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BI’PHENYL Aommsmmou on
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w m MINK(MUSTELA Visomfj. _,

Dissertation for the- Degree at Ph. D; .
MtCHIGAN STATE UNIVERSITY ' ~
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Influences of Polychlorinated Biphenyl Administration on
Reproduction and Thyroid Function in Mink (Mustela vison)
James Joseph Byrne

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ABSTRACT

INFLUENCES OF POLYCHLORINATED BIPHENYL
ADMINISTRATION ON REPRODUCTION AND
THYROID FUNCTION IN MINK (MUSTELA VISON)

 

BY

James J. Byrne

Studies were designed to determine the effects of

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[longwterm feeding of a polychlorinated_biphenyl (PCB) on

thyroid function and reproduction of female mink. Plasma
thyroxine (T4) was assessed over a 9 month period which
included the reproductive season in 32 mink fed PCB
Aroclor®1254llevels of 5 ppm, 2 ppm, 0.5 ppm and un-
treated controls. A second group of 16 mink fed 5 ppm PCB
{nus controls had quantitative measurements of thyroid
function assessed at diestrus, midgestation and lactation

131I-thyroxine degradation method for estimating

using the
thyroxine secretion rate (TSR), biological half-life (tk)'
thyroxine degradation rate constant (K), thyroxine distribu—
tion space (TDS/lOO gm b.w.) and extra thyroidal thyroxine
(Ett). Plasma thyroxine (T4), thyroxine binding globulin-
caPacities (TBG) and saturation index (SI) were also
measured at the same three dates.

PCB generally increased T4 levels and peripheral deg-

radation of thyroxine except during estrus and pregnancy.

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bunk fed at the two highest PCB levels were relatively hypo-
thyroid only at estrus and the reproductive season. They
failed to bear young. The PCB fed at 0.5 ppm consistently
increased T4 levels above those of the controls in which
large increases in T4 were observed during estrus, implanta-
tion and early pregnancy. Birth rate and weaning rate were
significantly higher in the 0.5 ppm PCB group than in the
controls. Birth rate was directly correlated to relative
1% levels during estrus, implantation and early gestation.
TBG-capacity changed little in the PCB-treated animals

tmt apparent binding to other T binding proteins did in-

4
cuease as shown by a large increase in the saturation index.
Liver weights and adrenal weights were significantly
lugher in the 5 ppm PCB group than in controls. Thyroid
weight was nonsignificantly higher in the 5 ppm PCB group

than in the controls. Histologically, the thyroid revealed

anatomical evidence of stimulation in the 5 ppm PCB group.

 

1Aroclor691254--Trade name for PCB containing 54% .
chlorine, Monsanto Chemical Company, St. Louis, Missouri.

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INFLUENCES OF POLYCHLORINATED BIPHENYL

ADMINISTRATION ON REPRODUCTION AND

THXROID FUNCTION IN MINK (MUSTELA VISON)

 

BY

C? ‘ '
James J. Byrne

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Physiology

1974

 

 

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Dedicated to my wife Leslie for her
patience, understanding and invaluable
support throughout this program, includ-
ing preparation of this dissertation.

Dedicated also to my children Kim
and Rich.

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ACKNOWLEDGEMENTS

I wish to express my sincere thanks and appreciation
to Dr. E. P. Reineke for his encouragement, guidance,
mummrt and friendship throughout my doctoral program and
:hlthe preparation of this dissertation.

I would also like to thank Dr. Robert K. Ringer for
Ins friendship, advice and encouragement during my time
here.

Thanks to Marilynn Baeyens for her assistance and
cxmperation in the preparation of this dissertation.

Thanks also to Dr. Duane Ullrey and Dr. P. O. Fromm
flnrtheir helpful suggestions regarding this dissertation.

Thanks to my typists--Nancy, Joyce, Joan and Mary for
their patience and cooperation.

The author is indebted to the College of Human Medi-
cfihe, Michigan State University, which provided funds
through a General Research Support Grant No. RR-05656-06
flmmlthe General Research Support Branch Division of
fiesearch Facilities and Resources National Institutes of

ealth.

I am also indebted to the Agricultural Experiment
Station, Michigan State University, which also provided
funds for this research and supported me with a research
assistantship.

. I am deeply indebted to my friends and colleagues at
Michigan State University for their friendship and moral
Support.

 

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IJST OF TABLES .

IJST OF FIGURES.‘

INTRODUCTION . .

REVIEW OF LITERATURE

TABLE

OF CONTENTS

 

I. Polychlorinated Biphenyls.
+- Toxicology of PCB's . .
Fetotoxicity of PCB's .
Reproductive Effects of

PCB's and Thyroid Function. . .
-» Storage and Elimination of PCB's. .

m o o o

PCB'

Effects of PCB's on Organ Weight and

Function

II. Thyroid Function .
Thyroid Secretion . . .
Thyroxine Binding Proteins. . . . . . .
Effects of Temperature,

on Thyroxine Binding
Effects of Estrogen and
on Thyroxine
Thyroid Hormone
Cycle.'.
Thyroid Hormone
Thyroid Hormone

STATEMENT OF THE PROBLEM..

‘MATERIALS AND METHODS.

Binding
and the

pH and Dilution
Proteins. . . .
Sexual Maturity
Proteins. . . .
Reproductive

and Pregnancy . . . . .
and Lactation . . . . .

I. Animal Treatment Protocol.

A. Animal Care and Reproductive

B. Experiment I . . . .
C. Experiment 11. . . .

iv

Page
vii

viii

13
14
18
20

22
23
23
29
31
32
33
37
39
42
43
43
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Thyroxine Level (pg/100 ml Plasma) of Female
Mink Receiving Four Treatments (5 ppm PCB,
2 ppm PCB, 0.5 ppm PCB and a control)
from Experiment I . . . . . . . . . . . .

TABLE OF CONTENTS--continued Page
D. Experiment III . . . . . . . . . . . . 45
E 0 Experiment IV I O O I O O O I O O O O O 4 5
II. Serum Thyroxine (T4) Determination . . . . . 46
III. Thyroxine Secretion: Degradation Rate . . . 47
A. SOlutionS. I O O O O O O O O O O O O O 47
B. Injection Procedure. . . . . . . . . . 47
C. Sample Collection. . . . . . . . . . . 49
D O computations O C O O O I O O O C O O O 4 9
IV. Thyroxine Binding Globulin (TBG) Capacity
and Saturation Index. . . . . . . . . . . 52
V. Statistical Analysis . . . . . . . . . . . . 53
RESULTS I O O O C O O I O C O O O O O O O O O O O O O 54
Experiment I--Plasma Thyroxine Level Changes
with Three Levels of PCB. . . . . . . . . 54
Experiment II--Estimation of Thyroxine Secre-
tion Rate and Associated Factors. . . . . 62
Experiment III-—Male and Female Thyroxine
Levels C O O O C O O O O O O I O O O O C O 8 6
Experiment IV--Thyroxine Binding and Saturation
Index I I C O O O O I O I O O I O C O O O 8 6
Reproductive Performance. . . . . . . . . . . . 91
Anatomical Parameters . . . . . . . . . . . . . 95
DISCUSSION 0 C O | O O O O O O O O O O O O O O C O I 0 lo 3
APPENDICES O O O ‘ O O O O O O C O O O O O O I O O O O 114
A. Chemical Structure of Polychlorinated Bi-
phenyl (PCB) and Related Compounds. . . . 114
B. BaSiC Mink Diet. 0 C O O I O O C O O O O O O 115
C. Technique for Measuring Binding Capacity of
TBG O I O O O O C O O C O I O O O O O O O 116

124

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TABLE OF CONTENTS--continued Page

E. Thyroid Parameters of Female Mink from
Experiment II o o o o o o o o o o o o o 0 126

F. Thyroxine Levels (pg/100 m1 Plasma) of Male
and Female Mink from Experiment III. . . 132

G. Female Mink Body Weights (Grams) of Animals
in Experiment I. . . . . . . . . . . . . 134

H. Formula Used in the Scheffé F Ratio . . . . 135

REFERENCES. 0 0 O O O O O O O O O O O O O O O O O O 136

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LIST OF TABLES

Factorial Analysis of Variance Table of Data
in Experiment I O O O O O O O O O O O I O O O 0

Statistical Comparison of Data in Experiment I
Using the Scheffé F Test . . . . . . . . . . .

Summary of Thyroid Parameters. .Means and
Standard Errors of the Means from Experiment I

Factorial Analysis of Variance Table: TSR—TDR
Data ' Experiment II 0 O O O O O O O O O O I O 0

Statistical Comparison of Data in Experiment
II (TSR-TDR) Using the Scheffé F Ratio . . . .

Saturation Index (SI) and TBS-Capacity of
Thyroxine Binding Globulin (Experiment IV) . .

Reproduction of Mink in Experiments I and II .

Body Weights and Organ Weights of Mink in
Experiment II 0 O O O O O C O O O O O O O O O 0

vii

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56

63

64

72

94

96

97

 

 

 

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LIST OF FIGURES

FIGURE

1.

Effects of long term ingestion of polychlori-
nated biphenyl upon circulating thyroxine in
female mink. Each point equals the mean
plasma thyroxine (Hg/100 m1 plasma) : the
standard error of the mean of 8 animals . . .

131I-thyroxine degradation regression line
and associated parameters of female mink con-
trols not receiving polychlorinated biphenyl.

131I-thyfoxine degradation regression line
and associated parameters of pre-estrus
female mink controls and those receiving 5
ppm polychlorinated biphenyl. . . . . . . . .

131I-thyroxine degradation regression line

and associated parameters of pregnant female
mink controls and those receiving 5 ppm poly-
chlorinated biphenyl. . . . . . . . . . . . .

l3J'I-thyroxine degradation regression line
and associated parameters of lactating and
non-lactating female mink controls and those
receiving 5 ppm polychlorinated biphenyl. . .

Summary of 131I-thyroxine degradation regres-
sion lines for control mink . . . . . . . . .

Summary of 131I-thyroxine degradation regres-
sion lines for mink receiving 5 ppm poly-
chlorinated biphenyl. . . . . . . . . . . . .

Comparison of plasma thyroxine (Hg/100 ml
plasma) of male and female mink measured at
periodic intervals of the year. . . . . . . .

Thyroxine binding curve of plasma measured on
five mink in December. Each point represents
the mean : standard error for thyroxine (ug/

100 m1 plasma) bound to protein . . . . . . .

viii

Page

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68

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LIST OF FIGURES—“continued

FIGURE ' Page

10. Saturation index of plasma from female mink.
SI = plasma thyroxine level in ug/lOO ml
plasma/thyroxine binding globulin capacity. . 93

ll. Thyroid gland photomicrograph from a control
mink during mid-lactation (4-18-74) (magni-
fied 250 timeS) o o o o o o o o o o o o o o o 100

12. Thyroid gland photomicrograph from a female

mink receiving 5 ppm polychlorinated biphenyl
(4-18-74) (magnified 250 times) . . . . . . . 101

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INTRODUCTION

The thyroid gland affects the structure and function
of nearly every tissue within the body. Thyroid hormones
have an effect on both major control systems--the nervous
system and the endocrine system. Both systems are depend-
ent upon thyroxine complementation for their "target"
tissue integrity and also for their own cellular integrity.
Thyroid hormones are involved in major biological events
such as growth and development, basal metabolism, tempera-
ture regulation, reproduction, lactation and numerous
intracellular events upon which the gross events depend.

Because of the many bioeffects of thyroid hormones,
estimations of thyroid function are an excellent index of
an animal's well-being.

During the brief history of endocrinology several
methods have been used in an attempt to establish reliable
indices of thyroid function. Among them thyroid secretion
rate, serum thyroxine, thyroxine degradation rates,

thyroxine-binding capacity of T binding proteins, protein

4
bound iodine and thyroidal uptake of iodine have been the
most successfully employed. Effects of external factors

on the thyroid when mirrored by the indirect estimates of

thyroid function provide insight into the probable effects

 

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upon the body of a treatment and its associated biological
events. Because the thyroid is so essential in modifying
the major systems, any factor suspected of affecting its
function or that of its hormones should be investigated.
Polychlorinated biphenyls (PCB's) are one such factor.

PCB's have been implicated in morphological changes in
the thyroid gland of lower animals and are known to affect
liver enzymes in diverse species. Although PCB's chemically
resemble such pesticides as DDT, DDE and dieldrin they are
used for quite different purposes and their physiological

effects are probably not identical.

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REVIEW OF LITERATURE

I. Polychlorinated Biphenyls

 

During the last three decades the world has been under—
going a synthetic chemical revolution. Large quantities
of synthetic organic compounds have been discharged into the
kflosphere. Reliance has been placed on dilution and degra—
dation to dispose of them, but with insufficient knowledge
of their natural degradation rates. Within the last half
dozen years the production of these chemicals has exceeded
their dilutability. Many of the man-made chemicals have
effects far beyond their original intent. Many have bio-
logical and toxicological effects upon vertebrates as well
as invertebrates. The ability to synthesize and put these
chemicals into use has by far exceeded the research into
their apparent multifold biological effects. For a number
of years research centered around DDT, DDE, dieldrin and
related pesticides which are naturally suspected to cause
environmental problems. Most recently the scope of the
investigations has been enlarged, and new culprits and many
new suspects are now being investigated. Polychlorinated
biphenyls (PCB's) along with other halogenated biphenyls

comprise such a group.

 

 

 

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PCB's have found their way into the environment by
nmny routes. They are transported throughout the ecosystem
tmth aquatically and terrestrially. Their properties of
lipid solubility and chemical solubility have contributed
'UJPCB's ubiquitous presence in the environment and allows

fitmem to enter easily into food chains.

Polychlorinated biphenyls, as the name implies, are

{cmmprised of a biphenyl (2 covalently bonded benzene rings)

ith chlorine substitutions. There are ten possible sites

 
    

3 Lfbr attachment of chlorine; however, anywhere from.four to

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\j eight of the available carbons may be chlorinated (Jensen,
‘4970). With four to eight sites available there is a po-
hential for 102 different isomers.

The environmental hazard of a chemical does not neces-
sarily lie in its benzene ring structure and chlorine atoms
alone (Bitman and Cecil, 1970). Dustman §E_§l. (1971)
pointed out that DDT, DDE, and methoxychlor (see Appendix A)
all have similar basic structures (dibenzene) but methoxy~
chlor is rapidly broken down and is rarely found in mammals.
It seems that toxicity of a chemical is then a function of
its specific structure not necessarily the quantity of ben-
zene, chlorine or carbon present.

Identification of PCB's in the environment came about
slowly. Part of the problem was the confounding of PCB's

with known pesticides, and also because PCB's are comprised

 

 

 

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of many different compounds and isomers which produce many
(fifferent peaks upon gas chromatography and spectrographic
analysis. Albro and Fishbein (1972) and Zitko & Choi (1972),
reported that variation in detection response to each com-
pmnent on chromatograms complicated the number of peaks
pmesent in quantitative analysis for PCB's. Although a
specific mixture of PCB's will elicit a given set of peaks,
any variation in the composition will unpredictably change
the total peak response. This problem becomes further
complicated when analyzing biological material for PCB's
for the mixtures are variable and random and are not easily
Hatched with commercial PCB mixtures. Simmons and Tatton
(1967) reported that organochlorine pesticides produce gas
chromatographic peaks which coincide with the peak produced
by PCB's, further complicating analysis for PCB. DDT and
DDE have peaks very much like PCB's. Soren Jensen (1966)
while looking at DDT and DDE was the first to identify

PCB's as a "troublesome unknown" which some previous authors

had reported but had not identified. The separation of
{other organochlorides from PCB's was done by several methods

:Simmons and Tatton, 1967; Holden and Marsden (1969). It is

more difficult than analysis for organochloride, by_gas
chromatography (Risebrough et al., 1969) and by thin-layer
chromatography procedures (Mulhern et al., 1971).

Mass spectrography was the first technique used to

iconfirm and identify a given gas liquid chromatographic

 

 

 

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{flGLC) peak as due to a PCB and determine the number of

f‘

hflflprine atoms per molecule. Jensen (1966,1970), was the

\
first to identify PCB's in biological tissue. Koeman

2343;. (1969), using Jensen's method, affirmed PCB presence
in Norwegian wildlife. Stalling (1971) used the mass
spectrograph but preceded it with a chromatograph to effect
aiseparation of the various PCB isomers. Bagley gt_al.
U970), identified 18 PCB isomers and Biros et_§1. (1970)
identified PCB's in human tissue and hair. Both used mass
spectrography. Confoundment of DDT by PCB's is not simply
a function of their equally widespread distribution but
recent studies by Maugh (1973) have shown that irradiation
of DDT vapor using ultra violet light, approximating sun-
light at ambient intensity, precipitates conversion of DDT
to PCB. The major step of this organic transformation
seems to be the removal of the ethane group from between
the benzene rings (see Appendix A for comparative struc-

tures).

X, Polychlorinated biphenyls were first described by
ESchmidt and Schultz in 1881 (Peakall and Lincer, 1970) and
Ewere first introduced in 1929 as flame resistant electrical

ES ,;transformers and conducters (Dustman et al., 1971).
I +

Penning in 1930 (Peakall and Lincer, 1970) expounded on
their physical and commercial characteristics and additional
practical applications. Since that time and particularly

since WOrld war II the use of various PCB's has expanded in

 

 

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explosive proportions. They are widely used as plasticizers,

‘ in all types of plastics, heat transfer agents, hydraulic

fluids, protective coatings (Rhee and Plapp, 1973) marine

:mnfifouling paints, in cardboard cartons (Bailey et al.,

1970), as "inert" ingredients or carriers for insecticides,
as dustallayers in detergents, and as vapor suppressors in

spray compounds, carbon paper, mimeograph fluids and typing

ink.

  

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PCB's are highly valued for their qualities of viscosity,
éat conduction, water resistance, flexibility, and adhesion
pupperties. They are marketed and manufactured by companies
throughout the world. Monsanto is the major United States
producer under the tradename Aroclor. France produces
Clophen and Japan produces Kanechlor. Great Britian, Italy,

USSR, and Czechoslovakia also have PCB manufacturers. PCB's

 

are marketed using differing percentages of chlorine depend-

_...1A.__.-._

 

ing primarily on the_purpose. An increase in percent

 

chlorine increases the viscosity of the PCB. Above 60%
chlorine content, the PCB's pass from the amorphous state

to a pliable resin. Monsanto's Aroclor is designated by a

4 digit number, the first two digits are a trade designation,
the second two digits indicate the percent by weight of
chlorine. For example, Aroclor 1254 contains 54% chlorine.

Q
The range of chlorine in commercially available chlorinated

abiphenyls is from 28% to 68%.

 

 

 

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The United States produces hundreds of millions of
gmunds of PCB's annually and because few product manufan
turers list the ingredients of their products, PCB uses are
not well-known. Production secrets also contribute to

some of the mystery surrounding this widely sold product.
oy
$54,1he particular environmental disadvantage of PCB's is a

4
“fix“ gresult of their lipid solubility and their low degradabilv
ivy ity, both of which contribute to their longevity and inv
creased concentration of the food chain.
, / S Pwlahsiayax3Riotshensnvironmenfgén emperous
K \mgys, many of which are extremely difficult to trace;

however, with their widespread use it is easy to guess some
of the routes of contamination. The major cause is probably
slovenly handling of individual, industrial and municipal
wastes. In several areas of the world, PCB contamination
has been traced back to this source. Koeman et_gl, (1969),
in the Netherlands, found that PCB's present in seabirds
and fish on the European Atlantic coast came from discharge
of the highly polluted Rhine River. Duke g£_gl, (1970),

in the United States, found the source of mollusk and fish
PCB contamination at a factory which was leaking PCB's into
the river. Holden (1970), on Scotlands Atlantic coast,
found that sludge from sewage treatment plants which was
being dumped in a deep water estuary contaminated shellfish
and marine life nearby. PCB's have been detected in both

raw and processed foodstuffs (Gustafson, 1970) and in milk

 

 

 

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1973) .

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from both cows and humans (Westwoo et al., 1970; Fries
et al., 1971; Platonow and Funnel, 1971; and Platonow and

Chen, 1973).

The incidence of PCB's and other halogenated biphenyls

resulting in contamination of animal species, including

Inmn are both numerous and world-wide. The following are a
ifew of the species contaminated: Fish and sea life (Jensen,

51966; Jensen et al., 1969; Koeman, 1969), brook trout

(Hutzinger et al., 1972), sediment and biota of lakes

(Duke et al., 1970), cormorants, pelicans (Anderson et al.,

‘1969), pheasants (Dahlgren et al., 1971), bald eagles

(Mulhern et al., 1970; Reichel et al., 1969), ducks (Friend

;& Trainer, 1970), seals and porpoises (Holdin and Marsden,

'1967), cattle and swine (Platonow & Tunnel, 1971; Platonow

get al., 1972), mink (Aulerich et al., 1972) and in human

ladipose tissue (Biros et al., 1970).

(

Toxicology of PCB's

Toxicity of PCB's to industrial workers has been known
for a number of years. Jones and Alden (1936), Schwartz
(1936) and Meigs et a1. (1954) described a disease named

chloracne which was an occupational disease of PCB-industry

,workers. Crow (1970) and Kuratsune et al. (1972) reported

that consumption of rice-bran oil accidently contaminated

‘~+\ with PCB's produced the disease "yusho" in Fukuska-Ken,

“Mi-“’4‘

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1.

\Japan. The most common symptoms were eye discharge,

 

 

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”'1 :follicular accentuation, acnevform eruptions, sweating
\+”%*igmlms, weakness and pigmentation of the skin and nails.

It was not until recently, however, when it was found
{that PCB's are toxic at much lower concentrations than had
‘2;' ibeen previously imagined. Voss and Notenboom-Ram (1972)
K95“; administered PCB's topically to rabbits upon a small shaved
iarea of approximately 5 by 10 cm. After 4 weeks of Aroclor
!1260 (20 applications of 120 mg PCB) microsc0pic examina-

: tion of the liver showed degeneration of some cell membranes
2 and cytoplasm with decreased glycogen stores and damaged
‘endoplasmic reticulum. Liver weight also increased and acne
(chloracne) was noticed.

./ Heath et_§1. (1972) tested in birds 6 PCB mixtures

(ranging from 32 to 62 percent chlorine. Although each

x
y

L// a species had a specific sensitivity to PCB's, increased
_%y§3 (toxicity was associated with increased chlorine percent in
E the mixtures. Toxicities of PCB were also similar to those

of DDE. Bobwhite quail were most sensitive, followed in

-_

iturn by pheasants, mallards, and coturnix. Birds became
Elethargic during PCB administration and tended to assume a
‘crouching position. They displayed mild tremors during the
last hours of the experiment. Heath gt_§l. also found that
the toxic effects of PCB 1254 were additive to those of DDE
when fed concurrently. Aulerich et_al. (1973) and Platon0w

c t. H

/I\4 * 3and Karstad (1972) reported that very low levels of PCB
W“ ' "

1(below 5 ppm) resulted in mortality in mink fed PCB's for

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11

several months.

Prestt gE_§l. (1970) found that PCB 1254 was about
one—third as toxic as DDT in Bengalese finches. Dahlgren
eE_a£. (1972) found that the degree of PCB sensitivity in
a species was variable and depended upon the individual
animal. For example, some birds died 30 days following a
10 mg per day dose while a few birds of the same species
lived until sacrificed. Eight months later they noted that
brain tissue levels of PCB's were much more useful for
diagnosing cause of death than either.musc1e or liver levels.
They reported that brain PCB residues are 300 to 400 ppm
in a majority of deaths, which suggests a range or level to

use as an index of PCB—induced mortality.

W3k_ {—a- Hattula and Karlog (1972) studied veilvtailed goldfish

tax, (

M n a\ , 1..- Im- --¢. \Ja—‘W
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in aluminum-lined aquaria which continuously contained from
0, 0.5, 1.5, 2.0, 2.5 and 4.0 ppm Clophen A 50 (Bayer) (50%
chlorine). When PCB concentration was regressed against
time on a semi—log scale the 50% mortality was linear and
had a highly correlated negative slope. The LD50 at 20 days
was 0.5 ppm PCB 1254 and the LD50 at 5 days was 4.0 ppm PCB

1254. Harmful effects of PCB were ea31ly observed. The

—_~__ I “MM new.
h.

bright orange color turned pale yellow and the fish 195t

.J
; their appetiteSaf Nervous System effects were primarily
l ,. .,
\ uncoordinated movements.
Q.“ h( Koller and Zinkel (1973) administered PCB's 1221, 1242,
\ 089v

(and 1254 once each week for 14 weeks to adult rabbits.

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fLivers of the 1254“ and 1242-treated rabbits were signifi-
, cantly enlarged compared to the 1221 group and controls.

4 .@ Megalohepatocytosis and necrosis of the midzone of the

A.)
r .- '

' liver was observed in animals on the two highest treatment
1 levels. Fibrous connective tissue filled the necrotic

{ zones. Rough endoplasmic reticulum in the ¥254 group livers

A A. w _ .. un-
——-— ——-v-—-—4-- u-._..---

: appeared to have been destroyed. Uteri of the 1254 group

-_...”c -.-—n 4“”.‘ .424»;th II >..-.

‘had also atrophied.

 

Lichtenstein et al.(l969) found that in Dipteran in-

, \ sects (flies) PCB's were toxic but percent chlorination was

‘2)
\ ' - .
§\ ‘ X inversely related to mortality which is opposite that found

\\in birds. The toxic effects of PCB's above 48% are very
low in house flies. PCB toxicity was more than just addi-
tive to those of DDT or dieldrin. Rhee and Plapp (1973),
found that induction of microsomal enzymes of some strains
of house flies was directly proportional to the percent

chlorine in the PCB.

) f Crustaceans and mollusks are extremely sensitive to

if ;
HNiyi PCB's. Duke et al. (1970) found that 48 hour exposure to

1 .
(100 ppb of Aroclor 1254 was a lethal dose for shrimp. In

3
124 hours 80% were dead. Accumulation of 3.9 ppm PCB was

found in the tissues, but in shrimp accumulating 1.3 ppm
PCB at a treatment level of 10 ppb none died. Exposure for
20 days at 5 ppb Aroclor 1254 killed 72 percent of juvenile
shrimp even though the tissues had accumulated 16 ppm.

Crabs were less sensitive for they received 5 ppb for 28 days

 

 

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and accumulated 23 ppm but did not die. Oyster shell

“ifiw—ufi w.-.

/,g¢/'§ growth was completely stOpped after 96 hours at 100 ppb,

«-. ..
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v L_but a 10 fold reduction in PCB only reduced shell growth to
40% of normal.

Tissue cell cultures from Chinese hamster (quasi-
diploid epithelial cells) were found to be most sensitive
to PCB 1016 which was a distillate of PCB 1254. As the
percent chlorine was decreased, toxicity increased. In
human lymphocyte cultures Aroclor 1254 at 100 ppm caused
no apparent effect upon chromosomal integrity as measured

by cytological evidence (Hoopingarner et al., 1972).

Fetotoxicity of PCB's

.-
ll

Fetotoxicity of PCB 1254 has been demonstrated by

dka ‘jVilleneuve et a1. (1971). Doses of 12.5 to 50 mg/kg/day,

(“4* administered orally, induced abortions and were toxic to

«70

\
1

_rabbit fetuses during the first 28 days of gestation.
Doses of 10 mg/kg/day were insufficient to induce abortion.
Aroclor 1221 at up to 25 mg/kg/day produced no fetotoxic
effects. Dead fetuses whose mothers had received 12.5
mg/kg/day showed no skeletal abnormalities. Rats treated
with up to 100 mg/kg/day PCB 1254 did not have fetal deaths
/nor malformations. McLaughlin gg_al. (1963) injected PCB

(
r
/(&V ) 1242 into chicken eggs at concentrations of 10 and 25 mg

(‘3-

sru! and found that growth was retarded, beak development was

\\ \ affected and they had only 0 to 5% hatchability.

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Grant gE_gl. (1971) found that PCB*S 1221 and 1254 crossed
rabbit placentae when administered orally during gestation.
There was also a direct correlation between amounts of PCB
given and the amount concentrated in the liver and fat
depots of fetuses and the does. Fetal liver had much
higher concentrations of PCB than the doe's liver. Aroclor
1254 accumulated to a much greater extent than did Aroclor
1221. The difference in fetal and doe liver levels might
be a result of the fetus not possessing the full comple-
ment of enzymes necessary for degradation of PCB's.
Villeneuve gE_§l. (1971) found that adult and fetal livers
did not differ in protein or carboxylesterase when dosed
for 28 days with 0, 1, or 10 mg/kg/day of Aroclor 1221 or
1254; however, 10 mg/kg/day of Aroclor 1254 induced micro-
somal enzymes (carboxylesterase, aniline hydroxylase and
aminopyrine n-demethylase) in the dam (Grand gt_§l., 1971).
Eckhoff (1972) reviewed the mechanisms of transplacental
passage of drugs and exogenous compounds and indicated that
most exogenous molecules, particularly lipid soluble ones,

cross the placenta by simple diffusion. Platanow and Chen

u...— an
“H .v' . 4.x

1;} (1973) reported transplacental transfer of PCB 1254 in the

cow and fetal PCB concentrations were higher than maternal

only in kidney tissue.

Reproductive Effects of PCB's

 

PCB's were first shown to affect reproduction in

mammals by Gilbert (1969) and later by Aulerich et a1.

 

 

 

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15

(1971), both in mink which had been fed PCBvcontaminated
fish. Ringer gt_al. (1972) fed PCB's to mink in approxiv
i mately the same levels as those contained in Lake Michigan
~k~ ) Coho salmon (10 ppm), demonstrating and confirming previous
Poi; { suspicions that PCB's were a causative agent in reproduc-
‘Vk \ tive failure of mink.
» Orberg & Kihlstrom (1973) and Kihlstrom g£_a1, (1973)
found that PCB as well as DDT significantly lengthened the

estrous cycle of rodents and suppressed the appearance of

vaginal cornified epithelial cells. In an experiment using

m"._h.._.u ,.
"""-~N- vu- ugNI WW '5. .-

,.mdce, Kihlstrom et a1. (1973) found that adult mice which

‘,IM,—-n—_

._.

had been suckling PCB- contaminated milk as neonates, when

pufg,. mated, demonstrated a significant decrease in the frequency
$34{ of implanted ova. Adult rats also maintained on PCB
V$J( (Clophen A 60, 20 mg/kg body weight) showed a decrease in

\¢JM implanted ova. These studies imply an estrogenic effect of
PCB's since any alteration in the sensitive balance of
estrogen and progesterone would interfere with either
implantation or estrus or both.

BitmgflwangflCecil'ilgzO) suggested that the geometric
similarity of PCB and DDT to the synthetic estrogen di-
ethylstilbestrol (DES) may be functional as well as struc-
tural. Using the sensitive 18 hour glycogen response of
the<ra£2u§erus, they found that DDT is about 1000 times

less estrogenic than natural estrogen. They found that

active estrogenicity is dependent upon the presence of at

 

 

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16

least one phenolic hydroxy ring structure. PCB's were found
to be at least as estrogenic as DDT. PCB's of lower chlori-
nation (21-48 percent) were more estrogenic in rats than

the higher chlorinated (54-68 percent).

In addition to being naturally estrogenic in nature

2 PCB' s affect enzyme systems which govern steroid levels.

_ *1.” .... ”‘4,
vw"

Plantnow et al. (1972) have found that PCB's markedly reduce

~r—- ._.. J’WW‘D

 

 

the urinary excretion level of gonadal steroids in subjects
receiving daily oral doses of PCB (1254, 10 mg/kg b.w.) and
receiving a single oral dose (100 mg/kg b.w.). PCB's also
have the capacitytoenhance steroidfhydroxylating enzyme
actiVltYEégféhe mer- R%sebr°ugh et a____1.-r 9968,!Beam???“
that PCB enzyme induction increased estradiol metabolism in

pigeons. Peakall and Lincer (1970) reported that PCB in-

creases in vitro metabolism of estradiol and also increases

 

cytoplasmic RNA in birds (American kestrels) which had been

Kmaintained on 0.5 and 5 ppm PCB.

Q ’5

’b

‘5

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'3

Birds have been found to be particularly sensitive to
PCB's. Rehfeld et al. (1971) and Plantnow and Funnel (1972)

have reported that PCB' s lnhlblted secondary sexual charac-

teristics in chickens Which was manifested in reduced comb

{and testicular development. Kolbye (1972) reported that

ngBécontaminated chicken eggs had alarmingly_reducedthatch-

? ability. Data from Plantnow and Reinhart (1973) showed that

~u-q-auuu-b‘ ’“'I‘ .w'

the concentrations of PCB in eggs inversely reflects the

rate at which eggs are produced. When 50 ppm PCB (1254) are

*.__~ ‘.

 

 

( '_.."

- '5‘" l“ n,"

 

 

aiiez' to the die‘
3.-- 953, no ef
£2: PCB levels

5:: L“. hatcnas 1

:::;::ed mainly

Heath et a:

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.:,::.: gct‘rcn c f

 

‘34

I

17

IQ¥*' added to the diet, hatchability is severely affected but at

5 ppm PCB, no effect is seen on hatchability of fertile eggs.
When PCB levels surpassed 15 ppm, an instantaneous depres-
sion in hatchability was noted. Embryonic mortality
occurred mainly in the latter stages of incubation during
the first 2 weeks PCB's were fed to hens. After 2 weeks,
embryo mortality occurred mainly in the early embryonic
stage.

Heath et_§l. (1972) reported that Aroclor 1242 affected
reproduction of White Leghorns at 10 ppm, but much higher
dosages were required to alter reproduction (100 ppm).
Neither egg shell thickness nor reproduction were affected
in Mallards or Bobwhite quail receiving 25 ppm and 50 ppm
(1254).

Peakall (1971) was first to report the lack of effect
of low level PCB upon egg shell thickness. Anderson gt_al,
(1969) in cormorants and wild pelicans reported that DDE
residues in eggs correlated better with shell thinning than
did residues of PCB or dieldrin. Dustman et_al. (1971)
reported evidence from the Industrial Bio-test Laboratories
which was conducting research for Monsanto Chemical Company.
They found Aroclor 1254 athO ppm and Aroclor 1242 at 10
ppm or 100 ppm in the diet of chickens, caused thinning of

we... .'...r,..

eggshells and reduced egghatchability and production. The

. -.' H..:,_.u--- .

information concerning the effects of PCB's upon bird repro-

duction, egg production, hatchability and shell strength is

t

 

 

'7‘ ‘mw ""14: "d"

 

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pd.

: :"‘""t of PC

many ‘0

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18

not simple or clear-cut. The following four factors are

H the determinants of PCB function: 1) which PCB is used,

.9.

* J 2) amount of PCB used, 3) species of test animal, 4) dura-
\

(\tion factor of administration.

PCB's and Thyroid Function

 

Very recently, PCB's have been implicated in avian
thyroid weight and 1311 uptake. Jeffries and Parslow (1972)
reported that thyroid weights of black billed gulls which
had been reared in pens and received daily dosages of 50,
100, 200, 400 mg/kg 1254, increased significantly from a
mean of 30.5 i 1.81 mg to 40.28 i 2.31, an increase of 32%
over controls. At these four high dosages, no difference in
effect from one PCB dosage to the next was observed. They
also reported that thyroid follicles were larger and con-
tained more colloidal space per follicle. This is differr
ent from DDT treated animals which have almost complete
colloidal loss and hyperplasia. The PCB-induced colloidal
increase amounted to a 22% increase in area. Most recently
Hurst §E_§l. (1974) found that Bobwhite quail responded to
PCB Aroclor 1260 by a decrease in thyroid size at low dosage
levels and to stimulate thyroid growth at high dose levels
(dosages 5, 50, 500 ppm). The enlarged glands of the high-
est PCB group took up more 131I than controls but when cal-

culated on the basis of thyroid weight, no difference was

observed. Thyroids of the two lowest level PCB groups took

 

 

’
n

-"‘::;5. tissue 1

:j::'.i size an

:2: the water

.332: (197 3) 1:

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19

up the same amount as the controls but each unit weight of

thyroid tissue took up less 131I. DDT and toxaphene stimu-

lated 1311 uptake in the Bobwhite and increased thyroid

weight. Channel catfish have also been known to increase

thyroid size and 131

I uptake upon introduction of PCB (1254)
into the water (Mayer et_al., 1972).

In addition to the numerous reports of PCB-caused liver
weight increases, PCB's also have been implicated in liver
enzyme induction. Risebrough gt_al. (1968) and Peakall and
Lincer (1970) have been cited earlier for their work on
steroid-hydroxylating enzyme and for ig_zit£g_estradiol
metabolism by liver tissue. Benthe §t_§l, (1972) reported
that PCB's and stress stimulate rat liver microsomal drug-
oxidizing enzymes and that tetrachlorobiphenyls are dis-

p'u’.

tinctly more effective enzyme inducers than dichlorobiphenyls.

11(emf/Rhee and Plapp (1973) found that PCB 1254 was a more effec-

. .— New
...1-"""‘" "

[ tive inducer of the microsomal enzyme aldrin epoxidase than

(“Jawm—J-fl 'c. a, _

 

“wt-d

 

1242 and 1221 in the house fly. The rate of 1nduct1on

.9141“ ‘ ’/ WNW. ”-

Lgdecreased with decreasing percent chlorine. They have also

.- “I“... H" _,1' ml (“W-Du
n.- W“ _‘

 

pointed out that differences among house fly strains exist

in 1) dose dependency, and 2) the time course of enzyme

induction.
3’ Liver microsomal cytochrome P-450 complex was signifi-
foiaéfi cantly increased by Aroclor 1254 (Konat and Clausen, 1973)
.§&?.E in mice. Mixed function oxygenase was also increased.
0*? .

{ PCB 1254 stimulated proliferation of liver endoplasmic

 

. .mii

 

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20

reticulum as ascertained by the evaluation of increased
microsomal protein content. Both lindane and PCB 1254
stimulated liver microsomal esterase. Aroclor 1254 was
found to be a better general stimulator of liver toxin
metabolism than lindane, and DDT was the least stimulatory.
PCB's were found not to change brain cytoplasmic esterases

but to act primarily on liver enzymes.

Storage and Elimination of PCB's

PCB 1254 was found to be concentrated in the liver of
the pheasant (Dahlgren gt_al., 1971) after feeding of
Aroclor 1254. The second and third highest levels were
found in brain and muscle,respectively. Administration of
Aroclor 1254 as a 50 mg capsule resulted in a 94% absorp-
tion during the first 24 hours and of the PCB absorbed,
410 mg was excreted in feces and 4.2 mg was excreted in the
eggs. In pheasants receiving a single 50 mg capsule of
1254, 1.5 mg PCB was found per egg 2 weeks after adminis-
tration. A total 40.5 mg of a single 50 mg PCB dose was
found by whole body analysis after 24 days. In mink,
Plantnow and Karstad (1972) found that PCB's were concen-
trated at the highest level in the liver and lowest in
blood. Heart usually had more than skeletal muscle.

Analysis of tissue extracts for Aroclor 1254 in the
Bobwhite quail, using mass spectrography, was found by
Bagley and Cromartie (1973) to contain all the relative

peak heights associated with Aroclor 1254, suggesting that

r!
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some

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21

all components of the Aroclor were readily absorbed.
Fourteen days after Aroclor 1254 feeding a methodical elim—
ination of certain components and an increase in others was
observed. Their mass spectroscopic (MS) findings were con-
firmed using gas liquid chromatography (GLC). The chroma-
tographs show that a dynamic system was at work completely
removing some chemical components while increasing others.
No foreign chlorinated isomers were observed, but isomeri—
zation of Aroclor 1254 is strongly suggested by the changing
MS and GLC peaks. Grant gt_al. (1971) in a study of Aroclor
1254 metabolism in male rats found that GLC-electron capture
patterns of the PCB residues were significantly different
from those of the standards.

Bagley and Cromartie (1973) reported that after 42
days post-treatment components of Aroclor 1254 were hardly
in evidence but several large peaks were observed. Mass
spectrographic analysis showed butyl esters of short-chain
fatty acids. It was thought that these were thermal degra-
dation products. They were observed only in PCB-treated
birds. Bagley and Cromartie (1973) suggested that they
might be related to the "fatty degeneration" observed by
Vos and Koeman (1970).

Biros §E_gl. (1970), reported that human adipose tis-
sue samples (2 humans) examined by combined gas chromatog-
raphy-mass spectrometry were found to contain substantial

quantities of PCB's ranging from pentachlorobiphenyl to

 

:;fl

 

WYATHAU '~ ‘ (l"f"" ‘ ‘—

 

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4‘: in forms

 

 

Lyn" .
T gorease as a function of PCB treatment, particularly with

22

decachlorobiphenyl including at least 14 isomers and homo-
logs; however, the origin of the PCB compounds was not
known.

Metabolism and excretion of pure PCB's was undertaken
by Hutzinger §E_al. (1972). They administered mono-, di-,
tetra-, and hexachlorinated biphenyl isomers to pigeons,
rats and brook trout. They found that excreta when examined
by chromatographic and mass spectrometric techniques showed
conversions of 4 chloro-, 4,4'-di-chloro and 2,2',5,5'-
tetrachlorobiphenyl isomers into monohydroxylated deriva-
tives by rats and pigeons but brook trout excreta contained
no hydroxymetabolites. None of the excreta of species
examined contained 2,2,4,4',5,5'-hexachlorinated biphenyls.
This study confirmed the suspicion that PCB's are elimi—
nated in forms other than those injected.

Effects of PCB's on Organ Weight and
Function

 

 

(r Liver weight has been shown in numerous studies to in-
) M-MAM"HIHH- .. , .

 

-

Aroclor 1242 and 1254 (Grant gt_al., 1971; Rehfeld gt_al.,
1971; Platonow and Funnel, 1971; Lincer and Peakall, 1973;
Bitman 2E_§l., 1972; Cecil gt_al., 1973; Abrahanson and
Allen, 1973; Dahlgren gt_§l., 1972; Voss and Beems, 1971).
The effect of PCB's in other organs is not so clear-
cut. The following organs were found to have weight changes

with PCB administration: The heart weight decreased

I

 

*—‘~'en et al.

.c
a“). ‘J O

 

 

2.; Panel , 1 97-

 

1:1 a most

 

 

 

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23

(Dahlgren et al., 1972; Iturri, 1974), spleen weight de-
creased (Dahlgren et al., 1972; Flick et al., 1965; Grant

et al., 1971), adrenal weight increased (Flick et al.,

 

1965), kidney weight increased (Dahlgren et_al., 1972;
Prestt §£_al., 1970), testis weight decreased (Platonow
and Funnel, 1971).

In a most recent study by Iturri (1974) PCB's were
implicated in cardiovascular and hematological problems in
birds. PCB's 1242 and 1254 at 100 ppm reduced heart rate
and the former also decreased blood pressure. Two PCB's
also produced abnormal electrocardiograms in White Leghorn
cockerals (inverted T-waves, prominent T or P waves and
lower 8 waves). Hematocrit, hemoglobin concentration and
total erythrocyte concentrations were decreased and the two
PCB's also produced observable anemia. Arterial blood pH
was found to decrease (1254) but plasma K+ concentration
was significantly higher (1242). Although Na+ did not
change in arterial blood, both Na+ and K+ concentrations

were increased in pericardial fluid. Hydropericardia was

also observed without producing bradycardia.

II. Thyroid Function

Thyroid Secretion

 

Estimation of thyroxine secretion rate (TSR) has been

attempted in vivo in numerous manners on many varied animal

 

 

 

 

 

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24

species. Bioassay technique by the goiter prevention method
was used first by Dempsey and Astwood (1943) and by Mixner
g£_§1. (1944). In this method a goitrogen is administered
to test animals; this suppresses thyroid hormone formation
and compensatorially results in an increased thyroid stimu-
lating hormone (TSH) release. Increased TSH then stimulates
thyroid growth. However, known quantities of thyroxine plus
unknowns can be administered to suppress TSH release and

the resultant change in thyroid weight of the goitrogen-
receiving animal can be compared with the controls. The
daily dosage of thyroxine to animals receiving the goitrogen
which results in thyroid weight equal to the untreated
groups is assumed to be the daily rate of thyroxine secre-
tion.

A thyroxine substitution method for estimating TSR was
first described by Perry (1951) in rats and Henneman,
Griffin and Reineke (1952) in sheep. A modified method
has been developed for rats (Reineke and Singh, 1955) and
several other species (Reineke,l959). It had great ad-
vantage over the goiter prevention method in that it used
fewer animals and it was the first method based on isotopes
of iodine. The method involves three important points:

1) the thyroid will rapidly accumulate "trap" a large
amount of exogenous iodine, 2) exogenous thyroxine will
block release of thyroxine from the thyroid in proportion

to the dose of thyroxine given, and 3) labeled iodine is

k 'V'
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dtOt

e

IA 5
0’" "
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vuv.\v .‘Vsk

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25

converted to thyroxine exactly as unlabeled iodine is con-

131

verted. Following injection of I, exogenous thyroxine

is administered at regular intervals until the point is

reached when no more labeled hormone is released from the

131

thyroid (i.e., when I from the thyroid is completely

inhibited). That amount is believed to be the daily thyroid
secretion rate.

The direct output method for TSR was first designed
by Reineke (1964). The method is free from the use of

goitrogens and exogenous thyroxine. It involves the measure-

131

ment of thyroidal I turnover and thyroidal iodine con-

tent. When the product obtained from multiplying these two
parameters was multiplied by a correction factor for the

different activities of T and T an estimate of daily TSR

4 3'
was computable. In practice external counts of the thyroid

were taken and were expressed as percent injected 13J'I dose

and plotted against time on semi-log paper. Thyroidal 1311

output constants were calculated as follows:

 

 

_ 0.693
X _ tk (days) (1)
K'4 = 1—e-x I (2)
_ K'4
K4 ‘ l-(U/lOO) (3)

where t = 1 day, e = base of natural logarithms, U = percent

131

of thyroidal I uptake extrapolated to zero time, K4 =

fractional 131I output rate per day corrected for recycling

 

:f :etabolized
'* rate cons

1
:21 ‘11 to c

'4

A. " ‘ ;
RCISEKE (J. .

 

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1:33.218 5.125 L...

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1 u
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N‘“ 1‘ Lu;

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97

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p
.A

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26

131

of metabolized I (Reineke and Lorscheider, 1967). The

output rate constant is multiplied by the amount of thy—

1311 to obtain the secretion rate.

roidal
Reineke (1964) and Singh gt_al. (1968) compared the
thyroxine substitution method with the direct output method
for evaluation of TSR. In domestic fowl the direct daily
output of thyroxine (1.2 pg t4/100 gm b.w. daily) is lower
than in the thyroxine substitution method (2.0 ug t4/100
gm b.w. daily). The goiter prevention method was also
found to be higher than the other two methods (2.32 mg t4/
100 gm b.w. daily). When examining the merits and demerits
of each method the authors were slightly more inclined to
believe the direct output method which they felt did not
overestimate TSR as the other methods had.

131

Thyroidal uptake and release of I has also been

used as a parameter for measuring thyroid function and
although it is not used to quantitatively measure TSR it
is a useful tool for clinical and qualitative recognition

of thyroid malfunction. Flamboe and Reineke (1959) found

no relationship (in goats) between TSR and either 131I %

uptake or 131I output rate. Hoersch, Henderson, Reineke

and Henneman (1961), using sheep, confirmed that because of

a low negative correlation between TSR and zero time % up-

131

take that I is not a reliable quantitative estimation of

T4 production.

 

 

 

Clinically,

. awn +- .—
e:::3ary h; :,o -..

The thyrox.

 

xii radioactix

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121-3“ data .

27

C1inica11y,Greenberg (1966) found that primary and
secondary hypothyroidism in humans can be differentiated
using 131I release rate. Goyings gt_al. (1962) used 24
hour 1311 uptake as a method for diagnosing hypothyroidism
in dogs.

The thyroxine degradation method for TSR evaluation
is predicated upon the validity of the assumption that the
rate of thyroid hormone degradation is equal to its rate
of secretion. In this method a known small amount of
labeled thyroid hormone is injected intravenously and blood
samples are withdrawn at regular intervals. The nonmetabol-
ized radioactive thyroxine is measured in the serum or
plasma. Biological half life, fractional turnover rate,
thyroxine distribution space and total extrathyroidal
thyroxine may also be determined from the thyroxine degra-
dation data. From these data an estimate of the amount
of T4 secreted daily can be made.

TSR's have been determined in numerous species using
the thyroxine degradation method. The first in sheep was
by Freinkel and Lewis (1957). Post and Mixner (1961) used
it on dairy cattle. It was used in man by Sterling g£_gl,
(1954), Ingbar and Freinkel (1955), Sterling and Chodos
(1956) and Gregerman gE_§1. (1962). Oddie gt_al. (1966)
reviewed the available literature on human TSR by TDR.

They pointed out that thyroxine distribution space (TDS)

did not change with height or age but did increase with

 

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iezreased in “y;
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Gregerman ;
:gher in the f'-
:z-rsa was als

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28

increased weight. TSR was lowered with aging and also
decreased in hypothyroidism and hypometabolism. TSR did
not change with weight or height in man.

Gregerman in 1963 found that in rats the TDR-TSR was
higher in the female than the male during cold exposure.
TDR-TSR was also found to increase in old rats along with
TDS. Singh, Reineke and Ringer (1968) and Singh (1966)
found that TDR-TSR for normal White Leghorn chickens was
2.03 ug/100 g/day and averaged 1.59 and 1.02 in 56 week old
chicks and goitrogen-treated chicks respectively. They
also found that the TDS values for the 7-week-old chick was
significantly higher than in the 56-week-old chicken.
Gregerman (1962) found that in euthyroid men thyroxine
degradation and TDS decreased with age at about the same
rate as basal metabolism (BMR)

Examination of thyroid function by Reineke and Singh
(1955) using the thyroxine substitution method indicated
that TSR for adult female rats was 2.21-2.56 ug/100 g b.w./
day. Lorscheider and Reineke (1972) using the "direct out-
put" method found that TSR was significantly reduced during
lactation or when dietary iodine is low. Beltz and Reineke
(1968) by the direct output method pointed out that neo-
natal rats have a very low TSR. At weights of approximately
22 gm they begin reaching adult TSR levels. Pipes gE_§1.
(1963) reported that TSR in beef cattle was 0.27 i 0.006

mg/100]1>b.w. which was lower than in dairy cattle

+ 0.013)

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‘
. s
45:.

:0
j:

.3

 

29

(0.40 i 0.013). Romack gE_§l, (1964), using the replace-
ment or substitution technique in swine found TSR to average
0.39 mg/100 1b b.w. Flamboe and Reineke (1959) using the
thyroxine substitution method in goats found that TSR ranged
between a high in October of 0.336 mg/100 lb b.w. to a low
in July of 0.178 mg/100 lb b.w.

In mink, using the thyroxine substitution method,
Reineke et_§1, (1960) reported that mink TSR was 0.95 ug/100

gm b.w.

Thyroxine Binding Proteins

 

It is generally agreed that circulating thyroxine
exists almost entirely in a dissociable complex with plasma
proteins. Gordon gg_al. (1952) were first to describe
thyroxine associated with an alpha-globulin in plasma which
was later termed thyroxine binding globulin (TBG). Numerous
investigations have described the TBG as having a high
affinity but occurring in low concentrations. Ingbar (1958)
described another plasma entity (albumin) with a capacity
to bind thyroxine. Albumin was found to be a thyroxine
binder but its characteristics were opposite those of TBG.
It had an extremely low affinity for thyroxine, but had a
high serum concentration. A third substance, pre-albumin,
was also discovered to have an affinity for thyroxine.

It was, therefore, assigned the name thyroxine-binding
prealbumin (TBPA) (Robbins and Rall, 1960; Ingbar, 1963;

Woeber and Ingbar, 1968). Thyroxine binding prealbumin has

 

 

 

 

1 :—
..nvv I

o-.~--

n‘a' vi

— a l

.U ‘1

("‘-
a

u.

I"

30

an affinity for T4 ranked between TBG and albumin.
Thyroxine binding globulin is the most important thyroxine
carrier and TBPA is recorded as second in line of impor-
tance. Serum albumin in many species has very little
function as a T4 carrier; deer are a notable exception,

because of very high serum T (Byrne, Reineke, Ullrey and

4
Youatt, 1974). In man and most other mammals 60-70% of the
T4 is carried by TBG, 30-40% by prealbumin and less than

10% by albumins. Zaninovich et_al. (1966) discerned that
thyroxine is the primary hormone bound to thyroxine bind-
ing globulin, whereas, triiodothyronine is not signifi-
cantly bound to TBG.

Almost all of the circulating thyroxine is bound to
carriers and only about 0.05% is present in the free form
(Robbins and Rall, 1960). It is generally agreed that only
the free form is able to affect the tissues. Hillier (1970,
1971) reports that the dissociation rate of thyroxine from
TBG to free T4 in solution has a biological halftime (t%)
of 38.6 i 2.1 sec. at 37°C. Binding of thyroxine to TBG
according to Hillier occurs "loosely" within 0.1 sec. but
to complete the normal binding some 20 seconds elapse. The
t35 for thyroxine dissociation from thyroxine binding pre—
albumin is 7.9 sec. at 37°C which is much faster than
is "looser" with TBPA than

4
with TBG and the carrying capacity is much lower. Dissoci-

release from TBG. Binding of T

ation as thyroxine from TBG and TBPA has its own separate

 

 

:r"' “

M .—

fl...u~
.

...- an.

s
.o-avd

a
..yano
- .-

u'. DO! I

 

 

. oz...
3' .n 2",

 

 

 

 

-‘

Hr

31

exponential decline which suggests that each has character-
istically different types of binding sites each with dif-
ferent affinities.

Effects of Temperature, pH and Dilution
on Thyroxine Binding Proteins

 

 

Temperature greatly affects the ability of thyroxine
binding proteins to carry thyroxine. Both quantity and
rate of dissociation are affected by temperature. Hillier
(1971) found that t35 for thyroxine dissociation from TBG
was 38.6 seconds at 37°C but was 8.1 minutes at room
temperature (25°C), a 12 fold increase. The t;5 for thy-
roxine dissociation from thyroxine—binding prealbumin was
7.9 sec. at 37°C but was 53 seconds at 25°C which equals
more than a 7 fold increase in rate of dissociation. At
31°C (pH 7.4) prealbumin binding was 30% greater than at
37°C (Lutz and Gregerman, 1969). Etta (1971) also found
that a temperature optimum of 37°C was necessary to restrict
binding of thyroxine to TBG in several species including
man.

The effect of pH has been examined in ziE£g_using
several buffering systems and is thought to play a role in
T4 transfer to the tissues. It is generally agreed that
the buffering system employed is as important in effecting
thyroxine binding specificity as the pH. Summarizing the
work of several authors who used barbital buffering sys-

tems of pH 8.6 and above, they reported inhibition of

_w 71".“ ~ *3: vw--m u

   

 

 

..
_..q~a

.-no‘-ll

 

 

 

'. ‘0-

“-n-

"I'u

.
‘-

~.--

I o.
In

(I!

Q

('1

l‘.’/

 

32

binding to albumins and prealbumins but no reduction in
binding to globulins (Robbins and Rall, 1960; Lutz and
Gregerman, 1969; Braverman et_§1. 1967; Keane'gt;§1., 1969;
and Coutsoftides and Gordon, 1970). Binding to albumin
was maximal at pH 8.6 when sodium phOSphate buffer was
used (Antoniades, 1960). Barbital buffer had the quality
of interfering with the albumin and prealbumin binding
sites at pH 8.6 and was therefore uniquely suited for use
in quantifying TBG capacities.

High dilution of thyroxine decreases binding to al«
bumins in blood and serum in_vi£rg_but has little effect
upon binding to globulins. Murphy and Pattee (1964)
reported that a dilution of 1:32 was sufficient to reduce
the feeble albumin binding to almost nothing. Murphy and
Pattee (1964) and Etta (1971) have shown that a dilution
factor of above 1:32 plus barbital at pH 8.6 will effective—
ly inhibit binding to albumins and prealbumins but have no
significant effect upon TBG binding.

Effects of Estrogen and Sexual Maturity
on Thyroxine Binding Proteins

Sex-related differences in thyroxine binding to its
serum receptors is often a function of other non-thyroidal
hormones. Dowling gt_al. (1956) demonstrated that pharma-
cological doses of estrogenic hormones increase the T4—
binding capaCity to TBG. Pharmacological doses of andro—

genic or anabolic hormones, however, decrease the binding

 

 

. _
- 4
.. ‘4
‘

.-.~~‘

 

Ui".
. .
n
U.
a
...-
l...‘

u..,

q...

es

0.‘

15..

In
I

33

capacity of TBG. It has been demonstrated by Braverman
§E_al. (1967) that sex related differences do naturally
occur in humans. Females were described as having a higher
T4 binding capacity for TBG and a lower capacity for T4—
binding prealbumin than males. Hyperestrogenicity either
due to exogenous administration of estrogen (Dowling §E_31.,
1956a, Zaninovich gt_al,, 1966) or due to increased engo-
genous levels as in gestation (Dowling, 1956b; Musa 35:31.,
1969) is followed by an increase in TBG—capacity. Estrogen
affects other factors in thyroxine regulation. It enhances
iodine trapping (T/S) and radioiodine uptake by intact,
hypophysectomized, or gonadectomized rats without necessar-
ily altering release of thyroxine. Furthermore, TSH secre-
tion is enhanced by low doses of estradiol but is inhibited
by high doses (Fisher and D'Angelo, 1971).

Puberty effects a marked change upon TBG capacity.
TBG was shown by Riecansky (1967) to be independent of sex
and age factors in prepubertal children. Females, because
they entered puberty much sooner than boys, showed a higher
TBG-binding capacity at age 15 than boys. The sex steroids

therefore modify the binding behavior of the thyroxine

binding proteins.

Thyroid Hormone and the Reproductive Cycle
It is well-established that severe hyperthyroidism or
hypothyroidism is detrimental to reproductive performance

and will often precipitate aberrations in the estrous cycle

nu...

 

Ir
0...-

,...'p

.,v.v
-

fin.

Ii...
u

 

‘a.
‘ V'

c I‘.
u

...

.1

 

 

c;-
.—

‘II
III

A-
‘5

 

ll)

34

(Reineke and Soliman, 1953). The causes of thyroid-induced
reproductive disfunction are at least four fold: 1) T4
affects primary sex tissues, 2) effect of thyroid hormones
upon the central nervous system and gonadotropins, 3) effect
of steroids upon thyroxine concentration, and 4) thyroxine-
induced changes in metabolism or a breakdown of reproductive
steroids. Experimental evidence for these mechanisms has
been completely reviewed through 1952 by Reineke and Soliman
(1953). Marked species differences in reproductive response
to hypothyroidism and hyperthyroidism also exist (Nalbandov,
1964).

Hypothyroidism affects secondary sex characteristics
as early as puberty. Vaginal cornification and ovulation
in rats occurs on the day of vaginal opening (34 days of
age). Thyroidectomized rats, however, experienced vaginal
cornification and ovulation much later than the day of
vaginal opening (Hagino, 1971). Anesthetization with pento-
barbital followed by an injection of PMS (pregnant mare
serum) caused ovulation at a normal age in thyroidectomized
pubertal rats. Hagino concluded that continuous exposure
of the central nervous system to thyroid hormone is neces-
sary for regulation of gonadotropin secretion.

Schultze and Noonan (1970) found that exogenous thy-
roxine increased uterine metabolism at the proestrus stage
but decreased estrogen-induced high uterine metabolic rate.

They also described an enhanced ovulation rate, implantation

 

V‘

1.1".11 7.)! 7'»

 

v"‘

,o-v

.II

- to."

 

u-c-
.-

-.vv

up. n

‘.

~~

35

rate and litter size with a thirty microgram dose of
L-thyroxine 6 days prior to breeding and discontinued on
the day of breeding. Soliman and Reineke (1954) demon-

131

strated that I uptake by the thyroid gland of mature

mice varied with the estrous cycle and that thyroids

attained maximal 131

I uptake during proestrous.

The effect of thyroid activity upon delayed implanta-
tion of blastocysts in rats was studied by Holland gt_al.
(1967). Their technique for inducing delayed implantation
was to ovariectomize rats on the third day after mating
and maintain them on progesterone during the delay period.
Implantation was subsequently induced by the administration
of 1 ug of estrogen daily following the 9th day after
mating. Upon autopsy on day 14, hyperthyroid rats possessed
more implantation sites than controls; however, hypothyroid
rats had fewer implantation sites than the controls. Their
results suggested that a moderate level of hyperthyroidism
is beneficial to implantation while hypothyroidism tends to
be detrimental to blastocyst implantation.

Non-pregnant rats show the highest thyroid gland activ—
ity, which occurs during pro-estrous in association with an
increase in pituitary TSH. This increase like the increase
in thyroid gland activity does not occur when ovulation is

blocked (Newcomer and Brown-Grant, 1971). TSH is also ob-

served to increase in early pregnancy.

v- 1“

 

 

(fl

lAuAR F
v- 5‘
"I. .

ono~AF
P‘ ,
—-'MVC

.‘ .
§§I~!.Q

o-u‘. pa‘

"--0 A.-
- poi
’VIIV .-
.
II-DA a.
y-
an... .4

.-~- ..
\u' I
"~-- 1‘

 

R'-

1..

":3 I
\

o...

27..

I~‘.'
“ V
o. " b.

I“.
v. v
6-.
.
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00.: ‘
-4

:"

 

 

 

 

36

Schreiber et_al. (1967) pointed out that thyroid hor—
mones play a decisive role in regulating hypertrophy and
metabolic activity of the anterior pituitary under various
conditions. Thyroxine administration was observed to pre-
vent estrogen-induced anterior pituitary hypertrophy, but
none of the other actions associated with estrogen were
observed to be altered. Their group also found that 125I-
thyroxine binding to pituitary proteins in vitro was in-
creased under estrogen administration. Furthermore, large
doses of thyroxine not only blocked anterior pituitary

125I-thyroxine binding

hypertrophy but increased in gitrg
to pituitary proteins. Testosterone was reported to stimu-
late the same thyroxine phenomena as estrogen in the rat
(Schreiber gt_al., 1967).

Association of thyroxine with anterior pituitary
mitochondria was observed by Schreiber gt_al. (1971).
Anterior pituitaries were incubated with labeled thyroxine
first, then by centrifugation the following 4 fractions
were separated: nuclear (2,000 g), mitochondrial (20,000 g),
microsomal (105,000 g), and cytosol. Counting of the 4
fractions indicated the preferential association of thy-
roxine with the mitochondria. Significantly higher amounts

of 125

I-thyroxine were found in the mitochondrial fractions
from animals receiving estrogen treatment than those without
exogenous estrogen. This association of thyroxine for the

anterior pituitary mitochondria is exceptional compared with

 

 

 

 

 

--~.‘

on.-
I

- n
.‘v

--'I
h .
.‘fi!

Q"

a-

-

n-

(I!

/

u.
‘s

 

 

 

 

37

other tissues where the attraction was not observed (liver,

kidneys, brain, heart).

Thyroid Hormone and Pregnancy

 

Pregnancy is accompanied by important modifications in
thyroid function. Hypertrophy and hyperplasia are observed
first in early pregnancy and many investigators have con-
sidered the thyroid goitrous during pregnancy. In addition
to these anatomical changes the following physiological
parameters are also increased: thyroid secretion rate,
thyroxine binding globulin, protein bound iodine, thyroid
iodide uptake, iodine pool and butanol extractable iodine
(Heineman et_al., 1948; Man et_al., 1969; Robbins and Nelson,
1958). Absolute free thyroxine and thyroxine turnover rate
were not altered by gestation (Ingbar et_al,, 1965; Dowling
32431., 1967). Plasma TSH levels are found to increase at
the sixth week of gestation in humans and remain higher than
normal until after parturition when they return to normal
(0.24 mU/ml normal and 0.48 mU/ml during pregnancy)
(Lemarchard-Beraud and Mean, 1970). It was also found that
TSH of slightly hypothyroid pregnant women is lower than in
controls both before and after parturition. Pregnant rats
and controls were found to have nearly identical thyroid
1311 regression slopes but uptake rates were slightly but

consistently lower in pregnant rats (Iino and Greer, 1961).

 

(I.

.-o

0.1.

I“.

rob-

u...

 

9}—

v...

5-.

 

(-

1‘

 

38

In contrast to the human and rat the golden hamster

131

has reduced PBI and thyroidal accumulation of I during

the last half of pregnancy (Galton, 1968). Urinary excre-

tion of l311-T4 was greatly increased when examined during

the late pregnancy in the hamster. The increased excretion

of iodide from T was associated with a fall in the con-

4
centration of serum T4. After delivery, urinary iodide
excretion and serum T4 concentration returned to normal.

Galton (1968) studied T metabolism in vitro and found that

4
deiodination in liver of 20 day pregnant hamsters was norm-
al, however, it was very high in comparable preparations
of fetal tissue. The rat pregnancy was associated with an
increased fractional turnover and probably an increased
absolute turnover of T4 in the tissues. The increase in
rat serum is probably due in part to the increase in thy-
roxine binding globulin in the serum and may also be
attributed in part to normal deiodination in fetal tissue.
Hernandez, Etta, Reineke, Oxender and Hafs (1972)

reported that fetal T in cattle serum was approximately

4
twice maternal levels between 90 and 180 days. TBG was
completely saturated in fetal serum but only 2/3 saturated
in maternal serum. Information concerning transport of
thyroxine across the placenta is often contradictory and is
probably dependent upon species. Some species may derive

fetal thyroxine crossing the placental barrier while others

have extremely active fetal thyroids which produce a higher

 

 

 

<‘v'

.o-

. v.
(I!

 

.—

39

circulating level of thyroxine than the mother.

Human and rabbits during the last trimester and par-
ticularly near term show significantly larger maternal to
fetal transPlacental differences in thyroxine (Fisher
e£_gl., 1964). Mice, sheep and bovine feti (Waterman,

1958; Hernandez et_al,, 1972) have very high serum thyroxine
levels and anatomically the fetal thyroids appear large and
much more active than maternal thyroids (Fisher et_al.,
1964; Fisher and Lam, 1974).

Hotelling and Sherwood (1971) found that circulating
triiodothyronine (T3) in humans increases from 196 ng/100
ml during the first trimester to 299 ng/100 ml in the third
trimester. The increase in T parallels that of T4 and TBG.

3

They also pointed out that T in vivo is significantly bound

3

to TBG. At birth umbilical levels of T3 are only 193 ng/ml
or approximately equal to the first trimester maternal

levels.

Thyroid Hormone and Lactation

 

Circulating thyroxine levels of the young neonate seems
to be a function of the neonates ability to mimic adult
locomotion and feeding behavior. Ungulates and guinea pigs
seem to be quite active when born and their serum thyroxine
levels are also elevated above maternal levels for some
time after parturition. Pals, Reineke and Shaw (1973)

showed that in young guinea pigs (Cavia porcellus) which is

 

 

nun

ncv

nu

to.

q.-

0"-

 

I:

‘-

 

 

40

one of the most precocious neonates to have serum thyroxine
levels rise 4 times the prenatal level within 1 hour after
birth. Thyroxine level declined exponentally at the rate
of 2.3% per hour. They reached adult T4 levels at 3-6
weeks of age at which time they were almost adult in size.

Hernandez et_al. (1972), reported that serum thyroxine
levels of bovine calves at birth were about twice adult
levels and that adult values were approached at the end of
6 days. TBG also declined following birth, releasing more
T4 for metabolic purposes. It was suggested that the days
of high thyroxine levels following birth were necessary
for the adjustment of the newborn calf to its new environ-
ment.

Maternal reaction to the lactating offspring is often
quite severe. Some cattle have even become severely hypo-
thyroid and in areas of low iodine such as the goiter belt
in the Northern Midwest and Pacific Northwest have even
died from the lack of replacement of iodine removed by the
suckling calf. Hernandez et_al. (1972) found that during

early lactation maternal T is approximately one-half neo-

4
natal levels. Lorscheider and Reineke (1972) reported that
during heavy lactation in rats thyroidal iodine supplies
were depleted, but that accumulation became more efficient.
Flamboe and Reineke (1959) noted substantial losses of
iodine in the milk of goats. They suggested that under

iodine deficient conditions a functional thyroid deficiency

 

K

 

 

 

 

..q

“nu

 

DU‘44!

 

o
...
c_ ‘
--v..-'

I)
(II

|.

.._

u...

I!-

o._.

‘N w

u
s"

41

could occur during lactation.

Indications,are that thyroxine is actively secreted
into milk. Flamboe and Reineke (1959) and Reineke (1961)
found that administered 131I was secreted into milk. The
phenomenon has been seen in numerous other species (see

Reineke, 1961). About 92% of the 131

I found in goats milk
was found as iodide and the remaining 8% was protein bound.
Reineke (1963) reports that under conditions of lactation
mammae compete with the thyroid for available iodine,
resulting in reduced thyroxine formation. He found that
the major component of iodine secreted into milk was mono-
iodotyrosine (52%). It is assumed that much more of the
ingested iodinated proteins reach the neonate than would
reach an adult since gastric acids of newborn are not in
abundance during lactation particularly in carnivorous
animals.

It has been found by Byrne et;21, (1974) that deer
lactation is similar to cattle. The extremely high T4 at
birth (26 ug%) drOps to almost adult levels during the sixty

plus days of lactation.

 

o.

 

STATEMENT OF THE PROBLEM

It is well-known that mink are one of the most sensi-
tive mammalian species to many toxins such as diethyl-
stilbestrol, DDT, DDE, dieldrin and other pesticides.

PCB's chemically resemble these compounds. Mink are there-
fore excellent models for describing the mechanisms of
action of these substances and might point the way of
possible harm to man.

Mink reproduction has been found to be severely re-
duced by PCB's; however, the physiological mechanisms were
not known.

Hypo- and hyperthyroidism have been known for years to
cause reproductive failure in many species and it is known
that PCB's have induced weight and morphological changes in
the thyroid gland of lower animals.

It was thought that PCB's might alter the balance of
hormones necessary for normal reproduction and that since
PCB's have been seen to alter thyroid anatomy, changes in
production and circulation of thyroid hormone might be

associated with PCB's.

42

 

 

 

“I 0..
—-
0.. ‘

.«C .

 

.p‘.

<.;
t.~“

."A

—
u
u.‘

III!

 

 

u...

 

MATERIALS AND METHODS

I. Animal Treatment Protocol

A. Animal Care and Reproductive Cycle

All mink in this study were housed and cared for at
the Michigan State University Fur Animal Project Ranch on
the south campus of Michigan State University.

The estrous cycle of mink begins with estrus on about
March lst and extends to March 15th. Estrus is followed
by a period of delayed implantation which occurs between
March lst and April lst. Implantation on April lst is
followed by 5 weeks of gestation up to May 8th. Lactation
continues from approximately May 8th to June lst. Weaning
takes place around June lst. Diestrus extends throughout

the year until pre-estrus in late February.

B. Experiment I

Thirty-two adult female mink (Mustella vison) were

 

randomly assigned to four groups of eight. All animals
were fed a high protein balanced diet, ad libitwm
(Appendix B). The groups received 3 concentrations of
polychlorinated biphenyl (PCB) (Aroclor<>12541; Monsanto

Chemical Co., St. Louis, Mo.) added to their normal daily

 

1@ Registered trademark for polychlorinated biphenyl
compoun s, Monsanto Chemical Co., St. Louis, Missouri.

43

 

., «V.
._ .1

‘5'

-a‘.‘ .,

um”

 

 

.. _l
I\;§ -
D‘vv \-

FAR‘ ‘N
‘ r- ,-
Ivvod.

.u ‘vz.
.4
WI". ' _
‘ L-
nun.“_
! -
I‘I’F
" um...

If)

my,

: ”V

.‘v “
1

'.‘V\
r

l’
3'“.

.=
'VA,,~
I.“ ‘1‘.

i':‘
.,_u t

 

44

diet (5 ppm, 2 ppm, 0.5 ppm) and a control.

Blood samples and body weights were taken at 28—day
intervals from September 19, 1973, through June 26, 1974.
Special diets were initiated on September 21, 1973, two
days after the first blood sampling, and were continued
through the last sampling date in June. Plasma was
separated from the blood and analyzed for circulating thy—

roxine (see Serum Thyroxine Determination).

C. Experiment II

 

Sixteen adult female mink were randomly assigned to
two groups of eight. Each was fed as described in Experi-
ment 1, except that Group I received 5 ppm (Aroclor 1254)
PCB. The PCB diet was begun in late October and continued
throughout the course of the experiment. On February 19th,
when the animals were pre-estrus, a sample of blood was
collected by toe clipping for determination of plasma thy-
roxine level and circulating thyroxine binding globulin
(TBG) capacity. Three days later on February 22, 1974,
thyroxine secretion rate (TSR) experiments were performed
on the mink by the thyroxine degradation method. These
same animals were again bled for a TSR experiment on April
12th, during the gestation period of mink, and again on
May 15th during lactation. In each instance blood for T4

analyses was withdrawn 3 days prior to the TSR experiment

and body weights were recorded to i_5.0 gram. An additional

 

 

 

"O- A‘F
‘
““fivs.

'Ihan.

....:D _

o-~ K.

.ui “A
o

' Qt

"‘ Av

--‘-..,,\

t.“ ‘ u a
“ F
"‘in\

q
h‘Dv-
J. ~Olh

'1!

45

group of five mink were added to experiment II in January,
1974, to be used to assess the TSR procedure in mink.

Two lactating controls were also added to the May 15th TSR
experiment. One week following the last TSR experiment,

on May let, the original group of 16 females plus two extra
lactating controls were killed and autopsied. Body weights
were recorded to the nearest 5.0 g. A cursory visual exami—
nation was also made of the heart, lungs, stomach, and
intestines. The thyroids were placed in glass vials_contain-
ing Dietrich Fixative and were imbedded in paraffin the
following day. They were later sectioned at 6 microns and

stained with hematoxylin and eosin.

D. Experiment III

This experiment was designed to delineate the differ-
ence in thyroxine level between female and male mink.
Blood samples were drawn by the toe clipping from mink of
both sexes at irregular intervals between autumn 1972 1
through summer 1973. Plasma was stored at -20°C until

ready for thyroxine analysis.

E. Experiment IV

Five female mink which had been maintained on a normal
ranch diet, were killed in late December during late
diestrus. Sufficient blood was collected to supply that
needed to run a thyroxine binding curve (see Appendix C for

the method). Plasma samples from the animals used for the

L

 

Ion—rm .- 1".‘1- ‘mr

 

 

pa-

\
.5“ U UJV

.-.R
‘v

”!|

u . 3'
.cv' -

a
'r‘
D:

VA.
N A.

 

 

5%: S

o

’1'
{l'

1'

46

TSR study in experiment II were used to assess the T bind-

4
ing capacity of the circulating thyroxine binding globulin

(TBG) and the saturation index.

II. Serum Thyroxine (TA) Determination

Serum thyroxine levels were measured by the Tetrasorb-
125 method (Abbott Laboratories, Radio-Pharmaceutical Divi-
sion). The method is a competitive protein binding assay
specific for total serum or plasma L-thyroxine. Briefly,
the principle of the method is that thyroxine extracted

from plasma competes with tracer 125

I-thyroxine for binding
sites in a given quantity of thyroxine binding globulin
(TBG). Levels of "cold" endogenous thyroxine will compete
with labeled T4 for the TBG sites with the assumption that
both have equal affinity for the available sites. When
equilibrium is reached, the addition of a resin-impregnated
sponge will bind the unbound thyroxine but not the TBG-
bound thyroxine. This renders the two components separable.
Therefore, as cold T

or plasma T increases it displaces

4 4
additional labeled T from the TBG which is then absorbed

4
by resin sponge. The TBG—bound thyroxine is washed from
the sponge and the ratio of the radioactive counts within
the resin sponge to the initial radioactive counts is pro-
portional to the unlabeled thyroxine.

Blood samples of approximately 2 ml were obtained by

the toe clip method. Plasma was separated from the formed

 

 

 

 

..n‘4.fl 1

 

47

elements within 4 hours after drawing the samples.

III. Thyroxine Secretion: Degradation Rate

 

A. Solutions
131

 

I-L-thyroxine in 50% aqueous propylene glycol
(Amersham/Searle Radiopharmaceuticals, Arlington Heights,
Illinois), because of its short half-life of 8 days, was
purchased fresh for each of 4 TSR experiments. It was di-
luted in glass-distilled water so that a 1 ml injection
would administer a dose of approximately 45 uCi. Although
most of the glassware had been siliconized, 0.25% bovine
serum albumin (Fraction V) was added as a carrier to pre-
vent thyroxine adhesion to the glassware.

Standards were prepared such that a 45 uCi/ml dose
was diluted 200, 250, 300, 350 and 400 times to approximate
the dilution which a dose of 45 uCi/ml would undergo in an

adult female mink of approximately 1000 grams.

B. Injection Procedure

Thirty minutes before each injected dose of 131I-L-T4
was aministered, a 1 ml subcutaneous injection of sodium
thiocyanate at 50 mg/ml was administered to each animal in
order to block the iodine trap of the thyroid, thereby pre-
venting recycling of labeled T4.

To anesthetize the animals 0.2 ml C-I 744 (Parke Davis,

Ann Arbor, Michigan) was injected intramuscularly (IM).

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48

Each animal was unconscious for approximately 10 minutes
and another 15 minutes elapsed for complete recovery.

Hair was removed and a small incision was made in the neck.
131I-thyroxine solution was then injected intravenously
(IV) into the exposed jugular. Extreme caution was taken
to assure that all of the labeled thyroxine was injected
directly into the vein. Sutures were then applied to
muscle and dermal layers. The thick hair covering all
superficial veins made the minor operation necessary for IV
injection.

After each animal had become active following anesv
thesia each was taken back to its individual cage within
the colony to prevent any additional stress. After a four«
hour distribution time, blood samples were taken at two«
hour (:_1 min.) intervals up to 12 hours.

Plasma samples were then frozen and stored at -4°C
until used. Because mink have very high hemotocrits
(approximately 60%) only about 0.8 to 0.9 ml of plasma was
obtained at each sampling period.

Procedure for T4 analysis was modified from the method
found in the Abbott Laboratory Tebrasorb Manual. Plasma
volumes of 0.6 ml were used instead of 0.3 ml to compensate

for the comparatively low T level in mink. A standard curve

4
was made from a working standard solution of L-thyroxine
that was purified in our laboratory. Standards contained

2.5, 5 and 10 mg/100 ml plasma. Uncorrected thyroxine values

 

 

 

 

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49

were obtained from the slope and intercept of the standards
and corrected T4 values were obtained by dividing the un-
corrected values by 0.79 which is the extraction efficiency

of 95% ethanol.

C. Sample Collection

At each sampling period a toe was clipped and approxi-
mately 1 ml of blood was collected in a heparinized paraffin
cup, transferred to a polyprOpylene microcentrifuge tube
and plasma was separated by centrifugation in a Beckman 152
microcentrifuge. Each animal was then returned to its cage.
A 20 microliter aliquot of plasma was transferred to a 1.3
cm x 8.6 cm polypropylene tube and set aside for counting.
All tubes were then held at room temperature until all
samples had been drawn and were summarily counted along with
standards for one minute in a gamma well Counter, model DS-5
(Nuclear Chicago), with a sealer—analyzer (Nuclear Chicago,

Des Plains, Ill.) set to count at the 1311 peak.

D. Computations
Computation of percent dose per ml plasma
a. Counts per microliter were converted to counts/ml
as follows: CPM-bkg/ZO pl plasma x 50 = CPM-bkg/ml
b. Injected dose = UCi experimental dose (e.g., 45 uCi
in standard)
c. Injected count = (standard cpm-background) x

(injeCted dose)

 

 

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50

d. Percent 131I-L-T4 dose/ml serum =

Counts-background/ml plasma (a)
Injected count (c)

Percent dose/m1 serum was then calculated for each of 16

animals at intervals of 4, 6, 8, 10, and 12 hours after

injection. The percent 1311 dose per 100 m1 serum was

regressed against time by the equation:

log y = a + bx

where log y = percent dose, x = time.
a = the log intercept at injection time.
b = the slope of the regression line.

Computation of Degradation Rate Constant

Data were converted to the log n form by the equation:
y = e I.

where x = slope, e = the base of the natural
logarithm.

y = percent injected dose/ml plasma at time t.
(The quantity of ex is most easily obtained from
a table of descending exponentials.*)

(l—e-x) (2.302) = Degradation Rate
Constant/hr. II.

where 2.302 = factor used to transform log10
to natural logarithms.

Computation of Biological half-life ty

_ 0.301
8 - slope (b) (the numerical slope, not
the algebraic log10 slope)

t = hours III.

 

*Tables of the Exponential Function ex National Bureau
of Standards, Applied Mathematics Series 14,.4th ed., U. S.
Government Printing Office, Washington 25, D. C., 1961.

‘4-

 

 

.

51

where:

tk is defined at the amount of time in hours required to

131

degrade half of the I-L-thyroxine concentration present

at time zero. The theoretical percent of 131I-L-T4 present
at time zero is obtained by extrapolating back from the
data points or was commonly computed from the intercept of

the linear regression line.

Computation of thyroxine distribution space
TDS/mls = 100% dose at time zero IV.

or TDS/ml - lOO/Anti-Log10 intercept
TDS/100 gm body weight = TDS/gm body weight/100 V.

Computation of Extra Thyroidal Thyroxine

Ett = (TDS/100 gm body weight) (T4 ug/ml serum) =
ug T4 IV.

Computation of Thyroxine Secretion Rate

TSR = (Ett) (Rate constant K) (24 hrs.) = TSR/100
gm/day IIV.

For TSR computations in this manner at least 4 assump-

tions must be made. First, secretion rate must equal

131

degradation rate. Second, I-thyroxine must be distrib4

uted to all the pools. Third, 131I-T4 is degraded just as

non-labeled thyroxine is degraded. Fourth, the NaSCN has

no peripheral effect on iodine metabolism as it does in the

thyroid.

52

IV. Thyroxine Bindinnglobulin (TBG) Capacity
and Saturation Index
Samples were first analyzed for serum thyroxine and
the remaining serum was frozen for later use. Briefly the
method, modified from a method by Etta (1971), involves
first a determination of the level of cold hormone which

125I labeled

is required to saturate and "compete out" the
thyroxine. The total amount of cold hormone required to

saturate the TBG is the sum of the endogenous T plus the

4
exogenous T4, both labeled and cold.

A thyroxine binding curve was made for the TBG capacity
at varying thyroxine concentrations. On the ordinate was

" ercent T bound" and on the abscissa was "T used pg per.
P 4

4
cent (exogenous and endogenous)". From the plateau of the
binding curve, the concentration of total T4 necessary to
saturate or exceed the TBG-capacity at 37° was observed
(see Appendix C for a detailed description of this method).
This concentration was used to determine the binding capaci—
ties of the TBG (see Appendix C for procedure and calcula—
tions) of mink at selected times under conditions of 5 ppm
PCB 1254 and control samples.

Three critical factors are necessary for the TBG i2_
yi££g_to specifically bind thyroxine. They are: 1) the
barbital buffering system at pH 8.6 completely inhibits

binding to albumins and prealbumins, 2) by maintaining a

high dilution factor of 30-35, binding to albumin which is

53

usually weak is reduced to null, and 3) incubation at 37°
during equilibration of the binding system maximizes bind-
ing of thyroxine to TBG and minimizes binding to all other
proteins.

The determination of saturation index is simply the
serum T4 divided by the TBG-capacity.

V. Statistical Analysis

 

Data for the experiments was statistically analyzed
using split plot factorial designs with block treatment.
Analysis of several sources of variance was undertaken using
a program adapted from one written by Edward Cogger. The
main IBM 6500 computer at the Michigan State University
Computer Center was used to handle the ANOVA analyses.

Hartley‘s F max test was used to assess the homogeniety
of the variances. Scheffé's F test was used to separate
mean differences. All P values reported herein are at the
0.05 level of confidence. See Appendix H for the equations
of the Scheffé F test.

Coefficient of correlation was used to test the
strength of the linear relationship in the thyroxine degra-

dation regression line.

RESULTS

Experiment I--Plasma Thyroxine Level Changes
with Three Levels of PCB

Throughout the course of this nine-month study sig-
nificant differences were found between the PCB treatment -
effects upon T4 levels and also between the time effects
(B) (see Statistics Table 1). Interactions between the two
factors (AB interactions) were also significant. The ob—
served differences in T4 levels (see Figure l) at various
time periods were, therefore, often the result of both dif-
ferences in PCB concentration and annual cycles. The
appearance of the lines meandering in Figure 1 may be a
function of the changes associated with the estrous cycle,
reproduction, gestation and lactation interacting with those
of the PCB treatments.

Thyroxine levels of all four groups were approximately
equal to 2.10 ug/100 ml at the initiation of the experiment
prior to PCB feeding. All four groups increased signifi-
cantly through September; however, the controls and 5 ppm
PCB groups were significantly higher than the remaining two
groups in October (see Table 2 for mean comparisons using
Scheffé F test). At 3.26 mg percent the controls were at

their highest measured levels in October and the difference

54

 

 

 

 

55

Table l. Factorial Analysis of Variance Table of Data in

Experiment I

 

 

 

 

Source SS df MS

1 Between subjects 18.3828 31 1.4141
2 A (type of signal) 9.84539 3 9.8453
3 Subj. w. groups 8.53742 28 0.71145
4 Within subjects 347.2331 288 12.4012
5 B 242.803 9 21.4015
6 AB interaction 34.6132 27 17.3066
7 B x subj. w. groups 69.8165 252 2.9090
8 Total 365.6159 320 8.9175
FA = 10.763234*/2.95

FB = 97.37664*/l.88

... *
FAB 4.62724 /1.48

 

*P< 0.05

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Table 2. Statistical Comparison of Data in Experiment I
Using the Scheffé F Test

 

 

F Test Critical
Treatment Comparison (A Effect) Statistics/Value,

 

Oct. 5ppm + control vs. 2 ppm + 0.5ppm 31.33 / 10.02
Nov. 2ppm vs. control + 0.5ppm 8.43 / 6.68
Nov. 5ppm vs. 0.5ppm 60.83 / 3.34

Dec. Control vs. 5ppm + 2ppm + 0.5ppm 177.6 / 10.02

Jan. Control vs. 5ppm + 2ppm + 0.5ppm 32.76 / 10.02
Jan. 5ppm vs. 0.5ppm 0.165/ 3.34 NS
Jan. 5ppm vs. 2ppm 7.09 / 3.34 NS
Jan. 5ppm vs. control 10.89 / 3.34
Feb. 2ppm vs. control 36.02 / 3.34
March 0.5ppm + 2 ppm vs. 5ppm + control 15.15 / 10.02
April Control + 0.5ppm vs. 2ppm + 5ppm 13.06 / 10.02
May Control + 0.5ppm vs. 2ppm + 5ppm 5.56 / 10.02 NS
May Control vs. 5ppm 1.49 / 3.35 NS
May 0.5ppm vs. 2ppm 4.45 / 3.34
June Control vs. 5ppm + 2ppm + 0.5ppm 26.40 / 10.02

 

MS Subj w. group = 0.71145; V1 = 2, V2 = 28

P< 0.05

continued

 

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Table 2--continued

57

 

 

Time Comparison (B Effect)

 

 

 

 

 

 

 

 

 

 

 

Controls F Critical 5ppm F Critical
Value Value
Sept. vs. Oct. F = 3.29 Sept. vs. Nov. F = 62.7
Sept. vs. Nov. 7.05 Nov. vs. Feb. 0.176 NS
Feb. vs. Mar. 4.54 Jan. vs. Feb. 12.65
Mar. vs. April 17.54 Feb. vs. Mar. 28.21
Feb. vs. April 34.02 Mar. vs. April+May 1.84 NS
April vs. May 19.87 May vs. June 0.99 NS
May vs. March 0.57 NS June vs. Feb. 0.99 .NS
May vs. June 13.27 June vs. Sept. 31.31
April vs. June 20.29 May vs. Sept. 3.96
April vs. Sept. 20.72
April vs. Oct. 0.44 NS
2ppm F Critical 0.5ppm F Critical
Value Value
Sept. vs. Oct. F = 11.02 Sept. vs. Oct. 15.74
Sept. vs. Nov. 2.75 Feb. vs. March 0.324 NS
Nov. vs. Dec. 2.30 Feb. vs. April 0.991 NS
Dec. vs. Feb. 0.32 NS Feb. vs. May 0.143 NS
Dec. vs. Jan. 3.59 April vs. May 0.38 NS
Jan. vs. April 12.65 April vs. June 12.65
March vs. April 2.45 May vs. June 8.97
April vs. June 12.65 Nov. + Dec. + Jan. 17.49
vs. Feb.
MS Bx Subj. w. group = 2.909; Vl = 9, V2 = 252

P< 0.05

Critical Value = 1.88

58

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60

between October and the second highest control level of
3.12 ug percent during April is significant. T4 of the

5 ppm PCB group and the controls was almost identical in
October. Similarly, the 2 ppm and 0.5 ppm PCB groups were
not significantly different in October.

In November the 5 ppm PCB group reached a seasonal
high of 3.76 pg percent which is the highest T4 level of
any group and month recorded throughout the duration of
the study. The other three groups were significantly lower
than the 5 ppm PCB group. Throughout the diestrous period
until estrus in March the 5 ppm PCB group remained signifi-
cantly higher than the control. The controls dropped from
their seasonal high in October to their lowest point in
December (1.61 Hg Percent T4)

During December all three PCB groups were approximately

equal at 2.8 pg percent T however, they were significantly

4.
higher than the control. Controls were still significantly
lower in February at 1.84 ug percent than the other three
groups. Controls were similarly reduced in February.
February is the only month when a direct dose response
relationship is seen between the groups. All four groups
are significantly different and are arranged such that the
control group is lowest and the 5ppm PCB group is highest.
At this period the full effect of the dosages is apparent.
Estrus begins in early March in mink and its influence

apparently alters the T levels quite drastically (see

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61

Appendix B for the mink estrous cycle). The 5 ppm PCB
group dropped by 30% between February and March to equal
the control at 2.3 pg percent. The control increased sig-
nificantly between February and March from 1.89 to 2.34 pg
percent. Groups receiving 0.5 ppm and 2 ppm PCB were sig-
nificantly higher than the other two groups but were them-
selves not significantly different. The 0.5 ppm PCB group
increased significantly between February and March and
reached its annual high (3.47 pg percent) during implanta-
tion and early gestation in April.

Between estrus in March through delayed implantation
in late March to gestation in early April, the two high
PCB groups had significantly lower plasma T4 concentrations
than the control and the 0.5 ppm PCB groups.. The wide
difference in T4 at this point exactly coincides with the
reproductive performance of these animals (see Reproductive
Performance Results, Table 7) where the 0.5 ppm PCB group
had the best reproductive performance followed closely by
the controls. The two groups on the highest PCB levels
had only one kit for 16 females. This one did not survive.

During gestation, between April and May, the T4 levels
in the control and 0.5 ppm PCB groups decreased by 30% and
36% respectively. Decreases such as this are not common
during pregnancy in many species. In the 5 ppm and 2 ppm

PCB groups thyroxine levels did not change. This would be

expected if indeed they were not pregnant.

 

 

 

 

 

 

 

 

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62

During lactation, in May, the 3 PCB groups had T4
levels significantly higher than the control groups. In
the 0.5 ppm PCB group T4 was approximately equal to the 5
ppm and 2 ppm groups. During late lactation, T4 in the

group receiving 0.5 ppm PCB rose to the same level as the
other treatment groups. This may be due to a cumulative

buildup of PCB's. (See Appendix D for data summarized in

Figure 1.)

Experiment II--Estimation of Thyroxine Secretion
Rate and Associated Factors

 

 

A factorial analysis of variance for each calculated
parameter in this study (Table 4) shows that treatment
effects (A) were significantly different in all except body
weight. In all parameters there were significant differ-
ences with time (B). There were significant dose vs. time
interactions in TSR, T4, tk, body weight and percent dose
at time zero. (See Table 5 for mean comparison using the
Scheffé F test.)

In January, 1974 (see Figure 2), when mink were in
late diestrus, thyroxine secretion rate of control mink was

0.854 pg/lOO g body weight/day. The T was 1.69 pg/lOO m1

4

plasma which was not significantly different from the T4 in
control mink at a comparable period in experiment I. The
degradation rate constant K equals 0.1172 percent dose

131I-T4/m1 plasma per hour. The biological half life (t8)

 

 

 

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Figure 2.

67

131I-thyroxine degradation regression line and

associated parameters of female mink controls
not receiving polychlorinated biphenyl.

 

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CONTROL

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TDS/I009 BM. t l8.l9
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K 8 O.|l72

T4 =l.69pq %
Efl=3l.9pq T4
TSR=0854 p9/IOOg/DAY

   

 

HOURS

Figure 2

69

equaled 6.002 hours. Thyroxine distribution space (TDS/
100 gm body weight) equaled 18.19 mls. The total amount
of thyroxine present in all of the pools outside of the
thyroid, extrathroidal thyroxine (Ett) equaled 31.9 pg.

By February 22nd (see Figure 3), thyroxine secretion
rate of the controls was 0.44 ug/lOO g/day which is approxi-
mately one-half that observed in the other group of controls
in experiment II sampled in January. The 5 ppm PCB group
was found to be over 5 times higher than the control at

'2.35 ug/lOO g/day. Plasma thyroxine at 3.69 ug/lOO ml in
the 5 ppm PCB group was over 2.5 times higher than controls
at 1.28 ug/lOO m1. Degradation rate constant, K was sig-
nificantly greater in the 5 ppm PCB group than the controls
(0.1338 and 0.1109, respectively), and although the slopes
d0 rust appear visually different, they are significantly
dififierent (see Summary Table 5). Biological half-life also
was; significantly shorter in the 5 ppm group than controls
5°16; hrs. vs. 6.17 hrs. respectively. TDS/100 g body
weiSJht was also significantly larger in the 5 ppm PCB group
than controls, 20.04 ml vs. 12.68 ml respectively. Ett was
approximately four times larger in the 5 ppm PCB group
than it was in the controls, 66.7 pg T4 vs. 16.46 ug T4
respectively.

During mid-gestation in April (4-12-74), TSR in both
gI‘Oups decreased significantly from their previous sampling

‘3ate in February (see Figure 4). The 5 ppm PCB group TSR

 

 

Figure 3.

70

131I-thyroxine degradation regression line

and associated parameters of pre-estrus
female mink controls and those receiving
5 ppm polychlorinated biphenyl.

9.0.-
0500

.0
.5

.0

PERCENT '3'! DOSE lml PLASMA
N

.0

71

 

FEBRUARY l974

 

 

 

 

 

 

 

.— CONTROL .
N 8?
TDS/IOO 9 B.W.= I2.68
fg/Z'GJT hrs.
K= O.|l09
_ ' T4=I.28 pg 96
EH'=I6.46119 T4
b TSR-0.44 pq/IOO g/DAY
PC 885 ppm
-N '8
TDS/ IOO q 8.W.820.O ' ‘
f'lz'SJG "'3.
"K8 O.l338
T48 3.69119 ‘56
E‘H’8 66.? pg T4
TSR!I 2.35 yo/IOO 9 [DAY
I l | 1 l
2 4 6 8 IO I2

HOURS

Figure 3

 

72

 

 

 

 

 

Table 5. Statistical Comparison of Data in Experiment II
(TSR-TDR) Using the Scheffé F Ratio
Treatment Comparison (A Effect)

MS Feb. Apr. May (L) May (NL)
15.622 TDS F= 121.26 21.93 1.22 NS 35.59
0.7634 TLi 46.72 242.1 88.49 231.0
0.0037 K 25.4 145.9 10.40 35.0

0.1816 T4 11.3 220.0 220.0 132.64
0.0206 ETT 4.28 49.64 123.76 184.8
0.3468 TSR 36.78 68.23 75.50 8.58
MS Subj. W. Group; Vl = 1, V = 12
P< 0.05 Critical Value 4.75

continued

73

Table 5--continued

 

 

Time Comparison (B Effect)

 

 

 

 

 

 

TDS (MS - 15.804) TL5 (MS = 1.2926)
PCB Feb. vs. April 28.30 PCB Feb. vs. April 0.520 NS
PCB May vs. April 108.02 PCB May vs. April 14.57
PCB May vs. Feb. 25.74 PCB Feb. vs. May 9.58
C Jan. vs. Feb. 13.49 C Feb. vs. April 5.20
C Feb. vs. April 38.11 C April vs. May NL 13.69
C Feb. vs. May L 46.24 C April vs. MayL 5.58
C Feb. vs. May NL 53.38 C Feb. vs. May L 35.77
C April vs. May L 96.19
C May vs. Jan. 13.20
K (MS = 0.0007) T4 (MS = 0.1883
PAC Feb. vs. April 14.14 PCB Feb. vs. April 36.33
PCB May vs. April 14.65 PCB May vs. Feb. 18.23
PCB May vs. Feb. 13.59 PCB May vs. Feb. 13.60
C Feb. vs. April 195. C Jan. vs. Feb. 7.03
C April vs. May L 1.07 NS C Feb. vs. April 0.506 NS
C April vs. May NL 3.47 NS C Feb. vs. May L 24.81
C Feb. vs. May L 244.4 C Feb. vs. May NL 42.20
C Jan. vs. Feb. 0.390NS C April vs. May L 18.23
April vs. May NL 33.90
Jan. vs. May L 5.42
Jan. vs. MayNL 15.07

 

 

MS BX Sub. W. Group; V = 2, V = 24

P<<0.05 Critical Value = 4.32
continued

Table

5--continued

74

 

 

 

 

 

ETT (MS = 0.0215) TSR (MS = 0.1819)
PCB Feb. vs. April 44.71 PCB Feb. vs. April 76.05
PCB May vs. April 105.83 PCB May vs. April 38.57
PCB May vs. Feb. 13.029 PCB May vs. Feb. 6.53
C Jan. vs. Feb. 7.94 C Jan. vs. Feb. 6.34
C Feb. vs. April 0.499 C Feb. vs. April 0.82 NS
C Feb. vs. May NL 47.42 C Feb. vs. May L 24.29
C April vs. May NL 55.38 C Feb. vs. May NL 11.90
C May vs. Jan. NL 16.19 C April vs. May NL 18.98
Jan. vs. May NL 0.86 NS
Jan. vs. May L 5.808
MS BX Subj. W. Group; Vl = 2, V2 = 24

P< 0.05

Critical Value

\.

4.32

 

Figure 4.

75

131I-thyroxine degradation regression line and
associated parameters of pregnant female mink
controls and those receiving 5 ppm polychlori-
nated biphenyl.

PERCENT '3': DOSE/ml PLASMA

.09:-
(D 00 C)

.o .o
N «h

.0

76

 

 

 

 

APRfl.I974
CONTROL .
N38
TDS/IOOg 8308.93
t =7.l5 hrs.
l/2
K'0.097O
,.. T4=L391Jq 96
E'H'l2.59»9 T3
t Tsaaozslugn cg/oAv
_ PCBtSppm
N87
TDS/l009 B.w.=I2.06
- 71,234.85 "'8.
K=0J352
T482.4699 1,
" afiszssug T4
TSR- 0.909109/I009/0AY
I I I I I I

 

2 4 6 8 IO l2
HOURS

Figure 4

 

77

group decreased by approximately 2.5 times to 0.909
Ug/100 g/day and the controls decreased 30% to 0.291 ug/lOO
g/day but a three-fold difference was still observed be-
tween the control and 5 ppm PCB group. The small increase
in T4 between controls in February and April was not sig-
nificant but the difference between the 5 ppm PCB group at
2.46 ug percent and control of 1.39 ug percent was signifi-
cant. The rate constant K decreased significantly between
February and April to 0.0970 in the controls but increased
slightly but significantly to 0.1352 in the 5 ppm PCB group.
The 5 ppm PCB group was significantly higher than the con-
trols in April. The tk's lengthened significantly to 7.15
hrs. in the controls between February and April and de-
creased in the 5 ppm PCB group to 4.85 hrs. The 5 ppm PCB
group's tk was significantly shorter than in the controls.
The TDS/100 g body weight decreased significantly in
both controls and 5 ppm PCB group to 8.93 ml and 12.6 ml
respectively between February and April. The 5 ppm PCB
group was significantly higher than the controls. Ett in
the 5 ppm PCB group was reduced to 29.6 ug T4 which is one-
half of its February level. Control Ett also decreased sig-

nificantly to 12.59 ug T Ett of the 5 ppm group remained

40
significantly higher than in the control.

At mid-lactation in May (5-16-74) (see Figure 5), TSR
in the control mink, both lactating and non-lactating,

equaled 1.25 and 1.00% ug/lOO g/day, respectively.

"JJ

 

Figure 5.

78

131I-thyroxine degradation regression line and
associated parameters of lactating and non-
lactating female mink controls and those
receiving 5 ppm polychlorinated biphenyl.

PERCENT '3' I DOSE/ml PLASMA

L0
(18

(16

0A?

F;

 

 

79

 

IMAY

LACTATINO
CONTROL A

Iii-5
T080009 81'.- 28.38
*Ilz' 7.88 Im.

10008886

1’4- 2.0599 7.
Err-60.20;” T4
TSR-LZS flfl/lOOQ/DAY

|974

uouucnrmo
CONTROL 0

"'3
TDS/l009 awn-23.64
1""2' 8.7‘ "'8.

K-0.0784
T43 2.29 99 %
err-54.27” T4

TSRs l.00 pQIIOOQIDAY

PCB-59pm I

NI?

TDS/l009 B.\V.- 27. 86
{IR-6.49 hrs.
K-0.097ll

T4-3.|2 yo %

EH" 87.2 no T4

TSR- I.93po I I00 olDAY

 

3
n 1 1 1 1 n
2 ‘1 6 8 IO l2
HOURS

Figure 5

 

80

The control TSR values were significantly higher than at
any other time in the study. The TSR's during lactation
increased over the gestational rate in April by a factor<mf
4 in lactating controls and a factor of 3 for the none
lactating controls. The TSR of the 5 ppm PCB group doubled
from April to 1.93 ug/lOO g/day which is still significantly
higher than both lactating and non-lactating control groups
in Experiment II. In all the parameters measured between
the two control groups in May in Experiment II, only non-
significant differences were found. The plasma T4 level
increased significantly between April and May to 2.05 pg
percent for lactating and 2.29 ug percent for non-lactating
controls. T4 levels of the PCB groups significantly in-
creased to 3.12% which is still significantly lower than
the high observed in January of 3.69 ug percent. T4 levels
of both control groups were found to be significantly lower
than in the treated group. T8 increased significantly to a
rate higher than at any other time in both lactating (7.88
hrs.) and non-lactating (8.74 hrs.) controls; however, the
difference between the two controls were not significant.
The 5 ppm PCB groups t8 increased significantly between
April and May but remained significantly lower than in
February when the most rapid rate of degradation and secre-

tion was observed. The 5 ppm PCB groups t% (6.49 hrs.)

remained.significantly lower than the controls.

81

No significant changes occurred in rate constants K
in the controls between April and May but at 0.0866 for
lactating controls and 0.0784 for non-lactating controls
they were significantly lower than the K = 0.1109 for con-
trols in February. The rate constant of the 5 ppm PCB
group at 0.0971 was significantly lower than in April but
was significantly higher than in both controls in May.

TDS in May controls increased significantly over the
other sampling dates to 28.39 m1/100 g body weight for
lactating and 23,64 for non-lactating groups. The 5 ppm
PCB group's TDS doubled between April and May to 27.65 ml
which is significantly higher than in the non-lactating
controls but not significantly different from the lactating
control.

Ett in controls, during May (60.24 pg T4 lactating,

54.2 pg T non-lactating) increased to 4 times the April

4

level of 12.59 pg T The 5 ppm PCB group's Ett increased

4.

in May to 87.2 pg T which is 3 times its April level and

4

significantly higher than in the two control groups (see

.Appendix E for data summarized in Figures 2-7 and Table I).
A summary of the degradation slopes may be observed

for the controls in Figure 6 and for the 5 ppm PCB group

in Figure 7.

 

 

Figure 6.

82

Summary of 131I-thyroxine degradation regres-
sion lines for control mink.

PERCENT '3': DOSE/ml PLASMA

LG

83

 

 

 

CONTROL
. JAN
I FEB
A APRIL
0 MAY NONLACTATING
A MAY LACTATING

 

 

HOURS

Figure 6

 

 

'7 4
v.
A .

 

Figure 7.

84

Summary of 131I-thyroxine degradation regres-
sion lines for mink receiving 5 ppm poly-
chlorinated biphenyl.

85

 

 

 

II APRfl.

 

AI FEB

PCE!
I

 

 

 

 

 

<53“. .5368 :m. szomma

nee. 4. 2 ...
I000 0 0

HOURS

Figure 7

 

t:

Figure 7.

84

Summary of 131I-thyroxine degradation regres-

sion lines for mink receiving 5 ppm poly-
chlorinated biphenyl.

85

 

     

PCB.

'0 ‘ A FEB
0-3 I APRIL
0.6 0 MAY
0.4

PERCENT '3'I DOSE/ml PLASMA
.0
N

 

 

 

.0

HOURS

Figure 7

 

 

86

EXEeriment III--Male and Female Thyroxine
Levels

Male and female mink plasma thyroxine was found to be
significantly different only in July, 1973 (see Figure 8),
when the female T4 level was 2.52 pg percent and the male
was 1.72 pg percent. Male T4 levels were highest in
February and females were highest in July at 2.52 pg per-
cent. Male T4's were higher, though not significantly so,
than in females in the months of August, February and April
whereas females' T4 was higher but not significantly, than
males in December, March, and May (see Appendix F for data
summarized in Figure 8). Seasonal changes were observed in
both males and females.

Experiment IV--Thyroxine Binding and
Saturation Index

The thyroxine binding curve in Figure 9 shows that a
plateau occurs at approximately 2.00 pg percent T4 which
indicates that December mink saturate their TBG at 2 pg per-
cent; however, more importantly, the curve reveals the
quantity of exogenous and endogenous thyroxine necessary to
saturate the thyroxine binding globulin without exceeding
the conditions of pH 8.6 barbital buffer and suppression of
binding to prealbumins and albumin.

The binding plateau remains constant to approximately
26 119 percent whereupon it rises steeply. Binding to other

proteins probably occurs at T 4 concentrations above 26 pg

Percent T4, most likely to thyroxine-binding pre-albumins

87

.Hmmh 85» mo mam>umuafi UHUOHHmm um consummfi xGHE mamamm can
mama mo AmemHm HE ooa\m:v mcflxoumnp saunas mo GOmHHmmfiou

 

 

.m musmflm

m whamflm

:55 >32 mad. mg). mm“. own. 034

 

88

 

 

 

 

\ x
\ \
x s-

i
L

 

\ _
.\.

K

 

 

 

 

 

345m“. D
“.342 §

 

 

VWSV‘Id Moon/171 5r?

89

Figure 9. Thyroxine binding curve of plasma measured

on five mink in December. Each point repre-

sents the mean : standard error for thyroxine
Ll (pg/100 ml plasma) bound to protein.

 

rM
epP
[mm

119% T4 BOUND
N or A on m «I

90

 

 

48

 

I I J

 

l I
l3 l7 2| 25 29 33 37
TOTAL T4 USED 09% (EXOG.+ENDOG.)

Figure 9

 

91

(TBPA) and albumins. Therefore, to remain within the limits
of the thyroxine binding globulin capacity, 19 pg Percent
total T4 (exogenous and endogenous) was used to assess the
saturation of the control and 5 ppm PCB treatment animals.
The saturation index (SI) (T4 binding capacity of TBG

divided into the plasma T level) of the 5 ppm PCB group was

4
significantly higher than the control at each sampling date
(see Figure 10 and Table 6). During pro-estrus, in February,
the saturation index of the 5 ppm PCB group at 3.0 was
three-fold higher than the controls at 1.0. Between

February and gestation, in April, there was a significant
decrease in both the controls and the 5 ppm PCB group SI.

The 5 ppm PCB group saturation index dropped by one-half to
1.318 and the control dropped significantly to 0.531 in
April. Despite the decrease in SI, the 5 ppm PCB group
remained significantly higher than the controls.

Between gestation in April and lactation in May no sig-
nificant changes occurred in SI although both the 5 ppm PCB
and control groups increased slightly to 1.441 and 0.634
respectively. The 5 ppm PCB group remained significantly

higher than the controls. TBG-capacity increased signifi-

cantly in April and remained high during lactation in May.

Reproductive Performance
‘The reproductive rate for mink on 5 ppm and 2 ppm PCB

diets in experiments I and II is essentially zero. One kit

92

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\mEmmHm HE ooa\m: GH Hm>mH msflxouanu mammam u Hm
.xcfls wamfimm Eoum mammam mo xmpcw coflpmusumm

 

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95

was produced by 23 animals and it died. All of the animals
in experiments I and II were mated and motile sperm were
found in the vagina. The 0.5 ppm PCB group significantly
outproduced the controls in both experiments I and II;
There were twice as many kits produced by the 0.5 ppm PCB
group as in the controls (16 and 35 respectively at birth).
For various reasons, after 8 weeks only three control kits
were alive, while the 0.5 ppm PCB group had weaned 15 kits.

Eight control mink in experiment II produced 26 kits
but since the adults were killed for autopsy, the young

were farmed out to adoptive mothers. (See Table 7.)

Anatomical Parameters

It was found that mink from experiment II, when
autopsied on May 18, 1974 (see Table 8), had thyroid weights
'not significantly different between controls and the 5 ppm
PCB group. Liver weights were significantly higher in the
PCB group, 28.78 gm vs. 27.45 gm for lactating controls
and 24.04 gm for non-lactating controls. When expressed as
grams liver weight/100 gm body weight, the differences are
Still significant (3.68 gm for the PCB group and 2.94 and
2.53 gm for lactating and non-lactating controls).
Differences in body weight were not significant. The PCB
group had adrenal weights (11.32 gm/lOO gm body weight)
significantly higher than the controls (8.19 gm for lactat-

ing and 6.88 gm for non-lactating). Body weights did not

96

Table 7. Reproduction of Mink in Experiments I andeI

 

 

 

 

 

Number Kits Kits
Number females observed alive
females 'to give alive at. at'8
Experiment I. mated birth birth weeks
Control 7 5 16 3
0.5 ppm 8 7 35 15
2 ppm 7 4 l 0
5 ppm 8 0 O 0
Experiment II
Control 7 6 26 *
5 ppm 8 O 0 *

 

(N = 8 for each group except controls in experiment I

where there were 7 females.)

*Adults were autopsied and kits were placed with foster

mothers.

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98

significantly differ in May, between controls and 5 ppm

PCB group animals. Body weights did differ with time in
both experiment I and experiment II (see Appendices E and
G for body weight by date). Analysis of variance of body
weights of animals in experiment II may be seen in Table 4,
‘where no significant differences exist between treatments
but do exist for time and time-treatment interaction.

Cursory examination of the heart, stomach, intestines,
lungs, liver, brain and pituitary of the 16 mink autopsied
from experiment II, revealed no obvious differences between
the controls and the PCB group.

Despite the lack of significant differences in thy-
roid weights, histological examination shows that thyroids
of PCB group animals were more active than the controls.
The PCB group had taller follicular cells generally larger
follicles and more vacuoles in the colloid than controls
(see Figures 11 and 12 for photo micrographs of the PCB

group and control thyroids).

 

Figure 11.

99

Thyroid gland photomicrograph from a control
mink during mid-lactation (4-18-74) (magni-
fied 250 times).

Figure 11

 

 

If"

Figure 12.

101

Thyroid gland photomicrograph from a female
mink receiving 5 ppm polychlorinated bi-
phenyl (4-18-74) (magnified 250 times).

{a
L

 

DISCUSSION

Generally PCB's have an overall stimulating effect
upon thyroid function in mink except during reproduction,
when dosages and events modify thyroid and thyroxine hor-
mone parameters. T4 modifications by PCB's at reproduction
apparently affect the biological response to breeding and

its associated biochemical events.

Feeding PCB's for a long term at 5 ppm and 2 ppm re-

sults in stimulation of thyroxine secretion rate (TSR) which

occurs throughout most of the winter and spring months;
however, TSR becomes quite low by mid-pregnancy. During
the time of high estrogen in April, the degradation rate
(rate constant K) remains elevated in the 5 ppm PCB group
(Figure 4). This creates a condition of high utilization
with concomitantly lower secretion. The result is seen in

Experiment I (Figure l) as a drop in T level in the 5 ppm

4
group at the time of high estrogen level. Control animals,

particularly in Experiment I, responded oppositely. Circu-

1atory T was elevated during estrus, implantation and also

4
early gestation (Figure 1). Through most of the autumn

and winter in Experiment I, very high T levels were found

4
in the 5 ppm and 2 ppm PCB groups which may indicate a high

103

L

l\'.~ I10 I. lbw “a IuI~WDM r

in 04.. m.

W‘zg ' .
u

104

thyroxine secretion rate as seen during mid-February in
Experiment II (Figure 3). TSR was probably high in the
PCB-treated animals throughout the diestrus months, since
the T4 level was high (Figure 1).

Interestingly, the very high T levels in the face of

4

high secretion rate indicate a probable malfunction of the

normal feedback system involved in TSH release. T4 levels

are sufficiently high to completely saturate the TBG and f “

 

also to precipitate binding of T4 to other carriers such 5

as prealbumin and albumin (Table 6). The saturation index

 

of the carrier proteins with T in the 5 ppm PCB groups is

4
three times that of the controls in February (Table 6) and
exceeds the thyroxine binding globulin capacity by a factor
of three. Since the TBG capacity in the 5 ppm PCB group is
essentially equal to the control TBG capacity high T4 levels
have not stimulated increases in the amount of circulating
TBG. Carrying capacity is probably expanded by the presence
of other carriers. The feedback mechanism, if it were op-
erating normally, would still be expected to respond to the
high free T4 levels which exist under such excess T4 condi-
tions.

Estrus and pregnancy and their associated behavior and
hormonal changes alter thyroid functions in both PCB groups

and controls. At implantation and early gestation when

estrogen levels are very high in most animals, T4 was high

105

in the controls (Figure l) but was low in the 5 ppm groups
(Figure l and Figure 4) two weeks later in mid-pregnancy.
Decreases in T4 and TSR are contrary to observations in
other species such as cattle and guinea pigs (Hernandez
§E_§l., 1972; Pals, Shaw, and Reineke, 1973) where T4 and
TSH are observed to increase during pregnancy.

A drop in the saturation index of both control and PCB
groups to one-half their pre-estrus level was also observed
during pregnancy (Figure 10 and Table 6). Part of the de-
crease may be attributed to (l) a more than two-fold
decrease in TSR, which in turn was probably reduced due to
a reduction in TSH, and (2) to a large increase in TBG-
capacity from 1.16 to 3.0 ug percent may have been stimu-
lated.by estrogen and progesterone.

Serum thyroxine decreased throughout pregnancy in the
controls which is opposite to that observed in such species
as rats and humans (Heineman §E_gl., 1948; Man et_§l., 1969;

Robbins & Nelson, 1958). The T in the PCB group (Figure l)

4
was not similarly affected since it had not risen during

estrus. The very low T levels probably contributed in part

4
to the high mortality of the fetuses. Ringer et a1. (1972)
reported low reproductivity in mink on 5 ppm PCB; further-
more, they observed that mink receiving PCB long term at 10

ppm had high maternal mortality.

106

Estrogen is known to increase thyroid activity.
Soliman and Reineke (1955) have shown that estrogen or
progesterone plus estrogen when administered to ovarectom-

131

ized rats stimulate I uptake by the thyroid. Progesterone

alone had quite the opposite effect. It reduced 131

I uptake
by the thyroid. For estrogen to affect thyroid function
they have found that the pituitary and its interrelationship
with the CNS must remain intact and functional. Progesterone
probably functions similarly. No information is available
concerning the tidal changes in steroid hormone levels dur-
ing mink reproduction; however, there must be some circu—
lating estrogen and progesterone in the PCB-fed groups since
ovulation and some implantation is known to occur. It is
not known whether PCB-fed animals in this stage were imv
planting but observation of the mink by experienced mink
breeders failed to reveal any externally visible signs of
advancing pregnancy in PCB treated mink.

Despite some apparently normal behavioral estrogenic
effects in PCB mink, no increased thyroid activity was ob«
served at estrus or proestrus as occurred in the controls
and many other species such as rats, mice, and humans
(Schreiber, 1967; Hotelling and Sherwood, 1971). In fact
the decrease in thyroxine levels which occurred during
estrus (Figure 1, Figure 4) in the two highest PCB groups

indicates that the impaired feedback system is at least in

part operable.

 

107

Additional supportive evidence of an impaired T nega-

4
tive feedback mechanism in PCB-fed animals was observed
with the thyroxine binding globulin. The saturation index
in May remained 1.5 times higher than the saturability of
TBG (Figure 10, Table 6). This occurred at a time when
control TBG was only 63% saturated. This implies a higher
than normal free T4 level since the free or loosely bound

to bound T4 equilibrium is apparently raised.

A noteworthy event occurred with regard to T levels

4
in the 0.5 ppm PCB group 4 (Figure 1). Throughout most of
the seasons preceding estrus this group had T4 levels which
could be described as stimulated but not as severely over-
stimulated as observed in the highest PCB group. At estrus,
in March, there was sufficient responsiveness remaining to
be highly stimulated by estrus and the reproduction events.
The T4 level of the 0.5 ppm PCB group exceeded that of the
control through the reproduction season. T stimulation at

4

this PCB level and at that time of year was apparently re-
productively optimal, for they whelped and weaned a signifi-
cantly large number of young than the controls (Table 7).
The excellent birthrate which was observed in mink receiving
0.5 ppm PCB contrasts exceedingly with the zero reproduction
observed in the two highest PCB treatment groups. T4 levels
in the 4 groups in Experiment I at implantation (Figure l)

correlate exactly with the reproduction performance observed

at parturition (Table 7). Iwamoto (1973) reported that

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108

PCB Aroclor 1254 was slightly stimulatory to reproduction
at 1 ppm.
Aulerich et_al. (1973), previously reported that high
PCB administration inhibits mink reproduction. They have
also found that fish from lake Michigan,containing PCB's,
/Freduced reproductive performance. Ringer gt_§l. (1972) and

Aulerich et al. (1972) reported that PCB's seem to be more

7‘“
WJ‘ highly concentrated in the brain than in any other organ.

_ {...-4...“.

\ Apparently the lipid soluble nature of PCB's causes it to

*1 I
fig“ +1 concentrate in fat deposits and in tissue with a high lipid

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h I :
i
.
’5 ~ _’
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ér; (content.

Dad The high content of lipid in the brain is the major
".‘reason for the very high PCB concentration found there. It
Pappears likely that the exceedingly high brain PCB levels

Linterfere with CNS Operation both humorally and electrically.
The high brain PCB appears to further enhance the contention

that the alteration in thyroid function may be attributed
to the impaired negative feedback mechanism. The feedback
system is highly dependent upon the hypothalamic, limbic
systems and higher brain centers.
A slightly below normal reproductive rate in controls
in Experiment I (Table 7) might be explained by disturb-
ances associated with drawing blood samples and handling

during breeding and implantation. These disturbances may

have induced some unnoticed abortions.

109

The fact that thyroxine levels differ between Experi-
ment I and II (Figures 1 and 4) during pregnancy may be
explained by the dates on which the samples were taken.
Experiment I thyroxine samples were taken on April 6th in

early pregnancy; whereas, T samples in Experiment II were

4

taken two weeks later on April 16th. Mink have an approxi-

mately 5 week gestation period extending from approximately

 

April lst to May 7th, and a 10 day difference in sample

dates equals approximately one trimester. Since circulating

 

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1 t1_-——— - .

T4 levels in Experiment I controls dropped one full pg per-
cent during pregnancy, T4 levels will not be identical at
any two sample dates during pregnancy.

No depletion in T was seen in the two highest PCB

4
groups during the usual gestation period (Figures 1 and 4)
since they were probably not pregnant or at least had begun
reabsorbing their fetuses. Fetal death may result from
fetotoxicity due to PCB's or from uteri unprimed for
receiving and supporting an embryo. Hypothyroidism as was
reported by Soliman and Reineke (1954) is closely associated
with fetal loss, abortion and impaired reproduction.
Thyroxine is known to be necessary for the preparation and
maintenance of receptive uteri; therefore, mink on high PCB
diets which were relatively hypothyroid during reproduction
may have had insufficiently prepared uteri even though they

had significantly greater thyroid activity than the controls

during the rest of the year.

I) fit

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a

110

Additionally, several months of hyperthyrodism in the
two high PCB groups may have led to a severe depletion in

fat reserves. High BMR's stimulated by T were not

4
measured but may have contributed to a reduced availability
of nutrients for the fetus. The energy depletion stress

may have further compounded the T and PCB factors in reduc-

4
ing reproduction to zero in the two highest PCB groups.
Excess thyroxine secretion which occurred (Figures 1
and 3) throughout autumn and winter in the two highest PCB
groups had by February saturated the T4 carriers far in
excess of the normal TBG-capacity by a factor of 3 times
(Table 6). Binding to prealbumin and albumins is the most
probable explanation for the excess bound T4. Through preg—
nancy and lactation in April and May the saturation index of
the 5 ppm PCB group remains higher than the control but
estrus, pregnancy and lactation has apparently stimulated
a higher concentration of TBG since the TBG capacity is
larger in the controls at that time. Dowling §E_§l,, 1956a;
Braverman §E_al., 1967; and Zaninovich gt_al., 1966, have
shown that estrogen increases TBG-capacity similar to these
results (Table 8).
The stimulation of adrenal glands which was observed
in the 5 ppm PCB (Table 8) group, could result,from two
sources. It may be due to the suppression of the negative

feedback mechanism for adrenal steroids but more likely it

results from adrenal gland stimulation by thyroxine.

.—

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111

Wallach and Reineke (1949) reported that exogenous thyroxine
in rats increased adrenal weight and concurrently caused
changes in adrenal ascorbic acid content. These are sensi-
tive indicators for adrenal function, thereby providing
evidence of a T4-induced stimulating effect upon the adrenal
gland. Iturri (1974) observed a PCB-induced adrenal weight
increase in chickens which is similar to the one we have

obServed in mink.

""Q/L dr-Liver weight was increased by PCB's (Table 8) probably

1" l.‘

as a result of increased demand for detoxifying enzymes or
as a result of metabolic stimulation by thyroxine or both.
Grant gt_al., 1971; Rehfeld gt_al., 1971; and Lincer and
Peakall, 1973, observed increases in liver weight in many
other species which had been treated with PCB. Induction of
liver enzymes by PCB's was also noted by Rhee and Plapp
(1973).

Circulating thyroxine levels in male and female mink
were essentially not different from each other throughout
most of the year (Figure 8) but differed in different months.
Female thyroxine secretion rates in January averaged 0.854
t 0.175 ug/loo gm B.W./day which agrees extremely closely
with that found in.males by Reineke §E_al,(1960). They
reported that TSR was equal to 0.95 ug/lOO g B.W./day using
the thyroxine substitution method of Reineke and Singh
(1955). The close agreement of the two methods for estimatv

ing TSR is noteworthy considering the differences in the

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112

thyroxine degradation method and the thyroxine substitution
method (see Literature Review and Methods section for
description of the two methods).

Distribution space for thyroxine was greater in the
5 ppm group than in the control groups (Figures 2-5). No
edema was noticeable upon autopsy of the 5 ppm PCB group as
reported in birds by Iturri (1974). It seems possible that
the PCB's may open additional pools to thyroxine. For
example, they may lower the blood brain barrier for T4 or

may expand the interstitial cell fluid space. Total extra

 

thyroidal thyroxine (ETT) was correspondingly large in PCB
treated animals (Figures 2-5). With more distribution

space available, the total T4 present within an animal would
be predictably expanded.

[“ Histology of the thyroids after 9 months on PCB's at

‘5 ppm revealed that thyroids were stimulated (Figure 12).

[They had numerous very large follicles, containing numerous

FKg, vacuoles within the colloid and tall cuboidal epithelium

 

(1 surrounded the follicles. In contrast controls (Figure 11)

P ‘01- had small follicles low cuboidal epithelium and few col-

 

Lloidal vacuoles.
These morphological data further support the chemical
data which suggest that stimulation of the thyroid produced

high T levels which were permitted to occur without slowing

4
the thyroxine output rate. This evidence further supports

113

the hypothesis that the PCB's affected the CNS and sup-
pressed the normal feedback system allowing high thyrotro~

pin output in the face of high circulating thyroxine.

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116

APPENDIX C
TECHNIQUE FOR MEASURING BINDING CAPACITY OF TBG

A. Reagents and Solutions for TBG—Capacities

125

1. I—L-thyroxine Solution

L—thyroxine 125

I in 50% aqueous propylene glycol
(Amersham/Searle Corp., Arlington Heights, Illinois) was
diluted in glass distilled water such that 0.05 ml of the
diluted standard would yield 10,000 to 50,000 cpm. The
contribution of this amount of labeled T4 during the first
2 half—lives was less than 0.1 pg percent per sample.

2. Cold Thyroxine Solution

Ten mg of purified L-thyroxine was weighed to the near—
est 0.1 mg and dissolved in glass distilled water with the
aid of NaOH to dissolve the crystals. All glassware was
siliconized to prevent T4 adhesion. The cold T4 was brought
to a concentration of 0.02'ug/m1 by serial volumetric dilu«
tion in glass distilled water at 25°C. A second concentra-
tion of 0.04 g/ml was also prepared to provide a more concen—
trated solution needed in the upper regions of the thyroxine

binding curve.

3. Barbital Buffer (pH 8.6)

 

(a) N/lO HCl solution:
9.857 gm of concentrated reagent grade HCL (37% HCl)
was weighed on a triple beam balance and was brought to 1000

ml volumetrically with glass distilled water.

117

(b) M/lO Sodium Barbital Solution:

20.62 gm of powdered sodium barbital was weighed to the
nearest 0.1 mg and diluted volumetrically to 1000 ml with'
glass distilled water.

(c) Preparation of Buffer:

129 m1 of the HCl solution (a) was added from a burette
into a 1000 m1 volumetric flask and brought to volume with
the sodium barbital solution (b). After thoroughly mixing
with a Magna Stirrer the pH was checked using a Beckman pH
meter with a Corning Semimicro electrode (Corning Glassworks,
Medford, Mass.) probe. Any slight deviations from pH 8.6
were adjusted with HCl or NaOH.

B. Resin-impregnated Sponges and Polypropylene
3.23.3.9;

Resin sponges with the capacity to absorb specifically
free thyroxine (Abbott Radiopharmaceuticals, North Chicago,
Illinois*) were cylindrical in shape and have the following
dimensions: 1.1 cm O.D. x 1.95 cm. Dispersed within the
polyurethane sponge is a finely divided ion exchange resin.
Unbound thyroxine is quickly and quantitatively bound to the
resin sponge while TBG-bound thyroxine is not. (IRA-400
anion-exchange resin may be substituted for the resin

sponge.)

 

*Thanks are due to Abbott Radiopharmaceutical for donat-
ing the sponges used for this procedure.

118

The polypropylene tubes had the following dimensions:
1.3 cm I.D. x 8.6 cm (Abbott Radiopharmaceuticals, North
Chicago, Illinois) and adhered no thyroxine as non-sili-
conized glass tubes would. They were also reusable upon
allowing the tracer to decay for a year or so and washing
with.Radiac Wash.
Computation of Thyroxine Concentration
for the Binding Curve

A range of total exogenous T4 levels was chosen from
12 to 34.6 pg percent in 1.0 pg increments. To compute the

total endogenous plus exogenous levels, the serum T4 level

was added to each increment. To calculate pg added:

pg percent cold _ pg T4/ml concentration volume

exogenous T4 of cold T4 X cold T

X
4.

100/0.05 VIII

The factor of 100/0.05 is used to transfer the units

of cold T4 into the same units as the 0.05 ml serum which

was added, i.e., to pg percent.

Total pg percent = pg percent cold + 125I T4 +

added exogenously
Serum T4 'IX
pg percent

To calculate the amount of barbital buffer needed,
1.65 ml total volume minus volume cold T4 = volume barbital

buffer (always more than 2/3 of the total volume).

 

119

Procedural Sequences for Binding Curve

Into a series of polypropylene tubes was measured a
sufficient quantity of barbital buffer (pH 8.6) to sum up
with the volume of cold T4 at a concentration of 0.02
Hg/ml or at 0.04 pg/ml to a volume of 1.65 ml per tube.
As the amount of cold thyroxine progressively increased in
small increments, the volume of buffer decreased.
Sequentially, to the barbital buffer was added 0.05 ml of
125I labeled L-thyroxine from disposable microliter pipettes
followed by a 15 second vortex mix.

Cold thyroxine was then added from a Hamilton 500

microliter syringe followed by another 15 seconds of vortex

mixing. Lastly, serum was added to each sample and vortex-

mixed for a minimum of 30 seconds. From previous experience,

binding to TBG will be erratic unless the tubes are thorough-

ly mixed immediately after adding each component. Tubes
were then immersed in a slowly shaking warm water bath at
37 i_0.5°C and the reaction mixture was allowed to equili-
brate for 1 hour, after which the resin impregnated sponges
were added. Each sponge was gently depressed three times
with a plastic plunger. Initial counts were taken using a
gamma counter and scaler analyzer set to count at the 1251
peak. The tubes were incubated for 30 more minutes at

37 i_0.5°C to allow the resin-impregnated sponges to absorb

free thyroxine. *Each tube was then immediately filled with

distilled water and the sponges were washed and aspirated

 

 

120

with a plastic suction apparatus. The washing was repeated
3 times and each time the sponges were depressed gently

with the suction apparatus and most of the liquid was re—
moved. The TBG-bound thyroxine was removed with the wash-
ing, leaving only free thyroxine adhering to the resin
impregnated sponges. A final count was then taken from each

tube.

Calculations for Thyroxine Binding Curve
For each level of cold exogenous thyroxine added to a
tube, pg percent T4 bound to protein is calculated from the

following equations:

(FCPM - Background cpm)

*- . O ' c = V '
pg percent bound .0 reSln sponge ICPM _ Background cpm x

(correction factor A) X
where the correction Factor A =

Blank (serum free) ICPM - Background cpm
Blank (serum free) FCPM - Background cpm

Correction factor for free
thyroxine not bound to resin XI
impregnated sponge.

(correction factor A was usually slightly less than

1.00)
pg percent T4 bound = l-percent pg x Total pg percent
to protein bound to resin cold T4
sponge

XII

When pg percent T4 bound to protein is plotted against

total T4 used the binding curve in Figure 9 results. From

 

 

121

the flat plateau portion of the binding curve the amount of
T4 necessary to saturate the TBG binding sites can be read.
T4 values above the plateau represent nonspecific binding,
probably to albumin or other proteins. At very high T4
concentrations, two criteria of the specificity of this
method are violated. The dilution factor is less than 30-
35 and the capacity of the barbital buffer (pH 8.6) to
suppress the normally feeble binding to protein is exceeded
at very high T4 concentrations. Therefore, a level of total

T4 about midway through the plateau will provide sufficient

thyroxine to saturate most, if not all, of the sites.

Procedural Sequences for TBG Capacity

Into each of 3 polypropylene tubes was measured a suf-
ficient quantity of barbital buffer (pH 8.6) to sum with
the volume of cold T4 at a concentration of 0.02 pg/ml to a
volume of 1.65 ml per tube and a dilution factor for serum
of 30-35. Two of the tubes were designated as duplicates
containing serum and the third was a serum-free blank.
Barbital buffer was added with a Hamilton microliter syringe.
To each tube was added 0.05 ml 125I-L-thyroxine using dis-
posable microliter pipettes followed by 15 seconds of vortex
mixing. Next in sequence, unlabeled thyroxine at 0.02 pg/ml
was added to each tube and vortexemixed for 15 seconds.

Lastly, serum was added to the two duplicates but none was

added to the blank. Each tube was then vortex-mixed for 30

122

seconds to ensure equal distribution of each component.
Tubes were immersed in a slowly shaking water bath at 37° :
0.5°C for 1 hour and were then treated from this step in
the same fashion as those tubes described in the binding
curve method.

Calculation of T,I Concentration Necessary
fOr Determining TBG Capacity

 

 

From the plateau region of the binding curve, a concen-
tration of total T4 was chosen which would saturate most, if
not all, of the available thyroxine binding sites but not
exceed the capacity of the barbital buffering system and the

dilution factor of 30-35 to inhibit nonspecific protein

binding.
(1) Amount (pg percent) of cold T4 needed =
Total T4, pg percent (exogenous + endogenous) -
serum T U9 Percent - 125I-T XIII

4' 4

(2) Volume cold T needed (ml) =

4
Amount cold T, needed (1)

 

 

 

 

 

Concentration’of cold T4, pg/ml XIV
(3) Buffer volume = 1.65 ml total volume -
(of buffer + cold T4)
ml cold T4 XV
Calculations for TBG-Capacity and
Saturation Index
iggfi : gzgfigigggg Egg x correction factor B =
pg percent bound to reSln XVI

sponge

123

where the correction factor B =

Blank (serum free) ICPM - background cpm =
Blank (serum free) FCPM - background cpm

 

correction factor for free
thyroxine not adhered to
resin impregnated sponge.

pg percent T4 bound to = l-pg percent bound
TBG to resin sponge
Total pg percent used XVII
Saturation Index (SI) = Serum thyroxine
pg percent/pg percent T4 bound to TBG XVIII

Correction for Thyroxine Not Bound by
ReSin Sponges

Correction factors A and B in the Calculation Section
were determined by preparing blank tubes to contain precisely
identical labeled and unlabeled thyroxine as do the dupli-
cates but contain no serum. Since there was no TBG in the
blanks, any observable difference between the blank ICPM and
the blank FCPM was a result of the amount of free thyroxine
not taken up by the sponges. The fractional correction of
ICPM/FCPM will correct for this usually small difference.

The correction fraction is used to correct the sample FCPM,
which assumably had also left unbound a similar quantity of

free T4.

 

 

 

 

 

 

124

 

 

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APPENDIX H

Formula used in the Scheffé F ratio

For comparison of the "A" effect between treatments:

F = [gj (A) + (C'j A2)]2

 

MS . 2 2—
Sub . W. Groups (C.) + (C'.)
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When Vl = (p - l) V

means compared

2 - P(n-l) and K = Number of

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, 2
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When V = (q - l), V = P (n - 1) (q - l) and

l
K

2

number of mean comparisons.

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