SOME EFFECTS OF
METHYLAZOXYM ETHANOL ACETATE ON.
THE NEUROENDOCRINOLOGICAL AND
NEUROLOGEGAL SYSTEMS lN THE RAT

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSHY
YOMM‘ MALEVSK!

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Michigan State

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This is to certify that the

thesis entitled

SOME EFFECTS OF METHYLAZOXYMETHANOL ACETATE
ON THE NEUROENDOCRINOLOGICAL AND
NEUROLOGICAL SYSTEMS IN THE RAT

presented by

YORAM MALEVSKI

has been accepted towards fulfillment
of the requirements for

Ph.D degreein Food Science and Human
Nutrition

 

 

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ABSTRACT
SOME EFFECTS OF METHYLAZOXYMETHANOL ACETATE
ON THE NEUROENDOCRINOLOGICAL AND
NEUROLOGICAL SYSTEMS IN THE RAT

BY

Yoram Malevski

Rats intoxicated with methylazoxymethanol acetate
(MAM-Ac) remained sterile for approximately 10 days post
injection. The sterility was associated with alterations
in the ovarian, pituitary and hypothalamus metabolism.

Normally cycling Sprague-Dawley rats at metestrus
were injected with 10 or 12 ul MAM-Ac or saline. The rats
injected with saline were divided into 2 groups: one group
was pair fed to the MAM-Ac injected rats and the second was
a positive control group fed ad libitum.

As a general reSponse to MAM-Ac intoxication the
adrenals underwent hypertrOphy associated with hemorrhages.
Food intake and body weights were reduced sharply, from
about 150 to about 35 g in the first week after injection.
At the same time the rats lost approximately 18% of their
body weight.

Estrous cycles were significantly prolonged (p<0.001)
when compared to the control rats. The first cycle after

the injection was prolonged from 4.5 days to 9.0—9.8 days

Yoram Malevski

in the MAM-Ac injected rats whereas in the pair fed controls
it only increased from 4.5 to 5.4 days. The cycles, however,
became normal in length in about 14 days and reproductive
performance became normal as evidenced by the number of pups
at delivery and at weaning for the treated and control
groups.

Ovarian, uterine and pituitary weights expressed as mg
per 100 g of body weight were similar in MAM-Ac injected
rats and in both control groups when they were categorized
according to the stage of the estrous cycle in which the
rats were sacrificed. However, when categorized according
to the sequence of sacrifice after treatment, it was found
that MAM-Ac injected rats had significantly lighter (p<0.05)
pituitaries (expressed as mg per 100 g of body weight) 4 to
12 days post injection and lighter ovaries and uteruses l to
3 days after the injection.

Histological examination of the ovaries and uteruses
of the MAM-Ac injected rats revealed that for approximately
8 days after the injections the ovaries were filled pri-
marily with well developed corpora lutea and the uteruses
had dense endometriums, narrow lumens and only few glands
were present.

MAM-Ac injected rats sacrificed at diestrus had
significantly less (p<0.05) blood LH and pituitary LH and

prolactin than did the controls. Pituitary LH was lower

Yoram Malevski

in the MAM-Ac injected rats sacrificed at proestrus, and
estrus than in the controls. One to six days after the
injection, MAM-Ac injected rats had significantly lower
(p<0.05) blood LH than controls and normal levels of blood
prolactin. Pituitary LH was lower in MAM-Ac injected rats
1 to.3 days and 8 to 16 days post injection and pituitary
prolactin was lower only 4 to 6 days after the injection
compared to controls. Prolactin inhibiting factor activity
was similar in all groups and luteinizing hormone releasing
factor activity was lower in MAM-Ac injected rats 1 to 6
days post injection, only when compared with the positive
control.

Pair fed controls, although their food intake was
reduced, had normal estrous cycles, normal organ weights
and organ histology, and normal blood and pituitary LH and
prolactin concentrations. Hypothalamic PIF and LHRF
activities were normal as well. Positive controls, fed ad
libitum, were found to be normal in all parameters examined.

The blood-brain barrier is not important in the
prevention of neurological disorders in weanling and mature
rats and mice since implantation with MAM-Ac or cycasin in
the third ventricle of the brain by stereotaxic technique,
did not cause neurological disorders or gross lesions in
the brain.

No brain lesions or neurological disorders were found

in rats injected with cycasin or MAM-Ac during their prenatal

Yoram Malevski

life. No tumors were found approximately 6 months after
cycasin treatments. However, about 2.5% of the MAM-Ac
injected rats had tumors at this time. The surgical pro-
cedure used for the injections of MAM-Ac or cycasin directly
in to the fetus was stressful, since only 30-41% of the
mothers was successful in raising pups and only 8-20% of

the pups were weaned.

SOME EFFECTS OF METHYLAZOXYMETHANOL ACETATE
ON THE NEUROENDOCRINOLOGICAL AND

NEUROLOGICAL SYSTEMS IN THE RAT

BY

Yoram Malevski

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1971

ii

To my parents

ACKNOWLEDGMENTS

I would like to thank many peOple who have helped to
make this dissertation possible. First of all, sincere
thanks to Dr. Modesto G. Yang for his inSpiration, guidance
and advice during the course of this research; and to
Dr. Olaf Mickelsen for his continuous interest and valuable
suggestions.

I am very grateful to the members of my guidance
committee: Dr. G. Borgstrom, my Chairman, Dr. L. E. Dawson
and Dr. D. E. Ullrey for helping me throughout my program.

My sincere thanks are extended to Dr. J. Meites for
making his laboratory available for my hormonal studies and
for his encouragement and interest; to Dr. C. W. Welsch for
his assistance with the stereotaxic experiments; and to
Dr. V. L. Sanger for his help with the histological
examinations.

I would like to eXpress my appreciation to Mr. A.
Sculthorpe and Mr. K. Krawford for their help with the
animal care, and to the students and faculty of the Food
Science and Human Nutrition Department, eSpecially
Mrs. Pat Shier and Mr. Nate Shier, for their interest and

help.

iii

Special thanks are due to my dear wife, Meira, for her
patience, understanding and support throughout my graduate

studies in the United States.

iv

TABLE OF CONTENTS

Page
INTRODUCTION 1
REVIEW OF LITERATURE 4
Plant Characteristics 4
The Uses of Cycads 4
The Isolation and Identification of the Toxic
Substances 6
The Conversion of Cycasin to MAM 8
Carcinogenicity 11
Other Biological Effects 13
Neurotoxicity 16
PART I. THE INFLUENCE OF MAM-Ac ON THE HYPOTHALAMUS
PITUITARY-OVARY AXIS IN THE RAT 19
Objective 19
Review of Literature 20
The Hypothalamus-Pituitary Axis 20
Releasing and Inhibiting Factors of the
Hypothalamus 20
Feedback of Hormones on the Hypothalamo-
Pituitary Axis 21
Prolactin, LH and FSH Levels During the Rat
Estrous Cycle 23
Prolactin, LH and FSH During Pseudopregnancy
in the Rat 24
Materials and Methods 24
Injections of MAM-Ac or Saline 25
Feeding Schedule 25
Determination of the Stage of the Cycle 28
Breeding 29
Fat and Moisture Determination 29
Histological Studies 29
LHRF and PIF Assays 30
Radioimmunoassays (RIA) 31
Spaying and Estrogen Injections 32
Statistical Analysis 32

Results
Post Injection Food Intake and Body Weights
Adrenal Weights and Histology
The Effect of MAM-Ac on the Length of
Estrus Cycle
The Effect of MAM-Ac on the Duration of
Time from Mating to Pregnancy and on
Number of Pups
Post Injection Ovarian, Pituitary and Uterine
Weights and Histology
Post Injection Prolactin and LH Levels
Post Injection PIF and LHRF Activity
MAM-Ac and Estrogen Interaction in Spayed Rats

Discussion

PART II. IMPLANTATION OF CYCASIN AND MAM-Ac IN THE
BRAIN THIRD VENTIRLCLE OF RATS AND MICE

Objective
Review of Literature

The Blood-Brain Barrier
Materials and Methods
Results and Discussion

PART III. PHYSIOLOGICAL AGE AND THE SENSITIVITY OF
RATS TO CYCASIN TOXICITY

Objective

Review of Literature

Materials and Methods
Operational Procedure
Treatments

Results and Discussion

SUMMARY AND CONCLUSIONS

LITERATURE CITED

vi

Page
32
32
34

41

41
47
53
57
63

66

102

Table

10

11

12

LIST OF TABLES

The division of the rats used into 8 trials

Moisture and fat content 4 days after the
injection of MAM-Ac or saline

Adrenal, ovarian and uterine weights 4 and
14 days after the injection of MAM-Ac or
saline

Lengths of estrous cycles (in days) before
and after injection of 10 ul MAM—Ac or
saline

Lengths of estrous cycles (in days) before
and after injection of 12 ul MAM-Ac or
saline to rats of trial 2

Lengths of estrous cycles (in days) before
and after injection of 12 ul MAM-Ac or
saline to rats of trial 6

Duration of time from mating to pregnancy and
number of born and weaned pups in trial 1

Duration of time from mating to pregnancy and
number of born and weaned pups in trial 2

Duration of time from mating to pregnancy and
number of born and weaned pups in trial 3

Duration of time from mating to pregnancy and
number of born and weaned pups in trial 4

Organ weights after the injection of 12 ul
MAM-Ac or saline

Organ weights after the injection of 12 ul
MAM-Ac or saline

vii

Page

26

38

39

44

45

46

48

49

50

51

52

54

Table

13

14

15

16

17

18

19

20

21

22

23

Blood and pituitary prolactin levels after
the injection of 12 ul MAM-Ac or saline

Blood and pituitary LH levels after the
injection of 12 ul MAM-Ac or saline

Blood and pituitary prolactin levels after
the injection of 12 ul MAM-Ac or saline

Blood and pituitary LH levels after the
injection of 12 ul MAM-Ac or saline

PIF and LHRF in the hypothalami of rats
injected with 12 ul MAM-Ac or saline

Body and organ weights of spayed rats
treated with estrogen and MAM-Ac

The division of rats into various experi-
ments of cycasin and MAM-Ac implantations

The division of mice into various experi-
ments of cycasin and MAM-Ac implantations

Summary data on rats undergoing laparatomy
injected with cycasin

Summary data on rats undergoing laparatomy
injected with MAM-Ac

Summary data on rats undergoing laparatomy
injected with saline

viii

Page

58

59

60

61

62

64

80

81

91

92

93

Figure

LIST OF FIGURES

Food consumption after injection of 10 ul

MAM-AC

Food consumption after injection of 12 ul

MAM-AC

Body weight changes
10 ul MAM-Ac and of

Body weight changes
12 ul MAM-Ac and of

Photomicroqraphs of

positive and a pair-

after the injection

of rats injected with
their pair fed controls

of rats injected with
their pair fed controls

adrenal glands from a
fed control rats 4 days
of saline

Photomicrograph of adrenal glands from 2
rats 4 days after the injection of 12 ul

MAM-AC

Photomicrograph of ovaries from representa-
tive rats 3 days after the injection of
12 pl MAM-Ac or saline

Photomicrograph of uteruses from rats 3
days after the injection of 12 ul MAM-Ac

or saline

Body weight changes

after implantation of

2-10 ul MAM-Ac to rats

ix

Page

33

35

36

37

42

43

55

56

83

INTRODUCTION

Poisonous substances occur in a wide variety of plants,
spread all over the world. These substances vary extremely
in their chemical and physiological prOperties. So far
many such substances were recognized, among these, the
best known are: antienzyme and antivitamin factors, metal
binding constituents, hepatotoxins, neurotoxins, hypo-
glycemic and hypothyroid agents, hemolysis inducing factors,
stimulants, depressants and estrogenic factors (Liener,
1969).

Toxic factors are present in many nutritionally
important plants. Legumes in particular have the ability
to inhibit the proteolytic activity of certain enzymes
(Liener and Kakade, 1969). Certain proteins extracted from
kidney beans or soybeans, have the prOperty to agglutinate
red blood cells (Jaffe, 1969). Thiocyanate and thiourea
were found to bear goitrogenic prOperties. Plants from the
genus Brassica are especially rich in such substances
(Van Ethen, 1969).

Some glucosides are very toxic to man and animals.
Cyanogenic glucosides, amygdalin, dhurrin and linamarin, are

mainly found in cassava, sweet potato, yam, maize, sugar

cane, peas, beans and the kernels of several fruits
(Montgomery, 1969). Different glucosides were isolated

from cycads. The active compounds were found to be cycasin
and methylazoxymethanol(MAM) which cause cancer, neurological
disturbances and other toxic effects (Yang and Mickelsen,
1969). Saponins are glucosides too. They have a bitter
taste, cause foaming in aqueous solutions and hemolyze red
blood cells. Soybeans, alfalfa and sugar beets are the most
important plants where saponins are found (Birk, 1969).

Gossypols which are polyphenolic pigments, were
isolated from cotton seeds. Besides their toxic prOperties
they are of economic significance since they are known to
cause dark discoloration of egg yolks (Beradi and
Goldblatt, 1969). Toxic amino acids were isolated from the
seeds of lathyrus species. When introduced to animals,
they were found to cause neurological disorders (Sarma and
Padmanaban, 1969). Hemolytic anemia following the ingestion
of Fava beans or inhalation of its pollen was documented
(Mager et al., 1969).

Some toxic factors present in plants, are of mold
origin. Toxic alimentary aleukia resulted from eating wheat
infested with Fusarium species. Cancer of the liver is
caused by aflatoxins--a group of mycotoxins produced by
Aspergillus flavus on peanuts. Rice and corn are infested
too by molds in particular Penicilhhm species (Friedman and

Shibko, 1969).

The widening gap between world supply of proteins and
the growth of pOpulation requires deve10pment of new protein
resources as well as extension and better utilization of the
existing resources. Plants, which are an important source
of protein are deficient in some essential amino acids and
may contain a wide variety of toxic substances. The
recognition and understanding of such toxic substances is
of most importance for maximum and safe utilization of plant
food stuffs. It is necessary that such substances are
isolated, identified, that the toxic prOperties are demon-
strated in several Species, in both sexes at different
ages. It is necessary that the lethal dose is determined
using different routes of administration, and methods for
qualitative and quantitative determination established.

Cycads which are used as a source of food in some
trOpic and sub-tr0pic areas of the world contain toxic
glucosides. Toxic effects of cycasin have already been
described (Laqueur and Spatz, 1968). However, no work was
done on the effects of MAM-Ac on the neuroendocrinological
system. For this reason research was started to determine
the effects of MAM-Ac on several pituitary hormones
associated with reproduction. Furthermore, research was
initiated to determine the effects of MAM-Ac or cycasin
implanted in the brain of rats or mice or injected directly

into the fetus of rats.

REVIEW OF LITERATURE

REVIEW OF LITERATURE

Plant Characteristics

 

During the Mesozoic Era, cycads or plants from the
Cycadaceae family were widely distributed on earth. Today
only 9 genera and about 100 species survive, mainly in the
tropic and subtr0pic areas of the world (Fosberg, 1964).
The genera, Macrozamia, Cycas, Zamia and Encephalartos
are of particular interest since they have been implicated
in toxic effects on human beings (Whiting et aZ., 1966).

The cycads belong to the gymnOSperms (Thieret, 1958).
They are palm like trees with a cylindric trunk and a
crown of large, stiff, feather-like leaves. In all
genera, the stamenate and pistillate organs are borne on
different plants. In most genera, the pollen and the ovule
bearing organs are borne in strobiles or "cones." The
fruit is a naked seed, the fleshy outer portion of which is
the outer layer of the seed coat, while the hard inner part

is the inner layer of the seed coat (Fosberg, 1964).

The Uses of Cycads

 

Cycads are mainly used as a source of starch which is
derived either from the seeds or from the stems. It is an
important source mainly during hard times, and in areas

where food supplies as a whole are limited. The kernels

4

that are rich in starch are made into flour or starch after
various treatments to remove the poisonous constituents.
The treatment given to the kernels differs in different
Islands of the Pacific. In the Andaman Islands Cycas seeds
are eaten after cooking. In the Key Islands the seeds are
roasted, and in Fiji the seeds are boiled and only the
softened kernels are eaten. In Guam, Cycas kernels are
soaked in water for several days with frequent changes in
water before they are used. On the Comoro Islands the seeds
are eaten after being put through a fermentation process
(Thieret, 1958; Whiting, 1963).

The leaves of the Cycads are used for several purposes.
Young leaves of several Cycas species are cooked and eaten
as a vegetable in some parts of the Philippines and
Indonesia. Leaves are also used as a raw material for
making hats, baskets, mats, fences and brooms. Dried
leaves of Cycas revoluta are used as a decorative material
in many countries. Surface fibers called "palm wool,"
found at the base of the leaves of several species of
Macrozomia and Cycas, are used for stuffing pillows and
mattresses. Fiber from other species is used for making
cloth (Thieret, 1958).

Gum is another product of cycads. Most of it is pro-
duced from the genus Cycas. The gum exudes through wounds
in the megasporophylls, stems and leaves. The gum is used
to treat malignant ulcers, or as an antidote for snake and

insect bites, and as an adhesive agent (Thieret, 1958).

Many diseases are believed to be cured by Cycads. The
stem of Cycas pectinala is used as a hair wash for diseased
hair roots. In Cambodia, the terminal buds of Cycas
circinalis, crushed in rice water, was used in the dressing
of ulcerated wounds, and to treat swollen glands and boils.
The juice of young Cycas leaves is reputedly a good agent
against vomiting blood. The pollen of Cycas rumphii is a
strong narcotic. Cycas seeds, pounded to a paste in
coconut oil, are used for sores and various skin complaints

(Thieret, 1958).

The Isolation and Identification of the Toxic Substances

The first record of the toxicity of Cycas dates back
to 1770. Captain Cook reported illness in his men when
they consumed C. media in Australia (Keegan and MacFarlane,
1963). However, it was not until 1941 that Cooper (1941)
obtained a crystalline substance from the seeds of
Macrozamia spiralis, a species native to Australia, and
called it macrozamin. Macrozamin, was later identified as a
glucoside of primeverose attached by a B-glucosidic link
to its aglycone (Lythgoe and Riggs, 1949). The aglycone
itself was identified a few years later (Langley et aZ.,
1951). The same glucoside was found to be present in
African cycad as well (Riggs, 1954).

A glucoside closely related to macrozamin was obtained
by Nishida et a1. (1955) from Cycas revoluta and by Riggs

(1956) from Cycas circinalis. Nishida named this

glucoside cycasin, which differs from the macrozamin only
in its sugar moiety, which is D-glucose in cycasin.

Once the carcinogenic prOperties of cycasin became
known (Laqueur et aZ., 1963), a need for quantitative
methods for the determination of the glucoside and its
aglycone became obvious. Cycasin can be assayed in several
ways, including a bioassay method by determining growth
rates (Campbell et aZ., 1966), colorimetrically, by using
a chromotropic acid assay (Matsumoto and Strong, 1963), or
by a paper chromatographic modification (Dastur and
Palekar, 1966). Recently, a direct method for cycasin
determination was developed (Wells et al., 1968) using gas
liquid chromatography. After a series of studies (Matsumoto
and Strong, 1963; Kobayashi and Matsumoto, 1964, 1965;
Matsumoto et aZ., 1965) methylazoxymethanol acetate (MAM-Ac)

14

was synthesized. More progress was achieved when C and

3H labelled MAM-Ac was synthesized (Horisberger and
Matsumoto, 1968). Methylazoxymethanol is very unstable.
Acidic hydrolysis yields nitrogen, methanol and formaldehyde,
whereas alkaline hydrolysis yields nitrogen, formic acid,
cyanide and traces of methylamine (Nishida et al., 1956).
MAM has a characteristic ultraviolet absorption Spectrum
with a maximum at 215 mu,which is identical with that of
cycasin (Matsumoto and Strong, 1963). Since MAM is

destroyed at boiling temperature, it is possible to measure

the absorption of a sample at 215 muybefore and after

boiling. The quantity of MAM present can be calculated from
the difference in the reading obtained before and after
boiling (Spatz and Laqueur, 1968a). Using chromotropic

acid reagent, MAM can be determined colorimetrically or by

thin layer chromatography (Spatz and Laqueur, 1968a).

The Conversion of Cycasin to MAM

 

Macrozamin was toxic to guinea pigs when given orally
but not when injected subcutaneously (Lythgoe and Riggs,
1949). Toxic properties of the African cycad
Encephalartos, where macrozamin is present too, were
described (Steyn et al., 1948). Cycasin was toxic for mice
and guinea pigs when given enterically, but not when
injected intraperitoneally (Nishida et al., 1956).

Because of the similarity in the toxicity of cycasin
and macrozamin, it was suggested that the aglycone-
methylazoxymethanol (MAM) which is present in both
glucosides is causing the toxic effects after being
released from the sugar moiety in the digestive system
(Nishida at al., 1956). This theory received support from
an experiment in which all the cycasin injected intraperi-
toneally was recovered in the urine of rats (Kobayashi and
Matsumoto, 1965). Additional support came from reports
indicating that the cells of the small intestine contained
glycosidase activity, using 6-Bromo-2-naphthyl as a

substrate (Dahlquist, 1965a, 1965b).

When high concentrations of cycasin were given to germ-
free rats for a period of 20 days, no toxic effects were
observed. On the other hand when the same amounts were
given to conventional rats, hepatic centrolobular necrosis
and demise resulted within a few days (Laqueur, 1964). Sub-
sequently, the excretion of cycasin from germ-free and
conventional rats was measured. It was found that the germ-
free rats excreted almost all the ingested cycasin, whereas
conventional rats excreted only part, and metabolized the
rest (Spatz et al., 1966). Thus, these data strongly
suggested that bacterial enzymes in the intestinal tract
might be responsible for cycasin hydrolysis. To support
this theory, germ-free rats were monocontaminated with
bacteria having varying B-glucosidase activities, and then
were given cycasin by stomach tube. It was found that the
cycasin was hydrolyzed only in the rats that were contami-
nated with B-glucosidase-containing bacteria. Rats
contaminated with the highest B-glucosidase containing
bacteria had the most severe liver necrosis, and the least
cycasin was excreted in their urine (Spatz et al., 1967b).

A single subcutaneous injection of cycasin to newborn
rats was reported to induce kidney tumors (Magee, 1965).
This experiment was repeated, and the report was confirmed
(Hirono et aZ., 1968a). The length of the postnatal
period in which subcutaneous injection might be toxic to

rats was found to be the first 17 days of life (Spatz,

10

1969). In this study some of the mothers also died of
cycasin toxicity, although they were not injected. This
suggested that the mothers must have licked the urine off
the pups and metabolized the cycasin present in this urine.
It has been shown that MAM can pass through milk (Mickelsen
and Yang, 1966); later, it was proven that cycasin cycles
from the newborn rats to their mother and back to the
newborn (Yang et aZ., 1969).

Further studies showed that the cycling mechanism is
not the only possible explanation for cycasin toxicity in
one day old rats. A subcutaneous injection of cycasin of
one day old germ-free rats, or of artificially fed one-day
old baby rats caused the toxic effects typical of cycasin
(Spatz, 1969). MAM itself was found in the homogenates of
cycasin injected conventional and germ-free rats, thus the
possibility of a direct cycasin effect was ruled out (Spatz,
1969). These observations suggested that enzymatic
hydrolysis of cycasin occurred in the baby rat. It was not
too long later that B-glucosidase activity was found in the
crude skin homogenate from both conventional and germ-free
baby rats, using cycasin and synthetic B-glucoside.‘ as
substrates. The greatest enzymatic activity was found
during the first few days of postnatal life. It then
decreased until the rats were 25 days of age, when it no
longer could be demonstrated (Spatz, 1968; Spatz et aZ.,

1968).

11

Carcinogenicity

 

The first evidence that crude cycad material is
carcinogenic was obtained in studies with rats in 1963
(Laqueur et al., 1963). Since then, tumors were induced
by single and repeated doses of cycasin or MAM in rats
(Laqueur et al., 1963, 1967; Laqueur and Matsumoto, 1966;
Hirono et aZ., 1968a; Yang at al., 1968; Spatz and Laqueur,
1967), guinea pigs (Spatz, 1964b), mice (O'Gara et al.,
1964; Hirono et aZ., 1969b), hamsters (Spatz, 1969), fish
(Stanton, 1966) and monkeys (Kelly and O'Gara, 1965).
However, cattle, horses, swine (Mickelsen et al., 1964)
and chickens (Newberne, 1965; Sanger et al., 1969), fed
unprocessed cycad, showed microsc0pic liver lesions, but
did not develOp tumors.

Cycasin is carcinogenic only after passage through
the digestive tract, whereas MAM is carcinogenic independ-
ent of the route of administration (Laqueur et aZ., 1967;
Laqueur and Matsumoto, 1966). MAM readily passes through
the placenta in pregnant hamsters and rats (Spatz and
Laqueur, 1968a) and also induces tumors by this route
(Spatz and Laqueur, 1967b). MAM induces all the tumors
that cycasin induces; but, in addition, when MAM is given
by the intraperitoneal route, it induces duodenal tumors as
well (Laqueur and Matsumoto, 1966; Laqueur et al., 1967).
Regardless of the cycad material used, the sex, and the age

of the animal, it takes approximately 6 months for the tumors

12

to develop (Spatz and Laqueur, 1967c). The rate of tumor
induction with MAM is 100%, but is variable with cycasin
(Spatz and Laqueur, 1967; Spatz, 1969; Laqueur and Spatz,
1968).

The site of tumor development depends on the duration
of the period of cycad feeding. Hepatomas require a longer
feeding period than renal tumors. Large intestinal neo-
plasmas are least dependent on the duration of exposure to
cycad (Laqueur, 1965). The age of the animals when cycasin
feeding is started determines the frequency of various
kinds of kidney tumors. Nephroblastomas, renal sarcomas
and the interstitial tumors of the kidney were more common
when immature rats were used. However, in mature and young
rats, the same frequency of renal adenomas was noticed
(Laqueur, 1964; Hirono et al., 1968b). No strain differ-
ences to cycad toxicity were described in rats. Similar
tumors were induced in Osborne-Mendle, Sprague-Dawley,
Fischer and Wistar rats (Gusek et al., 1967).

Tumors induced with crude cycad meal, cycasin, or MAM
can be grown in culture (Shimizu, 1969), and are trans-
plantable; but, in some nephroblastomas, the tumors grew
more rapidly when the recipient rat and the primary tumor
host-rat were of the same sex (Hirono et al., 1968b).

More evidence for sex dependence comes from studies in
which male Sprague-Dawley rats had a considerably higher

incidence of intestinal tumors than did the females of the

13

same strain under the same conditions (Laqueur, 1965; Hirono
et al., 1968a). Some sex differences were described in
kidney tumors as well. It was found that only female
Fischer rats had, among the interstitial tumors, smooth
muscle elements forming leiomyomas of substantial size
(Laqueur and Matsumoto, 1966). The cellular composition
during tumor induction by cycasin in the rat was determined.
In the tumor-bearing rats the DNA and RNA content of the
liver was greater than normal. RNA, DNA and nitrogen
concentrations were increased in kidney tumors, but not in

the uninvolved kidney tissue (Hoch-Ligeti et al., 1968).

Other Biological Effects

 

Intoxication of rats with cycasin causes liver
injuries. With mild injury, loss of cytoplasmic baSOphilia
and glycogen are noticed, whereas diffuse centrolobular
hemorrhagic necrosis occurs with severe injury (Laqueur
at al., 1963). Examination with an ultramicrosc0pe
revealed that, as early as 2 hours after intraperitoneal
injections of MAM-Ac, prominent changes occurred. The most
prominent lesions were found in the centrolobular zone,
while the cells in the periportal fields remained unchanged.
The lesions included segregation of nuclear components with
depletion of granular material, hypertrophy of the smooth
endoplasmic reticulum, decrease in the number of ribosomes,
and formation of membrane whorls (Ganote and Rosenthal,

1968).

14

Intoxication of rats with cycasin causes changes in the
normal patterns of protein, lipids and RNA synthesis. After
the introduction of cycasin, the levels of liver glucose-6-
phOSphatase (Spatz, 1964a), liver RNA, total phOSpholipids
(Williams and Laqueur, 1965) and the rate of liver catalase
synthesis (Rechcigl, 1964), were decreased. However, blood
cholinesterase (Orgell and Laqueur, 1964), hemoglobin and
hematocrit (Yang and Mickelsen, 1968) were elevated.
Deaggregation of liver polyribosomesoccurred after the
administration of MAM-Ac intraperitoneally (Shank, 1968),
and a reduction in incorporation of 14C leucine to liver
proteins of rats fed cycasin was noticed (Shank and Magee,
1967).

Cycasin and MAM were proven to be alkylating agents.

In in vitro (Matsumoto and Higa, 1966) and in in viva
(Shank and Magee, 1967) studies, it was found that liver
RNA and DNA were methylated. The purine base guanine was
found to be methylated to form 7-methyl guanine (Matsumoto
and Higa, 1966; Shank and Magee, 1967).

MAM was shown to be a mutagenic agent. MAM added to
the nutrient medium of Drosophila melanogaster caused a mark
rise in a sex-linked recessive lethal mutation (Teas and
Dyson, 1967). MAM caused an increase in the rate of mutation
from histidine requiring mutants to histidine independent
mutants of Salmonella typhimurium (Smith, 1966). When the

effect of MAM on insects was studied, it was found that

15

larvae of Seirovclia echo were able to detoxify MAM by
converting it back to cycasin. This conversion is done
apparently in the gut of the caterpillars by the enzyme
B-glucosidase, which was mentioned before as the cleaving
enzyme for cycasin (Teas, 1967).

Plants, too, are susceptible to cycasin toxicity.
Root-tip cells of onion, which have B-glucosidase activity,
showed chromosomal abberations, when exposed to cycasin
solution. The same radiomimetic effect was produced by
gamma rays (Teas et al., 1965). Compounds like 8-
mercaptoethylamine and 3-amino 1,2,4, triazole were pre-
viously shown to exert a protective effect against x-ray
irradiation (Bacq et aZ., 1953; Feinstein, 1957). When
these compounds were administered before the administration
of lethal doses of cycasin, the rats were partially pro-
tected, they did not die, but they developed tumors later
(Hirono and Laqueur, 1967; Hirono et aZ., 1968a).

MAM has been reported to have teratogenic effects.
When MAM was injected intravenously on the eighth day of
gestation, it produced a variety of malformations like
hydrocephalus, microcephalus and microphthalia in fetuses of
hamsters (Spatz et al., 1967a). In later studies, micro-
encephaly was produced in rats which had been exposed to
MAM on the 14th to 16th days of gestation. Brain tumors
were developed in about 10% of these rats (Spatz and

Laqueur, 1968b). Some intellectual deficit was noticed when

16

a Hebb-Williams maze was used. The rats treated with MAM-Ac
during prenatal life had significantly poorer scores than
their controls (Haddad et al., 1969). In microencephalic
rats, the cerebral hemispheres were diminished in size,

the parietal bone was narrowed, the suture lines were soft
and open at birth, and the total amount of cortical lipids

and proteins were less than in control rats (Spatz, 1969).

Neurotoxicity

 

A correlation between the consumption of Cycas
circinalis and high incidence of amyotrOphic lateral
sclerosis (ALS) on Guam has been suggested. It was found
that this disease is 100 times more prevalent in Guam than
in the United States and Europe (Kurland and Mulder, 1954;
Whiting, 1963). ALS affects the cells of the medulla and
cervical cord. It appears usually between the ages of
40 and 50. Weakness of the hands, difficulty in talking
and swallowing are the first symptoms. As the disease
progresses, the muscles of the hands, shoulders, pelvis,
legs and tongue become atrophied. The course of the
disease is rapidly progressive and always fatal. Mostly,
death occurs within 2-5 years (Cecil and Loeb, 1955).

Cycads were found to be associated with neurological
disorders in cattle. The symptoms are a progressive caudal
weakness of the hind limbs and paralysis (Britton and

Wilson, 1930; Mason and Whiting, 1968). Cattle that have

17

eaten leaves of Macrozamia Zucida and Bowenia serrulata
developed paralysis. When CNS and the spinal cord were
examined, it was found that demyelination of the spinal
columns occurred. The fasciculus gracilis and the dorsal
Spinocerebellar tracts were mainly affected (Mason and
Whiting, 1966; Hall and McGavin, 1968).

No neurological lesions were found in the brains or
spinal cords of over 500 rats fed crude cycad meal (Laqueur
et aZ., 1963). No apparent neurological disorders were
noticed when cycasin or MAM-Ac were implanted in the third
ventricles of female rats (Malevski et aZ., 1971). However,
a single subcutaneous injection of MAM into newborn mice
of the strain C57BL/6 caused hind leg paralysis in 80% of
the injected mice (Hirono and Shibuya, 1967). Lesions in
the brain of such mice were described (Hirono et aZ., 1969a).

Neurological disorders can also be induced by a toxic
amino acid isolated from C. circinalis and other plants
(Sarma and Padmanaban, 1969). a-amino-B-methylamino-
prOpionic acid isolated from the seeds of Cycas circinalis
when injected into chicks resulted in a loss of the ability
to extend legs and to stand erect. The birds bent their
heads down and tended to "run backwards" (Vega and Bell,
1967). A related compound, a-amino-B-oxaly1aminopropionic
acid is known to cause lathyrism. This amino acid was not

found in cycads (Bell, 1964).

18

It seems that cycasin and MAM exert several types of
mechanisms of action. The similarities between the chemical
structure and the biological effects of dimethylnitrosamine
(DMN) and cycasin were noted (Laqueur et aZ., 1963). Both
MAM and DMN were found to produce similar subcellular
changes when compared at equivalent doses and times (Ganote
and Rosenthal, 1968). Both were found to methylate liver
RNA and to cause polyribosomal deaggregation under the same
experimental conditions (Shank and Magee, 1967; Shank,
1968). It was suggested that both substances may have a
common metabolic pathway, forming diazomethane, which is
known for its alkylating and carcinogenic properties
(Miller, 1964). The pathway by which MAM and DMN are
converted to the active form was theorized (Miller and
Miller, 1965). Cycasin is deglucosylated to form MAM,
which is unstable and releases formaldehyde to form
azoxymethane, which in turn loses water to form
diazomethane.

This postulated mode of action can explain the carcino-
genic and alkylating properties of cycasin. However, no
mechanism of action has been suggested, so far, for the
neurotoxic, teratogenic, mutagenic and radiomimetic effects

of cycasin or MAM.

PART I
THE INFLUENCE OF MAM-AC ON THE HYPOTHALAMUS-

PITUITARY-OVARY AXIS IN THE RAT

PART I
THE INFLUENCE OF MAM-AC ON THE HYPOTHALAMUS-

PITUITARY-OVARY AXIS IN THE RAT

Objective

 

Pregnant rats fed cycad meal had fewer pups at birth
when compared to control rats (Yang, unpublished results).
In this experiment all rats were fed ad libitum and since
cycasin reduces food intake (Campbell et aZ., 1966) the
control rats consumed more than the cycasin fed rats. This
made the interpretation of the results difficult since it
is not possible to separate the effects of food intake
reduction from cycasin consumption.

The only indication available in the literature
suggesting alteration in gonadotrOpin metabolism comes
from studies by Spatz and Laqueur (1967c). They found that
when crude cycad meal was fed during pregnancy to rats,
the mammary glands were less developed, only small amounts
of milk escaped from incised mammary gland tissue and little
or no milk was found in the stomachs of the young.

For these reasons, we decided to determine the effects
of MAM-Ac, which is easily hydrolyzed by blood or tissue

esterases to MAM,on the hypothalamus-pituitary-ovary axis.

19

20

Review of Literature

The Hypothalamus-Pituitary Axis

No direct neural connections between the hypothalamus
and the anterior pituitary are evident. The only connection
between them is by the hypophyseal portal blood vessels
originating in the stalk and in the median eminence (POpa
and Fielding, 1930 a, b).

The first clear postulation that the hypothalamus
exerts a control over the anterior pituitary gland was made
by Harris (1937) in which he induced ovulation in rabbits
by electrical stimulation of the hypothalamus. Hinsey (1937)
was the first to postulate that this control was by neuro-
humoral substances produced in the hypothalamus and transferred
to the anterior pituitary gland by the portal vessels. This
postulate received further support when Harris and Jacobson
(1952) transplanted pituitary tissue to the median eminence
of hypOphysectomized rats. After the tranSplantation the

rats showed normal reproductive functions.

Releasing and Inhibiting_Factors of the Hypothalamus

The synthesis and release of the anterior pituitary
hormones is induced by hypothalamic hormones or releasing
factors. The only exception is prolactin which is inhibited
by hypothalamic hormone. Direct evidence of the presence
of a follicle stimulating hormone-releasing factor (FSH-RF)

in rat hypothalamus in an in vitro system was reported by

21

Mittler and Meites (1964) and in an in vivo system by
Igarashi and McCann (1964). Luteinizing hormone releasing
factor (LHRF) was first shown to exist by McCann et al.
(1960) in an in vitro system, and later in an in vivo
system (McCann, 1962).

Prolactin inhibiting factor (PIF) was first shown to
exist in the rat by Meites et al. (1961). Other Species
including sheep, swine and cattle were also Shown to
contain PIF in the hypothalamus (Talwalker et aZ., 1963;

Schally et aZ., 1965).

Feedback of Hormones on the Hypothalamo-Pituitary Axis

 

Ovarian hormones may induce or inhibit the secretion of
the gonadotrOpinS from the pituitary. They exert their
positive or negative feedback on the hypothalamus and/or
the pituitary gland. It has not yet been established
whether the hypothalamus or the pituitary is the major site
for the feedback. It appears that estrogen and progesterone
may inhibit FSH and LH secretion by a direct action on the
anterior pituitary (Bogdanove, 1963; Ramirez et aZ., 1964).
However, both steroid hormones induce the secretion of the
gonadotrOpins by a direct action on the hypothalamus
(Everett, 1964; McCann et aZ., 1968; Davidson, 1969, 1970).

Anterior pituitary hormones themselves act to control
their own secretion. These hormones act directly on the

hypothalamus to inhibit the secretion of the releasing

22

factors. Several pituitary hormones were found in the
median eminence (Guillemin et aZ., 1962; Schally et aZ.,
1962; Johnson and Nelson, 1966). These hormones reach the
median eminence area either via the general circulation or
by a reverse blood flow from the pituitary (Torok, 1964).

The first evidence that LH may be controlled in part
by itself was presented by Sawyer and Kawakami (1959). Later,
when small amounts of LH were placed into the medianeminence
of rats, a reduction in pituitary and serum LH levels
occurred. The decline in LH secretion was associated with
irregular vaginal cycling, few corpora lutea and normal
follicular growth. These effects were Specific to LH
implantation (David et aZ., 1966; Corbin and Cohen, 1966).
Recently, Terasawa and Sawyer (1968), reported that LH
administration activated the arcuate region, but depressed
the preoptic area. These two areas of the hypothalamus
are postulated to control LH secretion. Similar types of
experiments were conducted to Show that FSH too may inhibit
its own secretion through an action on the hypothalamus.

The first evidence that prolactin may control its own
secretion came from studies by Sgouris and Meites (1953).
They found decreased pituitary prolactin following injection
of prolactin in rats. These results were confirmed by Sinha
and Tucker (1968). Pituitary glands transplanted under-
neath the kidney capsule of ovariectomized rats secreted

mainly prolactin and only small amounts of the other

23

pituitary hormones (Welsch et aZ., 1968). Implantation of
prolactin into the median eminence of rats resulted in a
decrease in synthesis and release of prolactin which was
associated with regression of the mammary glands and fewer

corpora lutea (Clemens and Meites, 1968).

Prolactin, LH and FSH Levels During the Rat Estrous Cycle

 

The availability of radioimmunoassays for LH, FSH and
prolactin made it possible to measure these hormones during
the estrous cycle of rats. The pattern of gonadotropin
secretion during the estrous cycle was Shown by Gay et al.
(1970). Serum LH rose at least 50 fold during the afternoon
of proestrus but remained low at all other stages of the
cycle. LH content of the pituitary was highest on the day
of diestrus and it decreased late during proestrus (Monroe
et aZ., 1969). Serum FSH rose only 4-5 fold late in the
afternoon of diestrus. It remained low at all other stages
of the cycle (Gay et aZ., 1970). Serum prolactin increased
about 10 fold at late proestrus, but it remained high on the
morning of estrus (Gay et aZ., 1970; Amenomori et aZ.,
1970). The anterior pituitaries of estrous rats contain more
prolactin than the anterior pituitaries of diestrous rats.
Anterior pituitaries of proestrous and estrous rats released
more prolactin when incubated in viva and had less PIF when

compared to diestrous rats (Sar and Meites, 1967).

24

The half life for LH, FSH and prolactin on the after-
noon of diestrus was measured by the rate of their
disappearance from the blood. The half life for LH is
20 min, FSH is 110 min and prolactin is 13 min (Gay et aZ.,

1970).

Prolactin, LH and FSH During Pseud0pregnancy in the Rat

 

It appears that prolactin is the main pituitary hormone
responsible for luteal maintenance and progesterone
secretion in the rat. Fajer and Barraclough (1967)
reported that prolactin and not LH could stimulate progesterone
secretion by rat ovaries in an in viva system. Kwa and
Verhofstad (1967) reported a rise in serum prolactin levels
for the first 3 days of pseudOpregnancy and thereafter it
declined. Similar results were obtained in pregnant rats
(Amenomori et aZ., 1970). The question of luteal control

and maintenance is still unresolved.

Materials and Methods

 

Two hundred ninety-two, 3-5 month old female Sprague-
Dawley rats (Spartan Research Animals, Haslett, Mich.)
averaging 250 g were used in these experiments. All rats
were fed a grain ration (Campbell et aZ., 1966) and were
allowed free access to water. The rats were housed in a
laboratory where the temperature was kept near 24°C

throughout the experiments. Lighting was regulated to

25

provide 12 hours of illumination (8 a.m. to 8 p.m.) and 12
hours of darkness (8 p.m. to 8 a.m.) each day. All rats
were individually housed in suspended wire cages except
during the time of mating which was done in Trials 1-4.
The rats were divided into 8 different trials as indicated

in Table l.

Injections of MAM-Ac or Saline

 

Prior to the injection of MAM-Ac or saline all rats
except those in trials 5 and 8 were examined daily by
vaginal lavage to determine the stage of the estrous cycle.
The rats were allowed to cycle 2 to 3 times and then only
normally cycling rats (4-5 day cycles) at metestrus were
injected subcutaneously with MAM-Ac or saline near the left
rump area with a micro-syringe. The amounts injected are

Shown in Table l.

Feeding Schedule

 

Feeding cycad material causes a reduction in food
intake and as a result a reduction in body weight (Campbell
et aZ., 1966). This is why in all trials but trial 1, one
control group was pair fed to the MAM-Ac injected group.
The individual rats of the MAM-Ac group were injected 1-5
days ahead of their pair fed controls. This allowed time
for measuring the food intake of the MAM-Ac injected rats
before the injection of the pair fed mates with saline at

metestrus. In addition, in trials 5, 6 and 7 a positive

26

 

 

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unmews we» camuno oe .mmw
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cowumsflemxm HMUHmoHoumHm
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sesame one .unmamz seen

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.woon
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ousumHOE can mcHEHmumU 08 NH 0 o o m

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.H HMHHB CH mm mEmm NH AH OH I N

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Hogan: cam macho msouumw
mo eumcma .uemams seen

 

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Aumu\qne ~.oze A.ozv A.ozv
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cause: we ucsoea oauzmz cam seam m>fluflmom
mmomusm mm com: mumm .oz HMHHB

 

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27

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Hmoflmoaoumss we» no cows
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mamcmncm .msuwus on» NO

 

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:3 Tozr 105 705
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causes mo undead omIzaz pom ufimm o>fluemom
mmomnsm mm com: mumm .oz awake

 

AU.uGOUV H manna

28

control fed ad Zibitum was used. The only exception is
the control rats in trial 1, their food intake was arbitrarily
reduced approximately 40%.

Food intake was measured daily for the MAM—Ac injected
rats until the rats consumed pre-injection amounts for at
least 3 days, then the rats were fed ad libitum. Each pair
fed control rat was given only as much food as its experi-
mental mate had consumed during the preceding 24 hours.

Body weights were recorded in trials 1, 2, 3, 6 and 8
from the day of injection up to the day when the rats had
regained their initial body weight. Body weight gain was
recorded for the positive controls for the same period of

time.

Determination of the Stage of the Cycle

Vaginal smears were taken daily between 9 and 11 a.m.
as described previously (Evans and Bishop, 1922). The
smears which were taken with an eye drOpper were put on a
glass plate, dried and stained in 1% methylene blue solution
for 60 min. The type of cells in the smear was determined
using a light microsc0pe. In trials 1, 2 and 3 smear
examination was continued until the rats became pregnant and

in trials 6 and 8 unitl the rats were sacrificed.

29

Breeding

Breeding was done in trials 1 through 4. Two weeks
after the injection of MAM-Ac or saline, 3 to 4 female rats
from the same group were put together with a proven male.
Vaginal smears were continued daily until Sperms were
found. This was considered to be the first day of pregnancy
and at this stage the pregnant rats were transferred to
maternity cages. The time needed for each rat to become
pregnant and the number of the born and weaned pups were

recorded.

Fat and Moisture Determination

 

The rats from trial 5 were used for the determination
of fat and moisture. In previous trials it was found that
on the 4th day after the injection of MAM-Ac the rats were
at their lowest body weight. 0n the fifth day after inject
tion the rats started to recover, so it was decided to
sacrifice the rats on the 4th day after the injection. The
rats were overetherized and the contents of their G.I. tract
removed and weighed. Moisture and fat were determined as
described previously (Mickelsen and Andersen, 1959). The
G.I.content weight was .subtracted from the body weight for

the calculations of body fat and moisture.

Histological Studies

 

Rats from trials 6 and 7 were used for histological

studies. Ovaries and uteruses were obtained from the rats of

3O

trial 6 and adrenals and pituitaries from the rats of trial
7. The ovaries and the uteruses were obtained 1 to 6 days,
every day and 8 to 16 days, every second day after the
injection of MAM-Ac or saline. Adrenals and pituitaries were
obtained 4 and 14 days after the injections. All tissues
were fixed in 10% buffered formalin. Hematoxylin and eosin
stains were used and the examination of the Slides was done

with a light microscope.

LHRF and PIF Assays

 

The rats from trial 6 were used for this study. The
hypothalami were stored individually in 0.1 N HCl (0.2 ml/
hypothalamus) at -20° until the time of assay. Just prior
to the assay the hypothalami were pooled, homogenized, and
centrifuged at 12,000 g for 40 min at 4°. The hypothalami
from the rats killed 1-6 and 8-16 days after the injection
in each group were pooled. The acidic supernatants were
placed in protein free medium 199 (Difco Labs, Detroit,
Mich.) and the pH was adjusted to 7.4 by adding 0.1 N NaOH.
LHRF and PIF were measured by incubating the pituitary
halves from normal,mature male rats with pooled hypothalamic
extracts. The pituitaries were preincubated for 30 min and
the medium was discarded. Fresh medium containing the
neutralized acid extract was then added and the incubation
was terminated 2 hr later. The pituitary tissue was weighed
and the medium stored at -20° until assayed for LH and

prolactin (Piacsek and Meites, 1966; Kragt and Meites, 1967).

31

Radioimmunoassays (RIA)

 

Blood serum, pituitaries and hypothalamic extracts
from the rats of trial 6 were used for RIA. After injections
5 rats randomly selected from each group were sacrificed by
decapitation every day from 1 to 6 days and every second day
from days 8 to 16. The blood was collected from the neck
and the serum separated by centrifugation was kept frozen
at -20° until the time of assay. The anterior pituitaries
were individually weighed, placed in 0.01 M phOSphate
buffer in 0.14 M NaCl (pH = 7), homogenized with a cell
disruptor and stored at -20° until assayed. The
hypothalami were treated as indicated previously.

LH and prolactin from individual serum samples,
pituitaries and incubated medium were measured at 2-3 dose
levels by RIA for rat prolactin (Niswender et aZ., 1969),
and rat LH (Monroe et aZ., 1968). Purified rat prolactin
and LH were used as references. The purified prolactin
preparation was H-lO-lO-B (23 I.U/mg) obtained from Dr. S.
Ellis, National Aeronautics & Space Administration (NASA),
Ames Research Center, Moffett, Calif. 94035; and the LH
preparation was NIAMD-Rat-LH-RP-l (Biological potency equal
to 0.03 x NIH-LH-Sl), obtained from NIH, Rat Pituitary

Hormone Program, Bethesda, Maryland.

32

Spaying and Estrogen Injections

 

In trial 8, mature rats were anesthetized with sodium
pentobarbital (50 mg/kg body weight) and both ovaries were
removed. During the recovery period of 2 weeks, vaginal
smears were taken. The experimental group was then injected
daily with estrogen benzoate (15 pg/kg body weight) in 0.1 ml
corn oil. The control group was injected with equivalent
volumes of corn oil. Vaginal smears were continued and
after 2 weeks all rats were injected with 10 or 12 p1 of
MAM-Ac. A week later all rats were killed, their uteruses,

adrenals and thymuses removed and weighed.

Statistical Analysis

 

A "t" test (Sokal and Rohlf, 1969) was used to determine
the significance of differences between groups. Differences

are considered significant only when p is less than 0.05.

Results

Post Injection Food Intake and Body_WeightS

 

After the injection of 10 p1 MAM-Ac to rats in trial 1
and 3, daily food intake decreased Sharply from approximately
20 to 3 9. From the third day on food consumption started
to increase until it became normal again a week later
(Figure 1). Injection of 12 p1 MAM-Ac to rats in trials 2
and 6 caused a decrease in food consumption from a pre-

injection intake of 20 g/day to 1-2 g/day by the second

33

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a
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34

day after the injection. Thereafter the intake increased
until it returned to normal on the 9th day (Figure 2).

The reduction in food intake was accompanied by a
reduction in body weight. The rats injected with 10 p1
MAM—Ac lost 12-15% of their body weight by the third day
after the injection. Thereafter they gained weight until
they restored all loses by the tenth day. Pair fed controls
of trial 3 lost identical amounts of body weight as did the
MAM-Ac injected rats (Figure 3). Rats injected with 12 pl
MAM-Ac lost approximately 18% body weight by the fourth day
after the injection, and thereafter gained weight until
they restored all the weight lost by the twelfth day. Pair
fed controls lost the same amount of body weight, whereas
the positive controls continued to gain weight throughout
the entire period of the experiment (Figure 4). The
positive control rats had Significantly (p<0.05) more body
fat than the MAM-Ac injected rats and the pair fed controls.
The same amount of fat was found in the MAM-Ac injected and

the pair fed control rats (Table 2).

Adrenal Weights and Histology

 

Four days after the injection of MAM-Ac the weight of
the adrenals increased (p<0.1) in comparison with either the
pair fed rats or the positive controls. Fourteen days after
the injection, the adrenals of the MAM-Ac injected rats
remained heavier (p<0.05), compared with the pair fed controls

(Table 3).

35

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(6) exequr poog

Body weight (% of initial)

 

36

 

 

 

 

 

110 .
I Partial Food Trial 1
Restriction
100%.,
90’
. controls &-——4L————a
MAM-Ac injected a e —_ao
1: I;
110’
Pair-Fed Trial 3
0////e
//
100' A a ;
90
4 8

Figure 3.

Post injection (days)

Body weight changes of rats injected with 10 p1

37

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Hams» mo can odIzdz H: NH cufi3 pouooncfl mpmn mo momamso Ssmflo3 zoom

Amwmpv coauooncfi umom

NH m c

 

(J

OJ

’)

allilfillllq maouucoo com uflmm

o (0

1 u A

 

‘\

m Adana

 

e cmuomnfi canvas

 

91 AV

.9 Honucoo 0>Huflmom

N HMHHB

 

I

I4

 

 

.e musmflm

om

Apog

-.00H

bran

moa

(TQTQTUT 3° %) 4H

om

00H

moa

38

.Amo.ovmv maonpcoo o>fluamom Scum uqonommwp maucmowmwcmwmo

.ooa Scum ousumHoE w cam pom w mo coauomanSm hm

 

 

 

n
.ucmuaoo .H.O I .u3 moomm
on so
m.onm.hN w.ouN.HH m.onm.oo h.¢ Hm.N¢N n.me.HmN m owfluwmow
on so
o.HHm.m~ oo.HHS.m m.HHS.HS ~.m Hm.mo~ m.kfim.amm o ewmeflmw
. I . . I . . I . . I . . I . emuomflcH
m H+m hN om o+m m o N+m mm m Ha+o voN e n+5 NmN o odIzmz
Awe Awe Awe Ame Ame A.ozv
nusoucou ucoucou ucoucou movemenomm coauooflcH moonw
:flmuoum cam nmm umm onsumfioz pm .u3 zoom um .u3 moom mom mumm msonw
a.m.m H cmmzv
oSHHMm no o¢I2¢z mo cofluooncfl we» wanna mace v unopcoo new new ousumfloz .N magma

39

Table 3. Adrenal, ovarian and uterine weights 4 and 14
days after the injection of MAM-Ac or
saline (Mean i S.E.).

 

 

Group Day after Rats per Body wt. at Body wt. at

Injection Group Injection Sacrifice
(No.) (No.) (9) (g)

MAM-Ac

Injected 4 6 240.7:3.1 203.7i5.9

,Pair Fed

Control 4 3 240.7:3.8 208.0:2.3

P°Sltlve 4 2 246.0i6.9 255.5:4.5

Control

MAM-Ac

Injected 14 5 239.6:6.6 245.2:9.7

Pair-Fed

Control 14 3 235.0:4.9 250.7:8.1

P°Sltlve 14 2 231.5:9.5 260.0:9.o

Control

 

 

40

Table 3 (Cont'd).

 

 

Adrenal Wt. Ovarian Wt. Uterine Wt.
(mg/100 g B.W.) (mg/100 g B.W.) (mg/100 g B.W.)
50.8:4.3a 45.6il.8b 123.9: 3.4°
39.6:3.8 56.4:3.9 169.5:14.0
32.0:o.o 46.5:5.0 165.8: 9.8
36.2:2.5b 36.4:1.7 142.8112.6
24.0:o.9 4o.1:5.2 187.3i31.3
26.0il.5 41.3:2.2 155.3:16.0

aSignificantly different from both controls (p<0.1).
bSignificantly different from pair fed controls (p<0.05).

CSignificantly different from both controls (p<0.05).

41

Histological examination revealed that the adrenals
of both control groups were free of lesions and the
division of the different zones was Sharp and clear
(Figure 5a, b). However, the adrenals from MAM-Ac injected
rats had hemorrhages and the different zones were not

divided clearly (Figure 6).

The Effect of MAM-Ac on the Length of Estrous Cycle

 

Injection of 10 pl MAM-Ac caused prolongation in the
length of the estrous cycle from 4.2 to 7.6 days. This
prolongation was not statistically significant (Table 4).
The injection of 12 p1 MAM-Ac caused significant prolon-
gation (p<0.001) in the length of the first cycle after
the injection mainly by an increase in the diestrus stage.
In trial 2, this cycle was prolonged from 4.2 days to
9.0 days in the MAM-Ac injected group and from 4.5 to 5.4
days in the pair fed controls (Table 5). In trial 6 the
rats injected with MAM-Ac had a cycle of 9.8 days whereas
the pair fed rats and positive controls had cycles of 5.2

and 4.4 days respectively (Table 6).

The Effect of MAM-Ac on the Duration of Time from Mating to
Pregnancy and on the Number of Pups

The length of time from the day of mating to the first
day of pregnancy was not affected by the injection of MAM-
Ac. The number of pups in the uteruses of MAM-Ac injected

rats and the controls of trial 1 was found to be the same.

 

 

  

Figure 5. Photomicrographs of adrenal glands from a
positive (a) and a pair fed (b) control rats
4 days after the injection of saline. In both,
note clear division of zones and the absence
of pathological symptoms. (hematoxylin and
eosin stains x 40).

43

 

 

 

Figure 6. Photomicrograph of adrenal glands from 2 rats
days after the injection of 12 pl MAM-Ac.
Note unclear division of zones with an
extension of the zona fasciculata and presence
of hemorrhages (hematoxylin and eosin stain, x
40).

44

.usmoHMHcmHm no: we mcomflummEoo OBH mam cowsumn mucoummmflp anew

 

 

 

 

O. I O O .I I. C II C O I O C II C C II C HOHucOU

o o+o w m o+m v m o+m v m o+m v o o+o v o o+o w m o>fluwmom

. I . . I . . I . . I . . I . . I . emuomncH

m o+m v H H+m w m H+m h H o+N v N o+e v N o+m v oH odIzmz
lmsmac Immune Imamec lmamec Immune lmsmcc l.ozc
m cacao onHowo H odomu m mflmwu N macho H odomu macho

numcoa oHuNo cofluomncflIumom camcoa macaw coauomflsflIoum mom mumm macaw

m.1.m.w H amaze ocflamm so oaIS<z as ea
mo cowuoomsfl Hmumm cam muomon Amwmp SHV mmaomo msouumm mo mnumcoq .e magma

45

.AHo.ovmv pomGOHoum >HucmoHMHcmHm mH oHomo mean

 

 

 

 

on so

~.ofim.e m.osm.m m.oHe.m m.owm.e ~.onm.e ~.ofie.e SH owe Wemw

. I . . I . . I . . I . . I . . I . emuomncH

N o+¢ v n H+m 5 Mo H+o m H o+N v N o+m e N o+m w HH O¢I2¢S
lmsmec lmsmev lmsmec .msmuc lmsmec Images l.ozc
IMIoHomo m oHONU H oHomo m oHomu N mHowo H oHomo msowo

numsoH oHomo SOHuomwsHIumom numde oHUNo GOHDOMfisHImHm Mom mumm macho

.A.m.m H smozv N HmHHu mo mama on mcHHmm Ho odIzmz H: NH
mo mSOHuomnsH Hopmm can owommn Amamp ch mmHoho msouumm mo mnumsoq .m mHnme

46

.AHoo.ovmv pomSOHowm mHuSSOHMHcmHm mH oHoho one

Q

.mcoHuooncH can Houmm mmmp mH
can «H .NH .OH .m .o .m .v .m .N .H um poHHHx >HEopcmH ouo3 msoum mom much o>Hmm

 

 

 

 

on so
N.owm.v H.0Hv.v H.ow¢.¢ m.owh.v H.0Hv.w H.0Hv.v mm oMHuwmow
on so
o.owo.v v.oao.m N.OHN.m H.oav.v H.owm.v H.owm.v mm own WHSW
n
I I I I ~.oam.e am.OHm.m ~.oav.v m.ow~.e H.o“v.s we emwmwamm
Immune lmsmcv lmsmec Amsmec lmsmev Amsmec 1.ozc
m oHomo ,N oHomu IH oHomu m oHowo N oHomo H oHomU macaw
numsoH oHumo SOHuoothIumom ammcoH mHuNo GOHuoondHIon mom mumm msouw
.1.m.m H amaze m amass mo mums on meaamm no omIzaz a: ma
mo mGOHuommcH Houmm cam muomon Amwmp SHV moHoao msowumo mo maumsoq .o oHnme

47

No difference was found between the MAM-Ac injected rats
and their controls in trials 2, 3 and 4 in the number born

and number of pups weaned (Tables 7-10).

Post Injection Ovarian, Pituitary and Uterine Weights and

 

Histology

 

Organ. weights can be analyzed statistically according
to the stage of the cycle in which the rats were sacrificed
or to the post injection time of sacrifice. The weights
of the ovaries, uteruses and pituitaries per 100 g B.W.
of the MAM-Ac injected rats and of both controls were
comparable when similar organs were compared on either
proestrus or diestrus. MAM-Ac injected rats at metestrus
had significantly lighter pituitaries per 100 g body weight
and at estrus had significantly lighter ovaries (p<0.05).
Furthermore, MAM-Ac injected rats at diestrus had signifi-
cantly lighter ovaries, pituitaries and uteruses when
expressed on an absolute basis (Table 11).

It appears that the pituitaries per 100 g B.W. of the
MAM-Ac injected rats were lighter (p<0.05) than the pitui-
taries of the pair fed controls, 4 to 12 days after the
injection. They were significantly lighter than both
controls when expressed on absolute bases. MAM-Ac injected
rats had Significantly lighter ovaries (p<0.05) per 100 g
B.W. compared to the positive controls, 1-3 days after the

injection. Four'to Six days after the injection they were

.hno>HHo© nmoa mmam nHonn HHS one mnmn nonuoE m man mo mn

.momaumm mo noneaa can oaHEnouop
on moamammnm mo mwmp mH on mH aomznon woOHMHnUMm onm3 wnmn namamonm anew

 

48

 

. I . . I . I I I I . I . conconaH
m H+> m N H+m h nm b H+m m m oaIzaz
I I I I I I I I mo.Hno.o o.wno.o N Honnaoo
. I . . I . cmuomflan
I I I I I I I I am o+o H N H+o m e GNIZSS
1.0zc l.ozv 1.023 l.ozc lmsmoc 1.021
manmus Noamamonm
pmamm3 anon aH on maHnmE maono
mmam mmam maHno>HHop mnmm momauom Eonm mEHB nom mumm maonw

 

.H.m.m H amozv H HMHnB aH mmam poamoz
cam anon mo nonfiaa can moamamonm on maHumE Eonm mEHn mo aoHumnan .n mHnMB

49

.azoax noa mH HmHnn mHnn aH mHonnaoo can aH ooamenomnom
o>Hn05ponmon noom can no omamo one .xaHmHMmonpmn can m can >nm>HHop
maHnaU poHU N .namamonm uoa mnm3 m “no>HHop uoa UHw umnn oH can mo #50

n

.xaHmHmmonphn can m can wnm>HHop nmma mmam on» mum v .mno>HHo©
maHnap Uon N .namamonm noa mp3 H ”no>HHoU noa pHp umnn oH on» no naom

 

 

 

. . I . Honuaou
o o o o no N H+o s 0H pom nHmm
. I . empomflan
N v m w mH m H+N m HH oaIzaz
1.ozc l.ozc 1.021 1.023 1.021 lmnmec 1.ozc
moamammnm
onEmm mHmz Hmnoa anom maHnm>HHoU on maHumE maono
pmamm3 mmam mmam mumm Eonm mEHB nom mumm macnw

 

.A.m.m n amaze N HMHnB aH mmam coamm3
cam anon mo nonfisa paw woamamonm on maHnmE eonm oEHn mo aOHumnan .m oHnma

50

.mmam onu HHS one nan cono>HHop paooom mnn .enw>HHop maHnaU pmHo umn oaoo

.mmam on» HHm mum nan .ponm>HHop mumn N .uamamonm uoa onoB mnmn ose

n

.uamonHamHm noa mH masonm onn aoozuon ooaonoNMHc onem

 

 

 

. on ao
S.onm.v, s.onm.s ~.HHm.m e.one.nn onn e.nnm.m mn cw“ wnmw
. I . . I . . I . . I . . I . nanomnan
cm o+m e we o+m v cm H+H m Mm o+m OH nm cm H+o o NH oaIZNE
n.ozc n.ozv n.ozv n.ozc n.ozv Amnmoc n.ozv
hoamammnm
oHMEomI mez Hmuoe Emp\anon maHno>HHoa on maHnmz @aonw
pmamm3 mmam mmam mumm Eonm mEHe nmm mnmm maono

 

.A.m.m n amozv m HMHne aH
mmam poamm3 cam anon mo nonfiaa cam Noamamonm on maHnmE Eonm oEHu mo aOHnmnaa .m oHnme

51

.mno>HHo© nonmo mean on» HHo ono non oneo

.wno>HHop nonwo mean onn HHo one caooom onn .naoamonm nom noa pHU non oao

n

.naMOHMHamHm noa mH masonm on» aoo3non ooaonomme oneo

 

 

 

 

O I O O | O O I O O ll O O l O HOH“ ”H00
m o+ m m m H+ w v H H+ m m m H+ m m ow H N+ o m m comInHmm
. I . . I . . I . . I . . I . eouooflcn
m H+om v m N+oo m m H+om oH N H+oh OH nm N H+om n m oaIzmz

.H.ozc 1.ozc 1.ozc 1.ozc 1.023 lmnmec 1.021

evaoamonm

onEom oHoz Houoe Eoo\anom maHno>HHon on maHnoz msonw
poaoos mmam mmam muom Bonn oEHe nom mnom muonw

.H.m.m n aoozv v HoHne aH mesa
poaooS paw anon mo nonEaa cam hoaoamonm on maHnoE Eonm oEHn mo aOHnonaQ .OH oHnme

52

.mHmon onaHOmno a0 commonmxo aonz Hmo.ovmv mHonuaoo nnon Bonn uaonoMMHp mHuaooHMHamHm

.nmo.ovac nonueoo com unmm we» Scum econommne snuamonncmnmo

n

.Amo.ovmc mnonucoo soon scum uaonmmmno anuamonmncmnmm

 

 

 

N. mm+m. mvN N.mnN.Hv 0N.onmh.v m.¢nm.th HH mannmm

N. om+m. NNN N.Nnv.mm 5H.onvv.v m.vnH.OhN mH mannmoonm

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m.m +v. NNH m.NnH.¢m mH.onmm.¢ m.vnm.th mH manumonoz Honuaoo
o>HnHmom

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m.NmnN.NhN m.NHH.mm vv.on¢H.¢ o.mnm.h¢N NH mannmoonm

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com nHom

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oaIzaz

1.3Im m coaxmec ; .m m oon\mec 1.3.m m oon\mec Ame 1.021
.n3 oaHnouD .nz aoHno>o anonHanHm .uS hoom oHomo

nOHnonaa mnom mo omonm muonw.
.H.m.m n aoozv oaHHom no ONIZNS H: NH mo aOHnoonaH onn nonmm mnanoS aomno .HH oHnme

53

significantly lighter when compared with both controls and
14 to 16 days post injection they were lighter when compared
with the pair fed control. The uteruses of the MAM-Ac
injected rats compared with both controls per 100 g B.W.
were Significantly lighter (p<0.05) l to 3 days after

the injection. Expressed on an absolute basis the uteruses
of the MAM-Ac injected rats were lighter (p<0.05) 4 to 6
days after the injections (Table 12).

Histological examination of the ovaries revealed that
for a period of approximately 8 days after the injection,
MAM-Ac injected rats had ovaries filled primarily with well
developed corpora lutea. Pair fed and positive controls
had normal ovaries containing both corpora lutea and
follicles (Figure 7).

The uteruses of the MAM-Ac injected rats at the same
period of time were unstimulated. The endometrium was
dense, the lumen was narrow and only a few glands were
present. The pair fed and positive controls had normally

stimulated uteruses (Figure 8).

Post Injection Prolactin and LH Levels

Pair fed and positive controls sacrificed at the same
stage of the estrous cycle had the same concentrations of
prolactin and LH in the blood and pituitaries. The only
exception was the pair fed rats at diestrus which had

significantly more (p<0.05) prolactin and LH in their

54

.mHmon ouaHOmno ao commonmxo aon3 Hmo.ovmv mHonuaoo nnon Eonm

.Amo.ovmv Honnaoo o>HuHmom onn Bonn

.nmo.ovmv Honuaoo pomInHom onu Eonm

.nmo.ovmv mHonuaoo nuon sonm

ucmnmmmne snuemonmncmnm

econommne snucmonmnamnm

w

uaonoMMHp MHnaoonHamHmo

n

ucononmno mHuamoHanmem

 

 

 

m.anm.HNN a.mn a.mm mH.onNo.m o.wnh.veN 0H mHIwH
m.mNnN.hmH m.Nn h.o¢ oN.onmm.¢ «.wnv.th mH NHIm
>.m nN.NmH o.Nn o.Hm ¢H.onNm.¢ v.qnm.th mH m IH
H.mmnm.HmH H.Nn 0.0m NN.onN¢.¢ w.vno.NhN mH m IH Honuaou
o>HnHmom
N.mHnm.va N.Nn m.m¢ hm.onmo.m m.mnm.¢mN 0H mHIwH
m.mmnm.hmH m.HH m.mm mH.onmm.v m.vnm.HmN mH NHIm
m.omnm.th a.mn a.mm hN.onmh.v H.mnm.mmN mH m Iw
m.mHnH.mmH H.Nn m.o¢ mm.onon.v o.wno.meN mH m IH Honnaoo
comInHom
o.mmno.NNN o.mn>.Nm pH.onN.¢ m.vno.hoN m mHIvH
p.mmnm.mNN h.Nnm.mm a mN.onmm.m m.hno.mmN m NHIm
p m.h MN.HNH m.Nnm.mv a OH.onmo.v m.mnN.mNN vH m Ie
o m.e no.HHH N.mnm.vm ON.onom.¢ m.mnH.mmN mH m IH wouoonaH
odlzfiz
1.3.m m oon\mec 1.3.m m oon\mec 1.3.m m oon\mec lac 1.ozc lmnmev
.nz oaHnonD .nz aoHno>o anonHanHm aOHnoonaH
nOHnonad .nz epom mnom nmom moonw
.n.m.m H aoozv oaHHSm no oaIzaS H: NH mo aoHnoonaH onn nonmo muanoS aomno .NH oHnoe

Figure 7.

55

 

    

AA
a) Photomicrograph of ovary from a representative
rat 3 days after the injection of 12 pl MAM-Ac.
Note dominance of well developed corpora lutea.
b) Photomicrograph of ovary from a pair fed
control rat.
c) Photomicrograph of ovary from a positive control
rat.
In both controls note the presence of both corpora
lutea and mature follicles (hematoxylin and eosin
stains, x 40).

Figure 8.

56

 

a) Photomicrograph of uterus from a rat 3 days
after the injection of 12 p1 MAM-Ac. Note the
lack of estrogen stimulation.

b) Photomicrograph of uterus from a pair-fed
control rat.

c) Photomicrograph of uterus from a positive
control rat.

In both controls note the presence of estrogen

stimulation (hematoxylin and eosin stains, x

200).

57

pituitaries. MAM-Ac injected rats had significantly less
(p<0.05) prolactin in the pituitary during diestrus and
Significantly less (p<0.05) LH during diestrus, proestrus
and estrus. Blood LH was Significantly lower (p<0.05)
during metestrus and diestrus (Tables 13-14).

Pair fed and positive controls, 1 to 16 days after the
injections, had the same levels of blood and pituitary
prolactin except for the period of 8 to 12 days after the
injection when pair fed rats had Significantly more (p<0.05)
pituitary prolactin. Pair fed controls had significantly
more (p<0.05) blood and pituitary LH than the positive
controls during the period of 1-3 days after the injection.
However, 4 to 6 days post injection they contained more LH
in the blood and 14 to 16 days post injection they had
significantly more LH in the pituitary. MAM-Ac injected
rats had significantly less (p<0.05) pituitary prolactin
4 to 6 days post injection and less blood prolactin 14 to
16 days post injection. Pituitary LH was Significantly
lower (p<0.05) 1 to 3 days and 8 to 16 days post injection,
while blood LH was lower 1 to 6 and 14 to 16 days after

the injection (Tables 15-16).

Post Injection PIF and LHRF Activity

 

The activity of PIF appeared to be highest in the MAM-
Ac injected rats, but statistically it was not significantly
different from the activity in both control groups. Both

controls had the same PIF activity (Table 17).

58

I 1‘... . P

.Hmo.ovmv mHonnaoo o>HuHmom Eonm uaonoMMHp mHuaMOHMHamHm

 

v

.Amo.ovmv mHonnaoo nnon Eonm uaonoHMHp mHnaooHMHamHmo

.Hmo.ovmv mHonuaoo noMInHom Eonm naonoMMHc mHnaoonHamHm

n

.mIOHIOHIm aOHuonomonm ooaonomomo

 

 

 

N.¢HHN.NOH m.onh.h m. h H¢.vv HH mannmm
m.v “p.mm m.onm.m m. mmnh.HNH mH mannmoonm
m.v nm.m¢ N.onm.¢ N. H nm.mH wH manumoHo Honnaoo
o.m ne.Hh m.ono. m. H Hm.0N mH mannmouoz o>HnHmom
m.man.mNH m.on¢. m. m no.5m NH manumm
0.5 HN.m¢ m.onm. m. mvum.mmH vH mannmoonm

pm.m n¢.H> pm.onH.m w. H “a.mN mH manumoHo Honuaoo
m.e no.55 m.onm.w m. H Hm.vN NH mannmonoz com nHom
m.Man.HHH m.ono.m m. mHHm.hm m mannmm
m.m nm.mv m.onH.v N. omnm.hHH m manumoonm

o~.m He.am am.ona.m m. n HH.- mm manummna emuommcn
a.mNHm.hm m.onm.m o. v no.mH m mannmouoz oaIzdz
thouHanHm HenouHanHm

noHnonao\mnv nOHnouao mE\mnv Hfianom HE\maV H.ozv

oHomU
oaHuomHonm mnom mo omonm msonu
.H.m.m n amozv oaHHom no oaIzmz

H: NH «0 aoHnoonaH onn noumo mHo>oH aHnooHonm anonHanHm can UOOHm

.MH oHnme

59

.Amo.ovmv mHonnaoo o>HnHmom Eonm naonoHMHU wHuaMOHMHamHm

c

.Hmo.ovmv mHonnaoo poMInHom Eonm naonoHMHp mHnaooHMHamHmo

.Hmo.ovmv mHonuaoo nnon Eonm uaonomch mHuaooHMHamHm

n

.HImmImHIHMMIQzéHz I aOHumnomonm ooaonomomo

 

 

H.mNno.omm N.Nnm.Hm m.v «a.mv HH mannmm
a.mqnw.0Nm N.mn>.Nm N.¢H Hm.¢m mH mannmoonm
«.menN.hmm m.mnm.em N.N “a.mN mH mannmoHn Honnaou
H.Nmno.vom o.Nnm.mm m.m Ho.Hv mH mannmonoz o>HnHmom
m.wmnH.N¢m m.mnm.Hm >.N “N.NN NH mannmm
p.mmno.mm> o.mn¢.om N.NNNHm.Hom «H manumoonm

UH.NNHH.om> a.mnh.mm m.m nm.wm mH manumoHn Honuaou
p.mmnm.on m.mum.mv m.m HN.om mH mannmonoz poMInHmm
m.mhnv.mmm H.mHH.mv N.N ne.mN m mannmm
m.mmnv.mmv 0N.¢nm.mv o.womno.mhv m mannmoonm

oo.mmnv.oom on.mnm.om nm.N HH.HH mN mannmoHQ ponoonaH
N.NNHH.hHm w.vnm.ev no.o no.v m manumonoz oaIzaz
lmnmunsunm Annmunsunm

nOHnonao\m:v noHnonao mE\mnv Heanom HE\maV H.ozv

oHohu
own mumm mo omoum maonw

 

.n.m.m n aoozv oaHHmm
no onlzaz H: NH mo aOHnoonaH onu noumo mHo>oH 3H wnmuHanHm can poon

.vH oHnme

60

.Hmo.ovmv mHonuaoo o>HuHmom Eonm uaonoHMHU mHuaMOHMHamHmU

.lmo.ovmc mnonuqoo neon Scum unonmmmnu snuamonmnamnmo

.nmo.ovmv mHonuaoo pomInHom Eonm uaonoMMHp hHuamoHMHamHmn

.mIOHIOHIm aoHnmnomonm ooaonomomo

 

 

N.¢th.v0H m.onm.h o.m no.nm OH oHIvH
o.m nm.om m.onH.m ¢.hNno.wm mH NHIm
m.e “N.Nm m.onm.¢ H.N nm.NN mH m Iw Honuaou
N.NHnm.mm h.on¢.v N.NNHH.H> mH m IH o>HuHmom
N.NNn>.mNH v.Hnm.m m.mmnm.mOH 0H wHIvH
N.NHnm.mm om.onN.h a.mNHm.mh mH _ NHIm
N.0an.Oh m.onH.m m.thm.¢¢ mH m Ie Honnaou
N.oHnm.mw m.onm.v m.hNnm.>m mH m IH pom nHom
H.NHHN.00H o.th.m nN.m nN.NN m mHIHH
H.0Hnm.mm o.HHm.m v.NmHN.om m NHIm
om.m Ho.~m om.onm.m m.e Hm.om an a Is emuomflan
nN.m HH.mm N.onm.m N.mHHh.Hv mH m IH o<Izmz
nmnmunsuna Announsunm
nOHnouao\mnv nOHnouao mE\m:v Haanom HE\mav A.ozv Ammocv
aOHnoonaH
oaHHUMHonm mnom nmom maonu

 

.n.m.m n aoozv oaHHmm no oaIzaz
H: NH mo aOHnoonaH onn nonmo mHo>oH aHnooHonm mnmnHanHm can poon .mH oHnoe

61

.Hm0.0va mHonnaoo o>HnHmom Bonn uaonoHMHp mHnaoonHamHm

w

.Hm0.0vmv mHonnaoo oowInHom Eonm uaonoMMHn NHuamoHMHamHmo

.Hm0.0vmv mHonuaoo nnon Eonm naonoNMHp NHuaMOHwHamHm

n

.HINMImHInomIQZNHz aOHumnomonm ooaonomomm

 

 

N.Omnm.¢Nm m.mno.ow m.OH “H.Nh OH OHIOH

m.mmnh.mHO O.mHO.Nm N.NH “a.mm mH NHIO

0.0mnm.mNm H.¢Hv.mw h.m H0.0N mH m Iv Honnaoo

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pm.mmnm.~mv ©>.mnN.vm 0.00NHO.NON OH OHIvH

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m.vbnm.Nmm 0.0HO.mm U¢.bm Hm.mmH mH O Iw Honnaou
pm.mmnv.mom pm.mno.mo ch.mmmnm.omm mH m IH pom nHom

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a.mmnN.mom v.hnO.mm m.HHNuh.mmN m NHIm

a.menm.ome o.ono.m¢ oe.oH “a.mm an o IS oouoonan
o>.mmnh.mmm 0m.wnm.mv nb.N nm.m mH m IH oaIzaz

AmnouHauHm HenmnHauHm
nOHnonao\mnv nOHnonao mE\m:V HEanom HE\maV H.ozv Ammopv

aOHnoomaH
own mnom umom maono

 

.1.m.m H noose ocHHMm

no omIzaz H: NH «0 conuooHoH one noumm mHo>oH on mnmpnouno can ooon

.wH oHnme

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.HImmImHIuomIadez aOHumnomonm ooaonowomn

.mIOHIOHIm aOHnonmmonm ooaonomomo

 

62

 

O0.0nvO.m HmnvhN ON OHIO

. I . I Honuaoo

em O+Om O >m+th ON O IH o>HuHmom
N0.0nmN.m OOHOON ON OHIO

. I . I Honuaou

we O+OO m mm+ONN om O IH pom nHmm
N0.0neH.O mHnmNN 5H OHIO

. I . I oouooncn

0mm O+OH m NN+OOH ON O IH oaIzmz

oaOHa

om.onam.m Hmnome II II snounounm

AmnonHanHm AmnonHanHm
nOHnonam mE\mnv noHnonao mE\oav H.ozv Anemov
.poz aH OomooHom o.poz aH OomooHom poHoom aOHnoonaH
n 3H aHnomHonm HsoHonuomem Hmom muonw

 

.H.m.m n aoozv oaHHmm
no oaIzez n: NH suns oouoonan moon mo namnmeuoann on» an amen one mam .an onoma

63

LHRF was found to be Significantly lower (p<0.05) in
the MAM-Ac injected rats, but only when compared with the
positive controls. Both controls had the same LHRF

activity (Table 17).

MAM-Ac and Estrogen Interaction in Spayed Rats

 

Spaying rats caused a constant presence of leukocytes
in their vaginal smears. The injection of 0.2 pg estrogen
daily caused the disappearance of these cells and the
appearance of cornified cells typical of the estrous stage.
The injection of MAM-Ac into Spayed rats treated with
estrogen resulted in the reappearance of leukocytes which
lasted 3 days and then started to decrease. The injection
of MAM-Ac into rats treated with corn oil without estrogen
resulted in the appearance of leukocytes during the entire
period.

After the rats were sacrificed, it was found that the
injection of 10 or 12 pl MAM-Ac and estrogen resulted in
rats with significantly heavier uteruses (p<0.05) in
comparison to rats injected only with MAM-Ac. Rats injected
with MAM-Ac and estrogen also had significantly heavier
adrenals and Significantly lighter thymuses (p<0.05) when

compared with rats injected only with MAM-Ac (Table 18).

64

Table 18. Body and organ weights of spayed rats treated
with estrogen and MAM-Ac (Mean 1 S.E.).

 

 

Treatment Rats Initial Body wt. at Body wt. at
Per Body Wt. Injection of Sacrifice
Group MAM-Ac
(No.) (9) (g) (9)

Corn oil

+ 10 pl 3 225.3:6.3 291.0:9.5 287.7:13.4

MAM-Ac

Corn oil

+ 12 pl 3 236.3:0.3 303.3:4.5 294.0:12.5

MAM-Ac

Estrogen in

Corn oil + 6 229.5:4.3 255.8:4.5 243.0:5.8

10 p1 MAM-Ac

Estrogen in

Corn oil + 3 224.0:3.8 249.3i3.7 227.3:6.4

12 pl MAM-Ac

 

 

65

Table 18 (Cont'd.)

 

 

Uterine Wt. Adrenal Wt. Thymus Wt.
(mg/100 g B.W.) (mg/100 g B.W.) (mg/100 g B.W.)
38.7: 1.73' b 23.8:1.5a 196.6:23.2b
30.5: 1.2C 17.1:1.2C 193.4:11.1C
142.2: 9.4 28.4:1.4d 139.4: 9.3
147.3:11.5 46.2:5.2 112.0:3o.9

 

aSignificant difference (p<0.05)
rats injected with 10 and 12 pl

bSignificant difference (p<0.05)

injected, corn oil and estrogen

cSignificant difference (p<0.05)
injected, corn oil and estrogen

dSignificant difference (p<0.05)

between
MAM-Ac.

between
treated

between
treated

between

10 and 12 pl MAM-Ac injected rats.

corn oil treated

10 p1 MAM-Ac
rats.

12 pl MAMFAC
rats.

estrogen treated,

66

Discussion

 

It has been well established in rats that starvation or
reduction of food intake prolongs the estrous cycle and
decreases reproductive performance (Mulinos et aZ., 1939).
This is the reason why positive control and pair fed control
groups were used. Conducting the experiments this way
enabled the distinction between the effects of reduction in
food intake and reduction in food intake plus MAM-Ac
injection. Pair fed and MAM-Ac injected rats consuming
equal amounts of food were losing the same amount of body
weight (Figures 1-4). The Similar reduction in body weight
was accompanied by the same reduction in body fat (Table 2).
The positive control rats had significantly more body fat.
The amounts found in this experiment are in good agreement
with the amounts found in mature female rats (Schemmel et aZ.,
1969).

Dose response was achieved in the reduction in food
intake and body weight when different amounts of MAM-Ac
were injected. The higher the level of MAM-Ac injected
the more severe was the reduction in food intake and body
weight (Figures 1-4).

Yang and Mickelsen (1968) described some of the symptoms
appearing after cycad feeding. The rats were pale and had
an anemic appearance, very yellow urine, sometimes with
blood, and were cold to the touch. The same symptoms

appeared when MAM-Ac was injected.

67

Trauma, infection, intoxication, hemorrhagic shock,
cold exposure and other stressful stimuli produced an
"alarm reaction." The adrenals became hypertrOphic and
atrophy of the lymphoid tissues and lymphopenia occurred
(Selye, 1936). In these studies, the stress caused
hypertrophy of the adrenal and the atrophy of the thymus
(Tables 3 and 18). The weights of the adrenals of the
pair fed and positive controls were not increased. Signs
of general response to stress were noticed too in the
histological examination of the adrenals of the MAM-Ac
injected rats. In addition to hypertrcphy some hemorrhages
were noticed. The histological appearance of the adrenals
from both controls was normal (Figures 5-6).

MAM-Ac intoxication did not last long. Food intake
was restored to normal within a week (Figure l and 2) and
body weight within 2 weeks after the injection (Figure 3 and
4). Another proof for the Short lasting effects of MAM-Ac
intoxication comes from the reproductive performance
studies (Tables 7-10). Immediately after the injection of
MAM-Ac the rats did not become pregnant. However, after
the rats had recovered it took the MAM-Ac injected rats and
the pair fed controls the same period of time to become
pregnant. In addition in both, control and MAM-Ac injected
groups,the number of born and weaned pups was the same.

The normal length of an estrous cycle in rats is 4-5

days (Evans and Bishop, 1922). Reduction of food intake is

68

known to prolong the cycle (Mulinos et aZ., 1939). In these
experiments the reduction in food intake alone was not
severe enough to significantly prolong the cycle. The pair
fed controls in trials 2 and 6 did not have significantly
pnflonged cycles after the reduction in food intake (Tables
5 and 6). However, reduction in food intake plus MAM-Ac
injection caused a significant prolongation in the first
cycle after the injection. Since this cycle was completed
within 9.0 to 9.8 days and Since the intoxication period
was the same, it is expected that the following cycles
would be as long as they were in the pair fed controls.
The more MAM-Ac that was injected, the more prolonged was
the cycle. Rats injected with 10 p1 MAM-Ac prolonged their
cycles less than rats injected with 12 p1 (Tables 4, 5 and
6). The first cycle after injection was prolonged by an
increased length of the diestrus stage. Histological
examination of the ovaries and uteruses revealed that
MAM-Ac injected rats had ovaries and uteruses typical of
rats at diestrus. This lasted for approximately 8 days
after injection. Except for the predominance of corpora
lutea in the ovaries and the lack of estrogen stimulation of
the uteruses, no pathological lesions were observed. Pair
fed and positive controls had normally stimulated organs
throughout the experiment (Figures 7 and 8).

Pituitary, ovarian and uterine weights were statistically

the same in both pair fed and positive controls (Tables 11

69

and 12) and comparable to weights reported by others
(Minaguchi et aZ., 1968; VCogot et aZ., 1970). Pituitary
prolactin and LH were higher in pair fed controls (p<0.05)
compared to the positive controls only during diestrus. It
has been Shown that reduction in food intake reduces
production and release of the gonadotropins (Leathem, 1966).
It was also Shown that the maximum content of pituitary
prolactin and LH is on the afternoon of diestrus (Monroe

et aZ., 1969). Because of the fact that blood prolactin
and LH and the levels of PIF and LHRF were similar in both
control groups it does not seem that the pair fed rats

were affected by the reduced food intake or that the hormone
was discharged to the blood later in the afternoon in the
pair fed control rats. The reason for the differences
between both controls remains unknown at present. During
the period of l to 6 days after the injection the same
number of rats at the various stages of the cycle was
sacrificed in both control groups. This is the reason why
the differences in the concentration of LH in the blood
seems surprising (Tables 16 and 17). These differences can
be easily explained using the findings of Gay et a2. (1970).
They reported that LH is discharged to the blood on the
afternoon of diestrus and its half life is 20 min. The

rise is very Sharp and the increase may be of a magnitude of
50 fold. It is possible that more positive controls were

sacrificed before they had discharged the hormones to the

70

blood, and so pair fed controls had higher levels of hormones
in their blood. The large standard error obtained thus
supports this hypothesis. During the period of 1 to 3 and

14 to 16 days after the injection, pituitary LH in the pair
fed controls was higher than that in the positive controls.
This was also true for 8 to 12 days after the injection.

As for the higher LH concentrations in the pair fed controls
at diestrus, no explanation can be offered for these
findings.

Reduction of food intake plus MAM-Ac injection resulted
in more rats sacrificed at diestrus than in both control
groups. The MAM-Ac injected rats killed at diestrus had
Significantly lighter (p<0.05) pituitaries, ovaries and
uteruses when expressed on an absolute basis. Since MAM-Ac
injected and pair fed controls had similar body weights
after injections and Since such reduction in organ weights
was not noticed in the pair fed controls, the reduction in
organ weights can be attributed to the intoxication by
MAM-Ac. The uterus seems to be the first organ that responded
to MAM—Ac intoxication. Its decreased weight was noticed
one day following the injection and it remained lower than
control values for 6 days. Pituitary and ovarian weights
decreased starting from the fourth day after injection
(Tables 11 and 12).

Blood prolactin levels were similar in all groups 1 to

12 days after the injection and they became lower only during

71

14 to 16 days after the injection of MAM-Ac (Table 15).
It does not seem that this reduction has any physiological
significance. Apparently it happened that more rats of
the MAM-Ac group were killed before the rise in prolactin
late on the afternoon of diestrus. The relatively high
levels of prolactin l to 6 days post injection compared to
the levels of LH at this time are of great importance. The
corpora lutea are maintained and supported by prolactin in
the rat (MacDonald and Greep, 1968). As indicated before,
the rats of the MAM-Ac injected group had ovaries filled
primarily with corpora lutea for a period of 8 days. The
levels of prolactin needed for the support of corpora lutea
have not been established yet, but since prolactin did not
decrease as did LH (Tables 15 and 16) it is possible to
assume that the amounts present were able to support the
corpora lutea. Prolactin levels obtained in rats sacrificed
at diestrus were normal, whereas the levels of LH were
lower from those sacrificed at diestrus (Monroe et aZ.,
1969; Parlow et aZ., 1969). Since most of the rats injected
with MAM-Ac were sacrificed at diestrus during the first 6
days after the injection, it is expected that the levels of
prolactin would be unchanged and the levels of LH would
decrease during this period.

In contrast with pair fed controls, MAM-Ac injected
rats at diestrus had less prolactin and LH than pair fed

controls in their pituitaries. Pituitary LH was found to

72

be lower too in MAM-Ac injected rats at proestrus and
estrus. It is difficult to draw any conclusions regarding
the MAM-Ac injected rats at metestrus since only 3 rats
were in this stage at time of sacrifice (Tables 13 and 14).
The concentrations of prolactin in the pituitary were lower
1 to 3 days after the injection compared with the pair fed
control rats and the concentration of LH was lower 4 to 6
days after injection compared with both groups of control.
Because of the fact that the pituitaries were lighter after
the injection of MAM-Ac, the content of prolactin in the
pituitary was lower 1-6 days post injection and the LH
content was lower 1 to 3 days and 8 to 16 days post injec-
tion compared with at least one of the control groups.
Since PIF activity was Similar in all groups (Table 17),
one would expect equal pituitary and blood levels of
prolactin in the 3 groups. However, this is not the case
since the pituitary from MAM-Ac groups contained less
prolactin during diestrus and 1 to 6 days post injection.
This indicates that the pituitary was the organ affected
most by the injections of MAM-Ac. LHRF activity was found
to be higher in the positive controls than in the MAM-Ac
injected rats, 1 to 6 days post injection. AS a result
one would expect more LH in the pituitaries of the MAM-Ac
injected rats. Practically identical levels were found in
both groups. Concomitantly, Similar LHRF activities were

found in MAM-Ac injected and pair fed rats, however more

73

pituitary LH was found in the pair fed rats. The lower
levels of blood LH at this time can be explained on grounds
of less LHRF activity. From these results too, it seems
that the pituitary is the major gland intoxicated by MAM-Ac.

MAM-Ac was reported to exert radiomimetic effects (Teas
et aZ., 1965). It was found too, that x-ray irradiation of
ovaries caused sterility. The ovaries were less responsive
to gonadotropins and vaginal smears were continuously
cornified for 2 to 14 weeks, then the smears became similar
to smears found in spayed rats (Mandl and Zuckerman, 1956).
From these experiments it seems that MAM-Ac does not exert
the effects described by x-rays. The cells found in vaginal
smears after the injection of MAM—Ac were leukocytes which
are typical of diestrus and not cornified cells found after
the irradiation. Sterility or a reduction in ovarian
sensitivity to the gonadotrOpins causes an increase in the
levels of the gonadotropins (Flerko, 1963). In our experi-
ments we found identical blood prolactin in all 3 groups and
lower blood LH in the MAM-Ac injected rats than in the
controls. The presence of well developed corpora lutea and
the absence of mature folliclesin the ovaries of MAM-Ac
injected rats suggest the influence of low levels of
estrogen and FSH and high levels of progesterone.

As a response to MAM-Ac intoxication an increase in the
weight of the adrenals and a decrease in the weight of the

thymus occurred (Table 18). It has been reported that stresses

74

may cause an increase in the synthesis and release of
progesterone from the adrenals of ovariectomized rats.
Stress caused elevation of ACTH levels which in turn
increased progesterone secretion (Resko, 1969). In this
study it is Shown that injection of MAM-Ac to spayed rats
receiving estrogen daily caused a change in the histology
of the vaginal smears. The typical cells of the spayed
rats were replaced with leukocytes which lasted for 3 days
and then started to decline. Since the ovaries were removed
the only source of progesterone to stimulate the uterus was
the adrenals. How much progesterone is secreted from the
adrenals after MAM-Ac injections in the normal rat and what
is its influence on the ovaries is not known.

It is possible that MAM-Ac acts directly on the brain.
The reduction in food intake observed after MAM-Ac injection
(Figure 1 and 2) could have been caused by signals from the
stimulation of the satiety center in the ventro medial
nuclei or the depression of the hunger center in the lateral
hypothalamus (Mayer, 1970). The brain was reported to be
affected by MAM-Ac in another fashion too. Lesions in the
brain of several Species were reported after the administra-
tion of MAM-Ac to one day old rats or during pregnancy
(Spatz et aZ., l967c;Hirono et aZ., 1969a). PIF activity
was not affected by MAM-Ac while LHRF activity was lower
only when compared with the positive control but not the

pair fed control. The concentrations of prolactin and LH

75

in the pituitary were generally the same for all animals.
However, since pituitary weight was decreased in the MAM-Ac
injected rats, the total pituitary contents of prolactin and
LH were thus less in the MAM-Ac injected rats than in both
controls. Although these studies were aimed to examine the
mechanism of action of MAM-Ac on the ovary-pituitary-
hypothalamus axis, it remains unknown whether MAM-Ac is
affecting this axis directly or not. It is still unresolved
as to whether the effect is mediated through~ the ovary,

adrenals or brain.

PART II
IMPLANTATION OF CYCASIN AND MAM-Ac IN THE BRAIN

THIRD VENTRICLE OF RATS AND MICE

PART II
IMPLANTATION OF CYCASIN AND MAM-AC IN THE BRAIN

THIRD VENTRICLE OF RATS AND MICE

Objective

 

One day old mice and dogs injected with MAM-Ac develOped
lesions in the brain (Hirono and Shibuya, 1967; Hirono et aZ.,
1969a). MAM-Ac injections to one day old rats and to
mature mice did not result in such lesions. However, rats
developed microcephaly and hydrocephaly only when injected
with MAM-Ac in prenatal life (Spatz, 1969). Adult humans
and cattle developed neurological disorders after the con-
sumption of cycad material (Whiting, 1964; Hall and McGavin,
1968).

This evidence suggested to us that MAM-Ac may have a
different potential to enter the central nervous system
(CNS) at various stages in life. The purpose of the present
experiment was to determine whether the blood-brain barrier
prevents the toxicity of cycasin and MAM-Ac in weanling and

mature rats and mice.

76

77

Review of Literature

 

The Blood-Brain Barrier

 

The first observations concerning the blood-brain
barrier are attributed to Ehrlich during the years 1885-1887
(Bakay, 1956). In his studies he noted that intravenous
injection of Coerulein-S, an aniline dye into experimental
animals would stain most tissues of the body readily, while
the brain remained uncolored. During the following years
such observations were confirmed and the introduction of
radioisotopes helped explain the nature of the blood-brain
barrier.

The blood-brain barrier is not present early in life,
but it develOps gradually as the organism develops. It has
been shown that in most Species the brain undergoes a period
of rapid growth earlier than the corresponding period for
the rest of the body. The lack of barrier effect seen in
the neonatal brain is due to the increased need for sub-
stances by the developing brain (Dobbing, 1961). On the
other hand some reports suggest that the blood-brain barrier
develops early in embryonic development. Bakay (1953), by
using P32 showed that even during intrauterine life some
barrier activity was present. Dobbing (1963) points out that
the correct timing in the introduction of various substances
is of most importance. The lack of right timing can explain
the variation in results when different substances are used

at different stages in life.

78

It is generally accepted that the blood-brain barrier
is more permeable to basic dyes while the blood-cerebrOSpinal
fluid barrier is more permeable to acid dyes (Davson and
Danielli, 1943; Koenig and Hurbebaus, 1966). Bakay (1953)
summarized results of experiments done with radioactive

isotopes of ions. It was found that ions of C138, 1135,

K42, $35, Na24 and Br32 reach a relatively quick equilibrium
between the plasma and the tissues except brain. The
penetration of such ions to the brain is delayed and only

small amounts penetrate. D 0, on the other hand, was found

2
to penetrate this barrier easily. Rapid exchange exists
between plasma and brain amino acids (Lajtha, 1962).

However, the entry of radioactively labelled proteins from
the blood into the brain is much smaller in amount and

Slower than its entrance to other organs (Fishman, 1953;
Bering, 1955). Some drugs were found to be able to penetrate
the barrier. Sulfonamides and penicillin enter the brain
from the blood. Streptomycin does not enter (Quastel and
Quastel, 1961).

It has been reported that the lipid soluble substances
enter the brain faster than water soluble substances (Davson,
1955). Diseases of the central nervous system that are
severe enough to alter its structural organization result
in an increase in the permeability of the blood-brain barrier

(Bakay, 1968). Quadbeck (1968) reported on various drugs

affecting the penetration of several ions and dyes.

79

Materials and Methods

 

Sixty-two Sprague—Dawley female rats (Spartan Research
Animals, Haslett, Mich.) and 33 female C-57BL/6; mice
(Jackson Lab., Bar Harbor, Maine) were used in 8 experiments

as indicated in Tables 19 and 20. All rats were fed and
housed as described in Part I. The mice were housed in the
same laboratory as the rats but they were housed three to
four mice in one plastic cage. They were allowed free
access to Purina mouse chow and water.

Cycasin dissolved in water (547 mg cycasin/m1), liquid
MAM-Ac and MAM-Ac mixed with cholesterol were implanted in
rats and mice as indicated in Tables 19 and 20. The

procedure used was described by Rowland (1966).

Results and Discussion

 

The third ventricle was chosen as the site of implanta-i
tion because of its location in the brain and the fact that
from this ventricle the implanted material can easily reach
other parts of the brain.

Rats implanted with cholesterol pellets with or without
0.2 mg MAM-Ac, and 10-30 mg cycasin in water solution,
recovered quickly from the surgery and did not Show any
signs of MAM toxicity. MAM-Ac implantation resulted in the
appearance of the toxic Signs typical to MAM-Ac intoxication

which were described by Yang and Mickelsen (1968). The more

80

.UNISNE H: OIN nnH3 N\m uaoEHnomxo

aH paw odIzdz H: OHIN nqu ponaoHQEH ono3 N\m naoEHnomxo aH mnmn onen

.aHmoomo OE ONIOH nnH3 N\m uaoEHnomxo
aH paw aHmoomo me ONION nuHS ponaonEH ono3 N\m naoEHnomxo aH mnon oneo

 

 

 

 

on nn I II memo Hm Hm m\m
on on I II menace N ON m\m
II II O OH unnaoe N mH H\m
n.ozv nqozv n.ozv n.ozc n.ozv
umnnom nonnom Honoumonoeo
odIzdz admoowu HononmoHommI aH oaIzaz m8 N.O mnom OomD
n m nnHz ponaonEH mnom mo own mnom uaoEHnomxm
.maOHnouaonEH

omlzdz pan aHmooeo mo mnaoEHnomxo maoHnm> onaH mnmn mo aonH>HU one .OH oHnoe

.odIzflz H: OIN nnHz couaonEH ono3 oOHE onen

.aHmoomo OE NHIO nnH3 tonaOHmEH onoz ooHE oneo

 

81

 

 

 

N I I . I H N mxz

m N I I H m e\z

m I I I NIH m Nxz

m m I I «IN on N\S

I I N N vIN O H\z
n.ozv n.ozv n.ozv A.ozv Amnuaozv n.0av

umnnom nonnoa Honoumonoeo ommo
DNIZNZ aammomu HmnonmoHomu aH oaIZNZ m8 N.O oon poms
n m nnH3.OonaonEH oon no omd oOHz naoEHnomxm

.maOHnonaonEH

0¢I2¢z paw aHmoomo mo muaoEHnomxo mSOHno> onaH ooHE mo aOHmH>H© one .ON oHnoe

82

MAM-Ac implanted, the more severe was the toxicity. Although
general signs of toxicity appeared, no gross neurological
disorders were noticed.

Mice implanted with cholesterol pellets, cholesterol
pellets with MAM-Ac, and those implanted with 8 to 12 mg
cycasin, recovered quickly from the surgery and did not show
any Signs of cycasin or MAM toxicity. However, the
implantation of MAM-Ac resulted in severe toxicity and
sometimes death. Out of the 5 mice implanted with MAM-Ac in
experiment M/2, 3 died, 2 of which had trembled slightly
before death. In the following experiments, M/3, M/4 and
M/S, the same percentage of mice died, but none of them
showed trembling or other neurological disorders.

Because of the fact that rats and mice implanted with
0.2 p1 MAM-Ac and cycasin did not develop gross neurological
disorders following the implantations, it was decided not
to implant controls unless neurological disorders were seen
following MAM-Ac implantations.

It has been shown in the rat that the enzyme 8-
glucosidase which cleaves cycasin into MAM is present and
active only in the skin for a short period after birth
(Spatz, 1968). This is apparently the reason why the rats
and the mice were not intoxicated by the cycasin implanted.
The rats and the mice were not intoxicated by 0.2 mg MAM-Ac.
This amount is apparently too small to cause any damage

Since even 2 p1 MAM-Ac implanted had little effect (Figure 9).

Body weight (g)

250

200

150

83

 

 

 

 

 

—

J 1

-ll 0 3 6

Post injection (days)

Figure 9. Body weight changes after implantation of 2-10 pl
MAM-Ac to rats.

84

The more MAM-Ac implanted, the more severe the toxicity and
body weight loses recorded (Figure 9).

Only 2 mice out of 62 rats and 33 mice implanted, came
down with neurological disorders. Such disorders could not
have been reproduced although attempts were made. Thus, the
only conclusion must be that the implantation of MAM-Ac and
cycasin to the third ventricle of rats and mice, mature and
young,did not cause any gross neurological disorders.

One of the most important uses of stereotaxic techniques
is in experiments where the effects on the brain of substances
that can not pass the blood-brain barrier, are determined
(Rowland, 1966). So far it is not known how much of the
MAM-Ac injected to experimental animals subcutaneously
reaches the brain. It is still unknown how effective the
blood-brain barrier is in preventing MAM from entering the
brain in the different Species. These studies show that
the presence or absence of MAM in the brain is not the
determining factor for the development of neurological
disorders in mature rats and mice. It has been reported that
only during a limited period of time it is possible to
induce neurological disorders and lesions in rats, mice,
hamsters and dogs by injecting MAM-Ac (Spatz, 1969; Yang,
1971; Hirono and Shibuya, 1967). Thus, it appears that
the most important factor in the etiology of neurological
disorders is the stage of brain develOpment at the time of

its exposure to MAM. However, this explanation does not

85

seem to fit with findings in human and cattle studies. It

has been reported that adult humans and cattle may develop

neurological disorders after consuming cycad material

(Whiting, 1964; Hall and McGavin, 1968). Thus, it appears

that in human subjects and in cattle the blood-brain barrier

may have an important role in preventing greater MAM toxicity

to the neurological system.

PART III
PHYSIOLOGICAL AGE AND THE SENSITIVITY OF

RATS TO CYCASIN TOXICITY

PART III
PHYSIOLOGICAL AGE AND THE SENSITIVITY OF

RATS TO CYCASIN TOXICITY

Objective

 

One day old mice injected with MAM-Ac developed lesions
in their central nervous system (CNS) (Hirono and Shibuya,
1967). Rats at the same age did not develOp such disorders.
The fact that mice are born after 19 days of gestation
whereas rats after 21-23 days, suggested to us that a
possible reason for the differences in the reaction to MAM-
Ac injection was due to differences in the develOpmental
stage of the CNS at time of exposure to the toxin. Therefore,
we decided to examine whether rats at the same stage of CNS
development as mice would show the same neurotoxic symptoms

subsequent to MAM-Ac or cycasin injection.

Review of Literature

 

Three major stages of development can be distinguished
from the time of fertilization to birth. Embryogenesis is
the first stage lasting for 7.5 days in mice and 9.5 days
in rats. This stage is followed by organogenesis which lasts
up to the 16th day of gestation in the mouse and 18.5 in the
rat. Next is the developmental stage of the fetus which

lastsuntil the day of delivery. Parturition occurs on the

86

87

19th day of gestation in the mouse and during 21-23 days in
the rat (Christie, 1964; Edwards, 1966; Rugh, 1968).

Embryogenesis starts with the fertilization of the egg
and lasts until the mesoderm and the neural plate are
develOped. During this stage cell division, implantation
and heart appearance are the major events. Organogenesis
begins with the appearance of the neural plate. During this
stage all tissues and organs start their differentiation.

As a general rule the mouse reaches each develOpmental stage
l-2 days before the rat. The major developmental events
during this stage are the differentiation of nerves, brain
and sense organs from the ectoderm; digestive system, lungs
and glands from the endoderm; and bones, blood vessles and
gonads from the mesoderm. A detailed description of the
develOpment of mouse and rat embryo can be found in Hicks
(1968); Christie (1964); Edwards (1964); and Rugh (1968).
The final three days or so of gestation are the fetus stage
which is characterized more by growth than by differentiation.
This stage ends with parturition.

Most differentiation is in fact completed by the 16th
day in the mouse and by day 18.5 in rat. Certain organs such
as cerebellum do not mature until after birth. It has been
reported that in rats the brain reaches its maximum weight
per unit body weight only 10 days after birth. Similar
results were obtained in studies with mice (Davison and

Dobbing, 1966; Rugh, 1968).

88

By exposing the embryo to apprOpriate compounds at the
right time one can induce a variety of abnormalities.
According to Rice (1969) introduction of such compounds during
embryogenesis causes lethal damage to the embryo. Terato-
genesis results when such compounds are introduced in early
organogenesis and carcinogenesis results when appropriate
compounds are introduced during late organogenesis and
during the fetus stage.

The effects of diazo dyes on the embryo were studied
rather extensively in the past. A dye, trypan blue, was
reported to cause cardiovascular malformations in the rat
when introduced on the 7th, 8th or 9th day of gestation.
Abnormalities of the genito-urinary system were reported
when the dye was introduced during the 13th to the 17th day
of gestation. During days 16 and 17 of gestation stenosis
and occlusion were reported and during days 18 to 20 of
gestation the appearance of hydrocephalus was noticed (Beck
and Lloyd, 1967). Similar studies were conductedto study
the effects of X irradiation on the embryo. A dose of 200 r
on the 9th day of gestation kills the embryo whereas 100 r
causes cardiac malformation: Abnormalities of the forebrain,
Spinal cord and cerebral hemispheres are obtained after
irradiation during the period of days 13 to 19 of gestation.
The cerebellum on the other hand differentiates first on the
16th day of gestation and malformation in it appears if

irradiated on the 18th day of gestation and up to one week

89

after birth (Hicks and D'Amato, 1967). MAM was reported to
have similar effects on the brain when introduced during days
14 to 16 of gestation (Spatz, 1969).

Many different drugs and various treatments were studied
with relation to malformations in embryos. Cardiovascular
malformations in various mammals were reported after the
following treatments: administration of trypan blue,
thalidomide and streptonigrin; X irradiation, hypoxia and
hypercapina; and deficiency in folic acid or pantothenic
acid or riboflavin (Takas and Warkany, 1967). Teratogenic
effects were reported to be produced by X irradiation,
administration of diazo dyes or nitroso and azoxy compounds.
Such treatments as hyperthermia causes the same effects
(Beck and Lloyd, 1967; Hicks and D'Amato, 1967; Spatz and

Laqueur, 1967; Edwards, 1968; Rice, 1969).

Materials and Methods

 

Fifty-nine pregnant Sprague-Dawley rats were used on
days 13 through 20 of gestation. To obtain these timed
pregnant rats, 3-4 females were placed with a proven male
and vaginal smears taken daily to determine pregnancy. The
appearance of Sperm in the smear was designated as the
first day of gestation. At this time the pregnant rats were
transferred to maternity cages. All rats were fed ad libitum
and were housed in an animal laboratory under the conditions

described in Part I (Materials and Methods).

90

Operational Procedure

 

The rats were anesthetized with ether and the hairs
shaved off the abdomen. A midline abdominal incision was
made and the double horned uterus exteriorized. Each pup
was injected under the skin with MAM-Ac, saline or cycasin
using a micro-syringe. The number of injected fetuses was
recorded and the uterus returned to its normal location.
The muscles and the Skin were sutured separately with Silk
thread. The rats were allowed to recover and were returned

to their cages for continuation of gestation and delivery.

Treatments

 

The pups were injected in utero with 0.8-2.1 mg cycasin
or 3.1 ul MAM-Ac or 1 ul saline, as indicated in Tables 21-
23. The number of born and weaned pups was recorded. The
weaned pups were allowed to grow for 6 months and then all
of them were sacrificed and examined for presence of tumors
and malformation. The rats which died during the 6 months

were examined for cause of death.

Results and Discussion

 

After the injection of saline to fetus in utero, only
24% of the injected fetuses were weaned and only 30% of the
mothers that underwent laparatomy were able to wean pups.
When cycasin was injected, 41% of the mothers that underwent

laparatomy weaned pups and about 20% of the injected pups

91

 

 

 

 

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94

were weaned. In MAM-Ac injections only 8% of the injected
pups were weaned compared to 33% of the mothers that under-
went laparatomy being able to wean their pups (Tables 21-
23).

About 20% of the weaned rats of all treatments died
within the first 6 months of their life. Post mortem
examination revealed that the lungs were lesioned rather
severely. During the 6 months of the experiment no
impairment in coordination or other neurological functions
were noticed. When the rats were sacrificed at 6 months of
age no gross lesions were found in the brain. However, in
4 out of 167 rats injected in utero with MAM-Ac tumors
in the liver or kidneys were found. All rats injected in
utero with cycasin or saline were free from tumors.

It has been reported that feeding cycad meal to rats
at various periods of gestation caused tumors amounting to
18.5% of the weaned pups. In the same experiment, it was
found that 42% of the pups exposed to cycasin in their
prenatal life died soon after birth (Spatz and Laqueur,
1967). Injections of MAM-Ac to pregnant rats during days
14 to 16 of gestation resulted in brain lesions and brain
tumors in 10% of the pups. However, no information on the
percentage of the rats that died soon after birth is
available (Spatz, 1969). Because of the fact that same per-
centage of mothers in each group was able to wean pups, it

seems that the stressful surgical procedure was the reason

95

why some of the rats in each group died during delivery or
were consumed shortly after birth. The differences in the
percentage of survival among the groups can be attributed to
the type of compound used; cycasin and saline were much less
toxic than MAM-Ac (Tables 21-23). The lower percentage of
weaned rats found in this experiment compared with the one
reported by Spatz and Laqueur was apparently due to the
stressful surgical procedure used in this study.

In this study cycasin was found to be much less toxic
than MAM-Ac although equivalent amounts of the substances
in terms of MAM were given. It has been reported that cycasin
in order to be toxic, must be hydrolyzed first. It has been
found that the enzyme B-glucosidase which is responsible for
this reaction, is present in the subcutis of pups in their
prenatal life (Spatz, 1968). The reason why cycasin was
less toxic may be due to a Slow hydrolysis rate. It is
possible that the injected cycasin was removed via the
mother circulation and excreted in to her urine before it
was hydrolyzed into MAM in sufficient amounts to cause toxic
effects. As mentioned earlier, no brain lesions were found
and only about 2.5% of the rats exposed to MAM-Ac in utero
develOped tumors later in life. It has been reported that
introduction of cycasin during gestation resulted in tumors
found in 18.5% of the pups later in life. Introduction of
MAM-Ac resulted in 10% of the pups having brain tumors

(Spatz and Laqueur, 1967; Spatz, 1969). Because of the fact

96

that in this study many pups were consumed after birth
and many others died, it is possible that those rats which
would be tumor bearing were consumed selectively by their
mothers. It is possible too that the potential tumor
bearing rats died earlier because of lung lesions or other
reasons and thus the tumors did not have time to develop.
An attempt was made to inject rats in utero with MAM-
Ac or cycasin at the same develOpmental stage as mice are
at birth. Mice reach each developmental stage 1-2 days
before the rat, thus rats were injected at 19-20 days of
gestation. The neurological disorders seen in one day old
mice injected with MAM-Ac were not seen in rats at the
same developmental stage. Thus, the differences between
the response of rats and mice to MAM-Ac and cycasin cannot
be attributed to differences in develOpment at the time of
injection. It is more likely that what determines the
reSponse to MAM treatment is the Species of the animal

itself.

S UMMARY AND CON CLUS IONS

A total of 292 female Sprague-Dawley rats were used
in 8 trials to study the effects of MAM-Ac on the neuro-
endocrinological system associated with reproduction.
Normally cycling rats at metestrus were injected subcu-
taneously with 10-12 ul MAM-Ac or saline. Since MAM-Ac
injections causes a reduction in food intake, a pair fed
control group as well as a positive control was used.

The experimental procedure included post injection
determinations of food intake, body and organ weights,
body composition and reproductive performance. In
addition, adrenals, pituitaries, ovaries and uteruses were
histologically examined and the levels of LH and prolactin
were determined in the blood and pituitary by RIA.
Hypothalami were analyzed for PIF and LHRF activities.

Food intake dropped Sharply following the injection of
MAM—Ac. From a daily consumption of about 20 g, the intake
drOpped to 3 g one day post injection. Thereafter it
increased until it was back to normal one week later. As
a result of the reduced food consumption the rats lost about
12-18% of their initial body weight, which was restored only

10-12 days post injection. The same body weight loses and

97

98

body compositional changes were obtained in the MAM-Ac
injected and pair fed rats.

As a response to MAM-Ac injections the adrenals became
heavier and the thymuses lighter than in pair fed controls.
Histological examination of the adrenals revealed the
presence of hemorrhages and indistinct division of the
different zones.

The estrous cycle was Significantly prolonged (p<0.001)
following the administration of MAM-Ac. Before injection
all rats had cycles of 4-5 days. The first cycle after the
injection was completed within 9.0-9.8 days by the MAM-Ac
injected rats and within 5.2-5.4 days by the pair fed rats.
The second and third cycles after injection were similar in
length for all groups.

Reproductive performance as measured by the length of
time from mating to pregnancy, % fertility and the number
of born and weaned pups. All rats were pregnant within 9
days from mating and 10.8 pups per litter were born to MAM-
Ac injected rats versus 10.5 to the pair fed controls.

Organ weights expressed per 100 g body weight were
generally the same in all groups when analyzed according to
the stages of the estrous cycle in which the rats were sacri-
ficed. Since most of the rats treated with MAM-Ac were at
diestrus l to 6 days post injection and Since rats at
diestrus have lighter uteruses, thus MAM-Ac injected rats

had lighter uteruses during this time interval. The

99

ovaries and the pituitaries of the MAM-Ac injected rats
began to decrease in weight from the 4th day on.

Histological examination of the ovaries revealed that
for a period of approximately 8 days after the injection of
MAM-Ac, the rats had ovaries filled primarily with well
deve10ped corpora lutea and unstimulated uteruses with dense
endometrium, narrow lumen and few glands.

Gonadotropin metabolism as measured by RIA was impaired
in rats injected with MAM-Ac. LHRF activity measured in the
hypothalami of the MAM-Ac injected rats was lower when
compared with the positive control. However there was no
change in the PIF activity in the MAM-Ac injected rats when
compared with both controls. The anterior pituitary of
MAM-Ac injected rats sacrificed at diestrus was found to
contain less prolactin and LH. In addition MAM-Ac injected
rats sacrificed at proestrus and estrus contained less LH
in their pituitaries. Since most of the MAM-Ac injected
rats sacrificed 1 to 6 days post injection were at diestrus,
pituitary LH was lower 1 to 3 days post injection and
pituitary prolactin was lower 1 to 6 days post injection.
Blood LH was lower in MAM-Ac injected rats sacrificed at
metestrus and diestrus whereas blood prolactin levels were
the same in MAM-Ac injected rats and in both controls
sacrificed at the various stages of the cycle. AS a result,
1 to 6 days after the injection blood LH was lower in MAM-Ac
injected rats than in both controls whereas blood prolactin

did not decrease.

100

The significance of the blood-brain barrier in pre-
venting cycasin and MAM-Ac neurotoxicity was studied in rats
and mice. The experimental procedure included the implanta-
tion of cycasin or MAM-Ac in the brain third ventricle of
rats and mice, by stereotaxic instrument. Only 2 out of
the 33 implanted mice and none of the 62 implanted rats
Showed neurological disorders. No gross lesions were
observed in these animals.

The Significance of the physiological stage of the
developing brain in relation to MAM-Ac and cycasin toxicity
was studied during the gestation in rats. Laparatomy was
performed on pregnant rats during 13 to 20 days of gestation
and cycasin or MAM-Ac was injected subcutaneously to each
fetus. This procedure was found to be very stressful since
only 30-41% of the rats undergoing laparatomy weaned pups
and only 8-20% of the injected pups were weaned, regardless
of the treatment given. At the age of 6 months the rats
were examined for brain lesions and tumors. No gross
lesions were observed in these rats and only 2.5% of the

rats treated with MAM-Ac had liver or kidney tumors.

Conclusions

 

1. MAM-Ac injections caused disorders in the gonadotropin
metabolism and sterility prevailed for approximately

10 days.

101

2. The sequence of events from injection time to the
initiation of sterility and the primary organ affected
are unknown.

3. The blood-brain barrier is not the mechanism by which
neurological disorders are prevented in mature mice and

rats.

LITERATURE CITED

LITERATURE CITED

Amenomori, Y., C. L. Chen and J. Meites (1970). Serum
prolactin levels in rats during different reproductive
stages. Endocrinology, 86:506.

Bacq, Z. M., G. Dechamps, P. Fischer, A. Herve, H. LeBihan,
J. Lecomte, M. Pirotte and P. Rayet (1953). Protection
against X-rays and therapy of radiation sickness with
B-mercapto-ethylamine. Science,ll7:633.

Bakay, L. (1953). Studies on blood-brain barrier with
radioactive phOSphoruS. III. Embryonic development
of the barrier. Am. Med. Assoc. Arch. Neurol.
Psychiat.,70:30.

Bakay, L. (1956). The Blood-Brain Barrier. Charles C.
Thomas, Springfield, Illinois.

 

Bakay, L. (1968). Changes in barrier effect in pathological
states, in: Progress in Brain Research. VOl. 29,
Eds. A. Lajtha and D. H. Ford, Elsevier, N. Y.

Beck, F. and J. B. Lloyd (1966). The teratogenic effects
of azo-dyes, in: Advances in Teratology. V01. I,
Ed. D. H. M. Woollam, Academic Press, N. Y.

 

Bell, E. A. (1964). Relevance of biochemical taxonomy of
the problem of Lathyrism. Nature, 203:378.

Beradi, L. C. and L. A. Goldblatt (1969). Gossypol, in:
Toxic Constituents in Plant Foodstuffs. Ed. I. E.
Liener, Academic Press, N. Y.

 

Bering, Jr., E. A. (1955). Studies on the role of the choroid
plexus in tracer exchange between blood and cerebrospinal
fluid. J. Neurosurg., 12:385.

Birk, Y. (1969). Saponins, in: Toxic Constituents in Plant
Foodstuffs, Ed. I. E. Liener, Academic Press, N. Y.

 

 

Bogdanove, E. M. (1963). Direct gonad-putuitary feedback:
An analysis of effects of intracrnial estrogenic depots
on gonadotrOpin secretion. Endocrinology, 73:696.

102

103

Britton, N. L. and P. Wilson (1930). Scientific survey of
Puerto Rico and the Virgin Islands. N. Y. Acad. of
Sci., 6:1925.

Campbell, M. E., O. Mickelsen, M. G. Yang, G. L. Laqueur and
J. C. Keresztesy (1966). Effects of strain, age and diet
on the reSponse of rats to the ingestion of Cycas
circinalis. J. Nut., 88:115.

Cecil, R. L. and R. F. Loeb (1955). A Textbook of Medicine.
W. B. Saunders Co., Philadelphia, Penn.

 

Christie, G. A. (1964). DeveloPmental stage in somite and
post somite rat embryos, based on external appearance
and including some features of the macroscopic develop-
ment of the oral cavity. J. Morph. 114:263.

Clemens, J. A. and J. Meites (1968). Inhibition of
hypothalamic prolactin implants of prolactin secretion,
mammary growth and luteal function. Endocrinology, 82:
878.

C00per, J. M. (1941). Isolation of a toxic principle from
the seeds of Macrozamia spiralis. Proc. Roy. Soc. New
South Wales, 74:450.

Corbin, A. and A. I. Cohen (1966). Effects of median
eminence implants of LH on pituitary LH of female rats.
Endocrinology, 80:1006.

Dahlquist, A., B. Bull and B. E. Gustafsson (1956a). Rat
intestinal 6-bromo-2-naphthyl glycosidase and
disaccharidase activities. I. Enzymic properties
and distribution in the digestive tract of conventional
and germ-free animals. Arch. Biochem. BiOphys., 109:150.

Dahlquist, A., B. Bull and D. L. Thomson (1965b). Rat
intestinal 6-bromo-2-naphthyl glycosidase and
disaccharidase activities. II. Solubilization and
separation of the small intestinal enzymes. Arch. Biochem.
Biophys., 109:159.

Dastur, D. K. and R. S. Palekar (1966). Effect of boiling
and storing on cycasin content of Cycas circinalis L.
Nature, 210:841.

David, M. A., F. Fraschini and L. Martini (1966). Control
of LH secretion: Role of a "Short" feedback mechanism.
Endocrinology, 78:55.

104

Davidson, J. M. (1969). Feedback control of gonadotrOpin
secretion. in: Frontiers in Neuroendocrinology. Eds.
W. E. Ganong and L. Martini, Oxford Press, N. Y.

Davidson, J. M., R. F. Weick, E. R. Smith and R. Dominguez
(1970). Feedback mechanism in relation to ovulation.
Fed. Proc., 29:1900.

Davison, A. N. and J. Dobbing (1966). Myelination as a
vulnerable period in brain deve10pment. Br. Med. Bull.,
22:40.

Davson, H. and J. F. Danielli (1943). The Permeability of
Natural Membrane. Cambridge University Press, Cambridge,

 

 

England.

Davson, H. (1955). A comparative study of the aqueous humour
and cerebrOSpinal fluid in the rabbit. J. Physiol.,
129:111.

Dobbing, J. (1961). The blood brain barrier. Physiol.
Revs., 41:130.

Dobbing, J. (1963). The entry of cholesterol into rat brain
during development. J. Neurochem., 10:739.

Edwards, J. A. (1968). The external development of the
rabbit and rat embryo. in: Advances in Teratology.
V01. III. Ed. D. H. M. Woollam. Academic Press, N. Y.

 

Evans, H. M. and K. S. BiShOp (1922). On the relations
between fertility and nutrition. I. The ovulation
rhythm in the rat on a standard nutritional regime.
J. Met. Res., 1:319.

Everett, J. W. (1964). Central neural control of reproduc-
tive functions of the adenohypophysis. Physiol. Revs.,
44:373.

Fajer, A. B. and C. A. Barraclough (1967). Ovarian secretion
of progesterone and 20-a-hydroxpregn-4-ene-3-one during
pseudopregnancy and pregnancy in rats. Endocrinology,
81:617.

Feinstein, R. N. and S. Berliner (1957). Protection against
x irradiation by 3-amino-l,2,4, triazole. Science, 125:
936.

Fishman, R. A. (1953). Exchange of albumin between plasma
and cerebrOSpinal fluid. Am. J. Physiol., 175:96.

105

Flerko, B. (1963). The central nervous system and the
secretion and release of LH and FSH. in: Neuroendo-
crinology. Ed. A. V. Nalbandov. University of Illinois
Press, Urbana, Illinois.

 

 

Fosberg, F. R. (1964). Resume of the Cycadacea. Fed. Proc.,
23:1340.

Friedman, L. and S. I. Shibko (1969). Adventitious toxic
factors in processed foods. in: Toxic Constituents in
Plant Foodstuffs. Ed. I. E. Liener, Academic Press,

N. Y.

 

 

Ganote, C. E. and A. S. Rosental (1968). Characteristic
lesions of methylazoxymethanol induced liver damage.
Lab. Invest., 19:382.

Gay, V. L., A. P. Midgley, Jr. and G. D. Niswender (1970).
Patterns of gonadotropin secretion associated with
ovulation. Fed. Proc., 29:1880.

Guillemin, R., A. V. Schally, H. S. Lipscomb, R. N. Andersen
and J. M. Long (1962). On the presence in hog hypo-
thalamic of P-corticotropin releasing factor, a and B
melanocyte stimulating hormone, adrenocorticotropin,
lysin, vaSOpressin and oxytocin. Endocrinology, 70:
471.

Gusek, W., H. Buss and G. L. Laqueur (1967). Histologisch-
histochemische utersuchungen am "Interstitiellen cycasin
tumor der rattenniere". Beitr. Pathol. Anat. Allgen.
Pathol., 135:53.

Haddad, R. K., A. Rabe, G. L. Laqueur, M. Spatz and M. P.
Valsamis (1969). Intellectual deficit associated with
transplacentally induced microcephaly in rat. Science,
163:88.

Hall, W. T. and M. D. McGavin (1968). Clinical and neuro-
pathological changes in cattle eating the leaves of
Macrozamia Zucida and Bowenia serrulata (family
Zamiaceae). Pathol. vet., 5:26.

Harris, G. W. (1937). Induction of ovulation in the rabbit
by electrical stimulation of hypothalamo-hypOphysial
mechanism. Proc. Roy. Soc. (London) Ser. 3., 122:374.

Harris, G. W. and D. Jacobson (1952). Functional grafts of
the anterior pituitary gland. Proc. Roy. Soc. (London)
Ser. B., 139:263.

106

Hicks, S. P. (1958). Radiation as an experimental tool in
mammalian develOpmental neurology. Physiol. Revs.,
38:337.

Hicks. P. and C. J. D'Amato (1966). Effects of ionizing
radiations on mammalian development. in: Advances in
Teratology. V01. I. Ed. D. H. M. Woollam, Academic
Press, N. Y.

 

 

Hinsey, J. C. (1937). The relation of the nervous system to
ovulation and other phenomena of the female reproductive
tract. Cold Spring Harbor Symp. Quant. Biol., 5:269.

Hirono, I. and C. Shibuya (1967). Induction of a neurological
disorder by cycasin in mice. Nature, 216:1311.

Hirono, I. and G. L. Laqueur (1967). Protection against
acute toxicity of cycasin by B-mercapto-ethylamine and
3—amino-l,2,4, triazole. in: Fifth Conference on
Cycad Toxicity. Sponsored by the National Institute of
Arthritis and Metabolic Diseases, The National Institute
of Health, Bethesda, Maryland.

Hirono, I., G. L. Laqueur and M. Spatz (1968a). Tumor
induction in Fischer and Osborne Mendel rats by a single
administration of cycasin. J. Nat. Cancer Inst., 40:
1003.

Hirono, I. G. L. Laqueur and M. Spatz (1968b). Transplant-
ability of cycasin induced tumors in rats with emphasis
on nephroblastomas. J. Nat . Cancer Inst., 40:1011.

Hirono, I., C. Shibuya and K. Hayashi (1969a). Induction
of a cerebellar disorder with cycasin in newborn mice
and hamsters. Proc. Soc. Exp. Biol. Med., 131:593.

Hirono, I., C. Shibuya and K. Fushimi (1969b). Tumor
induction in C57BL/6 mice by a single administration of
cycasin. Cancer Res., 29:1658.

Hoch-Ligeti, C., E. Stutzman and J. M. Arvin (1968).
Cellular composition during tumor induction in rats by
cycad husk. J. Nat . Cancer Inst., 41:605.

Horisberger, M. and H. Matsumoto (1968)14 Studigs on
methylazoxymethanol: Synthesis of C and H labelled
methylazoxymethanol acetate. J. Labelled Compounds
IV:164.

(“In ..i II] Ill: 4::

107

Igarashi, M. and S. M. McCann (1964). A hypothalamic
follicle stimulating hormone releasing factor. Endo-
crinology: 74:446.

Jaffe, W. G. (1969). Hemagglutinins. in: Toxic Constituents
in Plant Foodstuffs. Ed. I. E. Liener, Academic Press,
N. Y.

 

 

Johnson, D. L. and D. M. Nelson (1966). Amount of luteinizing
hormone activity in extracts of Sheep hypothalamus.
Endocrinology, 78:901.

Keegan, H. L. and W. V. MacFarlane (1963). Venomous and
Poisonous Animals and Noxious Plants of the Pacific
Region. The Pergamon Press, MacMillan Co.

 

Kelly, M. and R. W. O'Gara (1965). Effects on monkeys. in:
Fourth Conference on Toxicity of Cycads. Ed. M. G.
Whiting, U. S. Department of Health, Education and
Welfare, Washington, D. C.

Kobayashi, A. and H. Matsumoto (1964). Methylazoxymethanol,
the aglycone of cycasin. Fed. Proc.,23:l354.

Kobayashi, A. and H. Matsumoto (1965). Studies on
methylazoxymethanol, the aglycone of cycasin:
Isolation, biological and chemical properties. Arch.
Biochem. Biophys., 110:373.

Koenig, H. and A. Hurbebaus (1966). Biochemical and cyto-
chemical studies of neutral red uptake into brain.
J. Neuropathol. Exp. Neurol., 25:172.

Kragt, C. L. and J. Meites (1967). Dose-reSponse relationship
between hypothalamic PIF and prolactin release by rat
pituitary tissue in vitro. Endocrinology, 80:1170.

Kurland, L. T. and D. W. Mulder (1954). Epidemiologic
investigation of amyotrophic lateral sclerosis. I.
Preliminary report on geographic distribution with
Special reference to the Mariana Islands. Neurology,
4:355.

Kwa, H. G. and F. Verhofstad (1967). Prolactin levels in
the plasma of female mice. J. Endocrinol., 38:81.

Lajtha, A. (1962). The brain barrier system. in: Neuro-
chemistry. Eds. K. A. Elliot, I. H. Page and J. H.
Questel. Charles C. Thomas, Springfield, Illinois.

 

108

Langley, B. W., B. Lythgoe and N. V. Riggs (1951).
Macrozamin, Part II. The aliphatic azoxy structure of
the aglycone part. J. Chem. Soc., 46:2309.

Laqueur, G. L., O. Mickelsen, M. G. Whiting and L. T. Kurland
(1963). Carcinogenic properties of nuts from Cycas
circinalis L. indigenous to Guam. J. Nat . Cancer Inst.,
31:919.

Laqueur, G. L. (1964). Carcinogenic effects of cycad meal
and cycasin (methylazoxymethanol glycoside) in rats and
effects of cycasin in germ-free rats. Fed. Proc.,
23:1386.

Laqueur, G. L. (1965). The induction of intestinal ne0plasms
in rats with the glycoside cycasin and its aglycone.
Virchous. Arch. Pathol. Anat. Physiol. Clin. Md., 340:
151.

Laqueur, G. L. and H. Matsumoto (1966). Neoplasms in female
Fischer rats following intraperitoneal injection of
methylazoxymethanol. J. Nat . Cancer Inst., 37:217.

Laqueur, G. L., E. G. McDaniel and H. Matsumoto (1967).
Tumor induction in germ-free rats with methylazoxy-
methanol (MAM) and synthetic MAM acetate. J. Nat .
Cancer Inst., 39:355.

Laqueur,G. L. and M. Spatz (1968). Toxicology of cycasin.
Cancer Research, 28:2262.

Leathem, J. H. (1966). Nutritional effects on hormone
production. J. Animal Sci., 25:685.

Liener, I. E. (1969). Toxic Constituents in Plant Food-
stuffs. Academic Press, N. Y.

Liener, I. E. and M. L. Kakade (1969). Protease inhibitors.
in: Toxic Constituents in Plant Foodstuffs. Ed. I. E.
Liener, Academic Press, N. Y.

 

Lythgoe, B. and N. V. Riggs (1949). Macrozamine Part I.
The identity of the carbohydrate component. J. Chem.
Soc., 4:2716.

Macdonald, G. J. and R. O. Greep (1968). Maintenance of
progestin secretion from rat corpora lutea. Perspect.
Biol. Med., 11:490.

109

Magee, P. N. (1965). Fourth conference on the toxicity of
cycads. Sponsored by the National Institute of
Neurological Diseases and Blindness and National Institute
of Arthritis and Metabolic Diseases, NIH, Bethesda,
Maryland.

Mager, J., A. Razin and A. Hershko (1969). Favism. in:
Toxic Constituents in Plant Foodstuffs. Ed. I. E. Liener.
Academic Press, N. Y.

Malevski, Y., M. G. Yang, C. W. Welsch and O. Mickelsen (1971).
Unpublished results.

Mandel, A. M. and S. Zuckerman (1956). The reactivity of
the X-irradiated ovary of the rat. J. Endocrin., 13:
243.

Mason, M. M. and M. G. Whiting (1966). Demyelination in the
bovine Spinal cord caused by zamia neurotoxicity. Fed.
Proc., 25:533.

Mason, M. M. and M. G. Whiting (1968). Caudal motor weakness
and ataxia in cattle in the Caribbean area following
ingestion of cycads. Cornell Vet., LVIII:541.

Matsumoto, H. and F. M. Strong (1963). The occurrence of
methylazoxymethanol in Cycas circinalis L. Arch.
Biochem. Bi0phys. 101:299.

Matsumoto, H., T. Nagahama and H. O. Larson (1965). Studies
on methylazoxymethanol, the aglycone of cycasin: A
synthesis of methylazoxymethanol acetate. Biochem. J.,
95:13c.

Matsumoto, H. and H. H. Higa (1966). Studies on
methylazoxymethanol, the aglycone of cycasin: Methylation
of nucleic acids in vitro. Biochem. J., 98:20c.

Mayer, J. (1970). Physiology of hunger and satiety;
regulation of food intake. in: Modern Nutrition in
Health and Disease. Eds. M. C. Wohl and R. S. Goodhart.
Lea and Febiger, Philadephia, Penn.

 

 

McCann, S. M., S. Taleisnik and M. M. Friedman (1960). LH
releasing activity in hypothalamic extracts. Proc. Soc.
Exp. Biol. Med., 104:432.

McCann, S. M. (1962). A hypothalamic luteinizing hormone
releasing factor (LHRF). Am. J. Physiol., 202:395.

110

McCann, S. M., A. P. S. Dhariwal and J. C. Porter (1968).
Regulation of adenohypophsis. Ann. Rev. Physiol.,
38:589.

Meites, J., R. H. Kahn and C. S. Nicoll (1961). Prolactin
production by the rat pituitary in vitro. Proc. Soc.
Exp. Biol. Med., 108:440.

Mickelsen, O. and A. A. Anderson (1959). A method for
preparing intact animals for carcass analysis. J. Lab.
Clin. Med., 53:282.

Mickelsen, O. and M. G. Yang (1966). Naturally occurring
toxicants in foods. Fed.Proc., 25:104.

Mickelsen,O., E. Campbell, M. G. Yang, G. Mugera and C. K.
Whitehair (1964). Studies with cycad. Fed. Proc.,
23:1363.

Miller, J. A. (1964). Comments on chemistry of cycads.
Fed. Proc., 23:1361.

Miller, J. A. and E. C. Miller (1965). Natural and synthetic
chemical carcinogens in the etiology of cancer. Cancer
Res., 25:1292.

Minaguchi, H., J. A. Clemens and J. Meites (1968). Changes
in serum and pituitary prolactin levels in rats from
weaning to adulthood. Endocrinology, 82:555.

Mittler, J. C. and J. Meites (1964). In vitro stimulation
of pituitary follicle-stimulating hormone release by
hypothalamic extract. Proc. Soc. Exp. Biol. Med., 117:
309.

Monroe, S. E., A. F. Parlow and A. R. Midgley, Jr. (1968).
Radioimmunoassay for rat luteinizing hormone.
Endocrinology, 83:1004.

Monroe, S. E., R. W. Rebar, V. L. Gay and A. R. Midgley, Jr.
(1969). Radioimmunoassay determination of luteinizing
hormone during the estrous cycle of the rat. Endo-
crinologY: 85:720.

Montgomery, R. D. (1969). Cyanogens. in: Toxic Constituents

 

in Plant Foodstuffs. Ed. I. E. Liener. Academic Press,
N. Y.

 

Mulinos, M. G., L. Pomerantz, J. Smelzer and R. Kurzrok
(1939). Estrus-inhibiting effects of inanition. Proc.
Soc. Exp. Biol. Med., 40:79.

111

Newberne, P. (1965). Effects on chickens. in: Fourth
Conference on the Toxicity of Cycads. Ed. M. G. Whiting.
U. S. Department of Health, Education and Welfare,
Washington, D. C.

Nishida, K., A. Kobayashi and T. Nagahama (1955). Studies on
cycasin, a new toxic glycoside of Cycas revoluta Thumb.
Part I. Isolation and the structure of cycasin. Bull.
Agr. Chem. Soc. Japan, 19:77.

Nishida, K., A. Kobayashi, T. Nagahama, K. Kojima and
M. Yamane (1956). Studies on cycasin, a new toxic
glycoside of Cycas revoluta Thumb. Part IV. Pharmacologi-
cal study of cycasin. Seikagaku, 28:218.

Niswender, G. D., C. L. Chen, A. R. Midgley, Jr., J. Meites
and S. Ellis (1969). Radioimmunoassay for rat prolactin.
Proc. Soc. Exp. Biol. Med., 130:793.

O'Gara, R. W., J. M. Brown and M. G. Whiting (1964). Induction
of hepatic and renal tumors by the t0pical application
of aqueous extracts of cycad nut to artificial skin
ulcers in mice. Fed. Proc., 23:1383.

Orgell, W. H. and G. L. Laqueur (1964). Effect of ingested
cycasin on rat blood plasma cholinesterase. Fed. Proc.,
23:1378.

Parlow, A. F., T. A. Daane and A. V. Schally (1969).
Qualitative differential measurement of rat serum FSH,
LH and hypothalamic FSH-RH and LH-RH with specific
radioimmunoassays. Slst Program of the Endocrine
Society, p. 83.

Piacsek, B. E. and J. Meites (1966). Effects of castration
and gonadal hormones on hypothalamic content of
luteinizing hormone releasing factor (LRF). Endocrin-
ology, 79:432.

P0pa, G. T. and U. Fielding (1930a). A portal circulation
from the pituitary to the hypothalamus region. J. Anat.,
65:88.

Popa, G. T. and U. Fielding (1930b). The vascular link
between the pituitary and the hypothalamus. Lancet, 2:
238.

Quastel, J. H. and D. M. J. Quastel (1961). The Chemistry of
Brain Metabolism in Health and Disease. Charles C.
Thomas, Springfield, Illinois.

 

 

112

Quadbeck, G. (1968). Drug influence on the barrier. in:
Progress in Brain Research, Vol. 29. Eds. A. Lajtha
and D. M. Ford. Elsevier, N. Y.

 

Ramirez, V. D., R. M. Abrams and S. M. McCann (1964). Effect
of estrodiol implants in the hypothalamo-hypophysial
region of the rat on the secretion of luteinizing
hormone. Endocrinology, 80:821.

Rechcigl, M., Jr. (1964). Rates and kinetics of catalase
synthesis and destruction in rats fed cycad and cycasin
in vivo. Fed. Proc., 23:1376.

Resko, J. A. (1969). Endocrine control of the adrenal
progesterone secretion in the ovariectomized rat.
Science, 164:70.

Rice, J. M. (1969). Transplacental carcinogenesis in mice
by 1-ethy1-l-nitrosourea. Annals N. Y. Acad. Sci., 163:
813.

Riggs, N. V. (1954). The occurrence of macrozamin in the
seeds of cycads. Australian J. Chem., 7:123.

Riggs, N. V. (1956). Glycosylazoxymethane, a constituent
of the seeds of Cycas circinalis L. Chem. and Industry,
35:926.

Rowland, V. (1966). Stereotaxic techniques and the produc-
tion of lesions. in: Neuroendocrinology. Eds. L.
Martini and W. F. Ganong. Academic Press, N. Y.

 

Rugh, R. (1968). The Mouse: Its Reproduction and Develop:
ment. The Burgess Publishing Co., Minneapolis, Minn.

 

Sanger, V. L., M. G. Yang and O. Mickelsen (1969). Cycad
toxicosis in chickens. J. Nat. Cancer Inst., 43:391.

Sar, M. and J. Meites (1967). Changes in pituitary prolactin
release and hypothalamic PIF content during the estrus
cycle of rats. Proc. Soc. Exp. Biol. Med., 125:1018.

Sarma, P. S. and G. Padamanaban (1969). Lathyrogens. in:
Toxic Constituents in Plant Foodstuffs. Ed. I. E. Liener.
Academic Press, N. Y.

 

Sawyer, C. H. and M. Kawakami (1959). Characteristics of
behavioral and electroencephalographic after reactions
to copulation and vaginal stimulation in the female
rabbit. Endocrinology, 65:622.

113

Schally, A. V., H. S. Lipscomb, J. M. Long, W. E. Dear and
R. Guillemin (1962). Chromatography and hormonal
activities of dog hypothalamus. Endocrinology, 70:
478.

Schally, A. V., A. Kurochuma, Y. Ishido, T. W. Redding and
C. Bowers (1965). The presence of prolactin inhibiting
factor (PIF) in extracts of beef, sheep and pig
hypothalamus. Proc. Soc. Exp. Biol. Med., 118:350.

Schemmel, R., O. Mickelsen and Z. Tolgay (1969). Dietary
obesity in rats: Influence of diet, weight, age and
sex on body composition. Am. J. Physiol., 216:373.

Selye, H. (1936). Thymus and adrenals in response of the
organism to injuries and intoxications. Brit. J. Exp.
Patol., 17:234.

Sgouris, J. T. and J. Meites (1953). Differential
inactivation of prolactin on mammary tissue from
pregnant and parturient rat. Am. J. Physiol., 175:319.

Shank, R. C. and P. N. Magee (1967). Similarities between
the biochemical actions of cycasin and dimethylnitros-
amine. Biochem. J., 105:521.

Shank, R. C. (1968). Effect of cycasin on protein synthesis.
Biochem. Biophys. Acta, 166:578.

Shimizu, M. (1969). Tissue culture of kidney tumors induces
by cycasin in rats with Special reference to Wilm's
tumors. Acta Sch. Med. Univ. Gifu, 17:35.

Sinha, Y. M. and H. A. Tucker (1968). Pituitary prolactin
content and mammary development after chronic
administration of prolactin. Proc. Soc. Exp. Biol.
Med., 138:84.

Smith, D. W. E. (1966). Mutagenicity of cycasin aglycone
(methylazoxymethanol), a naturally occurring carcinogen.
Science, 152:1273.

Spatz, M. (1964a). Effects of cycad meal and cycasin on
histochemically demonstrable liver phoSphatases and
nonSpecific esterases. Fed. Proc., 23:1381.

Spatz, M. (1964b). Carcinogenic effects of cycad meal in
guinea pigs. Fed. Proc. 23:1384.

114

Spatz, M., E. G. McDaniel and G. L. Laqueur (1966). Cycasin
excretion in conventional and germ-free rats. Proc. Soc.
Exp. Biol. Med., 121:417.

Spatz, M., W. Dougherty and D. W. E. Smith (l967a).
Teratogenic effects of methylazoxymethanol. Proc. Soc.
Exp. Biol. Med., 124:476.

Spatz, M., D. W. E. Smith, E. G. McDaniel and G. L. Laqueur
(1967b). Role of intestinal microorganisms in deter-
mining cycasin toxicity. Proc. Soc. Exp. Biol. Med.,
124:691.

Spatz, M. and G. L. Laqueur (1967). Transplacental induction
of tumors in Sprague—Dawley rats with crude cycad
material. J. Nat. Cancer Inst., 38:233.

Spatz, M. and G. L. Laqueur (1968a). Evidence for trans-
placental passage of the natural carcinogen cycasin and
its aglycone. Proc. Soc. Exp. Biol. Med., 127:281.

Spatz, M. and G. L. Laqueur (1968b). Chemical induction
of microencephaly in 2 strains of rats. Proc. Soc.
Exp. Biol. Med., 129:705.

Spatz, M. (1968). Hydrolysis of cycasin by B-D glucosidase
in Skin of newborn rats. Proc. Soc. Exp. Biol. Med.,
128:1005.

Spatz, M.,G. L. Laqueur and I. Hirono (1968). Hydrolysis
of cycasin by B-D glucosidase in subcutis of newborns.
Fed. Proc., 27:722.

Spatz, M. (1969). Toxic and carcinogenic alkylating agents
from cycads. Annals N. Y. Acad. Sci., 163:848.

Sokol, R. R. and F. J. Rohlf (1969). Biometry. W. H.
Freeman and Co., San Francisco, Cal.

Stanton, M. E. (1966). Hepatic neoplasms of aquarium fish
to Cycas circinalis. Fed. Proc., 25:661.

Steym, D. G., S. J. van der Walt and I. C. Verdoorn (1948).
The seeds of some Species of Encephalartos (cycads).
A report of their toxicity. S. African Med. J., 5:758.

Talwalker, P. K., J. Meites and H. Mizuno (1963). In vitro
inhibition of pituitary prolactin synthesis and release
by hypothalamic extracts. Am. J. Physiol., 205:213.

115

Teas, H. J., H. J. Sax and K. Sax (1965). Cycasin:
radiomimetic effect. Science, 149:541.

Teas, H. J. (1967). Cycasin synthesis in Seirarctia Echo
(LepidOptera) larve fed methylazoxymethanol. Biochem.
Biophys. Res. Commun., 26:686.

Teas, H. J. and J. D. Dyson (1967). Mutation in Dr0S0phila
by methylazoxymethanol, the aglycone of cycasin. Proc.
Soc. Exp. Biol. Med., 125:988.

Terasawa, E. and C. H. Sawyer (1968). Effects of luteinizing
hormone (LH) on multiple unit activity in the rat
hypothalamus. Fed. Proc., 27:269.

Thieret, J. W. (1958). Economic botany of the cycads.
Econ. Bot., 12:3.

Torok, B. (1964). Structure of the vascular connections
of the hypothalamo—hypophysial region. Acta. Anat.,
59:84.

van Ethen, C. H. (1969). Goitrogens. in: Toxic Constituents
in Plant Foodstuffs. Ed. I. E. Liener, Academic Press,
N. Y.

 

Vega, A. and E. A. Bell (1967). a-amino-B methylamino
pr0pionic acid, a new amino acid from seeds of Cycas
circinalis. Phytochem., 6:759.

voogot, J. L., C. L. Chen and J. Meites (1970). Serum and
pituitary prolactin levels before, during and after
puberty in female rats. Am. J. Physiol., 218:396.

Wells, W. W., M. G. Yang, W. Bolzer and O. Mickelsen (1968).
Gas liquid chromatographic analysis of cycasin in
cycad flour. Anal. Biochem., 25:325.

Welsch, C. W., A. Negro-Vilar and J. Meites (1968). Effects
of pituitary homographs on host pituitary prolactin and
hypothalamic PIF levels. Neuroendocrinology, 3:238.

Whiting, M. G. (1963). Toxicity of cycads. Econ. Bot., 17:
271.

Whiting, M. G. (1964). Food practices in ALS foci in Japan,
the Marianas and New Guinea. Fed. Proc., 23:1343.

Whiting, M. G., M. Spatz and H. Matsumoto (1966). Research
progress on cycads. Econ. Bot., 20:98.

I

116

Williams, J. N., Jr. and G. L. Laqueur (1965). Response of
liver nucleic acids and lipids in rats fed Cycas
circinalis L. endosperm or cycasin. Proc. Soc. Exp.
Biol. Med., 118:1.

Yang, M. G. and O. Mickelsen (1968). Cycad husk from Guam:
Its toxicity to rats. Econ. Bot., 22:149.

Yang, M. G., V. L. Sanger, O. Mickelsen and G. L. Laqueur
(1968). Carcinogenicity of long term feeding of cycad
husk to rats. Proc. Soc. Exp. Biol. Med., 127:1171.

Yang, M. G. and O. Mickelsen (1969). Cycads. in: Toxic
Constituents in Plant Foodstuffs. Ed. I. E. Liener,
Academic Press, N. Y.

Yang, M. G., O. Mickelsen and V. L. Sanger (1969). Cycling
of cycasin from newborn rats to their mother and back
to the newborn. Proc. Soc. Exp. Biol. Med., 131:135.

Yang, M. G. (1971). Unpublished results.

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