrus, a pre-treatment blood
sample (1 ml) was collected by cardiac puncture under light ether
anesthesia. At 11:30 A. M. the rats were injected intraperitoneally
with 1, 8, 12, 20 or 30 mg pyrogallol per 100 g body weights in O. 2
m1 physiological saline (0. 85% NaCl). Control rats were injected
with saline only. Serial blood samples of 1 ml each were collected
again by cardiac puncture under light ether anesthesia at 1, 2, 3,
and 4 hours after treatment. Blood was allowed to clot and serum
separated by centrifugation following the standard procedures
described earlier. The serum was stored in a freezer at -200C

until assayed for prolactin by radioimmunoassay.
Results

There were no significant changes in serum prolactin concen-
trations in the control rats at 1 or 2 hours after injection; however
at 3 and 4 hours after treatment there were slight but significant
increases in serum prolactin levels (Figure 9). These are believed
to be due to the repeated ether anesthesia and serial bleedings. .,
By contrast all rats injected with l, 8 or 12 mg of pyrogallol per
100 gm body weight showed significant reductions in serum prolactin
concentrations. The maximum effect of these doses of pyrogallol

was seen at 3 hours after injections when the serum prolactin

r_.
.g,‘"=l _‘I

Figure 9.

200

Effect of a single injection of pyrogallol on serum
prolactin concentrations in female Sprague-Dawley

rats.

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PRE-INJ

TIME AFTER INJECTION '

EFFECT OF WROGALLOI. ON SERUM PROLACTIN IN RATS

202

concentrations were at their lowest levels. By 4 hours after injec-
tions, prolactin concentrations began to increase again.

A dose-reSponse relationship was evident; 12 mg pyrogallol
per 100 g body weight caused greater reduction in serum prolactin
levels than either 8 or 1 mg pyrogallol per 100 g body weight. A
dose of 20 or 30 mg pyrogallol per 100 g body weight resulted in
several fold increases in serum prolactin levels (Figure 10). These
reached their maximum values at 2 hours after injection and started
to decline by the 3rd hour and returned to pretreatment levels by
4 hours after injection.

The actual serum prolactin concentrations for the various
doses of pyrogallol are given in Table 5. The percent changes in
serum prolactin levels as compared to the pre—treatment values are
given in Table 6. Doses of 1, 8 and 12 mg pyrogallol per 100 g body
weight reduced serum prolactin at 1 hour after injection by 7. 4-38%,
at 2 hours by 17. 6-52%, at 3 hours by 18. 9-50% and at 4 hours by

4. 8- 27%.

Conclusions

A single injection of pyrogallol, at dose levels of 1, 8 or 12
mg per 100 g body weight caused significant reductions in serum

prolactin levels in female Sprague-Dawley rats. Reductions in serum

 

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Figure 10.

203

Effect of a single injection of pyrogallol on serum
prolactin concentrations in female Sprague-Dawley

rats.

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207

prolactin were observed at 1 hour after injection and lasted for about
4 hours.

These results, as well as earlier reports from our laboratory,
further emphasize the role of neurotransmitters, particularly the
catecholamines in the regulation of the anterior pituitary hormones.
Earlier it was reported that three monoamine oxidase inhibitors, pargyline,
iproniazid or Lilly compound-15641, all of which inhibit degradation
of catecholamines in the hypothalamus, produced marked reductions
in serum prolactin values (Lu and Meites, 1971). It was further
demonstrated that these drugs and l-dopa also increased PIF activity
in the hypothalamus. Similar results were reported by Kamberi gt
El. (1970) who found that a single injection of dopamine into the third
ventricle decreased prolactin secretion in rats by increasing PIF
activity in the portal blood.

The present study provides evidence that inhibition of the
catecholamine degradative enzyme, catechol-o-methyltransferase,
by pyrogallol, also significantly decreases serum prolactin levels,
perhaps by increasing PIF activity in the hypothalamus. The
temporary increase in serum prolactin concentrations produced by
high doses of pyrogallol could have been due to the toxicity of

pyrogallol which is known to be a corrosive substance and undoubtedly

stresses the rats.

208

Effect of Pentobarbital and Ether on Plasma
GH Concentration in Sprague—Dawley Rats

Objectives

Some recent reports have described short term effects of
pentobarbital (PB) and ether anesthesia on growth hormone (GI-I)
secretion in the rat. Administration of PB produces, within 4 - 45
minutes, a sharp rise in plasma CH concentration, whereas brief
ether anesthesia results, within 2 - 30 minutes, in a marked
decrease in plasma GH concentrations (Howard and Martin, 1971;
Schalch and Reichlin, 1966; Takabashi e_t_a_1. , 1971; Kokka 23.3—1- ,
1972). In an earlier study Wuttke and Meites (1970) also reported
that administration of PB produced a significant rise in serum
prolactin concentration; however, this rise lasted for only about 30
minutes and was followed by a significant decrease in serum pro-
lactin levels. Therefore, it was of interest to determine the long
term effects of these two commonly used anesthetics on GH secre-
tion in rats kept under normal laboratory conditions and not sub-
jected to fasting, "gentling" or other procedures used in some of

the ea rlier studies.

209

Materials and Method 5

Sprague-Dawley male rats weighing between 250-275 g were
obtained from Spartan Research Animals, Inc. , Haslett, Mich.
They were housed in groups of 6 in steel cages in an air conditioned,
temperature (75 :t 1015‘) and light (14 hours of light from 5 A. M. to
7 P. M.) controlled room. The animals were fed a diet of Wayne Lab
Blox pellets (Allied Mills, Chicago, Ill. ) and given tap watergd

libitum. The animals Spent a week in our animal quarters before

being used in experiments. During this period no attempt was made
to ”gentle” the animals by handling or any other means. Experiments
were performed in the morning, beginning at 8 A. M. when all animals
used in the PB experiment were brought to the laboratory and not
given any feed or water till the end of the experiment about 4-5 hours
later. A similar schedule was followed for the ether experiment

In neither of the experiments were the animals

the next morning.

fasted prior to the experiment.

Expe riment No. l

A pre-treatment blood sample (1 ml) was collected by cardiac
puncture after a 45-60 second ether anesthesia. About half an hour
later, the rats were injected i. p. with a single dose of 1. O, 3. 1, or

5mg sodium pentobarbital/100 g BW in 0. 1 m1 of 0. 8570 NaCl

210

solution. Rats in the control group were injected with saline only.
Serial blood samples were obtained at 5 min, 30 min, 1 hr, 2 hr,
and 4 hrs after the injection. The rats which received 1. 0 mg
PB/lOO g BW were incompletely sedated and therefore had to be
given additional ether anesthesia for about 45-60 seconds before
each bleeding. Rats which received 3.1 mg PB/100 g BW needed
additional ether anesthesia for the last three bleedings whereas the

rats which received 5. 0 mg PB/100 g BW needed ether anesthesia for

the last bleeding only.
Effect of Ether Anesthesia on Plasma (3H

Rats were divided into 5 groups and treated as follows:

(1) 5 rats each were guillotined without ether at 0, 1/2, 2
and 4 hrs and blood was collected from the trunk.

(‘2) 5 rats each were guillotined after 45-60 seconds of ether
anesthesia at O, 1/2, 2 and 4 hrs and blood was collected from the
trunk.

(3) 10 rats were bled serially after 45-60 seconds ether
anesthesia by cardiac puncture at the same time intervals as above.

(4) 10 rats were bled serially as in (3) except after 15 min
ether anesthesia each time.

(5) 5 rats were bled by cardiac puncture after 45-60 seconds

ether anesthesia and then kept under continuous ether anesthesia

211

for 1 hr and bled again by cardiac puncture.

The 45-60 second ether anesthesia was given by placing the
animals in an ether chamber containing ether vapors while ether
anesthesias of longer durations were given by first anesthetizing the

animals in the ether chamber and then making them breathe from a

plastic cone containing cotton soaked in ether.

GH Radioimmunoa 5 say

Blood samples were collected in heparinized syringes or test
tubes and chilled in ice immediately. They were centrifuged 15-20
minutes later, plasma was separated and stored at ~200C till
assayed. Plasma (3H was measured in duplicate using a double—
antibody radioimmunoassay (Dickerman _e_t_al. , 1972). Details of the

GH radioimmunoassay have been described earlier.

Results
Effect of Pentobarbital on Plasma CH

The control rats which were injected with saline and bled
under ether anesthesia showed a continuous decline in plasma GH
concentrations from a pre-treatment level of 118. 2 :1: ll. 0 to 59. 7
:1: 5, 5 ng/ml at 4 hr after injection (Table 7). By contrast, the rats

which were injected with l. 0 mg PB/lOO g BW and also bled under

212

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213

ether anesthesia showed a sharp rise in plasma GH from a pre-
treatment concentration of 109. 3 :I: 9. 2 to 228. 5 :t 19. O at 5 min and
reached a maximum of 276. O :t 14.9 ng/ml at 30 min after PB injec-
tion. After this there was a continuous decline in plasma GH reach-
ing a minimum of 70. 5 :t 7. 7 ng/ml at 4 hr after injection. In the
rats injected with 3.1 mg PB/lOO g BW plasma GH rose from a pre-
treatment level of 103. 0 :i: 10. 7 to a maximum of 374. 0 :I: 29. 6 ng/ml
at 30 min, remained elevated up to 1 hr but declined to a low level
of 83. 2 :t 8. 3 ng/ml at 4 hr after PB injection. Plasma GH levels
in the rats treated with 5. 0 mg PB/lOO g BW increased markedly
from a pre-treatment value of 109. 3 :I: 9. 3 to 233.1 :1: 18. 2 and

410. 0 :L- 16. 5 at 5 and 30 min respectively, reached a maximum of

437. 6 :I: 23. 4 at 1 hr and declined to 76. 7 :1: 6. 3 at 4 hr after PB

injection.
Effect of Ether Anesthesia on Plasma GH

Plasma GH concentration in the rats guillotined after ether
anesthesia increased from 122. 3 d: 11. l in the rats killed at 0 hr to
150. 4 :t 20. 3 ng/ml in the rats killed at 4 hr but the increase was
not significant (Table 8). Plasma GH in the rats guillotined after
45-60 seconds of ether anesthesia also increased from 104. 6 :I: 7.1
in the group killed at 0 hr to 137. 4 :t 14. 1 ng/ml in the group killed

4 hrs later, but again the increase was not significant. Although

214

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215

plasma GH concentrations in the rats killed by guillotines were con-
sistently higher compared to those in the rats which were guillotined
at similar time intervals after ether anesthesia, the differences
were insignificant statistically. When the blood was collected
repeatedly by cardiac puncture following a 45-60 second ether
anesthesia each time, plasma GH declined from 132. 8 :I: ll. 2 at 0
hr to 74. 8 :1: 8.9 ng/ml at 4 hr. A similar decline in plasma GH
concentrations from 97. 2 :1: 5. 8 at 0 hr to 54. 5 d: 4.1 ng/ml at 4 hr
occurred when rats were exposed to a 15 min ether anesthesia before
each of the serial bleedings. The largest decrease in plasma GH,
from 109. 0 :I: 8. 8 to 40.1 d: 4. 9 ng/ml occurred when the rats were

kept under ether anesthesia for 1 hr.

Discussion

This study demonstrates that injection of PB caused an initial
rise followed by a fall in CH secretion in male rats. Although all
doses of PB elevated plasma 15 min after the injection, the extent
and duration of the rise depended on the dose of PB used, larger
doses causing higher rises which lasted for longer durations. How-
ever, in all cases plasma CH concentrations fell to below pre-
treatment levels by 4 hr after treatment. Ether was not able to
suppress the rise in CH secretion induced by PB. The lower plasma

GH levels observed at l, 2. and 4 hrs after injection of l. 0 or 3. 10

216

mg PB/lOO g BW could not have been due to the suppressive effect of
ether as plasma GH also decreased by 2 hr without exposure to ether
in the rats injected with 5 mg PB/lOO g BW.

Ether anesthesia produced a consistent decrease in CH secre-
tion. Although suppression of plasma GH due to 45-60 second ether
exposure immediately followed by decapitation was not significant
statistically, ether anesthesia of the same or longer duration caused
significant decreases in plasma GH in serial bleedings. A decrease
of more than 60% in plasma GH concentrations was noted when rats
were kept under ether anesthesia for one hour.

The mechanisms of action of PB and ether on GH secretion in
the rat have not been elucidated. Some recent reports have indicated
that alterations in CH secretion in the rat are perhaps mediated via
alterations in adrenalcorticoids. Takashi and co-workers (1971)
reported that a stress-induced decrease in plasma GH in the rat was
accompanied by a rise in plasma corticosterone whereas increases
in plasma GH due to gentling or injection of PB coincided with a
decrease in plasma corticosterone levels. Kokka 3311. (1972) were
not able to confirm these findings. They found that the PB-induced
rise in plasma GH was not accompanied by a decrease in plasma
corticosterone and that dexamethasone inhibited both GH and
corticosterone levels in blood. Wakabayshi 332.1. (1971) concluded

that the ether-induced reduction in plasma C3H was probably not

217

mediated via an increase in adrenal-corticoids. Recently Howard
and Martin (1972) reported that PB increased GH secretion by a
direct action on the rat anterior pituitary in y_i_t_r_g_. Earlier it was
reported from our laboratory that PB influenced prolactin in the rat
by a direct action on the anterior pituitary and also via the hypotha-
lamus (Wuttke EEfl° , 1971). It is possible that PB influences GH
secretion in the rat via similar mechanisms.

Relation of Size and ége of Pituitary Tumor (MtT. W15)

to Prolactin and Growth Hormone Secretion and
to Body Weight

 

Objectizes

Furth and co-workers (1961) have deve10ped several kinds of
transplantable pituitary tumors in rats. One of these tumors, the
MtT.W15, was originally induced by chronic estrogen treatment in
inbred Wistar-Furth ratsand has been maintained for several years
by serial transplantation in inbred rats of the same strain. It
secretes large quantities of prolactin and growth hormone (GH). It
was of interest to determine if the secretion of these hormones from
the tumor is influenced by the size and the age of the tumor,and also
if the gain in body weights of the tumor-bearing rats was related to the

increase in tumor size.

218

Materials and Methods

MtT.W15 pituitary tumor tissue was removed under sterile
technique from two Wistar-Furth rats and transplanted into 50-55
day inbred female rats of the same strain. About 6 weeks after
transplantation, when each tumor transplant was about 0. 4-0. 5 cm
in its largest diameter, each rat was placed under light ether anes-
thesia once a week and 1 ml of blood was removed by cardiac
puncture. Each blood sample was divided into 2 equal parts; 0. 5 ml
was used to obtain serum for prolactin assay and the remaining 0. 5
ml was immediately heparinized with O. 05 ml Na heparin solution
to obtain plasma for CH determination. Serum and plasma samples
were stored at -20°C for a period of 15-20 weeks before being
assayed. After blood collection, while the rats were still under
anesthesia, the largest diameter of each pituitary tumor was carefully
measured with a vernier caliper. Body weights were also recorded.
A total of 6 weekly blood samples were collected and corresponding
tumor sizes and body weights were recorded.

Each serum or plasma sample was assayed for prolactin and
GH in triplicate. The regression equation and line of best fit between
the pituitary tumor diameter and prolactin and GH concentrations
and body weight were determined by the method of least squares.

The significance of correlation coefficients also was determined.

‘ -
I3. 0004—...”

Figure 11.

219

Transplants of "mammosomatotropic" pituitary tumors
in inbred female rats of the Wistar-Furth strain. Both
transplants are of the same age. Note the difference

in size.

220

 

 

 

221

Results

Relation of Pituitary Tumor Size
to Serum Prolactin

There was considerable variability in serum prolactin con-
centration in rats carrying pituitary tumor transplants. The tumors
ranged in size from 0. 4 to l. 2 cm and in serum prolactin concentra-
tions from O. l to 1. 9 pg/ml at the beginning of the experiment
(Table 9). During the succeeding weeks as tumor size increased
there was a corresponding increase in serum prolactin levels. At
the end of 6 weeks tumor diameters ranged from 1. 7-4. 2 cm and
serum prolactin concentrations ranged from O. 4 to 8 ug/ml. Often
tumors of the same size secreted different amounts of prolactin but
within each rat there was always an increase in serum prolactin
with an increase in tumor diameter. A highly significant and positive
correlation (r = O. 9, P < . 001) was obtained between tumor diameter
and serum prolactin concentration. The line of best fit is given in
Figure 12. The regression application wasmost accurate when the
tumors were of medium size.
Relationship Between Pituitary Tumor Size and
Plasma GH Concentrations

There was more variability in plasma GH concentrations than

in serum prolactin concentrations. Plasma GH levels at the

Figure 12.

222

Correlation between serum prolactin concentrations
and size of pituitary tumor (MtT.W15) transplants in
female Wistar-Furth rats. Each dot represents a
serum prolactin determination at the tumor size
indicated on the abscissa. The correlation coeffici-
ent (y) was 0. 9 and highly significant (P < . 001).

223

6
5
SERUM
PROLACTIN
(ug/ml) p 4
3
2

 

1 2 3 4
TUMOR DIAMETER(cm)

224

beginning of the experiment ranged from 2. 5-5. 8 [lg/ml and increased
to 6. 0—14. 9 (lg/ml by the end of 6 weeks (Table 9). In a subsequent
trial we found that some tumors in the same size category were
secreting as high as 135 to 180 pg GH per m1 of plasma. Like serum
prolactin levels, there was always an increase in plasma GH concen-
trations with an increase in tumor diameter, and a significant and
positive correlation (r = 0. 6, P< . 001) was obtained between these
parameters (Figure 13).
Relationship Between Pituitary Tumor Size and
Body Weight of the Rats

Body weight of the tumor bearing rats increased from 205-300 g
at the beginning of the experiment to 335-415 at the end of 6 weeks
(Table 9). The correlation between tumor sizes and body weights
was positive and highly significant (r - 0.9, P< . 001). The line
of best fit between these two parameters (Figure 14) was better than
that between tumor size and serum prolactin concentration or between

tumor size and plasma GH concentration.

Conclusions

These results indicate that the rate of prolactin and GH secre-
tion from the pituitary tumors and the body weights of the tumor

bearing rats are influenced by the size of the tumor. Other

225

Table 9. Influence of pituitary tumor size on prolactin and growth
hormone secretion and on body weight of rats.

 

 

Week 0 Week 6

Tumor diameter 0. 4-1. 2 1. 7—4. 2
(cm)

Serum prolactin 0. 1-1. 9 0. 4-8. 0
(big/m1)

Plasma GH 2. 5-5. 8 6. 0-14. 9
(Hg/m1)

Body weight 205-300 325-425

(g)

Figure 13.

226

Correlation between plasma GH concentrations and
size of pituitary tumor (MtT. W15) transplants in
female Wistar-Furth rats. Each dot represents a
plasma GH determination at the tumor size indi-
cated on the abscissa. The correlation coefficient
(y) was 0. 6 and statistically significant (P < . 001).

227

 

o

14
12
1

PLASMA

G H 8

(HQ/ml)
6
4
2

1 2 3 4

TUMOR DIAMETER(Cm)

m.

f... .

Figure 14.

228

Correlation between body weight of female Wistar-
Furth rats and the size of pituitary tumors (MtT. W15).
Each dot represents a body weight determination at
the tumor size indicated on the abscissa. The cor-
relation coefficient (y = O. 9) was positive and highly
significant (P < . 001).

229

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aoov
WEIGHT

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Nd
“1

TUMOR DIAMETER (cm)

230

investigators have reported a gradual increase in body weight and

GH production after pituitary tumor implantation (MacLeod 9i a_l. ,
1966, Peake_t_1. , 1968). While our study was in progress, Ito gta_1.
reported that GH and prolactin concentrations in rats of the W15
strain, carrying transplanted pituitary tumors, increased in direct
relation to tumor weight and tumor age. The present study confirms
and extends these findings. We also found that tumors of the same
age may differ in their size and rate of prolactin and GH secretion.

However, there was a positive and highly significant correlation

between tumor size and prolactin and (3H secretion, and between

' tumor size and body weight.

Ergot-Induced Inhibition of Pituitary
Tumor Growth in Rats

@jectives

It was reported that ergocornine inhibited prolactin secretion
by a direct action on the pituitary gland and reduced its weight in the
normal rat (Lugtil. , 19 71). It was of interest to determine if
er800::ornine, ergocryptine and a related compound, ergonovine, could
inhibit growth of transplants of pituitary tumor MtT. W15 which

secretes large amounts of prolactin and growth hormone.

231

Materials and Methods

 

Pituitary tumor (MtT. W15) tissue was removed from 2 female
Wistar-Furth rats, minced in sterile physiological saline (0. 8570) and
injected subcutaneously into the post cervical area of 50-55 day old
virgin female rats of the same inbred strain. Details of the trans-
plantation technique have been described earlier in this thesis. About
8 weeks later, when each pituitary transplant was 1. 5 to 3. 0 cm in
diameter, the rats were divided into 5 groups and were injected
intraperitoneally once daily for 3 weeks as follows:

Group 1, controls, 0. 2 ml of the injection vehicle for the

ergot drugs which consisted of 9770 physiological saline
and 3% ethanol of 70% strength.

Group 2, 0.1 ml of ergocornine per 100 g body weight in

0. 2 ml saline-ethanol vehicle.
Group 3, 0. 2 mg of ergocornine per 100 g body weight in
0. 2 ml saline-ethanol vehicle.
Group 4, O. 2 mg ergonovine per 100 g body weight in
0. 2 ml saline-ethanol vehicle.
Group 5, 0. 3 mg ergocryptine per 100 g body weight in
O. 3 m1 saline-ethanol vehicle.
Once a week each rat was given light ether anesthesia by plac-

lng the rat in an ether chamber containing ether-air mixture and the

232

largest diameter of each tumor was carefully measured with the help
of vernier caliper. Body weights were recorded weekly. At the

end of 3 week-treatment period, the rats were killed and the pituitary
tumors removed, fixed in Bouin's fluid and stained with Masson's

trichrome stain for microsc0pic examination.
Results

Changes in Tumor Size

The percent changes in the pituitary tumor diameter are given
in Table 10. The tumors in the control group showed a steady
increase in tumor size throughout the treatment period with a maxi-
mum gain of 33. 6% in average tumor diameter by the end of 3 weeks.
By contrast, 0. 1 mg and 0. 2 mg ergocornine per 100 g body weight
completely inhibited pituitary tumor growth and caused a reduction
0f 14. 8% and 30. 67. respectively in tumor diameter by the end of the
treatment period. These reductions in tumor diameters were highly
Significant (P < . 0001). The dose of ergonovine used i.e. 0. 2 mg
per 100 g body weight slowed the growth of pituitary tumors but failed
to decrease pituitary tumor diameter. The pituitary tumors in the

ergonovine treated group gained only 11. 1% in diameter by the end of
3 Weteks compared to a 3-fold higher increase in the tumor diameter

Of the control rats. The dose of ergocryptine used had no effect on

233

Table 10. Percent changes in pituitary tumor diameters induced
by ergocornine, ergonovine and ergocryptine.

 

 

Week 1 Week 2 Week 3
Treatments (percent) (percent) (percent)
a/
Controls +11. 5 23. 6 33. 6—
Ergocornine a/
0.1mg/100gBW - 9.5 -11.5 -14.8—
Ergocornine a/
0.2mg/100 g BW -13.2 -20.1 -30.6-
Ergonovine a/
0.2mg/lOOgBW + 3.7 + 7.4 +11.1-
Ergocryptine
o. 3 mg/100 g BW +11.1 +25.9 +29. 6

+ = increase; - decrease;

3/p< .05- .0001

.UV'DI

3
l‘

234

pituitary tumor growth. The tumors in this group showed a gain of
29. 6% in diameter by the end of 3 weeks. The percent changes in
the pituitary tumor diameters induced by the different treatments
are plotted in Figure 15. The actual tumor diameters of the control
and 0. 2 mg ergocornine groups are given in Table 11. None of the
treatments induced any significant changes in the body weights of the

pituitary tumor-bearing rat 5 .

Histological Change 5

Microscopic examination revealed that the pituitary tumors
from the control rats consisted of numerous cells of different sizes
with prominent nuclei and many mitotic figures, whereas tumors
from ergocornine-treated rats consisted of relatively few, separated,

large cells in which nuclei were either absent or pycnotic.

Conclusions

These observations indicate that ergocornine can significantly
inhibit growth of MtT. W15 pituitary transplants in inbred female
Wistar-Furth rats. Ergonovine, at the dose level used, failed to
inhibit pituitary tumor growth although tumor growth rate was
considerably slowed down compared to that in the control group.
Ergocl‘yptine was not effective at the dose used and previously was

found to be less effective than ergocornine for inhibiting

1'}...

Figure 15.

235

Effects of ergocornine, ergonovine and ergocryptine
on growth of transplants of pituitary tumor MtT. W15
in inbred female Wistar-Furth rats.

236

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238

DMBA-induced mammary tumor growth in Sprague-Dawley rats
(Cassell 3311. , 19 71). V Associated with the tumor regression induced
by ergocornine was a marked disappearance of cells and pycnosis or
loss of nuclei, and a decrease in serum prolactin concentrations

(to be reported in the following experiment). The capacity of
ergocornine and ergonovine to inhibit growth of the W15 pituitary
tumor is apparently not dependent on any direct connection to the
hypothalamus suggesting that the major action of the drugs was
exerted directly on the tumor. This agrees with the observation

that ergocornine can act directly to decrease the size of a normal

rat pituitary grafted underneath the kidney capsule (Lu e_t_ 11. , 1971).
Prolactin secreting pituitary tumors have been'observed in human
patients (Peake gal. , 1969) and it would be of interest to determine
whether ergot drugs might be effective in treating such patients.

Effects of Ergocornine and CG 603 on Blood Prolactin and GH
in Rats Bearing a Pituitary Tumor

@jective s

The MtT.W15 pituitary tumor secretes large quantities of

prolactin and growth hormone (GH) (Kim e_tua_l_. , 1963; Quadri and
Me ites. unpublished data). Several ergot derivatives, including
erg°<=0rnine (Quadri Sifil- , 19 72) and ergotamine (MacLeod and

L h
e myer, 19 72) have been shown to induce regression of these

239

tumors. Presumably this is related to the ability of ergot drugs to
inhibit prolactin release (Wuttke £331. , 1971; Quadri and Meites,
1971a). Ergot drugs also induce regression of spontaneous (Quadri
and Meites, 19 71b) and carcinogen-induced (Cassell _e_t_al. , 19 71)
mammary tumors in rats. It was of interest therefore to determine
the effects of ergocornine on serum prolactin levels in rats carrying

a MtT. W15 pituitary tumor, and compare this with the effects of a
thalidomide-derived drug, CG 603, that also inhibits prolactin release
(Gelato e_t-3:1. , 1972) and inhibits growth and deve10pment of
carcinogen-induced mammary tumors in rats (Muckter 31331. , 1969).

The influence of these drugs on plasma GH levels also was measured

in these rats.
Materials and Methods

Jacob Furth and co-workers (1965) induced the MtT.W15
Pituitary tumor by chronic estrogen treatment and have maintained
this tumor for several years by serial transplantation into inbred
female Wistar-Furth rats (Kim _e_t__a_1_1_. , 1963). We originally obtained
tWO MtT.W15 pituitary tumor carrying rats from Dr. Jacob Furth
and have maintained the tumor for the last three years by trans-
plantation in inbred female rats of the same strain. Details of the

transplantation technique already have been described.

240

Treatment of Rats

About 6 weeks after transplantation, when each tumor had
reached 0. 5 cm or more in its largest diameter, the rats were
divided into 3 groups and injected subcutaneously once daily for 3
weeks as follows:

Group 1, controls, 0. 2 ml of the vehicle for ergocornine

consisting of 95% physiological saline (0. 85% NaCl) and
4% of a 70% ethanol solution.

Group 2, 0. 2 mg ergocornine methanesulfonate per 100 g
of body weight in 0. 2 ml saline-ethanol vehicle.
Ergocornine was kindly supplied to us by Dr. M.
Taeschler and Dr. E. Fluckiger, Sandoz Ltd. , Basel,
Switzerland.

Group 3, 60 mg phthalimide, (N-Cl-morpholinomethyl)2,
6-dioxo-4-piperidyl (CG 603) per 100 g of body weight.
This drug was injected as a suspension in 0. 85%
saline. CG 603 was a gift from Dr. Jane Taylor,

National Cancer Institute, USPHS.
Blood Collection and Tumor Measurement

Once a week the rats were given light ether anesthesia by plac-

mg them for a brief period (1-1. 5 minutes) in a glass chamber

241

containing a mixture of air and ether vapor. One ml blood was with-
drawn from the anesthetized rats by cardiac puncture by a 26 gauge
needle attached to a 1 ml plastic syringe. Each blood sample was
divided into equal portions: 0. 5 ml was expelled into a test tube
placed in an ice-bath and centrifuged later to obtain serum for
prolactin radioimmunoassay; the remaining 0. 5 ml was immediately
mixed in an ice-chilled test tube with O. 05 ml sodium heparin solu-
tion (100 mg/100 ml H20) and centrifuged to obtain plasma for CH
radioimmunoassay. Serum and plasma samples were stored at
-200C prior to assay.

As soon as the blood was withdrawn and while the rats were
still under anesthesia, the largest diameter of each pituitary tumor
was carefully measured with vernier calipers. Body weights also
were recorded weekly. At the end of the 3 week treatment period,
blood samples, tumor diameters and body weights were recorded

for an additional period of 3 weeks.

Radioimmunoassays

Serum prolactin (Niswender gt _a_l_. , 1969) and plasma GH
Dickerman gay, 19 72) concentrations were measured by double
antibody radioimmunoassays using NIAMD-RP-l rat prolactin and
NI‘AA’IDwRGH-RPI GH as standards. Each blood sample was assayed

in -
duplicate and average of the two values was obtained.

242

Results
Effects of Ergocornine and CG 603 on Serum Prolactin

Control rats showed a progressive and significant (P < . 01)
increase in mean serum prolactin concentrations from 0. 4 :t 0. 2
(lg/ml to l. 5 :1: 0. 4 (Lg/ml during the 3 week treatment period (Table
12). By contrast, daily injections of ergocornine completely
prevented the rise in serum prolactin seen in the controls and reduced
serum prolactin levels from 0. 4 :1: 0. 2 pg/ml to 0. 2 :I: O. 01 pig/m1
during the 3 week treatment period. CG 603 failed to inhibit pro-
lactin release which increased significantly in the serum (P < . 01)
from 1. 8 :I: 0. 4 to 3. '7 :I: 0. 2 pg/ml by the end of the three week
treatment period.

During the post-treatment period serum prolactin levels in the
control and CG 603 treated groups showed a continuous rise similar
to that observed during the treatment period, and reached levels of
3-9 i 1.1 and 6. 5 :l: 0. 5 (lg/ml, respectively, by the end of 3 weeks.
Serum prolactin in the group previously given ergocornine rose
rapidly by 1 week after termination of treatment and reached 5. 5 =1:

l

' 1 Hg/ml by the end of the post-treatment period. The changes in

serum prolactin induced by ergocornine and CG 603 are plotted

in Figul‘e 16,

243

 

 

 

 

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Figure 16.

244

Effects of daily injections of ergocornine and CG 603
on serum prolactin in inbred female Wistar-Furth
rats carrying a MtT. W15 pituitary tumor transplant.

245

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Treatment I Post -treatment

246

Effects of Ergocornine and CC 603
on Plasma GH

During the 3 week treatment period, mean plasma GH concen-
trations in the control group rose significantly (P < . 05) from 3. 6 :1:
0.4 pig/ml to 5.9 :I: 1.1 [lg/ml. Plasma GH showed a small but
insignificant decrease from 3. 5 i: O. 5 pig/ml to 2.9 :I: O. 5 ug/ml in
the ergocornine treated group (Table 13). CC 603 had no effect on
plasma GH concentration which increased significantly (P < . 02)
from 2. 8 :I: O. 6 ug/ml to 5. 8 :I: 0. 8 rig/ml, respectively, by the end
0f 3 weeks. Plasma GH concentrations in the rats previously given
ergocornine increased rapidly and reached a level of 8. 8 :1: 1. 4 ug/
ml by the end of the 3 weeks post-treatment period. The above

changes in plasma Gl—I are plotted in Figure 17.

Tumor Size

Mean tumor diameter in the control rats increased significantly
(P < . 05) during the treatment period from 0.9 :I: l to 1.9 :I: O. 3 cm
(Table 14), but showed a significant (P < . 05) reduction in size from
O- 8 i 0. 3 to 0. 3 :b 0.1 cm in the ergocornine treated group. There
was no effect on tumor growth in rats treated with CC 603, and mean
tumor diameter increased from 1. 6 :I: 0. 2 to 2.1 :I: 0. 2 cm. No dif-

fere ° . .
nce 1n tumor growth was observed after termination of treatment

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248

Effects of daily injections of ergocornine and CC 603
on plasma GH concentrations in inbred female Wistar-
Furth rats carrying a MtT. W15 pituitary tumor
transplant.

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251

in the control and CC 603 treated groups, but in the group previously
given ergocornine there was a rapid and significant (P < . 001)

increase in mean tumor diameter from O. 3 :I: 0.1 to 2. 6 :1: 0. 5 cm by
the end of 3 weeks. The effects of ergocornine and CC 603 on tumor

diameter are plotted in Figure 18.
Body Weight

Mean bodyweight in the control group increased significantly
(P < 0.1 - . 01) during the treatment period from 248. 2 :1: 12. 5 to
322.1 :I: 16.9 g, in the CC 603 treated group from 228 i 21 to 268 :1:
21. 5 g, and showed a slight but insignificant decrease in the ergo-
cornine treated group from 234.9 :I: 12. 9 to 230. 5 :I: 14. 5 g (Table
15). During the post-treatment period there was a rapid and signi-
ficant (P < . 05) increase from 230. 5 :1: l4. 5 to 343. 3 :I: 26. 3 g in the
rats previously given ergocornine, and smaller increases were

observed in the control rats and those previously given CG 603.

W

The results of this experiment demonstrate that treatment with
er30‘301‘nine for 3 weeks in rats carrying a MtT.W15 pituitary tumor
reduced serum prolactin levels by more than 50% and prevented any
increaSG in plasma CH, whereas in the controls serum prolactin

con
centrations increased by about 4007/0 and plasma GH increased

 

I;
. .
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Figure 18.

252

Effects of daily injections of ergocornine and CC 603
on average tumor diameter of MtT. W15 tumor trans-

plant in inbred female Wistar-Furth rats.

Av.
Tumor

Diam-
(CM)

253

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WEEKS

Treatment | Post - treatment

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255

about 100%. The decrease in serum prolactin in the ergocornine-
treated rats closely paralleled the reduction in tumor size of about
50% during the treatment period. We previously reported that
ergocornine produced degenerative changes in the MtT.W15 pituitary
tumor as indicated by dissolution of cells and disappearance of
nuclei (Quadri _e_t_a_1. , 19 72). The present results indicate that
ergocornine exerts its inhibiting action mainly on the prolactin
producing cells in the pituitary tumor since there was no significant
decrease in plasma GH levels. Ergocornine prevented any increase
in plasma GH by inhibiting growth of the pituitary tumor.

After termination of treatment the pituitary tumors in rats
previously given ergocornine showed a rapid increase in size, exceed-
ing that of the controls. This is believed to reflect removal of the
inhibitory influence of ergocornine. A similar resumption in growth
of mammary tumors was observed in rats after termination of treat—
ment with ergot drugs (Quadri and Meites, 19 71b; Casse11§_t__a_1. ,
1971). The rapid growth rate of the pituitary tumors after treatment
with ergocornine ceased was accompanied by an increase in body
weight and in serum prolactin and plasma GH values.

The present observations suggest that ergocornine acted
directly on the pituitary tumor to inhibit prolactin release and induce
regression in tumor size. A previous study in our laboratory

demonstrated that ergocornine can act directly on the normal i_n situ

 

256

pituitary, on the transplanted pituitary and on pituitary tissue in
y_i_t_r_o_ to inhibit prolactin release (Lu gal. , 1971). Ergot drugs also
can inhibit prolactin release via the hypothalamus by increasing PIF
activity (Wuttke e_tal. , 19 71), but this is believed to be of minor
importance in the present experiment since the pituitary tumor was
distant from the hypothalamus and secreted relatively enormous
amounts of prolactin as compared to the normal i_ns_it_:_u pituitary.

The thalidomide derived compound CC 603 had no significant
effect on pituitary tumor size or on prolactin or CH release. CC
603 was shown to inhibit prolactin release by the £5053 pituitary
(Gelato BEE}.- , 19 72) and induce regression of mammary tumors in
Sprague-Dawley rats (Muckter §_’c_a_1. , 1969). The present results sug-
gest that CO 603 has no direct action on the pituitary, and apparently
inhibits prolactin release from the normal .12 §_i_t£ pituitary via the
hypotha la mus .

Reinitiation of Estrous Cycles in Old Constant
Estrous Rats bLCentral-Acting Drugs

gbjectives

 

Regular estrous cycles rarely occur in old female rats. Many
Show continuous vaginal cornification (constant estrus), others
exhibit a repeated series of pseudopregnancies of irregular length,

and some show no predictable pattern (Mandl and Shelton, 1959:

257

Aschheim, 1961). Recent studies by our laboratory (Clemens £331. ,
1970; Clemens and Meites, 1971) provided preliminary evidence for
the hypothesis that changes in hypothalamic function may be at least
partially responsible for the disappearance of cycling in old female
rats. Female Sprague-Dawley rats approximately 20 months of age
and in constant estrus, were found to have low hypothalamic LRF
(luteninizing hormone releasing factor) and high FRF (follicle stimu-
lating hormone releasing factor) and low pituitary LH and high FSH
and prolactin as compared to cycling 3 month old female rats during
estrus (Clemens and Meites, 19 71). These old rats also showed well
developed ovarian follicles but no corpora lutea reflecting failure of
ovulation, and the mammary glands were hyperplastic reflecting
high prolactin secretion. Ovulation was induced in old constant
estrous rats by electrochemical stimulation of the preoptic hypo-
thalamus or by injections of progesterone for 3 days or epinephrine
in oil for 10 days (Clemens £331. , 19 70). In addition, the interesting
observation was made that daily injections of epinephrine resulted in
resumption of estrous cycles in some rats during treatment.

Other studies have indicated that pituitary release of LH, FSH
and prolactin are controlled by hypothalamic release of LRF-FRF
and PIF, resulting in increased release of LH and FSH and decreased
release of prolactin. Preliminary unpublished evidence from our

laboratory suggests that the hypothalamus of old rats may be deficient

”a! 0

258

in catecholamines, perhaps accounting for the low LH and high
prolactin found in these rats. Since epinephrine induced ovulation
and cycling in some old constant estrous rats (Clemens 9131. . 19 70),
it was of interest to confirm and extend this observation, and also to
determine the effects of l-dOpa (the immediate precursor of cate-

cholamines) on cycling in old constant estrous rats.

Materials and Method 3

 

Animals

Sprauge-Dawley female rats, 12-15 months old (retired
breeders) were obtained from Holtzman Co. , Madison, Wisconsin.
They were housed in steel cages in an air conditioned and tempera-
ture (75 :1: 20F) controlled room. The room was lighted for 14 hours
each day from 5 A. M. to 7 P. M. with artificial fluorescent light.
The animals were fed a diet of Wayne Lab Blox pellets (Allied Mills,
Chicago, Ill. ) and given tap water 3.2 libitum. Twice each week the
diet was supplemented with oranges and carrots. Once about every
4 months, for a period of 5-7 days, all animals were given oxytetra-
cycline (Pfizer) in their drinking water as a prophylactic measure

against infections.

259

Treatment

When the animals reached 20-23 months of age, daily vaginal
smears were taken at the same time each day for a period of 30
days with the help of a small glass dropper and lukewarm tap water.
The animals were handled gently and care was taken to avoid vaginal
injury. Only animals that showed constant vaginal cornification for
a continuous 30-day period were used in the present study. These
were randomly divided into 4 groups and treated as follows:

Group 1, controls, were injected sc twice daily at 9 A. M. and

5 P. M. with the vehicle used for injection of l-dOpa.
Group II were injected sc once daily at 5 P. M. with 4 mg of
l-epinephrine (K and K Labs. , Inc. , Plainview, N. Y.)

per rat in 0.1 cc corn oil.

Group III were injected with l-d0ps (kindly provided by

Dr. W. E. Scott, Hoffman-La Roche Company, Nutley,
N. J. ). L-dOpa was dissolved in warm 0. 5 N HCl, and
O. 5 N NaOH was added to achieve a pH of 3. 5-4. 0, and
injected sc at a dose of 20 mg twice daily at 9 A. M. and
5 P. M.

Group IV were injected sc once daily at 4 P. M. with iproniazid

phosphate (Hoffman-La Roche Company) at a dose of 10 mg

per 100 g‘ body weight in 0.15 ml of 0. 85% NaCl. These

260

treatments were continued for a period of 25 days. Vaginal smears
were taken daily during this period and during a post-treatment

period of 35 days.
Results

The results obtained in the control rats are given in Table 16.
None of the 13 rats exhibited estrous cycles. Seven rats showed
no change in the vaginal smears during the entire 25-day treatment
period, but two exhibited diestrus near the end of the post-treatment
period. Six rats exhibited vaginal cornification with intermittent
1-3 day diestrous periods during the 25-day treatment period; of
these, four continued to show vaginal cornification with intermittent
diestrus during the post-treatment period and two exhibited vaginal
cornification with one day of pros strus.

In the epinephrine treated group (Table 17), 10 out of 14 rats
showed one or more regular cycles during the 25-day treatment
period; 5 of these continued to cycle for 8-12 days during the post-
treatment period and ended with cornified or diestrous smears
in the later part of this period, while the remaining five rats returned
to the cornified state with four showing diestrous smears during the
last seven days of the post-treatment period. One rat showed 2

irregular cycles during the 25-day treatment period, one was in

261

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263

diestrus and two showed continuous vaginal cornification during the
entire 25-day treatment period.

In the l-dOpa treated group (Table 18), 14 of 18 rats showed
one or more regular estrous cycles during the 25 day treatment
period. One continued to cycle during the entire post-treatment
period but the majority went into diestrus. One rat exhibited a
regular cycle before going into a long diestrus, one returned to
constant estrus and one rat died. L-dopa had no effect on four rats,
and these remained in constant estrus throughout the treatment and
post—treatment periods.

In the iproniazid-treated group (Table 19), six rats out of
seven showed one or more regular cycles during the treatment. Of
these, one exhibited two cycles during the post-treatment period
while the others showed either diestrus or alternate periods of
diestrus and estrus. One rat in this group exhibited two regular
cycles interspaced with long diestrous periods during the treatment
period, and another showed alternate periods of diestrus and estrus.

Rats in the l-dopa-treated group appeared to be in better
health than any of the other groups. Only one out of 18, l-dopa
treated rats died in the post-treatment period, whereas three out of
seven died in the iproniazid-treated group. Rats in the latter group
appeared to be weak and were hypersensitive to handling. None of

the rats in the epinephrine-treated or control groups died.

264

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266

Conclusions

These results demonstrate that treatment of old constant
estrous rats with epinephrine, l-dopa or iproniazid can restore
regular estrous cycles in many rats during the treatment period.
Cycling continued in some of the rats during the post-treatment
period. The observations in the epinephrine-treated group confirm
our previous report that this drug can restore cycling in old constant
estrous rats (Clemens e_t_ai. , 1970), and shows that some of these
rats continued to cycle after termination of treatment. Treatment
with l-dopa appeared to be more effective and induced cycles earlier
than epinephrine or iproniazid. Iproniazid treatment restored nor-
mal cycles in several rats but resulted in high mortality after term-
ination of treatment. Only one rat in this group continued to cycle
during the post-treatment period and most rats went into a long
period of diestrus.

All three drugs used in this study increase catecholamines in
the body. Epinephrine is a catecholamine, l-dopa is the immediate
precursor of catecholamines and iproniazid increases accumulation
of catecholamines by inhibiting their metabolic degradation. L-dopa
and iproniazid are effective in increasing brain catecholamines
when administered systematically, but epinephrine usually is not

considered to be able to pass through the blood-brain barrier

267

(Innes and Nickerson, 19 71). However, the circulation to the hypo-
thalamus differs from the rest of the brain (Harris, 1955), and it is
possible that the blood-brain barrier in old rats is less resistant to
the entry of epinephrine than in younger rats. It also is possible
that epinephrine acted indirectly on the CNS. It appears reasonable
to assume that the drugs used in the present experiment restored
regular estrous cycles in the old rats by correcting a catecholamine
deficiency in the hypothalamus.

The effects of treatment with these drugs on blood levels of
LH, FSH and prolactin in the old rats remain to be investigated.
Preliminary results indicate that normal serum LH in old female
rats is low and serum prolactin is high as compared to young sexu-
ally mature female rats (Shaar, Euker, Riegle and Meites, unpub-
lished data). Also, l-dOpa increases serum LH in old constant
estrous rats (Euker, Riegle and Meites, unpublished). If the drugs
have similar effects in old as in young rats, they may be expected
to elevate serum LH and FSH and to depress serum prolactin values.
We are currently investigating whether reinitiation of estrous cycles
in old constant estrous rats by drugs can induce these animals to

mate and undergo fertilization, pregnancy and lactation.

268

Regression of Spontaneous Mammary Tumors
in Sprague-Dawley Female Rats
byErgot Drugs

Objectives

The ergot drugs, ergocornine mesylate and ergocryptine
mesylate were found to inhibit prolactin secretion in the rat .(Wuttke
9:531. , 1971; Quadri, Karande and Meites, unpublished data). These
drugs also induced profound regression of DMBA-induced mammary
cancers in rats (Nagasawa and Meites, 1970b; Cassellgtgl. , 19 71;
Quadri ital. , 1973c). It was the purpose of this experiment to inves-
tigate the effects of these compounds on spontaneous mammary
tumors in rats, which unlike the carcinOgen-induced multiple mam-
mary adenocarcinomas, are usually benign fibroadenomas and occur

as a single tumor per rat.
Materials and Methods

SPrague-Dawley female rats (Spartan Research Animals,
HaSIett, Michigan) 12 months of age or older, were obtained at
variable periods of time after appearance of a single spontaneous
mammary tumor. Some animals were allowed to grow old and
develop tumors in our own animal quarters. They were fed a stan-

dard labOratory diet supplemented with oranges and carrots.

269

The tumor-bearing rats were randomly divided into one control
group and eight experimental groups. Rats in the control group were
injected daily intraperitoneally for 4 weeks with O. 2 ml of a saline
ethanol vehicle which consisted of 96% physiological saline (0. 85%
NaCl) and 470 ethanol of 70% strength and was used to inject ergo-
cornine and ergocryptine. Rats in 4 experimental groups were
injected daily with O. 05, 0.1, or O. 4 mg ergocornine per 100 g of
body weight in O. 2 m1 of saline-ethanol vehicle. Rats in the remain-
ing 4 groups were injected similarly with O. 05, 0.1, 0. 2 or O. 4 mg
ergocryptine per 100 g body weight.

Beginning on the first day of treatment and once a week there-
after, the rats were placed under light ether anesthesia and the
largest diameter of each tumor was measured with the help of a
vernier caliper. After 4 weeks of treatment, all injections were
discontinued and the tumors were measured for an additional period
of 4 weeks. Body weights were recorded weekly throughout the

treatment and post-treatment period.

Results

The effects of different treatments on average tumor diameter
are shown in Table 20. The control rats showed a progressive
increase in tumor diameter throughout the treatment period and the

average tumor diameter increased from 2. 8 :h 0. 05 to 3.9 :L- O. 6 cm

270

Figure 19. A spontaneous mammary tumor in an old female rat
of the Sprague-Dawley strain.

271

 

 

 

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273

by the end of 4 weeks. By contrast, all doses of ergocornine induced
marked regression of mammary tumors. The average tumor diameter
in all groups treated with ergocornine decreased from Z. 8 :L- 0. 3 to
2. O :I: 0. 2 cm by the end of the 4 week treatment period. Treatment
with ergocryptine had a similar inhibitory effect on the mammary
tumor growth. The average mammary tumor diameter in all groups
treated with ergocryptine decreased from 2. 7 :1: 0.16 to l. 5 :l: O. 2
cm during the 4 week treatment period. There was no obvious rela-
tionship between dose of ergocornine or ergocryptine injected and
mammary tumor inhibition, but this may be due to considerable
variability in initial tumor size and to the small number of rats used
per treatment.

Growth of the mammary tumors during the post-treatment
period is shown in Table 21. Tumors in the control rats showed a
steady increase in size similar to the one noticed during the treatment
period. By contrast, tumor growth in all the ergocornine and ergo-
cryptine treated groups, except the group previously given 0. 05 mg
ergocornine per 100 g body weight, far exceeded that in the controls,
particularly during the first week after cessation of treatment. This
becomes more apparent when percent changes in average tumor
diameter induced by different treatments are calculated (Figures 20
and 21). Since there was considerable variation in the initial tumor

sizes of various groups, the percent changes in average tumor

274

 

 

 

 

 

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275

Effects of ergocornine on growth of spontaneous
mammary tumors in female Sprague-Dawley rats
(0.1, 0. 2 ..... = mg/100 g body weight;

( )= no. of animals).

 

+80

+60

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276

   

Controls (6)

 

 

 

 

 

 

 

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a.“ ....... 05(3)
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TREATMENT with POST-TREATMENT

Ergocornine

Figure 21.

277

Effects of ergocryptine on growth of spontaneous
mammary tumors in female Sprague-Dawley rats
(0.1, 0. 2 . . . . . = mg/100 g body weight;

( ) = no. of animals).

278

    

 

 

 

 

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TREATMENT with POST-TREATMENT
Ergokryptin

 

279

diameter provided a Valuable criterion for comparing the effects of

different treatment on mammary tumor growth.

Conclusions

Ergocornine and ergocryptine both induced significant regres-
sion of spontaneous mammary tumors in old female rats similar to
that previously observed in rats with carcinogen-induced mammary
tumors (Nagasawa and Meites, 1970b; Cassell 51.3.21- , 1971). Both
drugs appeared to be almost equally effective in decreasing size of
the spontaneous mammary tumors, in contrast to the greater effec-
tiveness of ergocornine in suppressing growth of carcinogen-induced
mammary tumors (Cassell _e_tal. , 19 71). Measurement of individual
tumors indicated that the 2 drugs produced greater inhibition of
ETOWth of the smaller than of the larger tumors, and in some cases
elicited complete disappearance of the smaller tumors. A similar
aCtion Of these drugs has been reported in relation to size of the
Carcinogen-induced mammary tumors (Cassell _e_t__a_l. , 19 71). Growth
Of the rnaotnmary tumors resumed quickly after termination of drug

tr
eatment and surpassed that of the control ratS. This is believed

to ref . . . .
lect increased prolactin secretion and action on the tumors

after drug removal.

280

Effects of LSD, Pargyline and Haloiieridol on Carcinogen-Induced
Mammary Tumor Growth in Rats

Obie ctive s

It was reported that the ergot derivative, lysergic acid diethyl-
amide (LSD), and the monoamineoxidase inhibitor, pargyline,
decrease prolactin release (Quadri and Meites, 1971a; Lu and Meites,
1971). The tranquilizing drug, haloperidol, caused a several-fold
increase in serum prolactin levels (Dickerman, S. 3’24. , 1972). It

was of interest to determine the effects of these drugs on growth of

DMBA-induced mammary tumors in rats.

Materials and Methods

Mammary cancers were induced in virgin female Sprague-
Dawley rats (Spartan Research Animals, Inc. , Haslett, Mich. ) by
a single injection of DMBA at the age of 55 days. About 10 weeks
after injection of DMBA when each rat had at least one mammary
tumor 1 cm in diameter, the rats were randomly divided into 4
groups and injected subcutaneously daily for 3 weeks as follows:

Group 1, controls 0. 1 ml saline-corn oil emulsion per 100 g

body weight.

Group 2, 2. 5 mg pargyline in 0. 1 m1 saline per 100 g body

281

weight. Pargyline was obtained from Abbott Laboratories,
North Chicago, Ill.

Group 3, 50 pg haloperidol in 0.1 m1 corn oil per 100 g body
weight. Haloperidol was supplied by McNeil Laboratories,
Inc. , Fort Washington, Pa.

Group 4, 0. 5, l and 2 pg LSD in 0.1 ml saline-corn oil
emulsion per 100 g body weight during the first, second,
and third weeks, respectively.

Body weights, number of tumors per rat and the largest dia-

meter of each tumor were recorded once weekly. Treatment with
the drugs was terminated at the end of 3 weeks and the same measure-

ments were recorded for an additional 3 weeks.

Results

The effects of the different treatments on tumor growth and
body weight are shown in Table 22. Mammary tumors in the control
group showed a steady increase in size and number during the treat-
ment period, reaching 6. 3 :I: O. 8 cm in average tumor diameter and
5 :I: l. 3 in average tumor number per rat by the end of the third week
of treatment. By contrast, pargyline treatment completely inhibited
tumor growth and caused a decrease in average tumor diameter
(4. Z :I: O. 8 cm) and average tumor number (2. 8 :1: 0. 4). There was

no inhibition of tumor growth in the LSD-treated group during the

Ur‘ré—w

Figure 22.

 

‘282

DMBA-induced mammary tumors in female Sprague-
Dawley rats.

283

 

 

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285

first 2 weeks of treatment, but during the third week the dose of LSD
was raised to 2. ug/lOO g body weight and no further increase was
observed in average tumor diameter or average tumor number. In
contrast to the pargyline and LSD treated groups, there was marked
stimulation of tumor growth in the ha10peridol-treated group. Aver-
age tumor diameter increased to 12. 2 :h 3. 2 cm and average tumor
number reached 8. O :L- l. 4 by the third week of treatment. None of
the treatments had any marked effects on body weights by the end of
3 weeks. Although a significant (P < . 05) decrease in average body
weight occurred in the pargyline-treated rats initially, most of the
rats showed a gain in body weight during the last week of treatment.

Tumor growth during the post-treatment period is shown in
Table 23. The control group continued to show a progressive increase
in average tumor diameter and average tumor number similar to that
observed during the treatment period. By contrast, there was a
complete reversal of the growth pattern in the experimental groups,
with a marked increase in both tumor diameter and tumor number
in the rats previously treated with pargyline or LSD, and only
minimal increases in tumor growth in the rats previously treated
with haloperidol.

Figures 23 and 24 show average percent changes in tumor
diameter and tumor number per rat during and after the treatments.

In the control group there was an average gain of 40. 8% in tumor

286

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287

Effects of LSD, pargyline (PR G) and haloperidol (Hal)
on diameter of DMBA-induced mammary tumors in
female Sprague-Dawley rats.

340

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Figure 24.

289

Effects of LSD, pargyline (PRG) and ha10peridol (Hal)
on number of DMBA-induced mammary tumors in
female Sprague-Dawley rats.

290

30

 

 

 

 

 

 

 

 

 

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diameter and 42. 37a in tumor number during the treatment period,
and similar gains during the post-treatment period. Tumors in the
pargyline-treated group showed a loss of 30. 87/0 in tumor diameter
and 26. 7% in tumor number during the treatment period, but gained
132. 4% and 132. 1% respectively during the post-treatment period.
Tumor diameter in the LSD-treated group increased by 27. 1% and
tumor number by 15. 4% during the first 2 weeks of treatment period,
but when the dose of LSD was raised to 2 pg. per 100 g of body weight
during the third week, it resulted in a 7% reduction in tumor diameter
and no further, increase in tumor number. On termination of LSD-
treatment, there was a gain of 108. 8% in tumor diameter and 97. 9%
in tumor number by the end of the 3rd week. Tumor diameter and
tumor number in the haloperidol-treated group increased by 340. 1%
and 255. 4% respectively, during the treatment period, but only by

7. 0% and 18. 3% reSpectively during the post-treatment period.

C onclus ions

 

The present observations provide further evidence for the
importance of prolactin in mammary tumor growth in rats. Pargyline
produced a significant reduction in tumor size and tumor number.

No new tumors appeared during the course of treatment with pargy-
line. It is possible that a more prolonged treatment with this drug

would have led to even a greater decrease in tumor size and number.

292

LSD in doses of 0. 5 and 1 pg per 100 gm body weight was not effec-
tive in reducing mammary tumor growth, but when the dose was
raised to 2 pg per 100 gm body weight there was a decrease in tumor
size and tumor number. In as much as rats are relatively resistant
to the actions of LSD (Jarvik, 1970), it is possible that higher doses
would have been even more effective in reducing tumor growth.
Ha10peridol produced a remarkable increase in tumor size (greater
than 300%) and tumor number (about 260%). Haloperidol was
reported to greatly increase prolactin release (Dickerman, S. e_t_gl. ,
19 72) and to stimulate mammary lobuloveolar growth and induce a
prolonged state of diestrus in rats (Tuchmann-Duplesis and Mercier-
Parot, 1966).

It is of interest that after treatment with these drugs was
terminated, the rats previously given pargyline and LSD showed a
pronounced acceleration in growth of mammary tumors that exceeded
that of the controls. This is believed to reflect the removal of
inhibition to prolactin secretion. On the other hand, the rats form-
erly given ha10peridol showed a marked reduction in tumor growth
that was below that of the controls. This undoubtedly reflects the
removal of the stimulus to prolactin secretion by haloperidol.

Pargyline and haloperidol are believed to influence prolactin
release by altering catecholamine activity in the hypothalamus,

thereby increasing or decreasing release of prolactin release

Z93

inhibiting factor (PIF) into the hypothalamo-pituitary portal vessels
(Lu and Meites, 19 71; Dickerman, S. 9_t__a_1. , 1972). Pargyline is a
monoamine oxidase inhibitor and therefore inhibits metabolism of
catecholamines (Jarvik, 1970), increases hypothalamic PIF levels
and reduces serum prolactin concentration (Lu and Meites, 19 71).
Haloperidol is a butraphenone that blocks the actions of catechol-
amines, reduces hypothalamic PIF levels and raises serum prolactin
values (Dickerman, S. _tgl. , 19 72). LSD is an ergot drug that
reduces serum prolactin concentration (Quadri and Meites, 1971a).
Ergot drugs such as ergocornine have been found to act on the hypo-
thalamus to increase PIF levels, and also act directly on the pituitary
to inhibit prolactin release (Wuttke e_t_a_l. , 1971; Lu e_t-fl. , 19 71).
Androgen-Induced Inhibition of DMBA-Induced

Mammary Tumor Growth in Rats;
Counteraction by Prolactin

 

 

 

Obiectives

 

Prolactin and estrogen are important for development and
growth of spontaneous and carcinogen-induced mammary tumors in
the rat (Meites, 1972, a, b, c). It has been mentioned earlier that
a reduction in prolactin secretion results in decreased mammary
tumor growth whereas an increase in prolactin secretion leads to
enhanced tumor growth. Estrogens have a dual effect on spontaneous

and DMBA-induced mammary tumor growth. Mammary tumor

294

growth is inhibited by ovariectomy but can be restored by treatment
with small doses of estrogen. On the other hand, large doses of
estrOgen produce regression of existing mammary tumors (Meites,

19 72a, b, c) and remission of breast cancer in about 20-25% of
human patients (Hayward, 19 70). Treatment with androgenic
steroids, including a modified testosterone compound, 2-methy1-17-
hydroxy-S-androstan-3-one (drostanolone), produces similar inhibi-
tory effects on mammary tumor growth in rats (Abe £331; , 1962;
Harda Eta}. , 1965) and man (Blackburn, 1962; Goldenburg and Hazes,
1962).

In an earlier study Meites _e_t_a_l_. (1971) reported that inhibition
of carcinogen-induced mammary tumor growth in rats by large doses
of estradiol benzoate could be completely overcome by daily injections
of large doses of prolactin. The present investigation was carried
out to determine whether administration of prolactin could similarly
counteract the inhibitory action of drostanolone propionate (DP) on

carcinogen-induced adenocarcinomas in rats.

Materials and Methods

 

Mammary cancers were induced in Sprague-Dawley (Holtzman
Co. , Madison, Wis.) female rats at the age of 55 days by a single

intravenous injection of DMBA as described earlier. After

295

development of tumors the rats were divided into 4 groups and
injected subcutaneously daily for 3 weeks as follows:

Group 1, controls, 0. 2 ml corn oil per rat.

Group 2, O. 5 mg DP per rat as a suSpension in 0. 2 ml corn
oil. DP was kindly supplied by Dr. R. I. Dorfrnan, Syntax
Research, Palo Alto, Calif.

Group 3, 1 mg ovine prolactin (NIH-P-S-B ovine prolactin,

28 1 p/mg) per rat dissolved in 0. 2 ml of 0. 01 M phosphate
buffer in 0.14 M NaCl, pH 7. 2

Group 4, 0. 5 mg DP and 1 mg ovine prolactin per rat admin-
istered as in groups 2 and 3.

The largest tumor diameter, tumor number per rat and body

weight was recorded weekly during the treatment period. All
measurements were continued for 3 weeks after termination of

treatment.

Results

The effects of the different daily treatments on mammary tumor
growth are given in Table 24. The control rats showed continuous
increments in mean total tumor diameter and mean tumor number,
whereas treatment with DP resulted in a decrease in these two
parameters to levels less than half of the pre-treatment values.

Prolactin alone produced an increase in mean total tumor diameter

296

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and mean tumor number to values more than twice as great as in the
controls. In the rats given both DP and prolactin there were small
but statistically insignificant increases in mean total tumor diameter
and mean tumor number, indicating that the inhibitory effects of DP
were completely overcome by prolactin. None of the treatments
produced any significant changes in body weight.

Mammary tumor growth during the post—treatment period is
shown in Table 2.5. Tumors in the control rats continued to show a
progressive increase in size and number after termination of treat-
ment, whereas the rats previously treated with DP showed a statistic-
ally insignificant gain in mean total tumor diameter and mean tumor
number per rat. Termination of prolactin treatment resulted in a
decrease in both mean total tumor diameter and mean tumor number.
Mammary tumors in the rats previously given both DP and prolactin
showed regression in size and number during the post-treatment
period.

The percent changes in mean total tumor diameter as affected
by different treatments are shown in Figure 25. By the end of the
3 week treatment period, mean total tumor diameter showed a gain
of 48. 4% in the controls, a loss of 65. 6% in the DP-treated rats,

a gain of 169. 8% in the prolactin-treated rats, and a 92. 5% gain in
the rats given both DP and prolactin. Mean tumor number per rat

showed a gain of 66. 5% in the controls, a loss of 60. 6% in the

298

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299

Effects of drostanolone prOpionate, prolactin and
their combination on percent change in mean
diameter of DMBA-induced mammary cancers in
female Sprague-Dawley rats.

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301

DP-treated rats, an increase of 248. 2% in the prolactin treated rats,
and a gain of 98. 0% in the rats given both DP and prolactin (Figure

26).

Conclusions

 

These results show that injections of 1. 0 mg ovine prolactin
daily completely overcame the inhibitory effects of 0. 5 mg DP daily
on growth of DMBA-induced mammary adenocarcinomas in Sprague-
Dawley rats. Treatment with DP alone resulted in a significant
reduction in mammary tumor size and number per rat. This is in
agreement with previously published reports (Abe EELI- , 1962;
Harda e_t_a_l_. , 1965). By contrast, prolactin markedly accelerated
mammary tumor growth as indicated by an increase of more than
150% in mean total tumor diameter and about 250% in average total
number of tumors per rat. This confirms earlier reports on stimula-
tion of mammary tumor growth produced by prolactin increases
induced by hypothalamic lesions, treatment with central acting drugs
or by direct administration of prolactin (Meites, 1972a; Pearson
_‘ggl. , 1969). In the present study prolactin not only completely
blocked the inhibitory effects of DP on mammary tumor growth, but
resulted in small but insignificant increases in average total tumor

diameter and average number of tumors per rat over the control

rats.

Figure 26.

 

302

Effects of drostanolone propionate, prolactin and
their combination on percent change in mean number
of DMBA-induced mammary cancers in female
Sprague-Dawley rats.

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304

Cessation of treatment with DP resulted in resumption of mam-
mary tumor growth. In earlier studies a similar reinitiation of
mammary tumor growth was observed after termination of treatment
with inhibitory doses of estradiol benzoate (Meites gt__a_l_. , 19 72) or
other drugs (Cassell _e_t_al. , 1971; Quadri e_t_a_l_. , 1973a, b, c). On
the other hand the rats previously treated with prolactin showed a
marked diminution of tumor growth during the post-treatment period,
as indicated by decreases in both mean total diameter and mean
number of tumors per rat. A similar reduction of mammary tumor
growth was observed after termination of treatment with drugs that
stimulated mammary tumor growth, such as haloperidol and methyl—
dopa (Quadri 5131. , 19 73a, b). In the present study, the rats which
were previously treated with both DP and prolactin showed decreases
in both tumor size and diameter during the post-treatment period,
presumably due to absence of the stimulating effect of the injected
prolactin.

It is not clear how steroids exert antitumor action on mam-
mary tumor tissue. Meites e_t_a_l_. (19 71) have discounted the pos-
sibility that large doses of estrogen inhibit tumor growth by inhibiting
prolactin secretion. This was based on the observations that large
doses of estradiol benzoate produced several fold increases in
prolactin secretion in ovariectomized rats and in rats bearing DMBA-

induced mammary tumors (Meites _e_til. , 19 72; Chen and Meites,

305

19 70). Meites _e_t_al. (19 71) have hypothesized that large doses of
estrogen interfere with peripheral action of prolactin on mammary
tumor tissue. It is possible that DP and other androgens act in

a similar way since testosterone has not been shown to inhibit
prolactin secretion.

Effects of L-Dopa and Methyldopa on Growth
of DMBA-Induced Mammary Tumors in Rats

Objective 5

 

L-dopa (3(3, 4-dihydroxypheny1)-L-alanine) is the immediate
precursor of catecholamines, and methyldopa (L-3-(3, 4-
dihydroxypheny1)-2-methylalanine) is a false precursor whose pres-
ence results in decreased catecholamine synthesis. L-dOpa has been
reported to significantly lower serum prolactin concentration in the
serum of rats and to increase hypothalamic synthesis and release of
PIF, whereas methy1d0pa was found to produce a decrease in hypo-
thalamic PIF and a rise in serum prolactin values ('Lu and Meites,
1971). It was of interest therefore, to investigate the effects of
these 2 drugs on the growth of carcinogen-induced mammary cancers

in rats.

306

Materials and Methods

Mammary tumors were induced in 55-day old Sprague-Dawley
female rats (Holtzman, Madison, Wisc.) by DMBA. After deve10p-
ment of tumors the rats were divided into 3 groups and treated for
4 weeks as follows:

Group 1, l-dopa was dissolved in 0. 5 N HCl and mixed with

0. 5 N NaOH to a pH of 3. 5-4 and injected subcutaneously
at the rate of 20 mg/rat, twice daily at 9 A. M. and 5 P. M.
during the first week, and 3 times daily at 9 A. M. , 5 P. M.
and 12 P. M. during the subsequent 3 weeks.

Group 2, methyldopa was dissolved in the same manner as

l-dopa and injected subcutaneously at the rate of 20 mg/rat
twice daily at 9 A. M. and 5 P. M.
Group 3, control rats were injected subcutaneously daily at
9 A. M. and 5 P. M. with the injection vehicle only.
Once a week during the treatment period the largest diameter
of each tumor was measured by calipers and the number of tumors

per rat and body weights were recorded. These measurements were

continued for 4 weeks after treatment.

307

Results

The effects of the different treatments on average tumor
diameter and average tumor number per rat are shown in Table 26.
The controls showed a progressive (P < . 01) increase in mean tumor
diameter and mean tumor number, reaching 6. 7 d: 1. 0 cm and 6. 2 +
l. 0 tumors per rat by the end of 4 weeks. Tumor growth in the l-
dopa group was not inhibited during the first week of treatment, but
when the dose was raised to 20 mg/rat injected three times daily
there was complete inhibition of growth in mean tumor diameter
(1. 6 :i: O. 4 cm) and mean tumor number (2. 0 :1: 0. 1) throughout the
remaining treatment period. Methyldopa produced an increase in
both size (10. 6 :t l. 5 cm) and number (10. 6 :1: 1. 3) of tumors as
compared to the control rats.

Figures 27 and 28 show the average percent change in tumor
diameter and tumor number during the treatment period. In the
control group there was a significant (P < . 01) increase in average
tumor diameter of 123. 6% and in average tumor number of 107% by
the end of 4Lweeks. In the l—dopa treated group there was a 51%
decrease in tumor diameter and a 30% reduction in tumor number
after the first week when the rats were given the higher dose of

l-dopa (P < . 05). Methyldopa produced significant (P < . 01)

308

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309

Effects of l-dopa and methyldopa on average
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increases of 164% in average tumor diameter and 171% in average
tumor number.

Table 27 shows the number of tumors in each category at the
end of the 4 week treatment period. The 9 rats in the control group
had a total of 55 actively growing tumors, and the same number of
rats treated with methyldopa had 95 actively growing tumors. By
contrast, the 8 rats treated with l-dopa had a total of only 16 tumors
by the end of 4 weeks and all were regressing; 5 tumors had dis-
appeared completely. At the end of the 4 week post-treatment period,
the rats previously treated with l-dOpa showed an average increase
in tumor diameter of 131% and in tumor number of 127%, whereas
the rats formerly treated with methyldopa exhibited an average
decrease in tumor diameter of 45% and of 61% in tumor number.

The controls gained 103% in tumor diameter and 93% in tumor number

during the post-treatment period.

Conclusions

 

The results of this study demonstrate that 1-dopa effectively
inhibits growth of DMBA-induced mammary cancers in rats. L-dopa
produced regression or disappearance of all tumors and prevented
development of new tumors. L-dopa is believed to act on mammary
tumors by lowering serum prolactin concentration in rats (Meites

_e_t_al. , 19 72; Lu and Meites, 19 71). Other drugs that increase

312

 

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313

catecholamines in the hypothalamus include monoamine oxidase
inhibitors such as iproniazid and pargyline. These also evoke an
increase in hypothalamic PIF and a decrease in release of prolactin
(Lu and Meites, 1971), and inhibit growth of DMBA-induced mam-
mary tumors in rats (Meites e_t a1. , 19 72).

In contrast to 1-dopa, methy1d0pa significantly increased
mammary tumor growth as compared to control rats. Methyldopa
has been shown to elevate serum prolactin levels (Lu and Meites,

19 72). Other drugs that reduce hypothalamic catecholamines and
evoke marked increases in serum prolactin include reserpine,
chlorpromazine and haloperidol (Meites fla_1. , 19 72b). Both
reserpine and haloperidol accelerate growth of DMBA-induced mam-
mary tumors in rats (Meites e_tal. , 19 72).

It is of interest that after cessation of treatment with l -dopa,
growth of the mammary tumors promptly resumed, whereas termina-
tion of treatment with methyldopa resulted in a rapid decline in tumor
growth. Similar resumption of mammary tumor growth in rats has
been observed after termination of treatment with ergot drugs, which
decrease prolactin release and inhibit growth of mammary tumors
(Nagasawa and Meites, 19 70b; Cassell ital. , 19 71; Quadri EEELL ,
1972). This emphasizes the importance of prolactin for mammary

tumor growth in rats.

314

Enhanced Regression of Mammary Tumors in Rats
by Combination of Ergocornine and Ovariectomy
or High Levels of Estrogen

 

Objectives

 

Prolactin and estrogen are the two most important hormones
involved in the development and growth of Spontaneous and carcinogen-
induced mammary tumors in the rat. Alterations in prolactin secre-
tion result in changes in mammary tumor growth. Procedures such
as grafting of extra pituitaries, lesions in the hypothalamus or
injections of haloperidol, reserpine, methyldopa or a mestranol-
norethynodrel combination, all of which increase prolactin secretion,
result in enhanced growth of carcinogen-induced mammary tumors
and increased incidence of spontaneous mammary tumors in rats
(Meites, 19 72a, b, c, d). On the other hand inhibition of prolactin
secretion by hyp0physectomy, or by administration of l-dopa,
iproniazid, pargyline or ergot drugs, result in marked regression
of mammary tumors in rats (Meites gg. , 19 72; Nagasawa and
Meites, 1970b; Quadri 213.51.}: , 1973 a, b).

Estrogen is the other key hormone involved in mammary tumor-
igenesis and growth. Long term treatment with estrogen alone
produces mammary tumors in intact rats, whereas ovariectomy (va)

or administration of large doses of estradiol benzoate (EB) result

315

in regression of mammary tumors in rats (Meites gal—1. , 1972;
Meites, 1972a, b, c) and remission of breast cancer in about 20%
of human patients (Hayward, 19 70).

The purpose of the present investigation was to determine if
treatment with various combinations of Ec, va, and large doses of
EB would result in greater regression of carcinogen—induced mam-

mary tumors in rats than produced by any single treatment alone.

Materials and Methods

 

Mammary tumors were induced in 55-56 day old Sprague-
Dawley female rats (Holtzman, Madison, Wis.) by a single intra-
venous injection of DMBA. Approximately 60 days later, when each
rat had developed at least one mammary tumor of 1 cm in diameter,
the rats were divided into 6 groups and injected subcutaneously daily
for 3 weeks as follows:

Group 1, intact controls, 0. 2 ml of a solution containing 95%
physiological saline (0. 85% NaCl) and 4% ethanol of 70%
strength.

Group 2, 0. 2 mg Ec/lOO g body weight; total dose for each rat
was injected in 0. 2 ml of the saline-ethanol mixture
described above.

Group 3, bilaterally va.

Group 4, 20 pg EB/rat as a suspension in 0. 1 m1 corn oil.

316

Group 5, 0. 2 mg Ec/100 g body weight + va.
Group 6, 0. 2 mg Ec/lOO g body weight + 20 pg EB/rat.
Group 7, 0. 2 mg Ec/lOO g body weight + 20 pg EB/rat + 1 mg
bovine prolactin/rat in physiolOgical saline.
Once a week during the treatment period the largest diameter of
each tumor, number of tumors per rat and body weight was recorded.
These measurements were continued during an additional period of

3 weeks after the termination of treatments.

Results

The effects of different treatments on mammary tumor growth
are shown in Table 28. In the intact control group mammary tumors
showed a steady growth in tumor diameter from 2. 8 :1: O. 6 to 3. 7 :t
0. 7 cm (Mean :1: SE) and in tumor number from 2. 7 :1: 0. 6 to 3. 9 :1:

0. 6 tumors per rat. By contrast, there was a regression of mam-
mary tumors, both in size and number, in all of the experimental
groups. The tumor diameter and tumor number decreased from

4. 2 :1: 1.0 to l. 8 :1: 0. 5 and from 2. 6 :1: 0. 5 to 1. 6 i O. 3 respectively
in the EC group, from 3. 7 :1: O. y to 2.1 :t 0. 7 and from 2.9 :1: 0. 5

to l. 5 :1: O. 4 in the va group, from 5. 0 :1: 0. 6 to 2. 0 :1: 0. 5 and from
3. 0 d: 0. 4 to l. 7 :1: 0. 2 in the EB group. The combination treatments
decreased tumor diameter and tumor number from 3. 9 :1: 0. 5 to 1.1 :h

0. 2 and from 3. 3 :1: O. 4 to 1. 4 :1: 0. 3 respectively in the EC + va

317

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group, and from 3. 7 :1: 0. 7 to 0.8 :1: 0. 2 and from 2.6 :1: 0. 5 to 0.9 :1:
0. 2 in the EC + EB group, and from 3. 4 :1: 1.1 to 1. 0 :1: 0. 6 and from
2. 4 :1: 0. 5 to 0.9 :1: 0. 4 in the EC + EB + prolactin group.

Percent changes in average total tumor diameter induced by
different treatments are shown in Figure 29. The combinations of
EC + va and EC = EB caused reductions of 72. 6% and 78. 970
respectively in average total tumor diameter by the end of 3 weeks.
These reductions are greater than those caused by EC (52. 2%), va
(41. 8%) 0r EB (60. 7%) alone. The combination of EC + EB + prolactin
decreased average tumor diameter by 70. 0%. The combination treat-
ments also caused greater decreases in average number of tumors
per rat, 56. 5% by EC + va, 65. 5% by EC + EB as compared to
'38. 7% by EC, 47. 9% by va and 43. 3% by EB alone by the end of the
treatment period (Figure 30). The combination of EC + EB + pro-
lactin decreased average tumor number per rat by 62. 5%.

The effects of different treatments on the state of individual
tumors are shown in Table 29. No tumors disappeared in the intact
control group during the treatment period, and only 0. 3 tumors per
rat were found to be regressing whereas 3. 1 tumors per rat were
in a state of active growth. By contrast, an overwhelming majority
of tumors had either disappeared or were in a state of regression in
all the experimental groups. However, a total of 6-8 tumors

remained unchanged in size or were actually growing in each of the

‘.. ...

Figure 29 .

319

Effects of ergocornine, ovariectomy and high levels
of estrogen and their combinations on size of
established 7, 12-dimethylbenz(a)anthracene-induced
mammary tumors in female Sprague-Dawley rats.

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three groups which received the single treatments of EC, va, or

EB. By contrast, no tumors were found to be growing in the groups
that received the combination treatments and only one tumor remained
unchanged in size in the EC + EB group.

After cessation of treatments a prompt resumption of tumor
growth was observed in all the experimental groups except in the va
group which showed a continued decline in average tumor size and
tumor number (Table 30). On the other hand, tumors in the control
group continued to show a progressive increase in size and number

in a manner similar to that observed during the treatment period.

C onc lus ion 5

This study shows that treatments with combinations of ergo-
cornine and ovariectomy, or ergocornine and estradiol benzoate,
produced more effective inhibition of mammary tumor growth than
treatment with ergocornine, ovariectomy or estradiol benzoate alone.
The combinations produced not only greater regression in average
tumor diameter and average tumor number of already established
tumors, but caused complete suppression of mammary tumor growth
and prevented development of new tumors. In the rats that received
the single treatments some of the tumors remained unresponsive to
treatments, while some showed growth stasis without any visible

signs of regression. By contrast, all tumors responded to

323

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combination treatments with a decrease in tumor size and number
except one tumor which remained unaffected in the ergocornine +
estradiol benzoate group. Thus the present study confirms earlier
reports that ergocornine, ovariectomy and high doses of estradiol
benzoate individually inhibit mammary tumor growth in rats and
presents evidence that combinations of these treatments result in a
more complete inhibition of mammary tumor growth.

Ergocornine and other ergot derivatives such as ergocryptine
and LSD are believed to suppress carcinogen-induced and spontaneous
mammary tumors by directly inhibiting prolactin secretion by the
pituitary gland and also by acting via the hypothalamus to increase
PIF activity (Meites ga_l_. , 19 72). Ovariectomy inhibits mammary
tumorigenesis by causing a marked decrease in serum prolactin levels
and also by removing the stimulatory action of estrogen on the mam—
mary gland (Meites, 1972c). Large doses of estrogen increase
prolactin secretion but prevent mammary tumor growth by blocking
the peripheral action of prolactin on mammary tumors (Meites gg. ,
1971). We believe that in the present study greater inhibition of
mammary tumor growth was achieved in the ergocornine + ovariectomy
group due to inhibition of prolactin secretion by ergocornine and
further reinforcement of this effect by ovariectomy, which also
denied the tumor tissue the stimulatory action of estrogen. The

combination of ergocornine and estradiol benzoate resulted in greater

325

suppression of mammary tumor growth due to a two-fold effect, one,
by decreasing prolactin secretion by ergocornine which has also been
shown to counteract the stimulatory action of estrogen on prolactin
secretion (Lugg. , 19 71), and two, the large doses ofestrogen
blocked the stimulatory action of prolactin on the mammary tumor
tissue.

In the rats treated with the combination of ergocornine, estra-
diol benzoate and prolactin, the percent decreases in the average
total tumor diameter and average number of tumors per rat were
less than those in the rats treated only with ergocornine and estradiol
benzoate. This indicates that prolactin was able to partially counter-
act the actions of estradiol and compensate for the inhibition of
prolactin release by ergocornine. We believe that a higher dose of
prolactin than used in this experiment would have more completely
counteracted the effects of ergocornine and estradiol benzoate.
Meites gg. (1971) reported complete counteraction by prolactin of
the inhibition of mammary tumor growth by EB alone.

It is interesting to note that after termination of treatment,
tumors in the control group continued to grow at a steady rate
similar to that observed during the treatment period. By contrast,

a marked acceleration of tumor growth was observed in all experi-
mentalgroups except in the ovariectomized group. We have observed

a similar resurgence of tumor growth after termination of

326

treatment with several drugs that inhibit prolactin secretion. This
is believed to be due to removal of inhibition to prolactin secretion.
This effectwas also observed in the ergocornine + estradiol benzoate
group, due to removal of the inhibition of tumor growth by the two

agents.

Figure 30.

327

Effects of ergocornine, ovariectomy and high levels of
estrogen on number of 7, 12, dimethylbenz(a)
anthracene-induced mammary tumors in female
Sprague-Dawley rats.

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YIIATMINY 'OST-YIIA‘IMINY

GENERA L DISCUSSION

The results described in this thesis demonstrate that the ergot
derivatives, LSD and ergocryptine, are potent inhibitors of prolactin
secretion. No studies have been done on the metabolic clearance
rate of ergot derivatives; however it appears that a single dose of
ergocryptine can remain effective for at least 24 hours after it is
administered. It should be possible to further prolong the action of
ergot derivatives by administration in oil or by subcutaneous implanta-
tion in a pellet form. This will be of particular value in view of the
anti-cancer properties of these drugs.

Although there is some evidence that ergot drugs can increase
PIF in the hypothalamus (Wuttke gal. , 1971), they appear to inhibit
prolactin secretion mainly by a direct action on the pituitary as was
demonstrated by their inhibitory effects on the size and secretory
activities of transplanted pituitaries and pituitary tumors (Lu ggl. ,
1971: Quadri 52.11- , 19 72). Results in this thesis show that the
inhibitory action of ergocryptine on the pituitary can be completely
overcome by perphenazine, which increases prolactin secretion by
inhibiting catecholamine activity in the hypothalamus. Thus, it
appears that the central nervous system is the dominant influence in
the control of prolactin secretion and can override the direct effects

of ergot drugs on the pituitary.
329

330

It has been reported earlier that the monoamine oxidase
inhibitors, iproniazid, pargyline and Lilly compound 5641, all of
which inhibit degradation of catecholamines in the hypothalamus,
produced marked reductions in serum prolactin levels (Meites gal. ,
19 72). Results obtained in this thesis indicate that inhibition of the
other catecholamine degradative enzyme, catechol-o-methyl trans-
ferase (COMT), by pyrogallol, can also markedly decrease prolactin
secretion. This reinforces our belief that catecholamines are
intimately involved in the control of prolactin secretion. It will be
interesting to investigate if pyrogallol, like monoamine oxidase
inhibitors, also increases PIF activity in the hypothalamus.

Investigation on the effects of sodium pentobarbital (PB) on GH
secretion revealed that it induces an initial rise followed by a fall in
CH secretion. Several investigators have reported the short-term
effects of PB on plasma GH levels (Howard and Martin, 1971;
Takabashi gal. , 1971; Kokka ga_l_. , 19 72). Results reported in
this thesis indicate that fuller investigation of the effects of PB show
that it has different effects depending on the time interval after its
administration. It was noteworthy that ether, also an anesthetic
and stressful agent, consistently decreased plasma GH levels.
Other stressful stimuli like exercise, cold exposure, insulin-induced
hypoglycemia, noise, vibration, have also been shown to decrease

plasma GH in rats (Garcia gal. , 1968; Schalch and Reichlin, 1968;

331

Takabashi gal. , 19 71). This is in contrast to their stimulating
effects on GH secretion in man and indicate a unique regulation of GH
secretion in the rat. It will be interesting to elucidate the effects of
catecholamines and other central acting drugs on GH secretion.
These observations will be published elsewhere.

Perhaps one of the most interesting findings described in this
thesis is that regular estrous cycles can be restored in old constant
estrous rats by chronic treatment with epinephrine, l-dopa or
iproniazid. These experiments were repeated and the same results
were obtained each time. Epinephrine is a catecholamine, l-dopa
is the immediate precursor of catecholamines and iproniazid is a
monoamine oxidase inhibitor. It is doubtful that systemically-
injected epinephrine can cross the blood—brain barrier (Innes and
Nickerson, 1970), but the latter two drugs reach the brain and
increase catecholamine activity in the hypothalamus. In an earlier
study (Clemens gal. , 1969), it was reported that epinephrine could
reinitiate estrous cycles in old constant estrous rats. At present
no explanation can be offered as to how epinephrine brings about
this action, except to suggest that it may be due to some indirect
action of epinephrine on the CNS or that the blood-brain barrier in
the old rats is less resistant to the entry of epinephrine than younger
rats. It appears reasonable to assume that both l-dopa and iproniazid

reinitiated regular estrous cycles in the old rats by correcting a

332

catecholamine deficiency in the hypothalamus. Frolkis (1966) has
reported that the brains of old rats are deficient in neurotransmitter
substances. It will be interesting to investigate whether the reinitia-
tion of estrous cycles in old constant estrous rats can lead to mating
and conception.

This thesis presents evidence that ergot derivatives are
powerful suppressors of pituitary tumors in rats. Ergocornine not
only markedly inhibited prolactin secretion but also produced exten-
sive degenerative changes in the tumor tissue as indicated by dissolu-
tion of cells and disappearance of nuclei. It was noteworthy that
ergocornine had little or no effect on GH secretion even though tumor
growth was completely suppressed. This indicated that ergocornine
selectively inhibited the growth of prolactin producing acidophils and
did not affect GH cells. It is possible that ergot derivatives might
prove to be of therapeutic importance in treatment of human pituitary
tumors (Peak gal. , 1969).

The thalidomide derivative, CG 603, inhibits prolactin secre-
tion by the normal _'_u_i__s_1_t_il pituitary, but failed to have any effect on
the growth or secretory activity of pituitary tumors. It will be
interesting to investigate whether CG 603 inhibits prolactin secretion
by increasing PIF in the hypothalamus and therefore may not influence
pituitary tumors removed from the hypothalamic influence due to

their distant location.

If?»

333

Although mammosomatotropic pituitary tumors have been
available for many years, little work has been done on the relation-
ship between size and age of the tumors and their secretory activities.
Macleod gal. (1966) and Peak gal (1968) reported that with the
increase in the age of tumors there was a gradual increase in the
body weight and plasma GH levels of the host rats. Ito _e_ta_l. (1972)
found that GH and prolactin concentrations increased in direct rela-
tion to tumor weight and age. The results of this thesis demonstrate
that age of a tumor is not always a good indicator of its secretory
activity. Rats carrying pituitary tumor transplants of exactly the
same age can have extremely wide differences in their blood prolactin
and GH levels. This is due to the fact that all tumor transplants of
the same age do not necessarily grow at the same rate because of
variations in the degree of immunological resistance offered by host
rats. It appears that size, rather than age, of the tumor is a good
indicator of its secretory activity since there was a highly significant
and positive correlation between these two parameters.

Work on mammary tumors produced several new findings. For
the first time it was reported that growth of spontaneous mammary
tumors could be suppressed by treatment with ergot drugs. This
also provided indirect evidence that spontaneous mammary tumors
are dependent on prolactin for growth. It has been reported earlier

that ergot derivatives are potent inhibitors of DMBA-induced

T

.VI'E: v

334

mammary tumors (Nagasawa and Meites, 19 70b; Cassell gal. , 1971).
Results obtained in this thesis indicate that another ergot derivative,
LSD, also shares this property. It was also found that the anti-
cancer property of ergot derivatives can be significantly enhanced by
simultaneous treatment with either large doses of estradiol benzoate
(EB) or ovariectomy. Thus, the combination of ergocornine with
EB or ovariectomy not only produced greater regression of mammary
tumor size and number but also completely prevented development of
new tumors. In the rats which were treated with ergocornine, EB
or ovariectomy alone, some tumors remained unresponsive to treat-
ment while others showed growth stasis without regression. By
contrast, almost all tumors responded to the combination treatments
and regressed or completely disappeared. It is suggested that
ovariectomy enhanced anti-tumor action of ergocornine by decreasing
prolactin secretion and also by removing the stimulatory effects of
estrogen on the tumor tissue, while large doses of EB interfered
with the peripheral actions of prolactin on mammary tumor paren-
chyma. Use of ergot drugs, particularly in combination with EB or
ovariectomy, remains to be tested in human breast cancer.

Evidence was presented that androgen-induced inhibition of
mammary tumor growth can be completely overcome by simultaneous
treatment with prolactin. In view of the lack of information about

the mechanism of action of androgens on the mammary tissues, it is

335

suggested that they interfere with the peripheral action of prolactin.
A similar explanation has been convincingly advanced for inhibition
of mammary tumor growth by large doses of estrogen (Meites gal. ,
1971).

This thesis provides convincing evidence that carcinogen-
induced mammary tumor growth can be profoundly altered by altering
catecholamine activity in the hypothalamus. Thus l-d0pa and pargy-
line, both of which decrease prolactin secretion by increasing hypo-
thalamic catecholamine activity, were found to produce marked sup-
pression of mammary tumor growth. The established tumors either
regressed in size and number or disappeared and deve10pment of new
tumors was completely suppressed. By contrast, drugs which
decrease hypothalamic catecholamine activity and increase prolactin
secretion had exactly the contrary effects on mammary tumor growth.
Thus, treatment with haloperidol, which blocks the actions of
catecholamines, or methyldopa, which decreases catecholamine
synthesis in the hypothalamus, produced marked enhancement of
mammary tumor growth. These results together with the previous
reports from our laboratory on the effect of hypothalamic lesions on
mammary tumors (Meites gal. , 19 72) provide a strong basis for
implicating the central nervous system in mammary tumorigenesis in
rats. It should not be surprising if it is proved in the future that

human breast cancer is also produced due in part to a change in

336

hypothalamic function leading to endocrine imbalance in the body.
Already there are reports that l-dopa and ergocryptine (CB 154)
have beneficial effects on metastatic carcinoma of the human breast.

This is ample reward for cancer research in laboratory animals.

337

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APPENDIX

Research Publications While a Ph. D. Student

Quadri, S. K. and J. Meites. 1971a. LSD-induced decrease in
serum prolactin in rats. Proc. Soc. Exp. Biol. Med. 137:
1242-1243. ...:

11.1.33. ‘.

Quadri, S. K. and Joseph Meites. 1971b. Regression of spontaneous
mammary tumors in rats by ergot drugs. Proc. Soc. Exp.
Biol. Med. 138:999-1001. I

 

 

 

Quadri, S. K. and J. Meites. 1973. Effects of ergocornine and
CG 603 on blood prolactin and GH in rats bearing a pituitary
tumor. Proc. Soc. Exp. Biol. Med. 142:837-841.

 

Quadri, S. K., K. H. Lu and J. Meites. 1972. Ergot-induced
inhibition of pituitary tumor growth in rats. Science. 176:
417-418.

Quadri, S. K., J. L. Clark and J. Meites. 1973a. Effects of LSD,
pargyline and ha10peridol on mammary tumor growth in rats.
Proc. Soc. Exg Biol. Med. 142:22-26.

 

Quadri, S. K., G. S. Kledzik and J. Meites. 1973b. Effects of
' l-dopa and methyldopa on growth of mammary cancers in rats.
Proc. Soc. Exp. Biol. Med. 142:759-761.

Quadri, S. K., G. S. Kledzik and J. Meites. 1973c. Enhanced
regression of mammary cancers in rats by combination of high
estrogen or ovariectomy and ergocornine. Proc. Am. Assn.
Cancer Res. Vol. 14:18. 7

 

 

Quadri, S. K., G. S. Kledzik and J. Meites. 1973d. Reinitiation of
estrous cycles in old constant estrous rats by central-acting
drugs. Neuroendocrinology. 11:248-255.

 

Quadri, S. K., E. Knecht and J. Meites. 1973. Inhibition of
prolactin release in rats by pyrogallol. Fed. Proc. 32:307.

 

381

382

Meites, J. K., H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa and
S. K. Quadri. 1972. Recent sutdies on functions and control
of prolactin secretion in rats. Rec. Prog. Hormone Res.

28:471.

 

Gelato, M., S. K. Quadri and J. Meites. 1972. Inhibition of pro-
lactin resease by a thalidomide related compound (CG 603).
Proc. Soc. Exp. Biol. Med. 140:319.