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This is to certify that the

dissertation entitled

Assessment of the Toxicologic, Pathologic and
Carcinogenic Effects of
3,h,3',4'-Tetrabromobiphenyl in Rats

presented by

Darlene Dixon

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Patho'logy

 

 

.fimz/ aye/a

 

Major professor /

Date 9/20/85

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

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FD

ASSESSMENT OF THE TOXICOLOGIC, PATHOLOGIC AND CARCINOGENIC

EFFECTS OF 3,4,3',4'-TETRABROMOBIPHENYL IN RATS

BY

Darlene Dixon

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Pathology

1985

39521713.

ABSTRACT

ASSESSMENT OF THE TOXICOLOGIC, PATHOLOGIC AND CARCINOGENIC
EFFECTS OF 3,4,3',4'-TETRABROMOBIPHENYL IN RATS

BY

Darlene Dixon

Pitot's bioassay for hepatocarcinogenesis was used to
determine :LE 3,4,334fl-tetrabromobiphenyl (3,4-TBB) can
promote or initiate gamma-glutamyl transpeptidase (GGT)
positive enzyme-altered foci (EAF). Groups of 6 female 180-
200 9 rats were used for initiation and promotion assays.
To test for initiation, rats were partially hepatectomized
(PH) and given 1, 5, or 10 mg of 3,4-TBB/kg orally, or 10 mg
of diethylnitrosamine (DEM/kg ip as an initiator. Thirty
days later, rats were promoted with 500 mg of phenobarbital
(PB)/kg for 180 days. Results indicated that 3,4-TBB may
have initiating potential as suggested by increased numbers
of EAF in rats initiated with 3,4-TBB and promoted by PB
compared to rats initiated with 3,4-TBB or DEN and fed basal
diets. To test for promotion, PH rats were initiated with
DEN and 30 days later diets containing 0.1, l, or 5 mg of
3,4-TBB/kg were fed for 180 days. 3,4-TBB increased the
number of EAF and thus appears to act as a hepatic tumor

promoter.

Darlene Dixon

3,4-TBB was not severely toxic in rats as evidenced by
the histologic appearance of the liver, spleen, thymus and
thyroid gland and ultrastructural changes in the liver.
Rats fed 5 mg of 3,4-TBB/kg had significantly decreased
hepatic retinyl esters concentrations compared to rats fed
diets containing 0.1 or 1 mg of 3,4-TBB/kg or 500 mg of
PB/kg. Serum thyroxine (T4) and free T4 concentrations were
also decreased in rats fed diets containing 3,4-TBB.

To determine the effects of 3,4-TBB on hepatic
glutathione (GSH) concentrations, groups of 3 male 160-180 9
rats were given a single oral dose (17 mg/kg) of 3,4-TBB or
a combination treatment consisting of a single oral dose (1
mg/kg) of 3,4,5,3',4',5'-hexabromobiphenyl (3,4,5-HBB) given
24 h before a single oral dose of 17 mg of 3,4—TBB/kg. 3,4-
TBB had no effect on hepatic GSH concentrations compared to
controls at 2, 4, 8, 24 or 48 h after dosing. Pretreatment
of rats with 3,4,5-HBB did not alter the effects of 3,4-TBB

on hepatic GSH concentrations.

DEDICATION

To my mother, Evelyn B. Dixon

"Guided by my heritage of a love of beauty and a
respect for strength - in search of my mother's
garden, I found my ownJ'

Alice Walker

ii ~

ACKNOWLEDGEMENTS

I would like to express my sincerest appreciation to
Dr. Stuart D. Sleight, my major professor, for his guidance
and support throughout my graduate studies. His friendship,
tolerance, advice and criticisms are appreciated.

I wish to thank Drs. Steven D. Aust, Allan L. Trapp,
Keiji Marushige and Tracie E. Bunton for serving as my
guidance committee members andikn:excellent direction in
completion of my research, academic program, and compilation
of this dissertation.

In preparation (Hf the research presented lJl this
dissertation, I have received faculty and technical
assistance from many persons at Michigan State University
and would like to acknowledge the following individuals for
their help: Dr. Ronald P. Slocombe, Dr; Gregory Fink, Dr.
Raymond Nachreiner, Dr. Richard IL Jensen, [ha Robert
Leader, Dr. Margit Rezabek, Cynthia Millis, Fran Whipple,
Mae Sunderlin.and Donna Craft. I would especially like to
thank Cheryl Assaff for typing this dissertation and Irene
Brett and Dr. Esther Roege for their technical assistance.

A special thanks to Dr. Albert W. Dade, Patricia Lowrey,
Dr. Charles Lowrey, [my Tuskegee colleagues, Dr. David

McConnell, Dr. Yasuko Marushige, Calvin and Deborah Moore,

iii

Joyce Wright, and Karen Wettlin for their encouragement,
advice and friendship.

My deepest appreciation to my mother for her unwithering

love, understanding and support.

iv

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . vii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . viii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . 5

Chemical and Physical Properties of Polybrominated
Biphenyls (PBB) . . . . . . . . . . . . . . . . . . . 5
PBB as Environmental Contaminants . . . . . . . . . . 7
Xenobiotics and Xenobiotic Metabolism by the Hepatic
Microsomal Monooxygenase System . . . . . . . . . . . 8
Pharmacokinetics of Polyhalogenated Aromatic
Hydrocarbons (PHAH) . . . . . . . . . . . . . . . . l3
PBB and Induction of Hepatic Microsomal Drug
Metabolizing Enzymes . . . . . . . . . . . . . . . . l6
Hepatic Monooxygenase Metabolism of PBB . . . . . . . 21
Toxicity and Hepatic Microsomal Enzyme Induction
Effects of PBB . . . . . . . . . . . . . . . . . . 25
Pathotoxicologic Effects of PBB . . . . . . . . . 25
Hepatotoxicosis . . . . . . . . . . . . . . . . . 26
Immunotoxicosis . . . . . . . . . . . . . . . . . 29
Thyroid Gland Toxicosis . . . . . . . . . . . . . 30
Mechanism(s) of PBB Toxicosis . . . . . . . . 31
Glutathione: Role in Detoxification and PBB Toxicity 36
Initiation and Promotion of Carcinogenesis: Pitot' 5
Model of Experimental Hepatocarcinogenesis . . . . . 40
PBB as Promoters in Experimental Hepatocarcino-
genesis . . . . . . . . . . . . . . . . . . . . . 44

CHAPTER I: ASSESSMENT OF 3,4,3',4'-TETRABROMOBIPHENYL
(3,4-TBB) AS A PROMOTER OR INITIATOR IN EXPERIMENTAL
HEPATOCARCINOGENESIS IN RATS . . . . . . . . . . . . . 47

Introduction . . . . . . . . . . . . . . . . . . . . 48
Materials and Methods . . . . . . . . . . . . . . . . 52
Results . . . . . . . . . . . . . . . . . . . . . . . 61
Discussion . . . . . . . . . . . . . . . . . . . . . 80
Summary . . . . . . . . . . . . . . . . . . . . . . . 84

Page

CHAPTER II: CHRONIC DIETARY ADMINISTRATION OF 3,4,3',4'-
TETRABROMOBIPHENYL (3,4-TBB) TO RATS: EFFECTS ON SERUM
AND HEPATIC VITAMIN A HOMEOSTASIS AND SERUM TRIIODOTHY-
RONINE (T3) AND THYROXINE (T4) CONCENTRATIONS . . . . . 86

Introduction . . . . . . . . . . . . . . . . . . . . 87
Materials and Methods . . . . . . . . . . . . . . . . 88
Results . . . . . . . . . . . . . . . . . . . . . . . 93
Discussion . . . . . . . . . . . . . . . . . . . . . 97
Summary . . . . . . . . . . . . . . . . . . . . . . . 98

CHAPTER III: ASSESSMENT OF THE EFFECTS OF 3,4,3',4'-
TETRABROMOBIPHENYL (3,4-TBB) ON HEPATIC GLUTATHIONE (GSH)
'CONCENTRAT IONS IN RATS C O O O O O C C O O O O O O C O 1 00

Introduction . . . . . . . . . . . . . . . . . . . . 101
Materials and Methods . . . . . . . . . . . . . . . . 103
Resu1ts O O O O O O O O O O 0 O O O O O O O O O O O O 108
DiscuSSion O O 0 O O O O O O O O O O O O O O O O O O 112
Summary . . . . . . . . . . . . . . . . . . . . . . . 115
CONCLUS IONS O O O O O O O O O 0 O O O O O O 0 O O O O O 1 l 7
LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . 120

VITA O O O O O O O O O O O O O O O O O O O O O O O O . . 137

vi

LIST OF TABLES

Table Page

1-1 Experimental design for 3,4,334'-tetrabromo-
biphenyl (3,4-TBB) initiation and promotion
assays . . . . . . . . . . . . . . . . . . . . . 53

1-2 Number of enzyme-altered foci (EAF)/cm3 of
liver in rats initiated with DEN and promoted
with PB or 3,4-TBB for 180 days . . . . . . . . 62

1-3 Number of EAF/cm3 of liver in rats initiated
with DEN or 3,4-TBB and promoted with PB for
180 days 0 O O O O O O O O 0 O O O 0 O O O O O O 64

1-4 Body and organ weights in rats initiated with
DEN or 3,4-TBB and promoted with PB or 3,4-
TBB for 180 days 0 O O O O O O O O O O O O O O O 65

1-5 Liver and adipose tissue concentrations of
3,4-TBB o o o o o o o o o o o o o o o o o o o o 79

2-1 Experimental design for dietary administra-
tion of 3,4,324'-tetrabromobiphenyl (3,4-
TBB) for 180 days . . . .. . .. . .. . .. . 89

2-2 Liver and serum retinol (ROH) and liver
retinyl esters (RE) concentrations in rats
fed 3,4-TBB for 180 days . .. . .. . .. . .. 94

2-3 Serunl triiodothyronine (T3 ), thyroxine (T ),
free (F)T and FT4 concentrations in rats ed
3,4-TBB fbr 180 days . . . . . . . . . . . . . . 96

3-1 Experimental design for assessment of the
effects of 3,4,3',4'-tetrabromobiphenyl (3,4-
TBB) on hepatic glutathione (GSH)
concentrations . . . . . . . . . . . . . . . . .104

3-2 Hepatic GSH concentrations in rats given a
single oral dose of 3,4-TBB, 3,4,5fi?,4',5“—
HBB (345-HBB) or a combination of both
congeners . . . . . . . . . . . . . . . . . . .109

3-3 Body and liver weights in rats given a single

oral dose of 3,4-TBB, 345—HBB or a
combination of both congeners . . . . . . . . .110

vii .

Figure

0-1

0-2

LIST OF FIGURES

Chemical structure of polybrominated
biphenYIS (PBB) O O O O O O O O O O O O O O 0

Modified hypothetical diagram of cytochrome
P-450 reductase and forms of cytochrome P-450
in membrane of endoplasmic reticulum .. ..

Modified microsomal electron transport chain

Pitot's two-stage model of experimental
hepatocarcinogenesis . . . . . . . . . . . .

Photomicrograph of a liver section from a rat
fed a diet containing 500 mg of PB/kg for 180
days 0 O O O O O O O O O O O O O O O O O O O

Photomicrograph of a liver section from a rat
fed a diet containing 5 mg of 3,4-TBB/kg for
180 days 0 O O O O O I O O O O O O O O O O O

Photomicrograph of an EAF within a liver
section from a rat initiated with 5 mg of
3,4-TBB/kg given orally and promoted with 500
mg of PB/kg for 180 days . .. . .. . .. .

Photomicrograph of an EAF within a liver
section from a rat initiated with 10 mg of
DEN/kg given ip and promoted with 5 mg of
3,4-TBB/kg for 180 days . . . . . . . . . . .

Photomicrograph of a histochemically-stained
EAF within a liver section from a rat
initiated with 10 mg of DEN/kg given ip and
promoted with 500 mg of PB/kg for 30 days ..

Photomicrograph of a neoplastic nodule within
a liver section from a rat initiated with 10
mg of DEN/kg given ip and promoted with 500
mg of PB/kg for 180 days . .. . .. . .. .

Photomicrograph of a liver section from a rat

initiated with 10 mg of DEN/kg given ip and
fed a basal diet for 180 days .. . .. . ..

viii

Page

11

12

52

68

68

71

71

73

73

75

Figure

Page

Electron micrograph of a hepatocyte from a
nonpartially hepatectomized and noninitiated
rat fed a diet containing 5 mg of 3,4-TBB/kg
for 180 days .. . .. .. .. .. .. .. .. 78

Higher magnification of hepatocyte in Figure
0 O O O 78

1-9 0 o o o o o o o o o o o o o o o o 0

ix

INTRODUCTION

Polybrominated.biphenyls (PBB) are lipophilic, water
insoluble, slowly metabolized polyhalogenated aromatic
hydrocarbons first used in the United States as flame
retardants in plastic materials in 1970 (Brinkman and deKok,
1980). Two companies, the Michigan Chemical Company (St.
Louis, MI), which later merged with the Velsicol Chemical
Corporation (Chicago, IL) and White Chemical Corporation
(Bayonne,rhn produced commercial quantities of PBB. The
Michigan Chemical Company produced approximately 11 million
pounds of a PBB mixture sold under the tradename Firemaster
(FM) BP-6 (Brinkman and deKok, 1980).

In 1973, approximately 1,000 pounds of FM BP-6 was
inadvertently mixed into livestock feed at a mill located in
Battle Creek, MI (Carter, 1976L. Consumption oftflflrsfeed
by cattle, swine and poultry resulted in contamination of
milk, meat, eggs and finished feed that was ultimately
consumed by many of Michigan's residents (Dunckel, 1975).
As a result of this accident, over 90% of Michigan's
residents have detectable levels of PBB in their tissues
(Selikoff and Anderson, 1979). To date, there is no
conclusive evidence that PBB cause acute or short term

illnesses in humans, but they pose possible long term or

chronic health risks due to their persistence in the body

1

and environment. The FM mixture has been reported to cause
hepatocellular carcinomas in rats (Kimbrough et al,, 1981).
FM and congeners of PBB have been reported to promote liver
cancer in rats, and in the future may be associated with
adverse long-term effects such as cancer in humans (Jensen
et 31” 1982, 1983b).

The FM BP-6 mixture is composed of a number of
congeners differing from one another in the number and
position of bromine substitutions on the biphenyl rings
(Aust 33 al., 1981). The structural configuration and
degree of bromination are important in predicting the
toxicologic, metabolic and carcinogenic properties of these
congeners. FM BP-6 and many congeners of PBB have been
found to cause toxic responses in many different mammalian
systems (Poland and Knutson, 1982). These include
hepatocellular hypertrophy, VaCUOlanJNl and necrosis,
porphyria, chloracne, a wasting syndrome, menorrhea, immune
suppression and thymic and splenic lymphoid depletion.

The first objective of the research described in this
dissertation was to assess the carcinogenic effects of
3,43?,4'-tetrabromobiphenyl (3,4-TBB), a minor component
tentatively identified in the commercial FM BP-6 (Robertson
et_;Lq 1982). 3,4-TBB differs from the other congeners of
PBB studied in our laboratory in that it is metabolized (£2
yitgg and $2 3139), and therefore it is not very persistent
in the tissues of the body (Millis gt, 1., 1985a; Mills et

al., 1985). This characteristic of 3,4-TBB allowed us to

evaluate the congener as both an initiator and promoter in a
two-stage initiation/promotion assay for
hepatocarcinogenesis in rats (Pitot gt al,, 1978). To date,
all other congeners of PBB studied in our laboratory have
acted as promoters in Pitotds assay (Jensen §£.ilw'1982)-
It is impossible to assess these congeners as initiators due
to their lipid solubility and persistence within fat and
liver parenchymal cells of mammals. It is hypothesized that
if persistent congeners capable of tumor promotion and a
nonpersistent congener or other congeners capable of tumor
initiation are within FM BP-6, this mixture can be
classified as a complete carcinogen. In the future this may
have significance in the development of cancer in Michigan
residents exposed to PBB.

The second objective of the research presented in this
dissertation was to evaluate the toxicologic effects of 3,4-
TBB in the rat as evidenced by light and electron
microscopic tissue changes and propose a mechanism for
toxicity. It is hypothesized that 3,4—TBB is metabolized by
the hepatic microsomal monooxygenase system to a toxic
intermediate, an arene oxide or epoxide. This metabolite is
very reactive and can be detoxified by enzymatic or
nonenzymatic conjugation with reduced glutathione (GSH). It
is suggested that in the event of hepatocellular GSH
depletion, acute toxicity may occur as a result of this
electrophilic intermediate binding to macromolecules within

the cellular cytosol, membrane, or nucleus.

The third objective of the research presented in this
dissertation was to characterize the effects of 3,4-TBB on
serum and hepatic vitamin A homeostasis and serum

triiodothyronine (T3) and thyroxine (T4) concentrations in

rats.

LI TERATURE REVIEW

Chemical and Physical Properties 9f
Polybrominated Biphenyls (PBB)

Congeners of PBB were introduced into the United States
in 1970 and were used as flame retardants in various plastic
and electrical materials found in business machines,
industrial equipment, thermostats and radio and television
parts (Brinkman and deKok, 1980). In.1974, the production
of FM BP—6, the only PBB product to reach large-scale
commercial production in the United States, was halted.

PBB are polyhalogenated aromatic hydrocarbons (PHAH).
Also belonging to this group of PHAH are the polychlorinated
dibenzo-p-dioxins, classically 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD), the polychlorinated biphenyls (PCB) and the
polychlorinated dibenzofurans (PCDF). These toxicants,
along with PBB, are all environmental contaminants and have
been shown to possess a similar structure-activity
relationship which dictates the carcinogenic, metabolic,
toxicologic and biochemical effects of these compounds in
mammalian systems (Poland and Knutson, 1982).

Congeners cu? PBB consist of 12 polycyclic ring
structures joined by a carbon-carbon bridge (Brinkman and
deKok, 1980). The empirical formula for PBB is C12H10_nBrn,

withrivarying from IIXDIO bromines. The bromines may be

5

ortho (2,2';6,6W, ‘mgtg (3,3';5,5W <1r pgtg (4,4') to the

carbon—carbon bridge as shown in Figure 0-1.

4',p

       

S'm 6,0 2',o 3',m

Figure 0-1. Chemical structure of polybrominated
biphenyls (PBB).

The average composition (wt. %) of the different
congeners within the FM BP-6 mixture is as follows: tetra
2-5; penta 5-10; hexa 70-80; hepta 12-18; remainder 0-1
(Brinkman and deKok, 1980). Gas chromatographic analysis of
FM BP-6 has shown the mixture to consist.of 12tx>14 major
congeners, 10 of these congeners have been structurally
characterized (Dannan gt a1”.1982b; Moore and Aust, 1978;

Moore gt _l., 1980). 2,4,5,234fl,5'-Hexabromobiphenyl
(2,4,5-HBB) is the major congener within FM BP-6 and
comprises approximately 50 to 70% of the mixture (Brinkman
and deKok, 1980; Jacobs gt gt”,1976; Moore and Aust, 1978;
Sundstrbm gt g_1_., 1976).

Commercial mixtures of PBB are typically white, off-
white or beige powdered solids. These chemicals are soluble
in organic solvents, insoluble in water, slowly metabolized

and highly lipophilic (Brinkman and deKok, 1980; Matthews,

1981; Tuey and Matthews, 198OL. Congeners of PBB are Very

persistent in the environment and upon U.V. irradiation
highly brominated, nontoxic congeners nmur be rapidly

photolyzed to lower brominated, toxic congeners (Millis gt

gt., 1985b).
PBB gg Environmental Contaminants

During the summer of 1973, FM BP-6 was accidently mixed
into livestock feed in place of "Nutrimaster" or magnesium
oxide, a compound with a similar physical appearance used as
a feed additive (Carter, 1976). At the Michigan Chemical
Company plant, wheree"Nutrimaster" also was manufactured,
there was :1 shortage of preprinted, red lettered bags
normally used to package FM BP-6. Both FM BP-6 and
"Nutrimaster" were packaged in brown bags on which
tradenames were stenciled in black. Ten to twenty 50 pound
bags of FM BP-6 were included into a truck load of
"Nutrimaster" and distributed to a large feed mill operated
by Farm Bureau Services, Inc. in Battle Creek, MI. This
incident went undetected until the spring of 1974 and
resulted.in widespread contamination ofrmafly.poultry and
milk products with ultimate human exposure (Bekesi gt gt.,
1978; Dunckel, 1975).

Some 30,000 livestock and 1,600,000 poultry on farms
throughout Michigan became contaminated at levels requiring
their destruction. Damages for livestock and poultry losses
were estimated at approximately 100 million dollars or more

(Carter, 1976; Dunckel, 1975).

8

The "PBB mix-up", as stated earlier, went undetected
for approximately 1 year. Within this time, not only was
there direct contamination of livestock through consumption

of tainted feed, but also cross-contamination occurred to

other livestock and poultry as a result of feed handled in
facilities that had been exposed to PBB. Many of the barns
and pastures on which the animals were kept were also
contaminated with PBB.

PBB may not be a problem to Michigan alone. To date,
PBB have been found in catfish in the Ohio River and in
plants, fish, soil, water and human hair in New York and New
Jersey (Culliton, 1977). FEB have not been conclusively
linked to short term illnesses in humans (Stross gt gt.,
1981; Kay, 1977), but because of their persistence in the
body and environment and their ubiquitousness in various
ecosystems, they pose a potential threat to human health.

Xenobiotics and Xenobiotic Metabolism
91 the Hepatic Microsomal Monooxygenase System

 

The prefix "xeno" is derived from tflua Greek term
"xenos" meaning stranger or foreigner. Therefore,
xenobiotics are compounds that are foreign to life or to an
organism. Examples of xenobiotics are environmental
contaminants such as PBB, PCB and TCDD. Animals are exposed
to many xenobiotics through ingestion of food and water, or
by inhalation of aerosols containing these foreign

compounds. Many xenobiotics may also enter an organism by

mere contact and absorption through the skin. Animals are

unable to separate foreign compounds from substances
utilized for production of energy or for building of tissue
components during the processes of ingestion, inhalation or
absorption, but through metabolism the animal is capable of
eliminating them from the body. In.some instances during
the process of elimination, some compounds are metabolized
to toxic intermediates that may do more damage to the host's
cells than the parent compound (Conney and Burns, 1972).

The liver is the primary site of metabolism of foreign
and endogenous compounds (Conney, 1967). Secondary sites of
metabolism are the lung, kidney, gastrointestinal tract and
the skin (Parke, 1968). The hepatic microsomal
monooxygenase system (MMS) is a nonspecific metabolizing
system which functionstx>convert lipid-soluble, nonpolar
substrates to more polar, water soluble, readily excretable
compounds. The MMS are contained in microsomes which
contain complex enzymes associated with the lipid bilayer of
endoplasmic reticulum of liver cells and cells of other
tissues. The MMS enzyme complex consists of a flavoprotein
(Fp), known as cytochrome P450 reductase (Yasukochi and
Masters, 1976; Guengerich, 1977), hemoproteins, collectively
termed cytochromes P450 (Cooper gt gt., 1965; Garfinkel,
1957; Klingenberg, 1958; Lu and west, 1980) and a
phospholipid, phosphatidylcholine (Strobel gt gl., 1970).

Cytochrome P450 reductaseifisthought tokmaexposed to
the exterior of the lipid bilayer of endoplasmic reticulum,

whereas the forms of cytochrome P450 are embedded in the

10

cellular membranes as shown in Figure 0-2 (Vermilion and
Coon, 1978). The reductase has one flavin adenine
dinucleotide (FAD) molecule and one flavin mononucleotide
(FMN) molecule and a molecular weight of approximately 78 K.
The exterior portion of the reductase molecule is considered
to be the active site. There is also a membrane binding
fraction (also called foot or tail) having a molecular
weight of 8-9 K, which is essential for reconstitution of
microsomal monooxygenase activity (Yasukochi and Masters,
1976).

The hemoproteins, cytochrome P450, is a carbon monoxide
“KN binding pigments, thatzhitheir reduced form binds CO
to form a difference spectrum with a maXimum around 450 nM,
hence P450 (Garfinkel, 1957; Klingenberg, 1958). Garfinkel
and Klingenberg each identified this pigment as a b-type
cytochrome with one protoporphyrin IX prosthetic group per
molecule (hemoprotein). A multiplicity of hepatic
microsomal cytochrome P450 isozymes have been purified and
characterized ixi'various rmmnmalian species (Dannan gt gg,,
1983; Lu and West, 1980; Nebert gt _t., 1981; Waxman and
Walsh, 1982; Welton and Aust, 1974L. Sonmaof the isozymic
forms of cytochrome P450 have been shown to have identical
residue sequences at the NH2 terminal which are thought to
be involved in anchoring these molecules to the lipid

bilayer of the endoplasmic reticulum as shown in Figure 0-2

(Waxman and Walsh, 1982L

 

Figure 0-2. Modified hypothetical diagram of the
relationship of cytochrome P450 reductase and forms of
cytochrome P450 in membrane of endoplasmic reticulum. Taken
from Nebert gt gt., 1981.

The microsomal monooxygenase system is an NADPH—
dependent transport chain that inserts one atom of
atmospheric oxygen (02) into their substrates (Conney,
1967). This electron transport pathway transfers electrons
or reducing equivalents through the reductase from NADPH to
the terminal oxidases, cytochromes P450 (Cooper, 1965; Lu gt
gt., 1969). Another name for this system is mixed function
oxidase (MFO) system, in that it requires both oxidizing and
reducing equivalents, not derived from the substrate, but
from NADPH (Mason, 1957L

During the initial processes of xenobiotic metabolism,
the foreign compound forms a complex with the oxidized form
of cytochrome P450, which is then reduced by a flow of
electrons from NADPH to cytochrome P450 reductase (Parke,
1968). The reduced cytochrome P450-substrate complex then

interacts with molecular oxygen to form a hydroxylated

12

substrate and water. During this reaction there is
simultaneous regeneration of oxidized P450 for further
substrate binding (Figure 0-3). There is also another
electron transport system in microsomes. This system is
NADH-dependent and is thought to play a role in the fatty
acid desaturase system (Sato gt gt” 1969). There appears
to be a relationship between this system and the microsomal
drug metabolizing systenlin.that there maylxea.process of
electron crossover from one pathway to the other and the
NADH—dependent system may supply some reducing equivalents
for the terminal oxidases (cytochromes P450) as shown in
Figure 0-3.

NADH——-—-ofp.———o Cytuhnmo It, ------------- -- O:

I
l
I

" Substrate (R)

" "‘:.... "s.060

NADPH
Q +
N Cytochrome
P- 430
”AD?

P-l:0”

Figure 0-3. Modified microsomal electron transport
chain. Taken from Wilkinson and Brattsten, 1972-73.

Hepatic xenobiotic Inetabolism {is divided into
microsomal and nonmicrosomal reactions (Ingelman-Sundberg,
1980; Kappas and Alvares, 1975; Parke, 1968). Phase I or
primary reactions occur within the microsomes and may be

accomplished by'cnua or a combination of the following

13

reactions: 1) side-chain oxidations, 2) hydroxylations, 3)
nitrogen oxidations, 4) sulfoxidations, 5) dealkylations, 6)
nitroreductions, 7) azoreductions, 8) dehalogenations, 9)
epoxidation and alcohol oxidations. Phase II (n: secondary
reactions occur subsequent to primary reactions. They
include reactions with enzymes such as epoxide hydratase,
conjugation reactions with UDP glucuronic acid, sulfate,
endogenous amines anui glutathione (GSH). The phase I
reactions are responsible for converting nonpolar foreign
substrates into less complexed polar substances that can be
further processed during phase II of xenobiotic metabolism
and excreted from the organism. There are many factors
which dictate the fate of a compound once introduced into an
organism. These factors will be discussed in more detail in
the following section.

Pharmacokinetics gt Polyhalogenated Aromatic Hydrocarbons
(PHAH)

 

 

Many factors must be considered when one looks at the
fate of xenobiotics upon entry into an organism. Of primary
importance are factors such as absorption and distribution
of a foreign compound prior to its metabolism and excretion
from the body. This section will briefly review the
pharmacokinetics of PHAH.

Congeners (ME PBB are lipid soluble PHAH. The
lipophilic properties of these compounds and other PHAH

allow their absorption across cellular membranes by passive

diffusion. In the gastrointestinal tract this absorption is

l4

favored by a concentration gradient and accounts for the
passage of these compounds from the gastrointestinal mucosa
into the blood (Surak and Bradley, 1976). Upon entry into
the blood, PHAH are almost completely adsorbed onto various
blood proteins (Matthews gt gt., 1977). Adsorbtion of PHAH
onto blood proteins does not interfere with the partitioning
of these compounds to other tissue proteins or lipid, but
does inhibit their excretion from the body.

Initial distribution of PHAH to various tissues is
determined by tissue volume, rate of tissue perfusion and
affinity of a given tissue for a specific compound
(Matthews, 1981). The liver has a high rate of bdood
perfusion and moderate to high affinity for specific PHAH
and is commonly a site of initial distribution for PHAH
compounds. On the other hand, muscle has a low affinity for
lipid soluble compounds and 51 moderate rate of tdood
perfusion, but due to its large tissue volume (40-50% total
tissue volume), it is also a primary site of initial
distribution of PHAH (Matthews and Anderson, 1975). Initial
distribution of PHAH to adipose tissue is surprisingly low.
This is due to the small tissue volume of fat in the body
(usually less than 10% of total tissue volume) and the slow
rate of blood perfusion (Matthews, 1981). However, it has
been shown that PHAH may accumulate in adipose tissue under
conditions of chronic exposure to high concentrations of a

compound, or if an acute high dose of a compound cannot be

15

effectively excreted from the body (Tuey and Matthews,
1980).

Once PHAH enter the body, there is establishment of.a
dynamic equilibrium between the PHAH, the blood and all
tissues of the body (Matthews, 1981). The rate at which
this equilibrium is reached is determined by the affinity of
the compound for a particular organ and the rate of
perfusion of that organ. (Other factors that may influence
the rate of equilibrium are receptor binding, lipid
solubility of the PHAH and lipid content of the tissue (Lutz
gt gt”.19770. Once equilibrium is reached, PHAH are free
to redistribute from one tissue compartment to another with
the blood. If there is an increase or decrease in the
concentration of PHAH in one tissue compartment, this change
‘will be reflected.:h1 the concentration of all tissues
(Matthews, 1981). The rate at which this change is seen is
proportional to the rate of tissue perfusion with blood,
hence once equilibrium is established, removal of PHAH from
slowly perfused sites like adipose tissue is extremely slow.
This factor attributes to the persistence of many PHAH in
the fat deposits of the body.

Excretion of PHAH occur via the feces and urine. As
mentioned earlier, the parent PHAH is usually a nonpolar and
lipid-soluble molecule which readily adsorbs to various
blood proteins. The protein-xenobiotic complex is too large
to enter the renal glomerular filtrate, so renal excretion

of the adsorbed PHAH does not occur (Matthews, 1981). If

16

the parent PHAH 1&5 metabolized to an intermediate
metabolite, the metabolite formed is usually more water
soluble and may enter the renal glomerular filtrate and be
excreted in the urine. Excretion of PHAH into the feces is
dependent upon the formation of amphipathic molecules during
metabolism (Smith, 1973). These molecules may then be
actively excreted from the liver into the bile. Fries
(1978) showed that PBB are also excreted from the body of
animals into fat containing products such as milk fat and
eggs. This author also found that molecular size and weight
of congeners of PBB made a major quantitative difference as
to the accumulation and/or excretion of these compounds in
the body.

PBB and Induction gt Hepatic Microsomal
Drug-Metabolizing Enzymes

 

 

 

 

The hepatic MMS is a nonspecific drug—metabolizing
system that functions through the elaboration of principally
oxidative emzymes ‘UD convert nonpolar, lipid soluble
substrates to more polar and readily excretable compounds.
There are numerous xenobiotic and endogenous substrates
known to stimulate the activity of drug-metabolizing enzymes
in liver microsomes, as well as microsomes located in other
tissues (Conney, 1967; Kappas and Alvares, 1975). Examples
of compounds that induce hepatic microsomal enzymes include
steroid hormones, carcinogens, environmental toxicants such

as PBB, PCB, TCDD and a variety of drugs including

l7

hypnotics, sedatives, anesthetic gases, anticonvulsants, and
muscle relaxants (Conney, 1967).

There are basically two types of hepatic microsomal
enzyme induction exemplified by phenobarbital UN” and 3-
methylcholanthrene (3-MC) which are designated "PB type" and
"3-MC type",:respectively (Conney, 1967L. These two types
of microsomal enzyme induction differ with respect to
substrate specificity and the absorbtion maxima of their
reduced cytochrome-CO complex and ethylisocyanide difference
spectra (Lu and West, 1978; Omura and Sato, 1964a,b).

Treatment of rat hepatic microsomes with PB results in
elevated synthesis of a hemoprotein that in its reduced form
bindsCKDat a maximum difference spectrum of 450rnL. This
hemoprotein is called cytochrome P450 and has the same
spectral characteristics as cytochrome P450 from untreated
rats (Lu and West, 1980). Conversely, treatment of rat
hepatic microsomes with 3-MC results in the synthesis of a
new hemoprotein, which in its reduced form binds CO at a
maximum difference spectrum of 448 nM. This new hemoprotein
is designated as cytochrome P448 (Alvares _t gt”.1967) or
cytochrome P1450 (Sladek and Mannering, 1966). Endogenous
or exogenous compounds that induce isozymes similar to those
induced by PB or 3-MC are classified as PB type or 3-MC type
of microsomal enzyme inducers.

There are multiple inducible isozymes of cytochrome
P450 and P448. These isozymes have been shown to have

different spectral properties, catalytic activity,

18

immunological properties and amino acid sequences (Dannan gt
gl., 1983; Lu and West, 1980; Nebert gt gt., 1981). The
major inducible isozymes of cytochromes P450 and P448 appear
to vary among mammalian species and may differ between
sexes, strains and individuals within a given species
(Dent gt gt., 1980; Guengerich gt gt., 1981; Kamataki gt
gt., 1983; Lu and West, 1980; Nebert gt _t., 1981). The
induction of polysubstrate monooxygenase activities is
thought to be genetically controlled by a combination of
regulatory, structural and possibly temporal genes located
at the Ah locus as shown in certain strains of mice
(Greenlee and Poland, 1979; Nebert.gtlgtu.l981; Poland and
Glover, 1980). This concept will be discussed later in this
dissertation under "Toxicity and Microsomal Enzyme Induction
- Effects of PBB".

The commercial PBB mixture, FM BP-6, can induce PB and
3-MC types of hepatic microsomal enzymes (Dent gt gt.,
1976a,b) and this type of microsomal enzyme induction is
termed mixed-type. The mixed-type of hepatic microsomal
enzyme induction seen with FM is due to its content of
individual congeners of PBB capable of inducing several
isozymes of cytochrome P450 or P448, or a combination of
both isozymes (Dannan _t__t” 1982d, 1983). The individual
congeners are classified as either strictly PB type (P450),

strictly 3—MC type (P448) or mixed-type (P450 and P448) of

microsomal enzyme inducers. There is a structure-activity

19
correlation suggested for congeners of PBBlandinicrosomal
enzyme induction.

Congeners of PBB such as 2,4,5-HBB, 2,3,4,5,254',5“—
heptabromobiphenyl,2L3,4,5,2'gT,4'—heptabromobiphenyl and
2,3,4,5,2fl3',435'-octabromobiphenyl are all strictly PB
type of hepatic microsomal enzyme inducers (Aust gt gt.,
1981; Besaw gt g_l_., 1978; Moore gt g_l_., 1978a,b, 1979;
Render gt gl., 1982). These congeners of PBB have 2
bromines at the gtttg (2A?) positions on the biphenyl rings
and this configuration is thought to be necessary for
strictly PB type of hepatic microsomal enzyme induction
(Besaw _t gt., 1978; Moore gt _t., 1978b, 1979; Render gt
al., 1982L. Dannan.gt__t.(1983) found that the presence of
bromine at carbons gtttg to the biphenyl bridge favored the
induction of several isozymes of P450 in rat hepatic
microsomes, but this structural arrangement did not
eliminate the ability of congeners to induce isozymes of
cytochrome P448 (P4SOBNF_B or P4SOBNF/ISF-Gi' PB type of
microsomal enzyme induction results in increased activity of
enzymes such as NADPH-cytochrome P450 reductase,
aminopyrine-n-demethylase and epoxide hydrase (Dannan gt
gt., 1982d, 1983; Moore gt gt., 1979). Other microsomal
enzymes induced. are UDP—glucuronyltransferase and
glutathione-S- transferases (Bock gt gt., 1973; Kaplowitz gt
gt,, 1975). Congeners of PBB that are strictly PB type of

microsomal enzyme inducers are not toxic, but have been

found 11) cause extensive proliferation cfif hepatic

20
endoplasmic reticulum as evidenced by electron microscopic

evaluation (Besaw gt a1” 1978; Moore gt aluyl978b, 1979;

Render gtg_l_., 1982).

A strictly 3-MC type of microsomal enzyme inducer, 3,4-
TBB has been tentatively identified within FM (Robertson gt
gl,, 1982L. These authors reported that 3,4,5-HBB, 3,4,4“-
tribromobiphenyl, 3,4,5uP-TBB and EL4,3',4Z5'-
pentabromobiphenyl were also capable of 3-MC type of
induction. They suggested for 3-MC type of hepatic
microsomal enzyme induction, congeners must possess halogen
substitutions at both pgtg (4MP) positions and at one, two,
three or four mgtg (Bfir,5fiT) positions. Poland and Glover
(1977) proposed two structural requirements for 3-MC type of
microsomal enzyme induction by halogenated biphenyls. These
authors found the presence of at least two adjacent halogen
atoms in the lateral position of each benzene ring
(positions 3,3',4,4',5,5') and the absence of 9.15.1222
(positions 2,256,6W halogenation allowed coplanarity of
these molecules which is important in receptor binding and
induction of aryl hydrocarbon hydroxylase (AHH), an enzyme
associated with 3-MC type of microsomal enzyme induction and
toxicity. Congeners of PBB that are 3-MC type microsomal
enzyme inducers increase AHH activity and are toxic as
evidenced by light and electron microscopic and functional
alterations in various tissues (Millis gt gt., 1985a; Render
gt gt., 1982; Robertson gt EH2! 1982). The

interrelationship between 3-MC type of microsomal enzyme

21

induction, receptor binding, increased AHH activity and
toxicity will be discussed in the section "Toxicity and
Hepatic Microsomal Enzyme Induction - Effects of PBB".
2,4,5fiV,4'-Pentabromobiphenyl, 2,3,4,5,234fl-HBB,
2,4,5,2'n¥,5'—HBB and 2,3,4,5fi?,4'—HBB are classified as
mixed-type of microsomal enzyme inducers (Aust gt gt,, 1981;
Dannan gt gt” 1978b, 1982a,b). These congeners of PBB are
present in.ETI‘BP-6 and have structural configurations that
allow induction of both P450 and P448 microsomal enzymes.
They have also been reported to cause toxic histologic and
ultrastructural changes in various organ systems in rats

(Akoso gt al., 1982a; Dannan et al., 1978a, 1982c,d).

Hepatic Monooxygenase Metabolism gt PBB

 

Congeners of PBB differ from one another in the number
and position.of bromines present;on the biphenyl rings and
these structural differences tend to dictate the type of
microsomal enzyme induction which in turn determines the
metabolic fate of these compounds. It has been proposed
that tg ytttg and tgflgtyg metabolism of PBB by cytochrome
P450-dependent monooxygenases occurs when there are adjacent
non-halogenated carbon atoms on at least one of the biphenyl
rings (Millis _t _a__l_., 1985a; Mills _t gt, 1985). Hepatic

microsomes isolated frcmi:hnmature male rats and pretreated

with 3-methylcholanthrene (3-MC) were found to increase tg

22

ytttg NADPH-dependent microsomal metabolism of 4M?-
dibromobiphenyl (DBB),3L4n4'-tribromobiphenyl, 3,4,324'-
tetrabromobiphenyl (TBB), 2,3,3fl4'-TBB, 2,5,354'-TBB and
2,4,225'-TBB in decreasing order (Mills g_ gt,, 1985). All
of the above listed congeners have nonhalogenated gttgg and
ggtg carbon atoms on at least one biphenyl ring, induce P448
type microsomal enzymes and are metabolized. Phenobarbital
pretreatment of isolated microsomes increased tg ztttg
metabolism of congeners of PBB possessing nonhalogenated
ggtg and pgtg carbon atoms on at least one biphenyl ring.
Examples of congeners found to have this type of structural
configuration are 2,2'-DBB, 2,4,2',5-TBB, 2,5,2',5'-TBB,
2,3,3',4'-TBB, 2,5,3',4'-TBB enui 2,4,5,2H5'-
pentabromobiphenyl. All of these congeners have been shown
to induce P450 type of microsomal enzymes and are
metabolized (Mills gt gt., 1985).

The importance of adjacent nonsubstituted carbon atoms
as a determinant of halogenated biphenyl metabolism has been
shown by many investigators in a series of studies using
congeners of either PBB or PCB (Kato gt al., 1980; Kohli gt

g_l_., 1978; Matthews and Anderson, 1975; Mills gt __l_., 1985;
Preston gt gt,, 1983; Tuey and Matthews, 1980; Van Miller gt
gt., 1975). It is postulated that aromatic hydrocarbons can
be metabolized to an unstable arene oxide by the hepatic
monooxygenase system (Jerina and Daly, 1974; Kohli gtggt”

1978; Matthews, 1981). Arene oxide formation requires the

availability of two adjacent unsubstituted carbon atoms.

23

Aryl halides having this type of structural arrangement are
more readily metabolized than highly halogenated congeners
lacking adjacent nonhalogenated carbon atoms. Mills gt gt.
(1985) found that increased bromination of congeners of PBB
inhibited metabolism even though they may possess adjacent
nonhalogenated.gttgg_and‘ggtg_carbons. 11:18 thought that
halogenation tends to inhibit oxidative metabolisniof the
aromatic rings because halogen atoms are large,
electronegative atoms vfluhfli sterically hinders the
microsomal enzymes and electronically hinders the electron
deficient oxygen involved in the oxidation (Matthews, 1981).
Once formed, the arene oxide may spontaneously isomerize to
a monohydroxylated product, react with epoxide hydrase to
yield aidihydroxylated product or possibly conjugate with
reduced GSH (Jerina and Daly, 1974; Matthews, 1981; Preston
gt gt., 1983).

An arene oxide has been identified as the intermediate
metabolite of 4-bromobiphenyl (Kohli gt gt”.l978). This
metabolite is very reactive and could possibly bind to
cellular macromolecules such as cytosolic proteins, membrane
lipids and/or structural components of DNA resulting in
cellular damage and toxicity. Adduct formation with
cellular DNA is thought to be one mechanism of tumor
initiation in chemical carcinogenesis and congeners capable
of being metabolized to electrophilic intermediates may act
as initiators of cancer (Conney and Burns, 1972; Miller and

Miller, 1977, 1981L. Prestrni t gt.(1983) have shown aryl

24

halide metabolism does not necessarily occur via an arene
oxide. The major intermediate metabolite of 2,3fl5,5“—
tetrachlorobiphenyl (TCB) was reported to be a non-arene
oxide, 3-hydroxy-TCB.

Dannan gt gt. (1982c) found congeners of PBB to be good
substrates for induction of hepatic microsomal enzymes.
This is important in that induction of the microsomal
monooxygenases by nonmetabolized congeners of PBB nuur
increase the rate of metabolism of metabolizable congeners.
3,4,5fiV,4H5'-Hexabromobiphenyl (3,4,5-HBB), a
nonmetabolized congener of PBB (not present in the FM BP-6)
with 3-MC type of microsomal enzyme induction has been shown
to increase the rate of metabolism of 3,4-TBB tg ytttg
(Mills gt gt., 1985). If congeners of PBB are indeed
metabolized to an arene oxide, metabolism of these compounds
could result in formation of electrophiles with subsequent
toxicity or possibly tumor initiation. (nithe other hand,
if the parent form of the congener is important in receptor
binding and that in turn is responsible for mediating
toxicity, increased metabolism would decrease the toxicity
of the metabolizable congener (Greenlee and Poland, 1979;
Millis gt gt., 1985; Poland gt gt., 1976; Poland and Glover,
1980). It has also been shown that metabolizable congeners
can induce their own metabolism, thereby, decreasing their
toxicologic effects (Millis gt gt., 1985a).

Inhibition.of metabolism can occur if nonmetabolized

anui metabolized congeners capable (n5 inducing similar

25

isozymes of cytochrome P450 are coadministered. When
isolated rat hepatic microsomes were treated with 3-MC
followed by coadministration of 3,4,5-HBB and 3,4-TBB,
3,4,5-HBB appeared to have an inhibitory effect on the
metabolism of 3,4-TBB (Mills gt gt., 1985). This is thought
to have occurred as a result of 3,4,5-HBB interacting with
microsomal enzymes at their binding sites and preventing the
normal metabolism of 3,4-TBB. It is also suggested that
nonmetabolized congeners such as 3,4,5-HBB may have a higher
affinity ffln: these binding sites, since equimolar
administration 3,4,5-HBB and 3,4-TBB almost completely
inhibited the metabolism of 3,4—TBB (Mills gt gt,, 1985).

Toxicity and Hepatic Microsomal Enzyme Induction -
Effects gt PBB

 

 

Pathotoxicologic Effects gt Egg

In the initial assessment of PBB,-crude-mixtures of FM
BP-6 were used to evaluate the potential toxicologic effects
of this mixture on various organ systems in different
laboratory and domestic animals. Upon the identification
and purification of individual congeners within FM, and with
the correlation of toxicity and microsomal enzyme induction,
research 1J1 this area veered toward assessing the
pathotoxicologic effects of the individual congeners. In
this section of this dissertation, I will attempt to give a
broad overview (M3 the significant toxicologic effects
associated with the administration of FM BP—6, 3,4-TBB,

3,4,5-HBB and 2,4,5-HBB as evidenced by light and electron

26

microscopic changes seen in theimn; Also, I will review
some of the current theories on the mechanism(s) of PBB

toxicosis.

Hepatotoxicosis

 

Initially, the liver is the primary site of PBB
distribution (Matthews, 1981). Understandably, this is also
one of themajor target organs affected in PBB texieosis in
several mammalian species including the rat, mouse, chick,

cattle, pig, monkey and guinea pig (Allen gt gt., 1978; Cook

IID

t l”.1978; Gupta gt alu,1983a4b; Gupta and Moore, 1979;

Kimbrough gt gt,, 1980; Render gt _t,, 1982; Ringer, 1978;
Sleight and Sanger, 1976; Werner and Sleight, 1981). Acute
and chronic administration of various dosescxfEWd(BP—6 or
FF-l) has been reported to cause increased liver weights,
hepatomegaly and hepatic microscopic changes characterized
by centrolobular to midzonal hepatocellular vacuolation,
intracytoplasmic fatty change of hepatocytes, hepatocellular
hypertrophy, bile ductule proliferation and necrosis (Gupta
and Moore, 1979; Kimbrough gt gt., 1980; Render gt gt.,
1982; Sleight and Sanger, 1976L. Hepatic ultrastructural
changes associated with dietary administration of 10 ppm or
100 ppm of FM BP-6 to rats for 9 days have been
characterized by increased proliferation and dilation of
smooth endoplasmic reticulum (SER) and increased cytoplasmic

lipid droplet accumulation (Render gtggt” 1982). Sleight

and Sanger (1976) found dietary administraticniof 1 ppm or

27

10 ppm of FM BP-6 for 30 days caused an increase in size of
hepatocyte mitochondria as evidenced by electron microscopic
evaluation. These authors also reported rats fed 100 ppm or
500 ppm of FWI for 30 days had ultrastructural hepatic
lesions characterized by 61 marked increase in SER
proliferation accompanied by hepatocellular vacuolation,
indistinct mitochondrial morphology and concentric whorling
of proliferated SER or myelin body formation. The severity
of the lesions seen in this study were dose-dependent, In
conclusion, FM is toxic and its toxicologic effects are
attributed to the individual toxic congeners of PBB within
the mixture capable of inducing 3-MC type microsomal
enzymes.

2,4,5-HBB, the major congener in FM BP-6, has been
reportedtxncause increased liver weight and hepatomegaly
when administered in the diet to rats for 9 days (Render gt
gt., 1982). These authors found that rats fed 10 ppm of
2,4,5-HBB had histologic hepatic lesions characterized by
centrolobular to midzonal hepatocellular enlargement and
vacuolation. Similar but more severe histologic hepatic
changes were observed when rats were fed 100 ppm of 2,4,5-
HBB. Ultrastructural changes observed with dietary
administration of 100 ppm of 2,4,5-HBB included extensive
dilation and proliferation of hepatocyte SER along with
accumulation of lipid droplets in these cells (Render gt
gt., 1982). 2,4,5-HBB is a strictly PB type of microsomal

enzyme inducer, is not metabolized and does not appear to be

28

toxic. The increase in liver size and weight and
microscopic and ultrastructural changes seen with the
administration of 2,4,5-HBB are thought to result from
stimulation of endoplasmic reticulum anxi are indicative of
enhanced enzyme activity (Hansell and Ecobichon, 1974) and
not true toxicity.

3,4,5-HBB,ainonmetabolizable congenerrunzpresent in
FM, has been reported to cause increased liver weight,
hepatomegaly’ and microscopic hepatic lesions 1J1 rats
characterized knr hepatocellular hypertrophy, increased
prominence of hepatocyte nucleoli, centrolobular to midzonal
hepatocellular vacuolation, and bile duct epithelial
hyperplasia when given at 100 ppm in the diet for 20 days
(Render gt gt,, 1982). Ultrastructural hepatic lesions seen
in these rats consisted of increased lipid droplets within
hepatocytes, SER proliferation with myelin body formation,
swollen. mitochondria. and disorganization cmf rough
endoplasmic reticulum. 3,4,5-HBB induces strictly 3-MC type
of microsomal enzymes and is considered a toxic congener of
PBB.

Another strictly 3-MC type of microsomal enzyme inducer
is 3,4-TBBu This congener has been tentatively identified
in FM and is metabolized (Mills _t _t”,1985; Robertson gt
gl,, 1982). 3,4-TBB is toxic, but less toxic than 3,4,5-HBB
(Millis gt gt., 1985a). Oral administration of 21 umoles/kg
of 3,4-TBB t1) rats caused uncroscopic hepatic lesions

characterized by mild, diffuse hepatocellular swelling with

29
decreased sinusoidal spaces in the midzonal regions of the
hepatic lobule (Millis gt gt., 1985a). These microscopic
changes were consistent with findings of Robertson gt _t.
(1983) in which rats given a single ip dose (150 umoles/kg)
of 3,4-TBB had similar hepatic alterations. The
histopathologic and ultrastructural hepatic changes

associated with chronic dietary administration<n53,4-TBB

will be discussed in Chapter I of this dissertation.

Immunotoxicosis

 

PBB have been reported to alter immune responses in

pigs (Howard gt 1., 1980), dogs (Farber t 1., 1978), mice

(Fraker, 1980), rats (Luster gt gt., 1978), non-human
primates (Allen gt gt., 1978), chickens (Ringer, 1978),
guinea pigs (Vos and van Genderon, 1973) and humans (Bekesi
gt gt,, 1978). The results obtained from the immune
function tests are not consistent within or between species
in many instances. These variations may be related to PBB
acting indirectly to alter the immune responses. The exact
mechanism of action or cellular predilection for the
immunotoxic effects observed with PBB are not known.

Many reports describe lymphoid cell depletion in the
spleen and thymus after administration of FM or congeners of
PBB to rats, but few studies have been performed to assess
the significance of the alterations seen. Microscopically,
splenic lesions seen with the oral administration of 100 mg,

300 mg or 1,000 mg of FM FF-l/kg/day'(5 days/wk” 22 doses)

consisted of depletion of the periarterial lymphoid cells of

30

the malpighian corpuscles, necrosis of lymphoblastic cells
1J1 germinal centers (of lymphoid. follicles and
hypocellularity of the red pulp (Gupta and Moore, 1979).
Thymic lesions were characterized by loss of demarcation
between the cortical and medullary regions and disappearance
of cortical lymphocytes. Render gt gt. (1982) found
lymphoid cell depletion in the cortex of the thymus and
follicles of the spleen in rats fed 10 ppm or 100 ppm of
3,4,5-HBB for 10 days. Millis g_ gt, (1985a) reported
severe thymic involution in rats which was characterized
histologically by loss of thymocytes from both the medullary
and cortical regions in rats given 21 umoles of 3,4-TBB or
3,4,5-HBB orally. Robertson gt gt. (1983) found that 150
Umoles of 3,4-TBB given ip also resulted in thymic

involution characterized histologically by marked depletion

of lymphoid cells in the cortex of the thymus in rats.

Thyroid Gland Toxicosis

 

Histologic changes in the thyroid gland have been
observed in male rats exposed to various doses of PBB
(FM BP-6 or FF-l) (Gupta and Moore, 1979; Gupta gt gt.,
1982a) or PBB congeners (Akoso _t_t., 1982b). Gupta _t _t.
(1983a) observed thyroid changes primarily in male rats
exposed to 10 mg/kg of PBB (FF-1) for 6 months. These
histologic alterations consisted of thyroid follicles lined

by columnar epithelium containing a few areas of epithelial

papillary projections. The follicular colloLd was sparse,

31

or bluish and had a stippled appearance. These authors also
found a significant decrease in serum thyroxine (T4) and
triiodothyronine (T3) concentrations and concluded that PBB
may interfere with thyroid hormone secretion. Akoso gt gt.
(1982b) also found decreased serum T4 concentrations in male
rats fed 100 ppm of FM or 10 ppm of 3,4,5-HBB.

Male rats fed 100 ppm of FM, 100 ppm 2,4,5-HBB or 10
ppm of 3,4,5-HBB had thyroid lesions characterized
microscopically by extensive hyperplasia and hypertrophy of
follicular cells and loss of follicular colloid. Rats given
100 ppm of FM or 10 ppm of 3,4,5-HBB in the diet for 30 days
were found to have ultrastructural thyroid gland lesions
characterized by increased lysosomal bodies, increased
cytoplasmic vacuoles and dilated cisternae (M3 the
endoplasmic reticulum (Akoso gt gt,, 1982b). These authors
also found similar ultrastructural lesions in the thyroid
gland of rats fed 100 ppm of 2,4,5-HBB for 30 days, but the
changes were less severe. Diets containing 100 ppm of FM
and 1.gnnn or 10 ppm of 3,4,5-HBB in this study caused
increased thyroid gland weights. Sleight gt gt. (1978)
reported similar histologic changes in male rats given 100

mg of FM in the diet for 30 or 60 days.

Mechanism(s) gt PBB Toxicosis

 

 

Congeners of PBB and other PHAH such as TCDD and PCB
have been found to produce a similar pattern of toxic

responses 111 mammalian species (Poland and Knutson, 1982).

Manycflfthese aromatic hydrocarbons are stereoisomers and

32

can stereotypically bind Useaprotein receptor UUIOI TCDD
receptor) that mediates 3—MC type microsomal enzyme
induction (P1450), increased AHH activity and toxicity. The
exact relationship between this particular type of
microsomal enzyme induction and the toxicologic effects
observed in mammalian species is unknown.

2,3,7,8-TCDD is the prototypical halogenated aromatic
hydrocarbon for Ah or TCDD receptor binding and is the most
potent known inducer<1ftflmaAHH systenn The current model
proposed for AHH induction by TCDD and other halogenated
hydrocarbons is similar to the molecular mechanism of
steroid hormone action, This model postulates a two-step
mechanisnl whereby the inducer (ligand) binds to a
"cytosolic" receptor, followed by a temperature dependent
"translocation" of the ligand-receptor complex from the
cytoplasm to the nucleus (Greenlee and Poland, 1979; Nebert
gt _t” 1981). This cytoplasmic to nuclear translocation
results in an accumulation of ligand-receptor complexes
within the nucleus with subsequent activation of structural
genes including P1450 specific mRNA and associated AHH
activity (Nebert gt gt., 1981).

Recently, it has been proposed that the protein
receptor is not a "cytosolic" receptor, but is nuclear in
location in the intact cell (Whitlock and Galeazzi, 1984).
These authors found that dilution factors contributed to the
redistribution cflf unoccupied receptors and ligand-receptor

complexes between the nuclear and cytoplasmic fractions of

33

broken cells and this phenomenon could account for the
appearance of receptors (and complexes) in cytosolic
preparations. It was further concluded by these authors
that minimization of this dilution effect caused 80-90% of
the receptors to associate with the nuclear fraction and
this was indicative of a nuclear location for the receptor.
These authors proposed a modified two-step model for the
mechanism of TCDD and other PHAH mechanism of action. The
first event is the binding of the ligand to the receptor
which is primarily a nuclear event. The second event is a
temperature dependent strengthening of the ligand-receptor
complex to the nucleus and not a translocation event as
proposed in the current theory, since the ligand-receptor
binding occurs in the nucleus. The binding of receptors (or
complexes) is thought.to be primarily to chromatin. This
results in the accumulation of ligand-receptor complexes
within the nucleus with transcripticWIof cytochrome P1450
gene.

The protein receptor,kxait nuclearcnrcytoplasmic in
location, is thought to regulate genes at the Ah locus as
seen in certain strains of mice (Greenlee and Poland, 1979;
Nebert gt gt., 1981). The murine Ah locus regulates the
induction by polyhalogenated and polycyclic compounds of
numerous drug-metabolizing enzymes in virtually all tissues
(Nebert gt gt., 1979). Following the prOposed preceding
events leading to accumulation of ligand-receptor complexes

within the nucleus, there is a pleiotypic response resulting

34

in activation of numerous structural genes atijmaAh.locus
(Nebert gt _t., 1981). AHH is one (Hi the several drug
metabolizing enzymes (structural gene products) induced upon
activation of structural genes at the Ah locus, and in many
mammalian systems its persistent induction is associated
with toxicity.

The ability of congeners of PHAH to bind the TCDD
receptor has been assessed by determining its ability to
induce 3-MC type of microsomal enzyme induction and increase
AHH activity. Congeners of PBB possessing this type of
microsomal enzyme induction also have been shown to be toxic
(Akoso gt gt., 1982a; Dannan, l982a,c,d; Millis gt gt.,
1985a; Moore gt gt., 1979; Render gt gt., 1982). It is
proposed that enzyme induction is an early event and that
toxicity occurs later as a result of persistent ligand
occupation of the receptor and subsequent gene expression
(Poland and Knutson, 1982). Congeners of PBB capable of
inducing increased AHH activity have structural
configurations which allow coplanarity, a prerequisite for
ligand-receptor binding and enzyme induction (Poland and
Glover, 1977).

Competitive receptor binding studies with 3,4,5-HBB and
3,4—TBB for the specific binding of (3H)TCDD to the mouse or
rat hepatic receptor showed 3,4-TBB to be a more potent
competitor for the TCDD receptor than 3,4,5-HBB (Millis gt

gt., 1985a). These authors also found significantly

elevated AHH activity in rats given an equimolar dose (21.3

35

umoles/kg) of 3,4,5-HBB or 3,4-TBB as compared to controls,
and on day 6 this enzyme activity decreased in rats given
3,4-TBB. It was concluded that 3,4-TBB is metabolized and
induces its own metabolism $2.2222 as evidenced by decreased
adipose tissue and liver concentrations of this congener.
These authors also suggested that the metabolism of 3,4-TBB
by hepatic microsomal enzymes could possibly explain the
decreased AHH activity on day 6.

An equimolar dose of 3,4,5-HBB was found to be more
toxic than 3,4-TBB as evidenced by histologic changes in the
thymus and liver of rats (Millis gt _t” 1985a). However,
competitive binding studies performed.tn{ these authors
indicated 3,4-TBB had.21 H) times greater affinity than
3,4,5-HBB for the TCDD receptor and if toxicity is mediated
through ligand receptor binding and induction of cytochrome
P1450 microsomal enzymes,ixxparticularlfifih then 3,4-TBB
should be considerably more toxic than 3,4,5-HBB. It was
suggested that metabolism of 3,4-TBB decreases its
intracellular concentration resulting in dissociation of the
congener from the TCDD receptor with subsequent decreased
gene expression and hence decreased toxicity. These
findings support the hypothesis of Poland and Knutson
(1983),jJ1that persistent occupation,cn:"hyperinduction"
is necessary for gene expression and toxicity. 3,4,5-HBB is
a nonmetabolizable congener of PBB and is toxic due to its
ability to persistently bind to the TCDD receptor which

results in gene expression and toxicity.

36

Alternativelyy formation of toxic intermediates (1&L
epoxides, arene oxides) during microsomal enzyme metabolism
has been proposed as a possible mechanism of toxicity by
metabolizable congeners of PBB and other PHAH (Jerina and
Daly, 1974; Kato gt gt”,1981; Kohli gt gt” 1978; Sweeney
gt gt., 1979). I}: is postulated that time toxicologic
effects observed with PHAH administration may occur through
the formation of electrophilic (reactive) intermediates
capable of binding DNA (Miller and Miller, 1977), cytosolic
proteins, or membrane and cytosolic lipids. This latter
event could possibly result in initiation of lipid
peroxidation, a proposed mechanism of cellular toxicity by
PCB (Kato gt gt., 1981), and TCDD (Sweeney gt _t., 1979).
Robertson gt gt. (1983) found that 3,4-TBB, a metabolizable
congener of PBB, does not exert its toxicologic effect via a
lipid peroxidation mechanism in rats given a single (150
umoles) ip dose of this congener, since antioxidants such as
butylated hydroxy toluene (BHT), butylated hydroxy anisole

(BHA) or vitamin E did not prevent histologic changes in the

liver.

Glutathione: Role tg Detoxification and PBB Toxicity

 

 

 

Glutathione (Y-glu-cys-gly) is a: thiol containing
tripeptide consisting of L-glutamate, Ircysteine and
glycine. Its gamnu1(Y) bond makes it resistant to normal
peptidase activity and permits its presence at high

concentrations in a variety of different cells (Kosower and

37

Kosower, 1978). Most of the cellular glutathione exists in
thiol form (reduced glutathione [GSH]), although various
disulfides (mainly G-SS-protein) and to a lesser extent,
glutathione disulfide (GSSG) contribute to the total
cellular pool of glutathione. GSH and GSSG are present in
various body fluids including plasma and bile, but at lower
concentrations than found intracellularly (Orrenius gt gt.,
1983). The liver is probably the major source of plasma GSH
that appears to originate from a steady efflux of GSH from
hepatocytes (Orrenius gt gt,, 1983).

Studies on the biosynthesis and degradation of
glutathione have led to the characterization of a series of
enzymes known as the y-glutamyl cycle (Reed and Beatty,
1980). Glutathione is assembled from its constituent amino
acids in two separate reactions (Snoke and Bloch, 1952),
each requiring one molecule of ATP. The first step involves
the formation ofaay-glutamyl linkage between.L-glutamate
and L-cysteine catalyzed by y-glutamyl-cysteine synthetase.
In the second step, glutathione synthetase catalyzes the
addition of glycine to y-glutamyl-cysteinyl to form reduced
GSH. Reduced (NH! is catabolized by ymglutamyl
transpeptidase (GGT) (Reed and Beatty, 1980). It is the
only known enzyme capable of hydrolyzing the y-glutamyl
peptide bond (Meister, 1975b. GGT cleaves the y-glutamyl
group, releasing glutamate enui leaving time remaining
cysteinyl-glycine peptide susceptible to cleavage by

aminopeptidase (Hanigan and Pitot, 1985).

38

The most widely known biological role of reduced GSH is
that of enzymatic or nonenzymatic conjugation with foreign
compounds or their metabolites. Many chemicals that
conjugate with GSH are excreted as mercapturic acids (Booth

gt a1” 1961). Enzymatic conjugation of GSH with several

aromatic compounds is catalyzed by a series of distinct

enzymes known as GSH-S-transferases (Kaplowitz e 1., 1975;

Ketterer gt gt., 1984). GSH-S-transferases (Baars gt al.,

1978), along with epoxide hydrase (Bresnick gt al., 1

KO

77)
and UDP-glucuronyl transferase (Bock gt gt., 1973) have been
reported to be induced in rats after administration of
xenobiotics. Conjugation with GSH does not only facilitate
the excretion of foreign compounds or their metabolites, but
serves as well to intercept highly reactive electrophilic
compounds before they bind to cellular macromolecules
leading to a toxic or mutagenic event (Conney and Burns,
1972; Reed and Beatty, 1980).

Conjugation with GSH does not always decrease the
reactivity of a compound resulting in decreased toxicity.
The compound 1,2 dichloroethane, a waste product derived
from vinyl chloride, has been shown to be activated through
conjugation with GSH to a reactive intermediate that is
mutagenic in the Ames Assay (Rannug gt gt., 1978). These
authors proposed.that.1,2<dichloroethane is activated tola
mutagenic intermediate 131 the presence (ME GSH and
glutathione-S—transferase.AenmlC when incubated with the

post mitochondrial liver fraction (S-9).

39

Glutathione also has a protective role in oxidative
processes (Mills, 1957). GSH peroxidase, linked to
glutathione reductase, and glucose-6-phosphate dehydrogenase
as a source of NADPH was found to be the actual
physiological detoxification mechanism for hydrogen peroxide
ijired blood cells HRflMNiand Hochstein, 1963L. There are
two forms of GSH peroxidase found in red blood cells; one is
a selenium (Se)-dependent enzyme whereas the other is Se-
independent (Lawrence and Burk, 1976; Mazzella gt gt.,
1983).

Glutathione peroxidase is also highly specific for GSH
as a sulfhydryl substrate in protection against nonspecific
hydroperoxides formed during lipid peroxidation of
membranes. It is suggested tg yttg GSH peroxidase inhibits
the initiation of lipid peroxide formation rather than
catalyzing the reduction of these peroxides (McCay gtjgt”
1976). Although the tg tttg mechanism of GSH peroxidase
activity against lipid peroxidation is unclear, it is
thought to be a critical factor in inhibiting lipid
peroxidation in biological membranes.

Many xenobiotics including acetaminophen, bromobenzene,
mycotoxins and benzo(a)pyrene are known to deplete liver GSH
levels after 3E1 tttg exposure (Mgbodile gt gt., 1975;

Mitchell gt gt., 1974; Raheja t., 1983; Thor gt gt.,

SE 2
1979; White, 1976; Zampaglione gt al., 1973). Depletion of

intracellular GSH concentrations has also been proposed as a

mechanism for hepatotoxicity observed in many mammalian

40

species exposed to PHAH. It is postulated that in the event
of intracellular glutathione depletion, a reactive
electrophile, such as an epoxide, would no longer be
detoxified by conjugation with GSH, and could exert its
toxic effects through macromolecule binding and possible
initiation of lipid peroxidation. Rifkind gt _t. (1984)
found 5 to 500 nanomoles/egg of 3,4,5-hexachlorobiphenyl or
3,4-tetrachlorobiphenyl had no significant effects on chick
embryo hepatic GSH levels, and concluded that GSH depletion
does not have a significant role in PCB hepatotoxicity.

Initiation and Promotion gt Carcinogenesis:
Pitot's Model gt Experimental Hepatocarcinogenesis

 

 

 

It is postulated that carcinogenesis in animal test
systems is a multistage process consisting of two major
phases: initiation and promotion (Berenblum and Shubik,
1947; Boutwell, 1974; Farber, 1981; Pitot and Sirica, 1980;
Rous and Kidd, 1941). Initiation is defined as a single
irreversible event that occurs rapidly after treatment with
a subcarcinogenic dose of a chemical, physical or biological
agent capable of directly or indirectly altering cellular
DNA (genotoxic) (Farber, 1981; Pitot gt gt., 1981; Pitot and
Sirica, 1980; Scribner and SUss, 1978). The altered DNA may
be a result of electrophilic binding (a mutational event) of
the initiators to purine and pyrimidine bases on the DNA
molecule (Farber, 1981) or as a result of damaged DNA—repair

enzyme systems (a non-mutational event) (Boutwell, 1974;

41

Farber, 1981; Pitot and Sirica, 1980; Scribner and 8655,
1978). Initiation results in modified cellular DNA that is
heritable and phenotypically unexpressed.(Boutwell, 1974;
Farber, 1981; Pitot gt gt”.1981; Pitot and Sirica, 1980).
The initiated cell and its immediate progeny can only be
identified by production of the promoted neoplasm (Boutwell,
1974; Pitot and Sirica, 1980). Initiation also requires
cell replication for "fixation" and perpetuation of the
altered, unexpressed genetic information (Farber, 1981;
Peraino, 1971; Pitot gt gt”,1981; Pitot and Sirica, 1980).
In Pitot's model of experimental hepatocarcinogenesis, rapid
cellular proliferation is achieved by a partial hepatectomy
(Higgins and Anderson, 1931), and this induces rapid
hepatocyte proliferation and increased DNA synthesis which
may increase the error rate in normal DNA replication and
DNA repair after initiator administration.

Promotion is defined as a reversible event that occurs
after repeated treatments with an agent that alters the
phenotypic expression of genetic information of a cell
(Boutwell, 1974; Pitot gt gt., 1981; Pitot and Sirica,
1980). The agent does not directly interfere with cellular
DNA (epigenetic), but may combine with target cell membrane
or cytoplasmic receptors (Boutwell; 1974; Pitot gt gt.,
1981; Pitot and Sirica, 1980) resulting in altered genetic
expression. (Changes in cell-cell communication.have been
proposed as a mechanism of tg ttttg tumor promotion whereby

neighboring initiated cells become unresponsive to contact

42

inhibition and begin to clonally proliferate (Newbold and
Amos, 1981; Trosko gt gt” 1981; Tsushimoto gt gtull982).
This may result from interference with gap junction function
(Trosko gt__t,, 1981). The exact mechanisms of promotion
are not completely understood and may include a combination
of processes such as activation of oncogenes or other
regulatory proteins or necrosis with subsequent compensatory
hyperplasia (Berenblum, 1944; Bishop, 1982; Jensen gt gt.,
1983b).

In experimental hepatocarcinogenesis, once the
initiated cell is; promoted, tine genetic alteration is
phenotypically expressed by production of preneoplastic and
neoplastic lesions (Pitot gt _l-r 1981; Pitot and Sirica,
1980; Williams, 1980). The preneoplastic lesions are
believed to be the initial stages of cancer formation and
may be clonal in origin (Pitot _t _t., 1981; Pitot and
Sirica, 1980; Rabes gt gt., 1972; Williams, 1980). These
preneoplastic lesions or enzyme—altered foci have been shown
microscopically to regress ix).a normal phenotype upon
cessation of administration of a tumor promoter (Rabes gt
gt., 1972). These lesions are resistant to iron uptake
(Williams, 1980), have increased GGT activity (Fiala and
Fiala, 1973; Kalengayi gt _t”.1975) and are deficient in
ATPase and glucose-6-phosphatase (Rabes e_t gt., 1972).
These characteristics shared by enzyme-altered foci and

hyperplastic nodules may be used to assess the initiating or

promoting abilities of an agent in experimental

43

hepatocarcinogenesis. In Pitot's model of experimental
hepatocarcinogenesis, increased fetal enzyme GGT is used as
one criterion for assessing initiators and promoters. GGT
is seen in actively proliferating and undifferentiated fetal
hepatocytes of rats and is a marker of cellular
dedifferentiation, signaling a reversion of the hepatocyte
to a fetal phenotype (Fiala and Fiala, 1973; Hanigan and
Pitot, 1985; Kalengayi gt gt,, 1975). In the adult rat, the
enzyme has decreased activity and is usually present in bile
ducts and canalicular regions of the liver, the convoluted
tubules of the kidney and the pancreas (Albert gt gt,, 1961;
Fiala and Fiala, 1973; Goldbarg gt gt”,1960; Rutenberg gt
gt., 1969).

Hanigan and Pitot (1985) suggested that compounds used
during the promotion phase CHE many hepatocarcinogenic
protocols utilize glutathione (GSH) and interfere with the
oxidation and reduction of GSH. These authors postulate
that increased GGT on membranes of altered hepatocytes
during hepatocarcinogenesis allows the replenishment of
intracellular GSH and gives these cells a selective growth
advantage during tumor promotion.

Pitot's model of experimental hepatocarcinogenesis is a
two-stage model consisting of an initiation and a promotion
phase (Pitot EE.il~'1978)- The initiation phase consists
cflfa partial hepatectomy'(PH) followed 24 hours later by a
subcarcinogenic dosecflfdiethylnitrosanujma(DEN),a.known

tumor initiator. The promotion phase in this model consists

44

of dietary administration of a test chemical beginning
approximately 30 days after initiation” The length of the
promotion phase is approximately 180 days. PB at 500 mg/kg
of feed is the standard promoting agent used in this
protocol.

The Pitot assay may be used to assess both initiators
and promoters of experimental hepatocarcinogenesis. One of
the advantages of this initiation/promotion protocol is that
it probably offers one of the best methods for promotion of
foci of altered hepatocytes, since interpretation is
uncompromised by inclusion.cfifaa complete carcinogenic dose
of DEN during the initiation phase (Leonard gt__t”.1982).
Also the Pitot assay can detect initiation by carcinogens
that do not significantly enhance hepatocyte replication,
which is required to fix initiating events, due to the
incorporation of PH as a mitotic stimulus during initiation.
An obvious disadvantage of this model is the length of the
promotion phase needed for the development of enzyme-altered
foci. An additional limitation is that the Pitot model is
not the most effective protocol with respect to the number
of foci produced at the end of 180 days (Leonard gt gt.,

1982).

PBB gg Promoters tg Experimental Hepatocarcinogenesis

 

 

Jensen gt gt. (1982) have demonstrated that FM BP-6 and
2,4,5-HBB are promoters of experimental hepatocarcinogenesis
tg vivo. The results indicated that there appeared to be a

dose-dependent response associated with 2,4,5-HBB tumor

45

promotion. This dose-dependent tumor promoting effect was
not seen with FM BP-6. The congener 3,4,5-HBB has also been
reported to possess tg ttgg tumor promoting ability in
Pitotfs two-stage hepatocarcinogenesis assay (Jensen gt gt,,
1983b». Dietary administration of 10 mg 3,4,5-HBB/kg for
140 days caused enhancement of enzyme-altered foci in rats.
Short term exposure to FM BP-6 has also been reported to be
as effective as long term exposure in enhancing the
development of enzyme-altered foci in Pitot's model of
hepatocarcinogenesis (Jensen gt gt,, 1983a).

FM BP-6 (Trosko gt gt., 1981), 2,4,5-HBB and
2,4,5,3U4'fiT—HBB (Tsushimoto gt gt., 1982) at nontoxic
doses are capable of inhibiting cell-cell communication tg
ytttg using Chinese hamster V79 cells, and this appears to
be a property of known tumor promoters (Newbold and Amos,
1981). Cytotoxic congeners of PBB such as 3,4,5-HBB tested
in this system at subtoxic doses were less effective
inhibitors of cell-cell communication than non-cytotoxic
congeners of PBB (Tsushimoto _tjgt”,1982). This suggests
that toxic PBB congeners possibly promote tumorogenesis
through other mechanisms such as cytotoxicity with
subsequent compensatory hyperplasia and neoplasia (Boutwell,
1944; Jensen gt gt., 1983b).

Reports on the assessment of the tg ttttg mutagenic
properties of congeners of PBB have varied (Kavanagh gt gt.,
1985; Kohli gt gt., 1978). Kohli gt gt. (1978) found the

congener 4-bromobiphenyl to have mutagenic activity in the

46

Ames Assay, whereas Kavanagh gt gt. (1985) reported that FM
BP-6, 2,4,5-HBB, 3,4,5-HBB and 3,4-TBB failed to induce
mutations in Chinese hamster V79 cells or WB rat liver
cells. More research is needed to assess the tg vitro and

1 _ttg mutagenic effects of PBB-congeners and also to

define a possible mechanism(s) of tumor promotion by these

compounds.

CHAPTER I

ASSESSMENT OF 3,4,3',4'-TETRABROMOBIPHENYL (3,4-TBB)
AS A PROMOTER OR INITIATOR

IN EXPERIMENTAL HEPATOCARCINOGENESIS IN RATS

CHAPTER I
ASSESSMENT OF 3MLS'MV-TETRABROMOBIPHENYL (3,4-TBB)

AS A PROMOTER OR INITIATOR
IN EXPERIMENTAL HEPATOCARCINOGENESIS IN RATS

Introduction

 

To date, there is no conclusive evidence that PBB
cause acute or short term illnesses in humans (Kay, 1977;
Stross gt gt., 1981), but it is possible they may cause long
term or chronic effects, such as cancer, due to their
persistence 111 the body enui ubiquitousness ill the
environment (Brinkman and deKok, 1981; Tuey and Matthews,
1980). Short term exposure to FM BP-6 has been found to be
as effective as long term exposure in enhancing the
development of preneoplastic lesions in livers of rats;
therefore, continuous exposure to PBB may not be necessary
for enhancement of tumorogenesis in humans (Jensen gt gt.,
1983a).

Carcinogenesis in animal test systems is a multistage
process consisting of two major phases, initiation and
promotion (Berenblum and Shubik, 1947; Boutwell, 1974;
Farber, 1981; Pitot and Sirica, 1980; Rous and Kidd, 1941).
Initiation is defined as a single irreversible, genetic
event that occurs rapidly after treatment 1N1U1 a

subcarcinogenic dose of a chemical, physical or biological

48

49

agent that is capable of indirectly or directly altering
cellular DNA (Farber, 1981; Pitot gt gt., 1981; Pitot and
Sirica, 1980; Scribner and 8855, 1978). Promotion is a
reversible, epigenetic event that occurs after repeated
treatment with an agent that alters phenotypic expression of
the genetic information of a cell (Boutwell, 1974; Pitot gt
gt., 1981; Pitot and Sirica, 1980; Scribner and Sflss, 1978;
Stott gt gt., 1981). Promotion occurs subsequent to
initiation and promoters of carcinogenesis appear to cause
selective proliferation of initiated cells (Pugh and
Goldfarb, 1978; Shulte-Hermann gt gt,, 1981).

In chronic carcinogenicity testing, single or multiple
doses of FM have been reported to cause hepatocellular
carcinomas in rats after 2 years (Kimbrough gt gt., 1981).
For FM to be a complete carcinogen, it should contain

congeners capable of tumor initiation and promotion (Pitot

I(D

E

n)

1., 1981; Pitot and Sirica, 1980; Scribner and 8855,
1978); however, the major congener found in FM BP-6 (2,4,5-
HBB) acts as a promoter in experimental hepatocarcinogenesis
in rats (Jensen gt gt., 1982). Also, PBB do not have
properties of known initiators (Dannan gt gt., 1978a; Trosko
gt gt., 1981; Tsushimoto gt gt., 1981) and it is speculated
that the carcinogenic effects observed in rats given FM
alone, may have resulted from promotion of environmentally
initiated cells (Jensen gt gt., 1982; Pitot gt 1., 1980;

Pivot and Sirica, 1980). Conversely, ea metabolizable

congener of PBB, 4-bromobiphenyl has been reported to be

50

metabolized.tx> a 4-bromobiphenyl arene oxide and is highly
mutagenic in the Ames Assay (Kohli gt gt,, 1978). Arene
oxides or epoxides formed during metabolism of aromatic
hydrocarbons can electrophilically bind to DNA with
subsequent adduct formation.(Hemminki, 1983), and this is
thought to be one mechanism of initiation by chemical
carcinogens (Miller and Miller, 1977).

The congener 3,4-TBB is 51 minor component tentatively
identified in PM BP-6 (Robertson gt gt., 1982). 3,4-TBB
differs from the other congeners of PBB tested for their
carcinogenic effects in rats, in that it is metabolized (tg
ttttg and tg xtyg), and therefore does not accumulate in
tissues of the body (Millis gt gt., 1985a; Mills gt gt.,
1985). This characteristic of 3,4-TBB allowed us to
evaluate the congener as both an initiator and promoter in a
two-stage initiation/promotion assay of hepatocarcinogenesis
in rats (Pitot gt _t,, 1978). It is impossible to assess
the initiating ability of other congeners of PBB tested thus
far due to their lipid solubility and persistence within fat
and liver parenchymal cells of mammals (Matthews, 1981; Tuey
and Matthews, 1980b. It:ushypothesized thatiifcongeners
capable of tumor promotion and a metabolizable congener
capable of tumor initiation through an intermediate epoxide
formed during metabolism are identified within FM, this
mixture can act as a complete carcinogen. This may have
significance ill the development (ME cancer 1J1 Michigan

residents exposed to PBB.

51

3,4—TBB is also a photoproduct of 2,4fiL2',425'-HBB
(245-HBB), the major congener in PM BP-6 (Millis gt gt.,
1985b), binds to the TCDD receptor and is a potent inducer
of aryl hydrocarbon hydroxylase (AHH) activity (Millis gt
gt., 1985a; Robertson gt gt., 1982). 3,4-TBB has a binding
affinity for the TCDD receptor 10 times greater than that of
3,4,5,3',4',5'-HBB (345-HBB), a slowly or nonmetabolized and
very toxic congener of PBB not present in FM BP-6. However,
3,4-TBB is less toxic than 3,4,5-HBB as evidenced by
histologic changes in the liver and thymus of rats (Millis
gt _t” 1985a). Toxic effects associated with short term
administration of 3,4-TBB include a dose dependent decrease
in thymic and splenic weight and increased liver weight

(Millis gt 1., 1985a; Robertson gt gt., 1982). Thymic and

hepatic alterations were characterized microscopicallyknz
Millis gt gt. (1985a) and consisted of lymphoid cell
depletion in the cortex and medulla of the thymus and
midzonal to periportal hepatocellular hypertrophy.

The mechanism of toxicity by PHAH is unknown, although
good evidence supports the hypothesis that toxicity of these
compounds is mediated through binding of a congener to a
polypeptide receptor, time TCDD receptor (Greenlee and
Poland, 1979; Poland and Glover, 1980; Poland gt gt., 1976).
Persistent ligand receptor binding is thought to result in
induction of cytochrome P1450 microsomal enzymes namely AHH,
an enzyme associated with PHAH toxicity. Congeners capable

of inducing increased hepatic AHH activity have been shown

52

to be toxic to various organ systems in rats (Akoso gt gt.,

1982a; Andres gt l”.1983; Millis gt _t”.1985a; Render gt

al., 1982; Robertson gt 1., 1982, 1983). However, the

exact correlation between increased hepatic AHH activity and
toxicity are unknown.

The objectives of this study were to determine if 3,4-
TBB acts as an initiator or promoter of hepatocarcinogenesis
in rats using Pitot's two-stage model of experimental
hepatocarcinogenesis and to characterize the histologic and
ultrastructural tissue changes associated with chronic

dietary administration of 3,4-TBB.

Materials and Methods

 

Experimental Design

 

Treatment groups for the initiation and promotion
assays consisted of 6 or 3 rats placed in groups as shown in
Table 1-1.

The promoting or initiating ability of 3,4-TBB was
assessed and compared to a standard promoter, phenobarbital
(PB), (n: to a standard initiator, diethylnitrosamine (DEN),
using PitotHs two-stage model CM? experimental
hepatocarcinogenesis as outlined in Figure 1-1.

Figure 1-1. Pitotis two—stage model of experimental
hepatocarcinogenesis.

Day: 0 1 31 211

 

TX: PH Initiator Promoter Kill

Table 1-1 .

53

Experimental design for 3,4,3',4'-tetrabromobiphenyl (3,4-
TBB) initiation and promotion assays.

 

 

Dose
Dose (mg/ kg) No . of

Group Tx Initiator (mg/ kg) Promoter of diet rats
Study I : Initiation assay

1 911a DE‘Nb 10 o o 6

2 ' PH DEN 10 1113C 500 6

3 PH 3,4-TBB 1 0 0 6

4 PH 3,4-TBB 1 PB 500 6

5 PH 3,4-TBB 5 0 0 6

6 PH 3,4-TBB 5 PB 500 6

7 PH 3,4-TBB 10 0 O 6

8 PH 3,4-TBB 10 PB 500 6
Study I I : Promotion assay

9 0 0 O 3 , 4-TBB O . 1 3

10 PH DEN 10 3 , 4-TBB 0 . l 6

ll 0 O 0 3 , 4-TBB l 3

12 PH DEN 10 3 , 4-TBB 1 6

l3 0 O 0 3 , 4-TBB 5 3

14 PH DEN 10 3 , 4-TBB 5 6

15 O O 0 PE 500 3

 

aPartial hepatectomy.
bDiethy 1nitrosamine .

CPhenobarbital .

54

In the initiation assay, rats were 2/3 partially
hepatectomized (PH) 24 hr prior to initiation with 10 mg of
DEN/kg given ip or 1, 5 or 10 mg of 3,4-TBB/kg given orally.
Promotion was with 500 mg of PB/kg diet fed from day 31 to
day 211. In the promotion assay, rats were 2/3 PH 24 hr
prior to initiation with 10 mg of DEN/kg given ip. Rats
used as controls were notlfiior'initiated. iPromotion was
with 500 mg of PB/kg diet or<qu 1, or 5 mg of 3,4-TBB/kg

diet'fed from day 31 to day 211.

tags

Female Sprague-Dawley (C/D) rats weighing 180-200 g
were obtained from Charles River Laboratories, Inc.,
Portage, MI. Rats were randomized and housed according to
groups in clear polypropylene cages, 3 rats per cage. Rats
were acclimated for 5 days. The room temperature was
maintained at 220 C with a 12 hr light/dark cycle» Cages
containing rats for the tumor promotion assay were placed in
filtered laminar flow units (Contamination Control Inc.,

Lansdale, PA).

Surgical Procedure for Partial Hepatectomy

Rats were partially hepatectomized by using the method
described by Higgins and Anderson (1931). Rats were
anesthetized by using ether and asepsis was maintained
throughout the surgery. A median-line incision was made
just prior to the xiphoid process of the sternum, that

extended 3 or 4 cm caudally to expose the underlying

55
abdominal muscles. The abdominal muscles and peritoneum
were bluntly dissected away to expose the median (right and
left) and left liver lobes. These lobes were lifted from
the abdominal cavity, ligated using sterile and absorbable 3
chrondrzgut.(Davis-Geck,.American.Cyanamid.Company, Pearl
River, NY) and excised. The abdominal muscles and
peritoneum were sutured by using 3-0 coated vicryl*,
absorbable suture (Ethicon, Inc., Jehnson anui Johnson
Company, Somerville, NJ). The skin incision was closed by
using metal clamps (Michel Clamps [18/8], George Tiemann and
Co., Plainview, NJ). There was no special postoperative

care, and rats had fully recovered by 24 h post surgery.

Chemicals

 

3,4-TBB 'was synthesized euui purified by
recrystallization and alumina chromatography (Millis, 1984).
Diethylnitrosamine UHHU was purchased from Eastman Kodak
Company, Rochester, NY, and phenobarbital was purchased from

Sigma Chemical (XL, St. Louis, MO.

Diet Preparation

 

 

The diets were prepared by adding appropriate amounts
of 3,4-TBB or PB in corn oil to a ground commercial feed
(Certified Rodent (Hunv #5002R, Ralston Purina Co., St.
Louis, MO). A premix containing 50 mg of 3,4-TBB/kg was
prepared by dissolving 100 mg of 3,4-TBB in corn oil and
adding it to.2J)kg'of feed. Diets containing 1 or Sing/kg

of 3,4-TBB/kg were prepared from the premix, and diet

56

containing 0.1 mg of 3,4-TBB/kg was prepared from the
mixture containing 1 mg of 3,4-TBB/kg. Rats were observed
daily for clinical signs of toxicity and body weights were

recorded once a week throughout the study.

Necropsy Procedure

 

 

Rats were taken off feed 16 h before necropsy and on
day 211 rats were weighed, anesthetized with ether, and
killed by decapitation. All organs were routinely examined
for gross lesions. The liver, spleen, thymus, kidneys,
adrenal glands and brain were removed from the carcass and
weighed.

Collection and Preparation gt ttgggg gggples for
Histopathologic Evaluation

 

 

 

 

Tissue: samples <mf liver, thymus, spleen, kidney,
adrenal gland, thyroid gland, heart, lung, stomach, small
intestine, pancreas, colon, urinary bladder, brain and
pituitary gland were collected and fixed in 10% neutral
buffered formalin. Formalin-fixed tissue samples were
processed, embedded in paraffin, cut by a microtome into 6
pm sections and stained with hematoxylin and eosin for

histologic examination.

Collection, Preparation and Histochemical Staining for Liver

Samples

At necropsy, representative liver samples were mounted
on corks by using OJLT. (Tissue-TekR, Miles Scientific,
Division (ME Miles Laboratories, Inc., Naperville, IL).

Liver samples were frozen in isopentane cooled in liquid

57

nitrogen. Five liver sections taken from the same:lobe of
each rat were cut at.E3 pm (IEC Microtome-Cryostat
Model/Minot Custom Microtome, International Equipment Co.,
Needham Heights, MA) and were stained for gamma glutamyl
transpeptidase (GGT) activity by the method of Rutenberg gt
‘gt.(1969). IEach section of frozen liver was placed onto a
glass slide, fixed in 100% acetone (40 C) for 10 to 15
minutes and air dried. Each section was incubated for 15 to
20 minutes in a freshly mixed and filtered substrate
solution (250 C) containing 1 rml of gamma-glutamyl-4-
methoxy-B-naphthylamide, 2.5 mg/ml (Vega Biotechnologies,
Inc., Tucson, AZ), 5 ml of 0.1 M Tris buffer, pH 7.5 (Sigma
Chemical Co., St. Louis, MO), 10 mg of Fast Blue BB salt
(Sigma Chemical Co.,EH; Louis, MO),]l)nm;of glycylglycine
(Aldrich Chemical Co., Milwaukee, WI) and 14 ml of 0.85%
saline. Each liver section was then placed in 0.85% saline
for 2 minutes, followed by a 2 minute incubation in.0.1 M
cupric sulfate. Each section was again placed in 0.85%
saline for 2 minutes, immersed in deionized water, allowed
to drain and counterstained with hematoxylin using three 1
second dips. Each section was rinsed with tap water, air
dried and mounted onto a glass slide and refrigerated.
Slides were evaluated within 8 h after histochemical
staining.

Histochemically stained liver sections were projected
and enzyme altered foci (EAF) were traced using a Leitz

Prado Projector (Ernst Leitz Wetzlar GMBH, Wetzlar, West

58

Germany) at a magnification of 90X. Approximately 40 fields
for each liver were evaluated, which was equivalent to an
area of 1.5-2.0 CW2 per rat. The area of each EAF/cm2 of
liver was determined with a planimeter (Lasico L-30, Los
Angeles Scientific Co., Inc., Los Angeles, CA) and the

methods of Scherer (1981) were used to compute the number of

EAF/cm3 of liver.

Chemical Analysis gt Liver and Adipose Tissue

 

Samples of liver and adipose tissue were taken from 2
or 3 rats from each group of rats fed diets containing 0,
0.1, 1 or 5 mg of 3,4-TBB/kg. Liver and adipose samples
were also taken from 3 rats initiated with 3,4-TBB and
promoted with PB. Tissue concentrations of 3,4-TBB were
determined tn; the following procedure. An electronic
analytical balance (Mettler AB 163, Mettler Instrument
Corporation, Highstown, NJ) was used to weigh 0.5 g of liver
and abdominal adipose tissue. Weighed samples were washed
in petroleum ether and ground with washed ignited sand
(Mallinckrodt, Inc., Paris, KY). The ground mixture was
dehydrated by the addition of 10 to 20 g of granular
anhydrous sodium sulfate (Mallinckrodt, Inc., Paris, KY).
3,4-TBB was extracted from the mixture by adding 15 m1 of
glass distilled hexane (J.T. Baker Chemical Co.,
Phillipsburg, NJ) and bringing the mixture to a boil over a
water bath (1000(3L. This mixture was filtered into a 100

ml volumetric flask and the hexane washes and subsequent

59

filtrations were repeated until a total of 4 extractions
were completed. After the fourth extraction, the 3,4-TBB
extract was brought to a volume of 100 ml by the addition of
hexane. Aliquots of 20 ml were taken from the 3,4-TBB
extract and evaporated with nitrogen.

The aliquot used for fat content determinations was
evaporated (N-EVAP, model III, Meyer Oranomation Assoc.,
Inc., Shrewsberry, MA) to 2 ml and rinsed with petroleum
ether into preweighed foil pans. Ilheated water bath was
used to evaporate the solvent in the foil pans. The pans
were then put in a vacuum desiccator for 24 h to promote
drying of the sample. The tissue fat content was determined
by subtracting the weight of the pan without the fat from
the weight of the pan with the fat.

The aliquot Luxxi for chemical determinations was
evaporated to 2 ml and put through a column (Chromaflex, 200
mm x 7 mm ID) that was prewashed with acetone and plugged at
the tapered end with glass wool. The column was filled with
1.6 g of activated magnesium silicate (Forisil, 60-100 mesh,
Fisher Scientific Co” Cleveland,(fln and 2cnncfifgranular
anhydrous sodium sulfate was added to the tOp of the column.
The column was washed with 5 ml of hexane and this was
discarded as the first 5 m1 of hexane. The 2 m1 evaporated
sample was added to the column and repeatedly rinsed with
hexane. The sample eluant was evaporated to 0.5 ml and
reconstituted to a total volume of 2 ml by adding iso-octane

(2,2,4—trimethyl-pentane) (Burdick and Jackson Laboratories,

60
Inc., Muskegon, MI). 3,4-TBB tissue concentrations were
then determined by injecting 2 ul of the sample eluant onto
a gas chromatograph equipped vwhfl1 an electron capture
detector (GAG. Model 3709, Varian Instrument Division, Palo
Alto, CA) utilizing an injector temperature of 2800 C, a
column temperature of 2500 C and a detector temperature of

3000 C. Nitrogen was used as the carrier gas at a flow rate

of 30 ml/min.

Gas chromatographic readings were recorded and tissue
concentrations of 3,4-TBB were compared to known standard
amounts of 3,4-TBB.

2211222122 222 11222122122 21 11221 2222122 122
Ultrastructural Examination

Liver sections were fixed in Karnovsky's fixative
(Karnovsky, 1965). These sections were trimnmxh placed in
Zetterqvist's wash (Pease, 1964) at pH 7.4 and postfixed in
1% osmium tetroxide in Zetterqvistls wash. Liver sections
were then dehydrated in graded alcohol (50, 70, 95, and
100%), placed in 2,2-dimethoxypropane (Baic and Baic, 1984)
and fixed in 100% acetone. An Araldite 502 plus DDSA
(dodecanyl succinic anhydride) mixture was used for plastic
embedding; IEmbedded liver sections were cured inalOOO C
oven for 24 h.

Plastic embedded samples were thick sectioned (1 um),
stained with toluidine blue and examined with a light
microscope. Representative areas vwnxa thin sectioned (900

A) and were stained with uranyl acetate and lead citrate.

61
The thin sections were Viewed by using an electron

microscope (EM 982, Carl Zeiss, Germany).

Statistical Analysis of Data
Data were statistically analyzed after logarithmic

transformation by the one-way analysis of variance (ANOVA)

and differences in group means were analyzed by the Student-
Newman-Keul's multiple comparison test at p<0.05, or the

comparison of percentages arcsin /‘———" test at tinfinity
(Steel and Torrie, 1980L. Organ and body weight data were
statistically analyzed by the one way ANOVA and the Student-

Newman-Keul's multiple comparison test at p<0.05.
Results

Enzyme-Altered Foci

 

The mean number of EAF/cm3 of liver for PH rats
initiated with 10 mg of DEN/kg given ip and promoted with
500 mg of PB/kg diet ortqu 1, or 5 mg of 3,4-TBB/kg diet
is given in Table 1-2. Dietary administration of 5 mg of
3,4-TBB produced significantly greater numberscnfhepatic

3

EAF/cm of liver than 500 mg of PB/kg diet or 0.1 or 1 mg of

3,4-TBB/kg. There appeared to be a dose dependent increase

in the number of hepatic EAF/cm3

of liver in rats fed 0.1,
1, or 5 mg of 3,4-TBB/kg diet, although there was no
statistically significant difference in time number of

EAF/cm3 of liver between rats fed 0.1 or 1 mg of 3,4-TBB/kg

diet. There was no significant difference in the number of

62

Table 1-2. Number of enzyme-altered foci (EAF)/cm3 of liver in rats
initiated with DEN and promoted with PB or 3,4-TBB for 180

 

 

days.

Day Day Day

0 1 Dose 31-211 Dose 3
—— (mg/kg —— ("IQ/kg Number of EAF/cm
'I‘x Initiator given ip) Promoter of diet) of liver

PH DEN 10 0 0 010

PH DEN 10 PE 500 4651355

PH DEN 10 3,4-TBB 0.1 1311121

PH DEN 10 3,4-TBB 1.0 217:158

PH 13m 10 3,4—TBB 5.0 1488:919a

 

Data are expressed as 2150 for groups of 6 rats.
aSignificantly different from rats promoted with PB or 0.1 or 1.0 mg of
3,4-TBB/kg or fed basal diets (p<0.05) .

63

EAF/cm3 of liver in rats initiated with DEN and promoted
with diets containing PB or 0.1 or 1 mg of 3,4-TBB/kg.

The mean number of EAF/cm3

of liver for PH rats
initiated with 10 mg of DEN/kg given ip or 1, 5, or 10 mg of
3,4-TBB given orally and promoted with 500 mg of PB/kg diet
or fed a basal diet for 180 days is given in Table 1-3.
3,4-TBB awn? have initiation potential as suggested by
increased numbers of hepatic EAF in rats initiated with 3,4-
TBB and fed PB, compared to rats initiated with 3,4-TBB and
fed a basal diet. A single oral dose of 1, 5, or 10 mg of
3,4-TBB/kg was less effective in initiating the formation of
hepatic EAF than 10 mg of DEN/kg given ip. in rats promoted
with PB for 180 days. The mean number of hepatic EAF/cm3 of

liver for rats initiated with 3,4-TBB or DEN and fed a basal

diet for 180 days was slightly greater than zero.

Body and Organ Weights

 

 

Data for body and organ weights in rats initiated with
DEN or 3,4—TBB and promoted with PB or 3,4-TBB are given in
Table 1-4. There was no significant differenceeinibodytor
splenic weights of rats in the various treatment groups.
Rats initiated with 5 or 10 mg of 3,4—TBB and promoted with
500 mg of PB/kg diet had significantly increased liver
weights compared to rats initiated with 10 mg of DEN and fed
a basal diet. Rats initiated with 10 mg of 3,4—TBB/kg and
promoted with 500 nmg<xf PB/kg diet and rats initiated with
10 mg of DEN/kg and promoted with 5 mg of 3,4-TBB/kg diet

had significantly decreased thymic weights compared to rats

64

Table 1-3. Number of EAF/cm3 of liver in rats initiated with DEN or
3,4dTBB and promoted with PB for 180 days.

 

 

Day Day Day
0 1 31-211 Dose 3
— Dose ————-— (mg/kg) Nunber of EAF/cm
Tx Initiator (mg/ kg) Promoter of diet of liver
PH DEN 10 o 0 0:0
PH um 10 PB 500 465135521
PH 3,4-TBB 1 0 0 0.83:2.84
PH 3,4-TBB 1 PB 500 22:32a
PH 3,4-TBB 5 0 0 214.9
PH 3,4-TBB 5 PB 500 36:21all
PH 3,4-TBB 10 0 0 0:0
PH 3,4-TBB 10 PB 500 1111706:1

 

Data are expressed as §:SD for groups of 6 rats.

aSignificantly different from rats initiated with DEN and fed a basal
diet using comparison of percentages arcsin f— at tinfinity ( co )
(p<0.05) .

65

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66

initiated with 5 mg of 3,4-TBB and fed a basal diet. Rats
initiated with 10 mg of DEN and promoted with 0.1,]q or 5
mg of 3,4—TBB had decreased thyroid gland weights compared
to rats initiated with 10 mg of DEN/kg and fed a basal diet.

There were no significant differences in body or organ
weights in non-PH or noninitiated rats fed diets containing
0.1, l, or 5 mg of 3,4-TBB/kg or 500 mg of PB/kg for 180

days.

Histologic Evaluation 9f Liver

 

PH rats initiated with DEN or 3,4-TBB and fed a diet
containing PB Emmi hepatic histologic changes characterized
by multifocal areas of moderate to severe centrolobular to
midzonal hepatocellular hypertrophy. The hepatocytes in
these regions had abundant acidophilic cytoplasm that caused
obliteration of the adjacent hepatic sinusoidal spaces as
shown in Figure 1-2. Similar changes were observed in non-
PH and non-initiated rats promoted with PB. Liver sections
from PH rats initiated with DEN and fed diets containing
3,4-TBB had mild periportal to midzonal hepatocellular
intracytoplasmic vacuolation. and Ihypertrophy with
obliteration of the adjacent hepatic sinusoidal spaces as
shown in Figure 1-3. Similar changes were observed in
groups of non-PH and noninitiated rats that were promoted
with diets containing 3,4-TBB. The severity of the
histologic lesions in rats fed diets containing 3,4—TBB

appeared to be dose-dependent. There were no significant

67

Figure 1-2. Photomicrograph of a liver section from a
rat fed a diet containing 500 mg of PB/kg for 180 days.
Notice areas of centrolobular hepatocellular hypertrophy
with obliteration of adjacent hepatic sinusoidal spaces (H &
E stain, 108X). '

Figure 1-3. Photomicrograph of a liver section from a
rat fed a diet containing 5 mg of 3,4-TBB/kg for 180 days.
Notice areas of periportal to midzonal hepatocellular
intracytoplasmic vacuolation and obliteration of adjacent
hepatic sinusoidal spaces (H & E stain, 200X).

68

 

Figure 1-2

 

Figure 1-3

69

changes observed in sections of thymus, spleen and thyroid
gland from rats in the various treatment groups.

The categories of preneoplastic lesions observed in
liver sections taken from PH rats initiated with DEN or_
3,4-TBB and promoted with diets containing 3,4—TBB or PB
consisted of INH? and neoplastic nodules (Institute of
Laboratory Animal Resources, National Research Council,
1980). The foci of altered cells were composed of
circumscribed regions of enlarged hepatocytes containing
abundant acidophilic to pale cytoplasm (Figure 1-4). These
cells had single to multiple vesicular nuclei containing
multiple prominent.nucleoli.(Figure I—SL. These EAF were
positive for GGT (Figure 1-6). The neoplastic nodules
consisted of large circumscribed regions occupying 2 or more
hepatic lobules. The nodules were composed of enlarged
hepatocytes having abundant acidophilic cytoplasm and
enlarged nuclei containing single or multiple prominent
nucleoli. There was a sharp demarcation of the periphery of
nodules from the surrounding liver tissue (Figure 1-7).
There were no preneoplastic changes observed in non-PH or
noninitiated rats :flafl diets containing 3,4-TBB. Rats
initiated with DEN or 3,4-TBB and fed a basal diet had no
apparent histologic or preneoplastic hepatic lesions (Figure
1-8). There were no hepatocellular carcinomas in any of the

livers from rats in the initiation or promotion assay.

 

70

Figure 1-4. Photomicrograph of an EAF within a liver
section from a rat initiated with 5 mg of 3,4-TBB/kg given
orally and promoted with 500 mg of PB/kg for 180 days.
Notice enlarged hepatocytes with abundant amounts of pale
cytoplasm (H & E stain, 200X).

Figure 1-5. Photomicrograph of an EAF within a liver
section from a rat initiated with 10 mg of DEN/kg given ip
and promoted with 5 mg of 3,4-TBB/kg for 180 days. Notice
enlarged hepatocytes with single to multiple vesicular
nuclei containing multiple prominent nucleoli (H & E stain,
180X).

71

 

Figure 1-5

72

Figure 1-6. Photomicrograph of a histochemically-
stained EAF within a liver section from a rat initiated with
10 mg of DEN/kg given ip and promoted with 500 mg of PB/kg
for 30 days (Gamma-glutamyl transpeptidase stain, 170X).

Figure 1-7. Photomicrograph of a neoplastic nodule
within a liver section from a rat initiated with 10 mg of
DEN/kg given ip and promoted with 500 mg of PB/kg for 180
days. Notice sharp demarcation of periphery of nodule from
the surrounding liver tissue (H & E stain, 76X).

.fl

 

73

 

Figure 1-7

 

74

Figure 1-8. Photomicrograph of a liver section from a
rat initiated with 10 mg of DEN/kg given ip and fed a basal
diet for 180 days. Notice normal appearing hepatocytes (H &
E stain, 210X).

75

 

Figure 1-8

76

Ultrastructural Evaluation of Liyer

Hepatocytes from non-PH and noninitiated rats given 5
mg of 3,4-TBB/kg for 180 days had abundant amounts of smooth
endoplasmic reticulum (SER) and Inihi intracytoplasmic
vacuolation. There was no evidence of cytotoxicity (Figures

1-9 and 1-10).

Chemical Analysis

 

Concentrations of 3,4-TBB in liver and adipose tissue
are given in Table 1-5. Results indicate that 3,4-TBB, when
administered to rats in the diet for 180 days, does not
persist in body tissues and is readily metabolized and

excreted from the body.

77

Figure 1-9. Electron micrograph of a hepatocyte from a
nonpartially hepatectomized, noninitiated rat fed a diet
containing 5 mg of 3,4-TBB/kg for 180 days. Notice the
abundant amounts of smooth endoplasmic reticulum (arrow) and
mild intracytoplasmic vacuolation and lack of cytotoxic
changes (Uranyl acetate-lead citrate stain, 6400X).

Figure 1-10. Higher magnification of
electronmicrograph of hepatocyte in Figure 1-10. Notice
abundant amounts of smooth endoplasmic reticulunl(arrow)
(Uranyl acetate-lead citrate stain, 12,800X).

78

 

1-9

igure

F

 

Figure 1-10

 

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79

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80

Discussion

 

The results of this study indicate that 0.1, l, or 5 mg
of 3,4-TBB/kg when given in the diet for 180 days, increases
the number of GGT-positive EAF and at these doses appears to
act.as a promoter in experimental hepatocarcinogenesis in
rats. Enhancement of hepatic EAF formation after initiation
with a subcarcinogenic dose of an initiator is a known
property of tumor promoters (Peraino gt gt., 1971, 1981;
Pitot, 1979; Watanabe and Williams, 1978L. The mechanism
whereby 3,4-TBB promotes hepatocarcinogenesis in rats is
unknown, but it appears to be more effective in enhancing
the number of hepatic EAF than PB, a known tumor promoter,
at a dose 100 times less than that of PB. It is suggested
that congeners of PBB nun? act as promoters in
hepatocarcinogenesis by inhibiting cell to cell
communication when givenan:noncytotoxic doses (Trosko gt
gt., 1981; Tsushimoto gt gt., 1982), or by chronic or
recurrent toxicity resulting in necrosis, cellular
regeneration and subsequent neOplasia (Berenblum, 1944) when
given in cytotoxic doses (Jensen gt gl,, 1983b).

3,4-TBB did IuN: cause hepatocellular necrosis and
regeneration in non-PH and noninitiated rats given 0.1, l or
5 mg/kg in the diet for 180 days, therefore it can be
concluded that 3,4-TBB probably does not promote by a
cytotoxic mechanism. To date, there is IN) conclusive
evidence to support the hypothesis that 3,4-TBB can inhibit

cell to cell communication lg vitro at noncytotoxic doses.

81

Speculatively, 3,4-TBB may promote tumorogenesis through
gene activation (Boutwell, 1974). It is suggested that
treatment with an initiator results in formation of
permanent and heritable unexpressed alteration in the cell
genome, and if promoters regulate nuclear gene
transcription, then treatment with a promoter would result
in increased synthesis of RNA and protein and these may in
part come from regions of the genome not normally expressed
(derepression). Gene activation (Boutwell, 1974) is, as is
toxicity of PHAH, thought to be regulated by a receptor
(Poland _t _t., 1976). The theory of gene activation in
part is invoked in the oncogene theory of carcinogenesis
(Cooper and Lane, 1984) and may be a credible theory of
promotion by 3,4-TBB and other congeners of PBB. Increased
expression of hepatic GGT in the adult rat liver is thought
to occur by derepression of a gene involved in the GGT
synthesis in the fetal liver that is normally repressed in
the adult state (Fiala and Fiala, 1970). Congeners of PBB
have been shown to enhance the expression of this fetal
enzyme in adult rat liver after initiation with DEN.

The results of this study also indicate that 3,4-TBB
may have weak initiation potential as suggested by increased
numbers of GGT-positive hepatic EAF in rats initiated with
3,4-TBB and fed PB, compared to rats initiated with 3,4-TBB
and fed a basal diet. However, a single oral dose of.l,EL
or 10 mg of 3,4-TBB was less effective in initiation of the

formation of hepatic EAF thanZHJnmJOf DEN given ip. ]H:is

82

suggested that metabolizable congeners of PBB such as 3,4-
TBB or 4-bromobiphenyl are metabolized by the hepatic
microsomal monooxygenase system to a toxic intermediate
Ufle. epoxide) that may electrophilically bind to cytosolic
macromolecules or structural components of DNA (Kohli gt
gl,, 1978). The latter event is thought to be one mechanism
of tumor initiation by chemical carcinogens (Conney and
Burns, 1972; Hemminki, 1983; Miller and Miller, 1977).

There have been conflicting reports as to the mutagenic
effects of metabolizable congeners of PBB. The congener
3,4-TBB has been reported to be nonmutagenic in Chinese
hamster V79 cells (Kavanagh g___ g_l_., 1985), whereas 4-
bromobiphenyl has been reported to be highly mutagenic in
the Ames Assay (Kohli gt‘ 1., 1978). Further tg ytttg and

in vivo studies are needed to assess the tumor initiating

 

potential and mechanism(s) of tumor promotion by 3,4-TBB.
Dietary administration of 1 mg 3,4,5-HBB/kg has been
reported to be hepatotoxic in rats (Jensen gt gt., 1983b),
whereas in this study chronic dietary administration of as
much as 5 mg of 3,4-TBB appeared to be relatively nontoxic
in rats as evidenced by histologic changes in the liver,
spleen, thymus and thyroid gland and ultrastructural changes
in the liver. It is postulated that enzyme induction is an
early event and that toxicity occurs later as a result of
persistent receptor occupation and gene expression (Poland

and Knutson, 1982). 3,4-TBB is a metabolizable congener and

the metabolism of congeners suchens3,4-TBB decreases the

83

toxicologic effects of these compounds in mammalian systems
(Mills gt gt., 1985). The decreased toxicologic effects of
metabolizable congeners is thought to be due to their
inability to exist in high intracellular concentrations to
allow persistent occupation of the TCDD receptor (Millis gt
gl,, 1985a). Nonmetabolizable congeners, such as 3,4,5-HBB,
are not readily excreted from the tissues of rats, and if
toxicity is mediated through persistent receptor binding
with subsequent gene expression, then 3,4,5-HBB, a
persistent congener, would be predicted to be more toxic
than the metabolizable congener 3,4-TBB when administered to
rats at lower or equimolar doses even though 3,4-TBB is a
better ligand for the TCDD receptor.

Liver and adipose tissue from rats fed diets containing
0.1, 1 cm: 5 mg of 3,4-TBB did not have appreciable
concentrations of this compound at the end of 180 days.
This indicates that chemical analysis of tissue is not a
good indicator of levels of exposure 1x) metabolizable
congeners of PBB.

Chronic administration of low dietary levels of 3,4-TBB
to non-PH and noninitiated rats did not cause a significant
increase in liver weights or decrease in thymic or splenic
weights as observed in rats given a single oral dose (21.3
umol/kg) (Millis gt gt., 1985a) or a single ip injection
(150 umol/kg) (Robertson g_ gt., 1982) of 3,4-TBB. These
authors gave rats single exposures to relatively high doses

of this compound, whereas in this study, 3,4-TBB was given

84

at lower doses over a longer period of time. 3,4-TBB
induces its (Hui metabolism anui during chronic dietary
administration, intracytoplasmic concentrations may never be
high enough at one time period to evoke a toxic response.
It is suggested that chronic administration of relatively
high doses of 3,4-TBB would increase intracytoplasmic
concentrations of this congener and produce toxic changes

analogous to those seen after acute administration.

Summary

Pitot's bioassay for hepatocarcinogenesis was used to
determine ii? 3,4gT,4'-tetrabromobipheny1 (3,4-TBB) can
promote or initiate the development of gamma-glutamyl
transpeptidase (GGT) positive enzyme-altered foci (EAF).
3,4-TBB is a minor component of Firemaster BP-6. It is
metabolized, binds to the TCDD receptor, and induces AHH
activity. Groups of 6 female, 180-200 g rats were used for
initiation and promotion assays. To test for initiation,
rats were partially hepatectomized (PH) and given 3,4-TBB or
diethylnitrosamine (DEN) as an initiator. Thirty days
later, rats were fed 500 ppm of phenobarbital (PB) as a

3 of liver

promoter for 180 days. The mean number of EAF/cm
was: DEN (10 mg/kg), 465; 3,4-TBB: (1 mg/kg), 22; (5
mg/kg), 36;(10 mg/kg), 111. The mean numberofEAF/cm3 of
liver for PH rats initiated with 3,4-TBB or DEN and fed a

basal diet for 180 days was slightly greater than zero. To

test for promotion, PH rats were initiated with 10 mg of

85

DEN/kg and 30 days later fed 3,4-TBB or PE for 180 days.
The mean number of EAF/cm3 of liver was: PB (500 ppm), 465;
3,4-TBB: “Ll ppm), 131;(1 ppm), 217;(5 ppm),.l488. 3,4-
TBB fed continually in the diet increased the number of EAF
and thus appears to act as a hepatic tumor promoter. Also,
3,4-TBB may have initiating potential as suggested by
increased numbers of EAF in rats initiated with 3,4-TBB and
promoted by PB.

Non-PH and noninitiated female Sprague-Dawley rats
weighing 180-200 g were used as controls to assess the
histologic and ultrastructural tissue changes associated
with chronic administration of 3,4-TBB. Dietary
administration of 0.1, 1 or 5 mg of 3,4-TBB/kg does not
appear to be severely toxic in rats as evidenced by light
and electron microscopic changes and alterations in organ
and body weights. 3,4-TBB is metabolized and does not

accumulate in the liver and adipose tissue of rats.

CHAPTER II

CHRONIC DIETARY ADMINISTRATION OF
3,4,3',4'-TETRABROMOBIPHENYL (3,4-TBB) TO RATS:
EFFECTS ON SERUM AND HEPATIC VITAMIN A HOMEOSTASIS
AND SERUM TRIIODOTHYRONINE (T3)

AND THYROXINE (T4) CONCENTRATIONS

CHAPTER II

CHRONIC DIETARY ADMINISTRATION OF
3,4,3',4'-TETRABROMOBIPHENYL (3,4-TBB) TO RATS:
EFFECTS ON SERUM AND HEPATIC VITAMIN A HOMEOSTASIS
AND SERUM TRIIODOTHYRONINE (T )

AND THYROXINE (T4) CONCENTRATIONS

Introduction

 

Polybrominated biphenyls (PBB) are compounds classified
as polyhalogenated aromatic hydrocarbons (PHAH) and are
components of Firemaster (FM) BP-6, the commercial mixture
of PBB that contaminated much of Michigan%slivestock and
residents in 1973 (Carter, 1976). Also belonging to the
PHAH group are the polychlorinated dibenzo-p-dioxins,
classically 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
polychlorinated biphenyls (PCB) and polychlorinated
dibenzofurans (TCDF). These chemicals, along with PBB, are
all environmental contaminants and have been shown to
produce a similar toxic syndrome and biochemical responses
1J1 mammalian species (Poland aumi Knutson, 1982). The
toxicity:Hscharacterizedknra wasting syndrome, lymphoid
involution, hepatotoxicity, chloracne, and gastric lesions.
Some of the biochemical responses that are affected by PHAH
administration include hepatic vitanUJIA levels, serum T3
and T4 concentrations and lipid metabolism (Poland and

Knutson, 1982). FM and 3,4,5,3‘n?,5'-HBB (3,4,5—HBB) have

87

88

been found 1x3 affect hepatic anui serum vitamin Ix
homeostasis. Alterations in vitamin A status were thought
to be related to 3-methylcholanthrene (3-MC) type microsomal
enzyme induction (Jensen, 1983). 3,4-TBB is also a 3-MC
type microsomal enzyme inducer and if there is a correlation
between this type of enzyme and vitamin A status, then 3,4-
TBB would be predicted to cause alterations in serum or
hepatic vitamin A concentrations. 3,4,5-HBB has also been
found to alter serum T4 concentrations in rats (Akoso gt gt,
1982b).

The objectives of this study were to assess the effects
of chronic dietary administrationcfif3,4-TBB on serum and
hepatic vitamin.1\ homeostasis and serunl'P3 and T4

concentrations in rats.
Materials and Methods

Experimental Design

 

Rats were placed in treatment groups as shown in Table
2-1. iRats had free access to 0.1,14 or 5 mg of 3,4-TBB/kg

or 500 mg of PB/kg administered in the diet for 180 days.

Chemical
3,4-TBB 'was synthesized euui purified by

recrystallization and alumina chromatography (Millis, 1984).

Rats
Female Sprague-Dawley rats weighing 180-200 g were

obtained from Charles River Laboratories, Inc., Portage, MI.

89

Table 2-1. Experimental design for dietary administration
of 3,4,3fl4'-tetrabromobiphenyl (3,4-TBB) for

 

 

180 days.
Concentration (mg/kg) Number
Group Tx in the diet of rats
1 3,4-TBB 0.1 3
2 3,4-TBB 1 3
3 3,4-TBB 5 3
4 PBa 500 3

 

aPhenobarbital.

 

90

Rats were randomized and housed according to groups in clear
polypropylene cages, 3 rats per cage. All rats were
acclimated for 5 days. The room temperature was maintained

at 220 C with a 12 hour light/dark cycle.

Diet Preparation

 

The diets were prepared by adding appropriate amounts
of 3,4-TBB or PB dissolved in corn oil to a ground
commercial feed (Certified Rodent Chow #SOOZR, Ralston
Purina Co., St. Louis, MO). Rats were fed the diets for 180

days before necropsy.

Collection gt Serum Samples for Vitamin A and Thyroid
Hormone Analysis

 

 

 

 

 

Approximately 5 ml of blood was obtained by
intracardiac puncture from anesthetized rats. The blood
samples were collected in VacutainerR, nonadditive,
disposable tubes (165 x 16 mm) (Vacutainer Systems,
Rutherford, NJ), placed in a test tube rack that was stored
in reduced light and allowed to clot. The samples were
centrifuged and approximately 2 ml of serum was removed from
each tube and frozen at -200 C until vitamin A or thyroid

hormone determinations were performed.

Extraction gt Serum Samples for Vitamin A Analysis

 

 

Plasma extractions for vitamin A analysis were done
using the method of Bieri gt gt, (1979). Serum samples were

thawed at room temperature (approximately ZSOCD and were

91

extracted.as follows: in 200 ulcflfserunland 12;fl.of l3-
cis-ethylretinamide (CER) (5.07 ng/ul) standard (gift from
Dr. YJR Shealy, Southern Research Institute, Birmingham,
AL) were placed into a glass disposable tube (10 x 75 mm)
and nitrogenized. 1D The contents of the tube were mixed

using a Vortex-GenieTM

(Scientific Industries, Inc.,
Bohemia, NY) for 5 seconds and was set on ice for at least 5
minutes. I” Five hundred.ul of ethanol (plus 10 mg/500 ml
of butylated hydroxy toluene [BHT]) was added to this
mixture and the contents of the tube were nitrogenized and
vortexed for 5 seconds. 4)].nfl.of hexane was added to the
tube and this mixture was nitrogenized and vortexed for
exactly 1 minute using several start and stOp motions. 5)
The contents of the tube were centrifuged for 5 minutes in a
refrigerated centrifuge and by using a pasteur pipet the
hexane phase was removed and placed intona conical bottom
tube. 6) The hexane phase was dried with nitrogen. Steps
4-6 were repeated once.

Prior to sample analysis, the hexane phase was
reconstituted with 200 pl of acetylnitrile and 100 pl of
extracted serunl‘was directly injected onto a high

performance liquid chromatograph.

Collection and Extraction gt Livgt Agmples for Vitamin A
Analysis

 

 

Approximately 2 gcfifliver was collected from rats at
necropsy, placed in aluminum foil and frozen at —200 until

liver extractions for vitamin A analysis were performed.

 

 

92

Liver extractions were done using a modified method of Olson
(1979). Samples of 100 mg of liver and 500 mg of anhydrous
sodium sulfate were weighed using an electronic analytical
balance (Mettler AB 160, Mettler Instrument Corporation,
Highstown, NJ), ground together (mgrtar and pestle), and
packed into the bottom of a 5 ml flat-bottomed scintillation
vial. Next, 65 ul of 13-CER (0.507 ug/ul) standard was
injected into the contents of the vial followed by the
addition of 1 ml of chloroform. The contents of the vial
were nitrogenized and stored in a -200 C freezer for 8 to 12
hours. At the end of this time, 4 m1 of methanol was mixed
into the frozen contents of the vial and this mixture was
centrifuged. Either 50 ml or 100 pl of extracted liver
sample was directly injected onto a high performance liquid

chromatograph.

Vitamin A Analysis gt Liver gt Serum Samples

 

Serum or liver vitamin A concentrations were determined
by using high performance liquid chromatography. A Waters
590 Programmable Solvent Delivery Module (Waters
Chromatography Division, Milford, MA) with a flow rate of 2
ml/minute and a Waters 440 absorbance detector utilizing a
340 nm wavelength filter was used for vitamin A analysis of
liver and serum extracts. The column was a reverse phase
Partisil 10 ODS-2 (Whatman, Inc., Clifton, NJ).

The chromatographic method (Hf Cullum euui Zile
(unpublished) using a solvent switching system was used as

follows: 88% methanol and 12% water for 13 minutes (retinol

93

phase); 93% methanol and 7% water for 5 minutes (retinyl
acetate phase);anu185% methanolenu115% chloroform for 12
minutes (retinyl esters phase). Chromatographed peaks were
integrated using a Hewlett Packard 339A integrator (Hewlett

Packard Co., Avondale, PA).

§gtgg T3 ggg EA Analysis

Serum T3, T4, free (F)T3 and FT4 were determined by
solid phase radioimmunoassay methods (Beckers gt gl,, 1973;
Yoshida gt _t” 1980). Serum thyroid hormone extractions
and.analysis were done:h1the endocrinology laboratory of
the Animal Health Diagnostic Laboratory (AHDL), Michigan
State University, using a solid phase radioimmunoassay kit

(Immunodiagnostics, Becton. Dickinson enui Company,

Orangeburg, NY).

Statistical Analysis gt Data

 

Data were statistically analyzed by the one-way
analysis of variance (ANOVA) amd the Student—Newman-Keule

multiple comparison test at p<0.05 (Steel and Torrie, 1980).

Results

Liver Vitamin A

 

Results of analysis for liver retinol (ROH)‘and liver
retinyl esters HUD are given in Table 2-2. Rats fed 5 mg
of 3,4-TBB/kg had significantly decreased concentrations of
liver RE compared to rats fed 0.1 or 1.0 mg of 3,4-TBB/kg or

500 mg of PB/kg’diet. There was no significant difference

 

 

94

Table 2-2. Liver and serum retinol (ROH) and liver retinyl esters (RE)

concentrations in rats fed 3,4-TBB for 180 days.

 

Treatment group
and

 

concentration
of test chemical Liver ROH Liver RE Serum ROH
in feed (1119/ liver) (ug/ liver) (ng/ml)
3,4-TBB (0.1 mg/kg) 7415:1725 10708519882 262.7:105
3,4-TBB (1 mg/kg) 3535179.? 7764.7:966.1 27551845
3.441133 (5 mg/kg) 241.5325 2342331232335l 176.0:185
PE (500 mg/kg) 5685:1665 58915111675b 237518.12

 

Data are expressed as §+SD. N=3.

aSignificantly differ-e-nt (p<0.05) from group if of rats fed 0.1 or 1 mg

gf 3,4-TBB/kg diet or 500 mg PB/kg diet.

Significantly different (p<0.05) from group 2 of rats fed 0.1, l or 5

mg of 3,4-TBB/kg diet.

95

in liver concentrations of RE between groups of rats fed
diets containing 0.1 or 1.0 mg of 3,4-TBB/kg diet. Rats fed
500 1mg of PB/kg diet had significantly lower liver RE
concentrations than groups of rats fed<LJ.or 1 mg of 3,4-
TBB/kg diet. There was no significant difference in the
liver ROH concentration in groups of rats fed the four
treatment diets, however there appeared to be a dose
dependent decrease in liver ROH concentrations in groups of

rats fed 0.1,]”0,<1rELO mg of 3,4-TBB/kg diet.

Serum Vitamin A

 

Results of serum ROH determinations are given in Table
2-2. There wasIN)significant.difference:UIthe serum ROH
concentrations in rats fed 0.1, 1.0, or 5.0 mg of 3,4-TBB/kg

diet or 500 mg of PB/kg diet.

Serum Thyroid Hormones

 

Results of serum T3, T4, FT3 and FT4 determinations are
given in Table 2-3. Rats fed diets containing 0.1,]u0, or
5.0 mg of 3,4-TBB/kg had significantly decreased serum
concentrations of T4 compared to rats fed 500 mg of PB/kg
diet. The decrease in serum T4 levels appeared to be a dose
dependent response. Serum FT4 levels were also
significantly decreased in the rats given 3,4-TBB compared
to rats fed PB. There was no significant difference in
concentrations of serum T3 or serum FT3 in groups of rats

given the four diets.

96

Table 2—3. Serum triiodothyronine (T3), thyroxine (T4), free (F)T3 and
FT4 concentrations in rats fed 3,4-TBB for 180 days.

 

Treatment group

 

and
concentration of
test chemical Serum T4 Serum T3 FT4 FT3
in feed (mg/ml) (ng/ml) (pg/m1) (pg/m1)

3,4433 (0.1 mg/kg) 27.4:25 1.74:0.20 15.810.75 3.4_+_o.5
3,4-TBB (1 mg/kg) 24.9155 1.35:0.50 16.2135 2.710.94
3,4-1'33 (5 mg/kg) 20.7125 2.04:0.20 13713.0 3.910.411

1:3 (500 mg/kg) 37.710.43 1.50:0.30 24.112.3a 3.410.233

 

Data are expressed §:SD for 3 rats .
aSignificantly different (p<0.05) from group :7: of rats fed 0.1, l or 5
mg of 3,4-TBB/kg diet.

97

Discussion

 

Serum T4 and FT4 were significantly decreased in groups
of rats fed 0.1 mg, 1.0 mg or 5.0 mg of 3,4-TBB/kg, however
there were no histologic lesions observed in sections of
thyroid gland examined in rats from these groups. 3,4,5-
HBB, a toxic, slowly or non-metabolized congener of PBB, was
found to alter thyroid gland structure and function when fed
to rats at 1.0 or 10.0 ppm of the diet for 30 days (Akoso gt
gt., 1982b). 3,4,5-HBB is a 3-MC type microsomal enzyme
inducer capable of increasing hepatic AHH activity and UDP-
glucuronyl transferase (UDP-GT) activity (Aust g_ g},,
1981), and has been found to decrease serum T4
concentrations in rats (Akoso gt gt., 1982b). It is
suggested that induction of increased UDP-GT activity could
be related in part to increased glucuronidation of T4 and
increased biliary excretion of this thyroid hormone (Akoso
gt At” 1982a). 3,4-TBB is also a 3-MC type of microsomal
enzyme inducer and has been found to significantly increase
UDP—GT activity 1J1 rats (Millis, 1984). Increased
glucuronidation and increased biliary excretion of T4 could
in part be a mechanism for the decreased serum thyroid
hormone concentrations observed in rats fed 3,4-TBB in this
study.

Hepatic concentrations of RE were significantly
decreased in rats fed 5 mg of 3,4-TBB/kg as compared to rats

fed diets containing 0.1cnrlu0 mg of 3,4-TBB.or 500 mg of

PB/kg. 3,4,5-HBB and FM BP-6 were found to cause decreased

 

98

liver concentrations of retinyl palmitate with concomitant
increases in serunlluni concentrations vnuni administered to
rats in the diet for 140 days (Jensen, 1983). This effect
on vitamin A homeostasis was observed only in rats treated
with FM or a congener cable of 3-MC type microsomal enzyme
induction. The mechanism(s) whereby this occurs and the
significance of the level of vitamin A depletion associated
with administration of PBB is unknown. It has been
suggested that PHAH can decrease hepatic vitamin A levels as
a result of increased conjugation with UDP-GT with
subsequent increased biliary excretion (Thunberg gt gt.,
1980) or by PHAH induction of microsomal enzymes capable of
metabolizing vitamin A to inactive metabolites (Kato gt gt.,

1978).

Summary

Female Sprague-Dawley rats weighing 180-200 g were fed
diets containing 0.1, 1J)cm'5.0 mg of 3,4,3fi44-TBB (3,4-
TBB)/kg diet for 180 days to assess the effects of this
congener on serum and hepatic vitamin A homeostasis and
serum thyroid hormone concentrations. 3,4—TBB is a minor
component of FM BP-6, it is metabolized (t_ ytttg and tg
ytyg), binds to the TCDD receptor and induces increased AHH
activity. 3,4-TBB has a binding affinity for the TCDD 10
times greater tjuni that of 3,4,5fi?,4'fi?-HBB (3,4,5-HBB), a
toxic, nonmetabolized congener of polybrominated biphenyls

(PBB) not present in FM, but an oral equimolar dose of 3,4-

99

TBB is less toxic than 3,4,5—HBB as evidenced by histologic
changes in the liver and thymus of rats. The results of
this study indicate that chronic dietary administration of
3,4-TBB does alter hepatic vitandrllx homeostasis and serum
thyroxine (T4) and free T4 concentrations in rats. The
significance of these alterations and the exact biochemical

mechanism(s) whereby these changes occur are unknown.

CHAPTER III

ASSESSMENT OF THE EFFECTS OF
3,4,3',4'-TETRABROMOBIPHENYL (3,4-TBB)
ON HEPATIC GLUTATHIONE (GSH) CONCENTRATIONS

IN RATS

 

 

CHAPTER III

ASSESSMENT OF THE EFFECTS OF
3,4,3',4'-TETRABROMOBIPHENYL (3,4-TBB)
ON HEPATIC GLUTATHIONE (GSH) CONCENTRATIONS
IN RATS

Introduction

 

Congeners of polybrominated biphenyls (PBB) and
chemically related polyhalogenated aromatic hydrocarbons
(PHAH) produce a variety of toxic effects which include a
wasting syndrome, thymic atrophy, chloracne, edema,
hyperkeratosis, alterations in biochemical responses and
hepatotoxicity (Poland and Knutson, 1982). The mechanism of
toxicity of PHAH is unknown, although there is evidence that
toxicity is caused by congeners of PHAH that are inducers of
cytochrome P448 mediated microsomal enzymes, namely aryl
hydrocarbon hydroxylase (AHH) (Poland and Knutson, 1982).
Toxicity and P448 type of microsomal enzyme induction are
thought to be coordinated through a protein receptor, known
as the TCDD receptor, which is regulated by a single gene
locus referred to as the Ah locus (Greenlee and Poland,
1979; Poland and Glover, 1980; Poland gt gt., 1976). To
date, a definite cause and effect relationship between
hepatotoxicity observed with the administration of PHAH and
P448 type of microsomal enzyme induction has not been

established and alternate mechanisms, such as lipid

101

peroxidation, have been proposed for the hepatotoxic changes
observed.j111mammalian species exposed to congeners of PHAH

(Albro t 1., 1978; Sato gt 1., 1981; Sweeney gt al.,

1979).

The congener 3,4-TBB is ginninor component tentatively
identified in the commercial mixture of PBB, Firemaster (FM)
BP-6 (Robertson gt _t., 1982). This congener is a good
ligand for the TCDD receptor, induces P448 type microsomal
enzymes, in particular hepatic AHH activity (Millis gt gt.,
1985a; Mills gt gt., 1985), and is hepatotoxic in rats
(Millis gt gt., 1985a; Robertson gt gt., 1982). This
congener is also metabolized (tg ytttg and tg ytyg) (Millis
gt gt., 1985a; Mills gt gt., 1985). It has been established
that oxidative metabolism of many aromatic hydrocarbons
occur primarily through an arene oxide mechanism with
possible formation of reactive intermediates (i.e. epoxides)
vfiflxfl1 may spontaneously isomerize 1x) monohydroxylated
products, react with epoxide hydrolase to yield
dihydroxylated products or possibly conjugate with reduced
GSH (Jerina and Daly, 1974; Preston _t g__l_., 1983; Matthews,
1981). In the event of hepatic GSH depletion, a reactive
electrophile, such as an epoxide formed as an intermediate
during the metabolism of a congener, would no longer be
detoxified by conjugation with GSH and could extert its
toxic effects by initiating lipid peroxidation, or tn?
binding to nuclear, cytosolic or membrane macromolecules of

cells (Conney and Burns, 1972; Kohli gt gt., 1978; Miller

103

and Miller, 1977). It is therefore of interest to assess
whether the hepatotoxic effects of 3,4-TBB are solely
mediated by the TCDD receptor, or whether other factors such
as glutathione depletion also contribute to the histologic
changes observed in rats.

The objectives of this study were to assess the acute
effects of a single oral dose of 3,4-TBB on rat hepatic GSH
concentrations and to determine if induction of cytochrome
P448 type microsomal enzymes by pretreatment of rats with a
single oral dose of 3,4,5,3',4',5'-HBB (3,4,5-HBB) would
alter the effects of 3,4-TBB on hepatic GSH concentrations.
Histologic evaluation of liver sections was done to
characterize the acute changes associated with the
administration of 3,4-TBB at 2, 4, 8, 24, or 48 h after

dosing.
Materials and Methods

Experimental Design

 

Rats were divided in groups as shown in Table 3-1.
Rats were given a single oral dose of corn oil, 17 mg of
3,4-TBB/kg, or 1 mg of 3,4,5-HBB/kg; A:single oral dose of
3,4,5-HBB/kg was given as a control to determine the effects
of this congener when given alone and to compare these
effects to those observed in the combination treatment
groups. A combination treatment consisted of 1 mg of 3,4,5—

HBB/kg given 24lignjrn:to administration of 17 mg of 3,4-

TBB/kg; both congeners were given in a single oral dose.

104

 

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.Hscmnmnnosounmxmmn.m..3..m.m.4.m
.HHO CHOU mo as H :H maamuo cm>Hm mmB HMUHEmcU

Q

 

 

 

m
ma m m m m m mg + a mmsuv.m + enmmmum.4.m >H
ma m m m m m H nmmmlm.v.m HHH
ma m m m m m RH mmeu¢.m HH
ma m m m m m HE a Hflo cuou H

mumu Mn wv u: «N u: m H: v H: Amx\@E. x9 msouu
mo mmmoo

.oz mcflaaflx mo mafia cam mumu no “@2552

 

.mcoflumuucmocoo Ammo. mcoflnumusam Uwummmn so AmmaI¢4mv chmnmfinoeounmuumu

I.v..m.v.m mo muommum 02.... mo ucmsmmmmmm

How cmammc HousmEHummxm .Hnm manna.

105

Rats were killed at 2, 4, 8, 24 or 48 h after dosing with

3,4-TBB.

Chemicals

 

The congener 3,4-TBB was synthesized and purified by
recrystallization and alumina chromatography (Millis, 1984).
3,4,5-HBB was purchased from RFR Corporation, Hope, RI and
was purified by personnel in the Department of Biochemistry,
Michigan State University. The procedure used for
purification of 3,4,5-HBB consisted of repeated alumina

chromatography (Aust gt gl., 1981).

3311.5.

Sixty male Sprague Dawley rats (C/D) weighing 70-90 g
were obtained from Charles River Laboratories, Inc.,
Portage, 141. Rats were randomized and housed according to
groups in clear polypropylene cages,23rats per cage. All
rats were acclimated for 5 days. The room temperature was

maintained at 220 C with a 12 hr light/dark cycle.

Dose Preparation

 

Oral doses were prepared by adding appropriate amounts
of 3,4-TBB or 3,4,5-HBB to corn oil. Rats were given 1 ml
of corn oil containing 17 mg of 3,4—TBB/kg or 1 mg of 3,4,5-
HBB/kg by using a 16 gauge, 3" curved feeding needle

(Harvard Bioscience, South Natick, MA).

 

106

Necropsy Procedure
Rats were weighed and were killed by using dry ice
(C02). All organs were routinely examined for gross

lesions. The liver was removed from the carcass and

weighed.

Preparation gt Liver for Histologic Evaluation

 

 

Liver samples were fixed le 10% neutral buffered
formalin. Formalin-fixed liver samples were processed
(Fisher HistomaticTM Tissue Processor Models 165 and 166,
Fisher Scientific Co., Pittsburgh, PA), embedded in
paraffin, cut by a microtome into 6 m sections and stained

with hematoxylin and eosin for histologic examination.

Hepgtic Glutathione Determinations

Hepatic GSH levels were measured by the enzymatic
recycling assay of Griffith (1980) in which GSH is
sequentially oxidized by EL5'-dithiobis-(2-nitrobenzoic
acid) (DTNB) and reduced by NADPH in the presence of
glutathione reductase.

Liver samples were placed into cryotubes and
immediately frozen by submersion into isopentane cooled in
liquid nitrogen. Frozen liver samples were kept in a
freezer at -800 C until liver glutathione assays were
performed.

One gram of frozen liver sample was weighed using a

top-loading'balancee(Mettler Instrument Corp”,Highstown,

 

107

NJ), placed in a 50 ml polycarbonate centrifuge tube and set
on ice.

Liver samples were homogenized (Polytron, Brinkmann
Instruments, Westburyy‘NY)iJI4 volumes of 1% WLMN picric
acid and centrifuged at 9,000 x g for 15 minutes. The 9,000
x g supernatant equivalent to 0.2 to 0.4 g wet weight liver,
0.3 mM NADPH (B-NAD phosphate, reduced form, type III) 6 mM
DNTB and glutathione reductase-type III (from Baker's yeast)
were combined in a 1.5 ml disposable cuvette in a total
volume of 1 ml. Enzymes and substrates were purchased from
Sigma Chemical Co., St.‘Louis, MO. The change in absorbance
over 2 optical density units at 412 nm at room temperature
was monitored using a spectrophotometer (Beckman Model 35,
Series 30 UV—Visable, Beckman Instruments, Inc., Fullerton,
CA). The glutathione content of the aliquot assayed was
determined by comparison of the change in adsorbance over
the linear part of the curve to the change in adsorbance

measured with known amounts of glutathione on the same day.

Statistical Analysis

 

Data from the glutathione assays were statistically
analyzed by using the factorial analysis of variance
(ANOVA). Organ and body weight data were statistically
analyzed by using the one-way ANOVA and the Student-Newman-
Keul's multiple comparison test at p<0.05 (Steel and Torrie,

1980).

108

Results

Hepatic GSH

 

Results for analysis of hepatic GSH levels are given in
Table 3-2. Hepatic GSH concentrations in rats treated with
3,4-TBB, 3,4,5-I-IBB or a combination of both congeners were
not significantly different from control GSH concentrations

at 2, 4,EL 24 or 4811after dosing.

Histopathology

 

Liver sections taken from rats given a single oral dose
of 17 mg of 3,4-TBB/kg, 1 rm; of 3,4,5eHBB/kg or a
combination of both congeners and killed at 2, 4, or 8 h
after dosing did notcdiffer from controls. iLiver sections
from rats treated with a combination of 3,4-TBB and of
3,4,5-HBB and killed at 24cn:48 h.after dosing had mild to
moderate intracytoplasmic vacuolation of hepatocytes in the
periportal regions. Rats treated with 3,4-TBB and killed at
24 h had similar histologic changes, however they were less
severe than changes observed in rats given the combination
treatment and killed at 24 (n: 48 hr after dosing.
Histologic changes observed in liver sections from rats
treated with 1 mg of 3,4,5-HBB and killed at 24 or 48 h
after dosing consisted of diffuse areas of mild to moderate

hepatocellular vacuolation.

Body and Liver Weights

 

 

Data for body and liver weights are given in Table 3-3.

Rats given ea single oral chum; of 3,4,5-HBB before

109

Table 3-2. Hepatic GSH concentrations in rats given a single oral dose
of 3,4-TBB, 3,4,5,3',4',5'-hexabromobiphenyl (3,4,5-HBB), or
a combination of both congeners.

 

Hepatic glutathione
Kil 1 time concentration

 

Tx (h) (nmol/g of liver)

Corn oil 2 2.901056
3,4-TBB 2 2.93:9.25
3,4,5-333 2 3.30:9.17
3,4,5-3333 + 3,4-TBB 2 2.92:9.45
Corn 011 4 2.13:0.80
3,4,5-333 4 2.20:9.50
3,4,5-333a + 3,4-TBB 4 2.18:9.29
Corn oil 8 1.67_+_0.40
3,4-TBB 8 2.00:0.26
3,4,5-333 8 2.33:9.50
3,4,5-333a + 3,4-TBB 8 1-9719-40
Corn oil 24 3.13:0.83
3,4-TBB 24 2-4Q:0-20
3,4,5-333 24 2.20:9.35
3,4,5-333a + 3,4-TBB 24 2.73:1.30
Corn oil 48 2.90:0.60
3,4-TBB 48 2.63:0.83
3,4,5-333 48 2.27:0.60
3,4,5-HBBa + 3,4-TBB 48 2.52:9.29

 

Data are expressed as group 3; and SD for 3 rats.

All rats were given either a single oral dose of 1 ml of corn oil, 17 mg
3,4-TBB/kg, 1 mg of 3,4,5-HBB/kg or a combination of 1 mg of 3,4,5-
HBB/kg and 17 mg 3,4-TBB/kg in 1 ml of corn oil.

33,4,5-HBB was given 24 h before administration of 3,4-TBB.

110

 

 

Table 3-3. Body and liver weights in rats given a single oral dose of
3,4-TBB, 3,4,5-HBB, or a combination of both congeners.
Absolute Absolute
Kill time body liver

Tx (h) weight (9) weight (9)

Corn oil 2 194.011.0 9.8111.21
3,4-TBB 2 189.014.4 10.6010.86
3,4,5-333 2 194-718-3 9.9411.03
3,4,5-333a + 3,4-TBB 2 185.619.0 9.54:9.37
Corn oil 4 197.7116.3 8.9010.71
3,4-TBB 4 184.3113.8 8.8811.06
3,4,5-333 4 187.316.5 8.8010.52
3,4,5-333a + 3,4-TBB 4 182.712.5 9.6710.99
Corn oil 8 183.7165 8.931054
3,4-TBB 8 184.3118.9 9.7911.03
3,4,S-HBB 8 182.7110.1 9.461050
3,4,5-3338 + 3,4-TBB 8 165.7118.2 8.8311.18
Corn oil 24 174.811o.4 10.2219.35

3,4-TBB 24 188.7110.4 10.6610.28b
3,4és-333 24 194.817.o 9.7119.34

3,4,5—333 + 3,4-TBB 24 182.719.0 10.8510.48b
Corn oil 48 208.718.1 11-0910-39
3,4-TBB 48 196.7116.8 11.2811.72
3,4,5-333 48 199.317.6 11.0910.81
3,4,5-333? + 3,4-TEB 48 199.018.7 12.5411.50

 

 

Data are expressed as group x and SD for 3 rats.

All rats were given either a single oral dose of 1 ml of corn oil, 17 mg
3,4-TBB/kg, 1 mg of 3,4,5-HBB/kg or a combination of 1 mg of 3,4,5-
HBB/kg and 17 mg 3,4-TBB/kg in 1 ml corn oil.

a3,4,5-I-IBB was given 24 h before administration of 3,4—TBB.
bSignificantly different (p<0.05) from groups of rats given 3,4,5-HBB
and killed at 24 h.

111

administration of a single oral dose of 3,4-TBB or rats
given a single oral dose of 3,4-TBB alone and killed at 24 h
had significantly increased liver weights compared to rats
given a single oral dose of 3,4,5-HBB alone and killed at 24
h. There were no significant differences in body weights
between rats killed at 24 h. Body and liver weights did not

differ significantly between rats killed at 2, 4, 8 or 48 h.

112

Discussion

 

Results from this study indicate that 3,4-TBB when
administered to rats in a single oral dose (17 mg/kg) had no
effect on hepatic GSH concentrations compared to controls at
2, 4,5L 24 or 4811after dosing. Liver'sections from rats
given 3,4-TBB and killed 24 h after dosing had mild
intracytoplasmic vacuolation and hypertrophy of hepatocytes
in the periportal regions, but at 2, 4, 8 or 48 h after
dosing there were no differences in liver sections from rats
given 3,4-TBB compared to controls. Therefore, depletion of
hepatic GSH did not appear to be a factor in causing the
histologic changes observed in rats given 3,4—TBB alone, and
killed at 24 h after dosing.

Dannan gt g1. (1982c) found congeners of PBB to be good
substrates for inducing hepatic microsomal enzymes. This is
important 1J1 that induction (ME the hepatic microsomal
monooxygenase system (MMS) by nonmetabolizable congeners may
increase the rate of metabolism of metabolizable congeners.
3,4,5-HBB, a slowly or nonmetabolizable congener of PBB
capable of P448 type microsomal enzyme induction, has been
found to increase the rate of metabolism of 3,4-TBB 1g y1ttg
(Mills g_ g1., 1985). If metabolizable congeners of PBB,
such as 3,4-TBB, are indeed metabolized to epoxide
intermediates by the hepatic MMS, then induction of
microsomal enzymes by pretreatment of rats with a
nonmetabolizable congener such as 3,4,5-HBB could potentiate

the toxic effects of congeners metabolized by P448 type

113

microsomal enzymes. Alternately, if the parent compound is
important in receptor binding and that in turn mediates
toxicity (Greenland and Poland, 1979; Poland and Glover,
1980; Poland gt g1., 1976), then increased rates of
metabolism would decrease the toxicity of metabolizable
congeners. In this study, pretreatment of rats with a
single oral dose (1 mg/kg) of 3,4,5-HBB 24 h before oral
administration of 3,4-TBB (17 mg/kg), did not potentiate the
effects of 3,4-TBB on hepatic GSH concentrations compared to
controls at 2, 4, 8, 24 or 48 h after dosing.

It is suggested that enzyme induction is an early event
and persistent occupation of the TCDD receptor by the ligand
is necessary for gene expression and subsequent toxicity
(Poland and Knutson, 1982). The congener 3,4-TBB induces
its own metabolism and is rapidly removed from the liver of
rats (Millis gt g1., 1985a; Mills gt g1., 1985), and if
persistent ligand-receptor binding is necessary for
toxicity, then single doses of 3,4-TBB should not be
severely hepatotoxic. Histologic examination of liver
sections from rats given 3,4-TBB alone and killed at 24 h
suggest that 3,4-TBB is not extremely hepatotoxic and that
the mild acute changes observedenz24 h were reversible as
evidenced by lack of histologic changes in rats given 3,4-
TBB and killed at 48 h compared to controls. It is
suggested that metabolism of congeners such as 3,4-TBB

decreases intrahepatic concentrations of the parent compound

 

 

 

114

resulting in decreased ligand-receptor binding and hence,
decreased toxicity.

The congener 3,4,5-HBB is run: metabolized and
accumulates in the liver of rats (Jensen gt _1., 1982;
Millis gt g1., 1985a; Mills gt g1., 1985). Again, if
toxicity associated with PHAH occurs as a result of
persistent ligand-receptor binding and AHH activity, then
the histologic changes in livers of rats fed the combination
treatment and killed at 24 h‘were primarily dueix>3q4,5-
HBB, and at 48 h can be solely attributed to the effects of
3,4,5-HBB due to rapid metabolism of 3,4-TBB by this time
period. Changes observed in rats given 3,4,5-HBB and killed
at 24 or 48 h after dosing were similar to those seen in the
combination group at 24 or 48 h, but were less severe.
Increased severity of hepatic lesions in the combination
group>is most likely duetx>the increased time of exposure
to 3,4,5-HBB. Rats in this group were given 3,4,5-HBB 24 h
prior to administration of 3,4-TBB, so that histologic
hepatic changes observed atlfllor 4811were representative
of 48 or 72 h, respectively.

In conclusion, the results from this study indicate
that hepatotoxicity associated with 3,4—TBB is not due to
depletion of hepatic GSH concentrations. Similar findings
have been reported for the chlorinated analog (3,4,3244-
tetrachlorobiphenyl) of 3,4-TBB (Rifkind gt g1., 1984).
These findings suggest that 3,4-TBB toxicity is most

probably coordinated with induction of P448 type microsomal

 

115

enzymes, namely AHH which is thought to be mediated by the
TCDD receptor (Poland and Glover, 1977, 1980). Results from
this study also indicate that it is important to consider
the manner of administration.of a metabolizable PHAH) the
number of observations after the compound is given, and also
at what time periods the observations are made. In this
study, histologic changes associated with the administration
of 3,4-TBB occurred by 24 h, and by 48 h rats treated with

3,4—TBB were similar to controls.

Summary

Male Sprague-Dawley rats weighing 160-180 g were given
a single oral dose of 17 mg of 3,4,3',4'-TBB (3,4-TBB)/kg, 1
mg of 3,4,5,3',4',5'-HBB (3,4,5-HBB)/kg or a combination of
both congeners to assess the effect of 3,4-TBB on hepatic
glutathione (GSH) concentrations and to determine if
induction of cytochrome P448 type microsomal enzymes by
pretreatment of rats with a single oral dose of 3,4,5-HBB
would alter the effects of 3,4-TBB on hepatic GSH
concentrations. Histologic evaluation of liver sections was
done to characterize the acute changes associated with the
administration of 3,4-TBB at 2, 4, 8, 24 or 48 h after
dosing. The congener 3,4-TBB when given to rats alone or 24
h after administration of 3,4,5-HBB had no effect of hepatic
GSH concentrations at 2, 4, 8, 24 or 48 h after dosing
compared to controls. Histologic changes were observed only

at 2411iJ1rats given 3,4-TBB alone and consisted of mild,

 

116

focal areas of hepatocellular hypertrophy and
intracytoplasmic vacuolation. These changes were absent in
rats given 3,4-TBB and killed at 481L In conclusion, GSH
depletion does not appear to be significant in the
hepatotoxicity observed with 3,4-TBB. The acute histologic
changes associated with the administration of a single oral
dose of 17 rm; of 3,4-TBB/kg appears to be rapidly

reversible.

CONCLUSIONS

The results from the research presented in this
dissertation indicate that:

JJ The metabolizable congener, 3,4,3H4'-
tetrabromobiphenyl (3,4-TBB), when administered to rats in
the diet for 180 days acts as a promoter in Pitotfis model of
experimental hepatocarcinogenesis. as levidenced by
enhancement of the number of gamma-glutamyl transpeptidase
(GGT) positive enzyme altered foci (EAF) after initiation
with diethylnitrosamine (DEN).

2) Dietary levels of S rm; of 3,4-TBB/kg are more
effective than 500 mg of phenobarbital (PB)/kg (standard
tumor promoter) in enhancing the number of hepatic GGT
positive EAF.

3) 3,4-TBB appearstx>have weak initiating potential
in Pitotfs model of experimental hepatocarcinogenesis.

4) Single oral doses of 1, 5 or 10 mg of 3,4-TBB are
less effective than 10 rm; of DEN (standard tumor
initiator)/kg given 14> in initiating the formation of
hepatic GGT positive EAF.

5) Dietary administration of 0.1, 1 or 5 mg of 3,4-

TBB/kg for 180 days is not severely toxic in rats as

117

 

118
evidenced by histologic and ultrastructural changes in the
liver.

6) Chronic dietary administration of 3,4-TBB does not
cause toxic changes intflmathyroid.gland, spleen or thymus
in rats as evidenced by lack of histologic changes in these
organs.

7) 3,4-TBB does not accumulate in the liver or adipose
tissue of rats when administered in the diet for 180 days.

8) Dietary administration of 3,4-TBB does not cause
significant changes in liver, thymic, splenic or thyroid
gland weights in rats.

9) Dietary administration of 3,4-TBB/kg for 180 days
causes decreased liver retinyl esters concentrationsin
rats, but has no effect on serum retinol concentrations.

10) Dietary administration 3,4-TBB for 180 days causes
significantly decreased serum thyroxine (T4) and free T4
concentrations in rats.

11) A single oral dose of 17 mg of 3,4-TBB/kg does not
cause alterations in hepatic reduced glutathione (GSH)
concentrations in rats, and GSH depletion does not appear to
play a significant role in 3,4-TBB hepatotoxicosis.

12) Pretreatment of rats with a single oral dose of 1
mg of 3,4,5,3HMV,5'-hexabromobiphenyl (3,4,5-HBB)/kg 24 h
before a single oral dose of 17 mg of 3,4-TBB/kg does not
potentiate the effects of 3,4-TBB on hepatic GSH

concentrations in rats.

 

119
13) 3,4-TBB causes mild hepatic lesions that appear to
be rapidly reversible when administered to rats in a single

oral dose.

 

LI ST OF REFERENCES

 

LI ST OF REFERENCES

Akoso, B.T., Sleight, S.D., Aust, S.D., and Stowe, H.D.
(1982a). Pathologic effects of purified polybrominated
biphenyl congeners in rats. iAm. Coll. Toxicol. 1, 1-
21.

 

Akoso, B.T., Sleight, S.D., Nachreiner, R.F., and Aust, S.D.
(1982b). Effects of purified polybrominated biphenyl
congeners on the thyroid and pituitary glands in rats.
it Am. Coll. Toxicol. 1, 23-36.

 

Albert, Z., Orlowski, M., and Szewczuk, A. (1961).
Ifistochemical demonstration of gamma-glutamyl
transpeptidase. Nature 191, 767-768.

Allen, J.R., Lambrecht, I~K., Barsotti, D.A. (1978).
Effects of polybrominated biphenyls in non-human
primates. g1 Am. Vet. Med. Assoc. 173, 1485-1489.

 

Alvares, A.P., Schilling, G., Levin, w., and Kuntzman, R.
(1967). Studies on the induction of CO-binding pigments
in liver’ microsomes kn! phenobarbital and 21-
methylcholanthrene. Biochem. Biophys. Res. Commun. 29,
521-526.

 

Andres, J., Lambert, L., Robertson, L., Bandiera, S.,
Sawyer, T., Lovering, S., and Safe, S. (1983). The
comparative biologic enui toxic potencies of
polychlorinated and polybrominated biphenyls. Toxicol.

 

 

Appl. Pharmacol. 70, 204-215.

 

Aust, S.D., Dannan, G.A., Sleight, S.D., Fraker, P.J.,
Ringer, RJK., and Polin, D. (1981). Toxicology of
polybrominated biphenyls. In Toxicity gt Halogenated
Hydrocarbons: Health and Ecological Effects (M.AJL
Khan, R.H. Stanton, eds.). Pergamon Press, New York.

 

 

 

 

Baars, AWJ., Jansen, M., and Breimer, D.D. (1978). The
influence of phenobarbital, 3-methylcholanthrene and
2,3,7,8—tetrachlorodibenzo-p-dioxin on glutathione S-
transferase activity of rat liver cytosol. Biochem.
Pharmacol. 27, 2487-2494.

 

120

121

Baic, D., and Baic, B. (1984). A fast method for
processing biOpsy material for electron microscopy.
Ultrastructural Pathol. 6, 347-349.

Beckers, C., Corrette, C., and Thalasso, M. (1973).

Evaluation of serum thyroxine by radioimmunoassay. .11

Nucl. Med. 14, 317-320.

Bekesi, J3G., Helland, CLF., Anderson, ILA.. Fischbein,
AnS., Rona, w., Wolff, M.S.,1and Selikoff, I.J. (1978).
Lymphocytic function of Michigan farmers exposed to
polybrominated biphenyls. Science 199, 1208-1209.

Berenblum, I. (1944). Irritation anui carcinogenesis.
Arch. Pathol. 38, 233-244.

 

Berenblum, I”,and Shubik, P. (1947). Alnew quantitative
approach to the study of the stages of chemical
carcinogenesis in mouse skin. Br. g1 Cancer 1, 383-390.

Besaw, L.C., Moore, R.W., Dannan, G.A., and Aust, S.D.
(1978). Effect of 2,2',3,3',4,4',5,5'-octabromobiphenyl
on microsomal drug metabolism enzymes. Pharmacologist
20, 251.

Bieri, J3G., Tolliver, T.J., and Catiynani, GuL. (1979).
Simultaneous determination of a-tocopherol and retinol in
plasma cm: red cells kur high pressure liquid
chromatography. Ag; g; Clin. Nutri. 32, 2143-2149.

 

Bishop, J;M. (1982). Oncogenes. Scientific American 246,
80-90.

 

Bock, R.W., Frbhling, w., Hemmer, H., and Rexer, B. (1973).
Effects of phenobarbital and 3-methylcholanthrene on
substrate specificity of rat liver microsomal UDP-
glucuronyltransferase. Biochim.Biophys.Acta 327,46-
56.

 

Booth, J., Boyland, E., and Sims, P. (1961). An enzyme
from rat liver catalyzing conjugations with glutathione.
Biochem. _J_._ 79, 516-523.

 

Boutwell, RJK. (1974). The function and mechanism of
promoters of carcinogenesis. CRC Crit. Rev. Toxicol. 2,
419-443.

 

Bresnick, E., Mukhtar, H., Stoming, T.A., Dansette, P.M.,
and Jerina, ELM. (1977). Effect of phenobarbital and 3-
methylcholanthrene administration on epoxide hydrase
levels in liver microsomes. Biochem. Pharmacol. 26, 891-
892.

 

 

122

Brinkman, U.A.Th., and deKok, A. (1980). Production,
properties, and usage. In Halogenated Biphenyls,
Terphenyls, Naphthalenes, Dibenzodioxins, and Related
Products (R.D. Kimbrough, ed.). Pp. 1-40, Elsevier/North
Holland, New York.

Carter, LJL (1976). Michiganfis PBB incident: Chemical
mix-up leads to disaster. Science 192, 240-243.

Cohen, (Lu and Hochstein, P. (1963). Glutathione

peroxidase: The primary agent for the elimination of
hydrogen peroxide in erythrocytes. Biochem. 2, 1420-

1428.

 

Conney, ANH. (1967). Pharmacological implications of

microsomal enzyme induction. Pharmacol. Rev. 19, 317-
366.

 

Conney, A.H., and Burns, J.J. (1972). Metabolic

interactions among environmental chemicals and drugs.

Cook, R.M., Prewitt, L.R., and Fries, G.F. (1978). Effects
of activated carbon, phenobarbital, and vitamin A, D and

E on polybrominated biphenyl excretion in cows. g; Dairy
Sci. 61, 414-419.

Coopery ELY., Levin, S., Narashimhulu, S., Rosenthal, 0.,
and Estabrook, RJL. (1965). Photochemical action
spectrum of the terminal oxidase of mixed-function
oxidase systems. Science 147, 400-402.

Cooper, G.M., and Lane, M.-A. (1984). Cellular
transforming genes and oncogenesis. Biochim. Biophys.

 

Culliton, BJL (1977). ‘Widespread PBB contamination can
affect immune system. Science 197, 849.

Dannan, G.A., Guengerich, F.P., Kaminsky, IuS., and Aust,
S.D. (1983). Regulation of cytochrome P-450
immunological quantitation of eight isozymes in liver
microsomes of rats treated with polybrominated biphenyl
congeners. g1 Biol. Chem. 258, 1282-1288.

 

Dannan, G.A., Mileski, G.J., and Aust, S.D. (1982a).
Reconstitution of some biochemical and toxicological
effects of commercial mixtures of polybrominated
biphenyls. Fund. Appl. Toxicol. 2, 322-326.

 

Dannan, G.A., Mileski, G.J., and Aust, S.D. (1982b).
Purification of polybrominated biphenyl congeners. g1
Toxicol. Environ. Health 9, 423-438.

 

 

123

Dannan, G.A., Moore, R.W., and Aust, S.D. (1978a). Studies
on the microsomal metabolism and binding of
polybrominated biphenyls (PBB's). Environ. Health
Perspect. 37, 179-182.

 

 

Dannan, G.A., Moore, R.W., Besaw, L.C., and Aust, S.D.
(1978b). 2,4,5,3',4',5'-Hexabromobiphenyl is both a 3-
methylcholanthrene- and a phenobarbital-type inducer of

microsomal drug metabolizing enzymes. Biochem. BiOphys.
Res. Comm. 85, 450-458.

 

Dannan, G.A., Sleight, S.D., and Aust, S.D. (1982c).
Toxicity and microsomal enzyme induction effects of
several polybrominated biphenyls of Firemaster. Fund.
Appl. Toxicol. 2, 313-321.

Dannan, G.A., Sleight, S.D., Fraker, P.J., Krehbiel, J.D.,
and Aust, S.D. (l982d). Liver microsomal enzyme
induction and toxicity studies with 2,4,5,3',4'-
pentabromobiphenyl. Toxicol.]fiuflu Pharmacol.64, 187-
203.

 

Dent, CLG., Graichen, M.E”, Schnell, S., and Lasker, J.
(1980). Constitutive and induced hepatic microsomal
cytochrome P-450 monooxygenase activities in male
Fischer-344 and CD rats. A comparative study. Toxicol.
Appl. Pharmacol. 52, 45-53.

 

Dent, J.G., Netter, K.J., and Gibson, J.E. (1976a). Effect
of chronic administration of polybrominated biphenyls on
parameters associated with hepatic drug metabolism. Res.
Commun. Chem. Pathol. Pharmacol. 13, 75-82.

 

Dent, J.G., Netter, K.J., and Gibson, JZE. (1976b). The
induction of hepatic microsomal metabolism in rats
following acute administration of a: mixture of
polybrominated biphenyls. Toxicol. Appl. Pharmacol. 38,
237-249.

 

 

Dunckel, AuE. (1975). {An updating on the polybrominated
biphenyl disaster in Michigan. gL_Ahu Vet. Med. Assoc.
167, 838-841.

 

Farber, E.. (1981). Chemical carcinogenesis. New Engl. g1
Med. 305, 1379-1389.

 

Farber, T”.Kasza, L”,Giovetti,.A”,Carter,(L, Earl,FhL.,
and Balazs, T. (1978). Effect of polybrominated
biphenyls (Firemaster BP-6) on the immunologic system of
the beagle dog. Toxicol. Appl. Pharmacol. 45, 343.

 

Fiala, S., and Fiala, A.E. (1970). Aquisition of an
embryonal biochemical feature tar rat hepatomas.
Experientia 26, 889-890.

 

124

Fiala, S., and Fiala, E5. (1973). Activation by chemical
carc1nogens of gamma-glutamyl transpeptidase in rat and
mouse liver. g1 Natl. Cancer Inst. 51, 151-158.

Fraker, PJL, (1980). Antibody mediated and delayed type
hypersenSitivity responses in mice to polybrominated
biphenyls. Toxicol. Appl. Pharmacol. 43, 1-7.

 

Fries, G.F. (1978). Distribution and kinetics of
polybrominated biphenyls and selected chlorinated
hydrocarbons in farm animals. g1 Am. Vet. Med. Assoc.
173, 1479-1484.

 

Garfinkel, D. (1957). Isolation and prOperties of

c tochrome b5 from pig liver. Arch. Biochem. BiOphys.
7 , 111-120.

 

Goldbarg, J.A., Friedman, G.M., Pineda, ELP., Smith, E.E.,
Chatterji, Tu, Stein,IEJL4 and Rutenberg, A.M. (1960).
The colorimetric determination of gamma-glutamyl
transpeptidase with a synthetic substrate. Atggt
Biochem. Biophys. 91, 61-70.

 

Greenlee, W.F., and Poland, A. (1979). Nuclear uptake of
2,3fin8-tetrachlorodibenzo-p-dioxin by hepatic cytosol.
g1 Biol. Chem. 254, 9814-9821.

 

Griffith, OJm. (1980). Determination of glutathione and
glutathione disulfide using glutathione reductase and 2-
vinylpyridine. Anal. Biochem. 106, 207-212.

 

Guengerich, FQP. (1977). Separation and purification of
multiple forms of microsomal cytochrome P-450. g; Biol.
Chem. 252, 3970-3979.

Guengerich, F.P., Wang, P., Mason, P.S., and Mitchell, M.B.
(1981). Immunological comparison of rat, rabbit and
human microsomal cytochrome P-450. Biochem.20, 2370-
2378.

 

Gupta, B.N., McConnell, E.E., Goldstein, J.A., Harris, M.W.,
and Moore, JxA. (1983a). Effects of a polybrominated
biphenyl mixture in the rat and mouse. I. Six-month
exposure. Toxicol. Appl. Pharmacol. 68, 1-18.

 

Gupta, B.N., McConnell, E.E., Moore, J.A., and Haseman, J.K.
(1983b). Effects of a polybrominated biphenyl mixture in
the rat and mouse. II. Lifetime study. Toxicol. Appl.
Pharmacol. 68, 19-35.

 

 

Gupta, BQN”, and Moore, CLA. (1979). Toxicologic

assessments of a commercial polybrominated biphenyl
mixture in the rat. Am. g1 Vet. Res. 40, 1458-1468.

 

 

125

Hanigan, M.H., and Pitot, 11.C. (1985). Gamma- -glutamyl

transpeptidase - its role in hepatocarcinogenesis.
Carc1nogenesis 6, 186- -202.

Hansell, M.M., and Ecobichon, [LJ. (1974). Effects of

chemically pure chlorobiphenyls on morphology of rat
liver. Toxicol. Appl. Pharmacol. 28, 418-427.

Hemminki, K. (1983). Nucleic acid adducts of chemical
carcinogens and mutagens. Arch. Toxicol. 52, 249-285.

Higgins, G.M., and Anderson, R.M. (1931). Experimental
pathology of the liver: Restoration of the liver of the

white rat following partial surgical removal. Arch.
Pathol. 12, 186- 202.

Howard, S.K., Werner, P.R., and Sleight, S.D. (1980). PBB
toxicosis in swine. Effects on some aspects of the
immune system in lactating sows and their offspring.
Toxicol. Appl. Pharmacol. 55, 146-153.

Ingelman-Sundberg, M. (1980). Bioactivation or
inactivation of toxic compounds? ttgggg _i_g
Pharmaceutical Sciences 1, 176-179.

Institute of Laboratory Animal Resources, National Research
Council, National Academy (ME Sciences. (1980).
Histologic typing of liver tumors of the rat. g1 Natl.
Cancer Inst. 64, 178-190.

 

Jacobs, L.W., Chou, S.-F., and Tiedje, J.M. (1976). Fate
of polybrominated biphenyls (PBB's) in soils.
Persistence and plant uptake. g_._ Agric. Food Chem. 24,
1198-1201.

 

Jensen, ELK. (1983). Pathologic effects and hepatic tumor
promoting ability of Firemaster BP-6, 3,3',4,4',5,5'-
hexabromobiphenyl and 2,2',4,4',5,5'-hexabromobiphenyl in
the rat. Ph.D. Dissertation, Michigan State University.

Jensen, R.K., Sleight, S.D., and Aust, S.D. (1983a).
Effect of varying the length of exposure to
polybrominated biphenyls in the development of gamma-
glutamyl transpeptidase enzyme-altered foci.
Carcinogenesis 5, 63-66.

 

Jensen, R.K., Sleight, S.D., Aust, S.D., Goodman, J.I., and
Trosko,.JJL (1983bL. Hepatic tumor-promoting ability
of 3,3',4,4H5,5'-hexabromobiphenyl: The
interrelationship between toxicity, induction of hepatic
microsomal drug metabolizing enzymes, and tumor-promoting
ability. Toxicol. Appl. Pharmacol. 71, 163-176.

 

 

126

Jensen, R.K., Sleight, S.D., Goodman, J.I., Aust, S.D., and
Trosko, J.E. (1982). Polybrominated biphenyls as
promoters 5J1 experimental hepatocarcinogenesis :hu rats.
Carcinogenesis 3, 1183-1186.

Jerina, D.M., and Daly, J.W. (1974). Arene oxides: A new
aspect of drug metabolisnu Science 185, 573-582.

Kalengayi, M.M.RH, Ronchi, G., and Desmet, VkJ. (1975).
Histochemistry of<ymnma-glutamyl transpeptidase in rat
liver during aflatoxin Bl-induced carcinogenesis. g;
Natl. Cancer Inst. 55, 579-588.

Kamataki, T., Maeda, K., Yamzoe, Y., Nagai, T., and Kato, R.
(1983). Sex difference of cytochrome P-450 in the rat:
Purification, characterization, anui quantitation of
constitutive forms of cytochrome P-450 from liver
microsomes of male and female rats. Arch. Biochem.

 

Biophys. 225, 758-770.

 

Kaplowitz, N., Kuhlenkamp, J., and Clifton, G. (1975).
Drug induction of hepatic glutathione S-transferase in
male and female rats. Biochem. g1 146, 351-356.

 

Kappas, A., and Alvares, Adm (1975). How the liver
metabolizes foreign substances. Sci. Amer. 232, 22-31.

 

Karnovsky, M.J. (1965). A formaldehyde-glutaraldehyde
fixative of high osmolality for use in electron
microscopy. Abstr. g1 Cell Biol. 27, 137A-138A.

 

Kato, N., Kato, M., Kimura, T., and Yoshida, A. (1978).
Effect of dietary addition of PCB, [NHL or BHT and
dietary protein on vitamin A and cholesterol metabolism.
Nutr. Report Int. 18, 437-445.

 

Kato, N., Kawai, K., and Yoshida, A. (1981). Effects of
dietary level of ascorbic acid on the growth, hepatic
lipid peroxidation, and serum lipids in guinea pigs fed
polychlorinated biphenyls. g1 Nutr. 111, 1727-1733.

Kato, S., McKinney, J3D., and Matthews, H.B. (1980).
Metabolism of symmetrical hexachlorobiphenyl isomers in
the rat. Toxicol. Appl. Pharmacol. 53, 389-398.

 

Kavanagh, T.J., Rubinstein, C., Liu, P., Chang, C.-C.,
Trosko, CLE., and Sleight, S.D. (1985). Failure to
induce mutations in Chinese hamster V79 cells and WB rat
liver cells by the polybrominated biphenyls Firemaster
BP-6, 2,2',4,4',5,5'-hexabromobiphenyl, 3,3',4,4',5,5'-
hexabromobiphenyl, and 3,3',4,4'-tetrabromobiphenyl.
Toxicol. Appl. Pharmacol. 79, 91-98.

 

127

Kay, K. (1977). Polybrominated biphenyls (PBB)
environmental contamination 111 Michigan, 1973-1976.
Environ. Res. 13, 74-93.

Ketterer, E., Meyer, D., Taylor, J., and Burchell, B.
(1984). Glutathione transferases and their role in
xenobiotic metabolism. Trends in Pharmaceutical Sciences
5, 319-320. ' —

Kimbrough, R.D., Groce, D.F., Korver, M.P., and Burse, V.W.
(1981). Induction of liver tumors in female Sherman rats
by polybrominated biphenyls. g1 Natl. Canc. Inst. 66,
535-542.

 

Kimbrough, R.D., Korver, M.P., Burse, V.W., and Groce, D.F.
(1980). The effect of different diets or mineral oil on
liver pathology and polybrominated biphenyl concentration
in tissues. Toxicol. Appl. Pharmacol. 52, 442-453.

 

Klingenberg, M. (1958). Pigments of rat liver microsomes.
Arch. Biochem. Biophys. 75, 376-386.

 

Kohli, J., Wyndham, C., Smylie, M., and Safe, S. (1978).
Metabolism of bromobiphenyls. Biochem. Pharmacol. 27,
1245-1249.

 

Kosower,‘NJ&N and Kosower, E.M. (1978). The glutathione
status of cells. Jinn Rev. Cytol. 54, 109-160.

 

Lawrence, R.A., and Burk, R.F. (1976). Glutathione
peroxidase activity in selenium deficient rat liver.
Biochem. Biophys. Res. Commun. 71, 952-958.

 

Leonard, 138., Dent, J3G., Graichen, M.B., Lyght, 0., and
Popp, JxA. (1982). Comparison of hepatic carcinogen
initiation-promotion systems. Carcinogenesis 3, 851-856.

 

Lu, Aszhq Junk, K.W., and Coon, M.J. (1969). Resolution
of the cytochrome P-450 containing w-hydroxylation system
of liver microsomes. lg; Biol. Chem. 244, 3714-3721.

 

Lu, A.Y.H., and West, S.B. (1978). Reconstituted mammalian
mixed-function oxidases: Requirements, specificities and
other properties. Pharmacol. Ther. Part A 2, 337-358.

 

Lu,1LY.H”. and West, S.B. (1980). Multiplicity of
mammalian microsomal cytochrome P-450. Pharmacol. Rev.

 

 

31, 277-295.

Luster, M., Faith, R., and Moore, J.A. (1978). Effects of
PBB on the immune response in rodents. Environ. Health
Perspect. 23, 227-232.

 

 

128

Lutz, R.J., Dedrick, R.L., Matthews, H.B., Eling, T.E., and
Anderson, MJL (1977L. A preliminary pharmacokinetic
model for several chlorinated biphenyls in the rat. Drug
Metab. Dispos. 5, 386-396.

Mason, ILS. (1957). Mechanisms of oxygen metabolism.
Science 125, 1185.

Matthews, ELB. (1981). Disposition CHE persistent
halogenated hydrocarbons in higher animals. In
Toxicology gt Halogenated Hydrocarbons: Health and
Ecological Effects (M.AJL Khan and RJL Stanton, edsJ.
Pp. 289-297, Pergamon Press, New York, pp. 289-297.

 

 

Matthews, ILB., and Anderson, M.W. (1975). Effect of
chlorination on the distribution and excretion of
polychlorinated biphenyls. Drug Metab. Dispos. 3, 371-
380.

 

Matthews, H;B., Surles, J3R., Carver, J3E., and Anderson,
M.W. (1977). Polychlorinated biphenyl transport by
blood components. Toxicol. Appl. Pharmacol. Abstract 41,
201.

Mazzella, GJL, Sinforiani,lE”.Savoldi, F”.Allegrini, M.,
Lanzola, E., and Scelsi, R. (1983). Blood cells
glutathione peroxidase activity and selenium in multiple
sclerosis. Eur. Neurol. 22, 442-446.

 

McCay, P.B., Gibson, D.D., Fong, K.-L., and Hornbrook, K.R.
(1976). Effect of glutathione peroxidase activity on
lipid peroxidation in biological membranes. Biochim.
Biophys. Acta 431, 459-468.

 

 

Meister, A. (1975L. Biochemistry of glutathione. Zhi
Metabolic Pathways (D.M. Greenberg, ed.). Vol. VII, pp.
101-188, Academic Press, New York.

 

Mgbodile, M.U.K., Holscher, M., and Neal, R.A. (1975). A
possible protective role for reduced glutathione in
aflatoxin B toxicity: Effect of treatment of rats with
phenobarbi a1 anui 3-methylcholanthrene (n1 aflatoxin
toxicity. Toxicol. Appl. Pharmacol. 34, 128-142.

 

Miller, ELC., and Miller, 03A. (1981). Mechanisms of
chemical carcinogenesis. Cancer 47,;1055-10644

Miller, J.A., and Miller, E.C. (1977). Ultimate chemical
carcinogens as reactive mutagenic electrophiles. In
Origins gt Human Cancer (H.H. Hiatt, J.D. Watson, J.A.
Winsten, edsJ. Pp. 605-627, Cold Spring Harbor, New
York.

 

129

Millis, C.D. (1984). Studies on the chemical and
pharmacotoxicological properties of polybrominated
biphenyls. M.S. Thesis, Michigan State University.

Millis, C.D, Mills, R.A., Sleight, S.D., and Aust, S.D.
(1985a). Toxicity of 3,4,5,3'uU,5'-hexabromobiphenyl
and 3,4,3fl4'-tetrabromobiphenyl. Toxicol. Appl.
Pharmacol. 78, 88-95.

 

 

Millis, C.Dr, Mills, ILA., Sleight, S.D., and Aust, S.D.
(1985b). Photolysis products of 2,4,5,2',4',5'-
hexabromobiphenyl: Hepatic microsomal enzyme induction
and toxicity in Sprague-Dawley rats. Fund. Appl.
Toxicol. 5, 555-567.

 

Mills, G.C. (1957). Hemoglobin catabolism. I.
Glutathione peroxidase, an erythrocyte enzyme which
protects hemoglobin from oxidative breakdown. gL_Biol.
Chem. 229, 189-197.

Mills, R.A., Millis, C.D., Dannan, G.A., Guengerich, F.P.,
and Aust, SAL (1985). Studies on the structure-
activity relationships for tflua metabolism of
polybrominated biphenyls by rat liver microsomes.
Toxicol. Appl. Pharmacol. 78, 96-104.

Mitchell, J.R., Thorgeirsson, S.S., Potter, W.Z., Jollow,
DAL, anui Kwiser, H. (1974). Acetaminophen-induced
hepatic injury: Protective role of glutathione in man
and rationale for therapy. Clin. Pharmacol. Therap. 16,
676-684.

Moore, R.W., and Aust, S.D. (1978). Purification and
structural characterization of polybrominated biphenyl
congeners. Biochem. Biophys. Res. Commun. 84, 936-942.

Moore, R.W., Dannan, G.AH, and Aust, ELD. (1980).
Structure-function relationships for the pharmacological
and toxicological effects and metabolism of
polybrominated biphenyl congeners. In Molecular Basis gt
Environmental Toxicity ”LS. Bhatnagar, edJ. Pp. 173-
212, Ann Arbor Science Publishers, Michigan.

Moore, R.W., O'Connor, J.V., and Aust, S.D. (1978a).
Identification of a major component of polybrominated
biphenyls as 2,2',3,4,4',5,5'-heptabromobiphenyl. Bull.
Environ. Contam. Toxicol. 20, 478-483.

Moore, R.W., Sleight, S.D., and Aust, S.D. (1978b).
Induction of liver microsomal drug metabolizing enzymes
by 2,234,4'fiL5'-hexabromobiphenyl. Toxicol. Appl.
Pharmacol. 44, 309-321.

 

 

130

Moore, R.W., Sleight, S.D., and Aust, S.D. (1979). Effects
of 2,2'-dibromobiphenyd. and 2,2',3,4,4',5,5'-
heptabromobiphenyl on liver microsomal drug metabolizing
enzymes. Toxicol. Appl. Pharmacol. 48, 73-86.

Nebert, D.W., Eisen, H.J., Negishi, M., Lang, M.A.,
Hjelmeland, L.M., and Okey, A.E. (1981). Genetic
mechanisms controlling the induction of polysubstrate
monooxygenase (P-450) activities. Ann. Rev. Pharmacol.
Toxicol. 21, 431-462.

 

Nebert, D.W., and Jensen, N.M. (1979). The Ah locus:
Genetic regulation of the metabolism of carcinogens,
drugs, and other environmental chemicals by cytochrome P-
450-mediated monooxygenases. In CRC Critical Reviews lg
Biochemistry (GAL Fasman, edJ. CRC Press, Cleveland,
Ohio, pp. 401-437.

 

Newbold, ILF., and Amos, J. (1981). Inhibition of
metabolic cooperation between Chinese mammalian cells in
culture by tumor promoters. Carcinogenesis 2, 243-249.

 

Olson, J.A. (1979). A simple dual assay for vitamin A and
carotenoids in human liver. Nutr. Rep. Internatl. 19,
807-813.

 

Orrenius, S., Ormstad, K.,-Thor, H., and Jewell, S. (1983).
Turnover and functions of glutathione studied with
isolated hepatic and renal cells. Fed. Proc. 42, 3177-
3188.

 

Omura, T., and Sato, R. (1964a). The carbon monoxide-
binding pigment of liver microsomes. 1. Evidence for
its hemOprotein nature. g1 Biol. Chem. 239, 2370-2378.

 

Omura, T., Sato, R., COOper, D.Y., Rosenthal, 0., and
Estabrook, RJm. (1965). Function of cytochrome P-450
microsomes. Fed. Proc. 24, 1181-1189.

 

Parke, ELV. (1968). The Biochemistry gt Foreign Compounds.
Pergamon Press, New York.

 

 

Pease, DAL (1964). Histological Technigue for Electron
Microscopy, 2nd ed. Pp. 38-39, New York.

 

 

Peraino, C., Fry, R., and Staffeldt, E. (1971). Reduction
and enhancement by phenobarbital of hepatocarcinogenesis

induced in the rat by 2-acetylaminofluorene. Cancer Res.
31, 1506-1512.

 

131

Peraino, C., Staffeldt, ELF., and Ludeman, VZA. (1981).
Early appearance:of histochemically altered hepatocyte
foci and liver tumors in female rats treated with

carcinogens one day after birth. Carcinogenesis 2, 463-
465.

 

Pitot, H.C. (1979). Drugs as promoters of carcinogenesis.
In The Induction gt Drug Metabolism (R.W. Estabrook and
E. Lindenlaub, edsJ. Pp. 471-483, FJL Schattauer
Verlag‘Stuttgart, New York.

 

Pitot, H.C., Barsness, L., Goldsworthy, T., and Kitagawa, T.
(1978). Biochemical stages of hepatocarcinogenesis after
a single dose of diethylnitrosamine. iNature 271, 456-
458.

Pitot, H.C., Goldsworthy, T., Campbell, H.A., and Poland, A.
(1980). Quantitative evaluation of the promotion by
2,3,7,8-tetrachlorodibenzo-p-dioxin of
hepatocarcinogenesis from diethylnitrosamine. Cancer
Res. 40, 3616-3620.

Pitotq ELC., Goldsworthy, T., and Moran, S. (1981). The
natural history of carcinogenesis: Implications of
experimental carcinogenesis in the genesis of human
cancer. g1 Supramol. Cell Biochem. 17, 133-146.

 

Pitot, H.C., and Sirica, A.E. (1980). The stages of
initiation and promotion in hepatocarcinogenesis.
Biochim. BiOphys. Acta 605, 191-215.

 

Poland, A., and Glover, E. (1977). Chlorinated biphenyl
induction of aryl hydrocarbon hydroxylase activity: A
study (M5 the structure-activity relationship. Ag11
Pharmacol. 13, 924-938.

 

Poland, A., and Glover, E. (1980). 2,3,7,8-
tetrachlorodibenzo-p-dioxin: Segregation of toxicity
with the Ah locus. Mol. Pharmacol. 17, 86-94.

 

 

Poland, A., Glover, E., and Kende, A.S. (1976).
Stereospecific, high affinity binding of 2,3,7,8-
tetrachlorodibenzo-p-dioxin by hepatic cytosol. g; Biol.
Chem. 251, 4936-4946.

Poland, 4A”. and Knutsonq J. (1982). 2,3,7,8-
Tetrachlorodibenzo-p-dioxin and related halogenated
aromatic hydrocarbons: Examination of the mechanism of
toxicity. Ann. Rev. Pharmacol. Toxicol. 22, 517-554.

 

132

Preston, B.D., Miller, J.A., and Miller, E.C. (1983). Non-
arene oxide aromatic ring hydroxylation of 2,235,5h-
tetrachlorobiphenyl as the major pathway catalyzed by
phenobarbital-induced rat liver microsomes. J. Biol.
Chem. 258, 8304-8311. ‘—

Pugh, T.D., and Goldfarb, S. (1978). Quantitative
histochemical and autoradiographic studies of
hepatocarcinogenesis in rats fed 2-acety1aminofluorene
followed by phenobarbital. Cancer Res. 38, 4450-4457.

 

Rabes, HAL” Scholze, P”.and Jantsch, B. (1972L. Growth
kinetics of diethylnitrosamine-induced enzyme-deficient
"preneoplastic" liver cell populations in vivo and 1g
vitro. Cancer Res. 32, 2577-2586. .—

 

Raheja, K;L., Linscheer, WRG., and Cho, C. (1983).
Prevention of acetaminOphen hepatotoxicity by
propylthiouracil in the glutathione depleted rat. Comp.
Biochem. Physiol. 76C, 9-14.

 

Rannug, U., Sundvall, A., and Ramel, C. (1978). The
mutagenic effect of 1,2-dichloroethane on Salmonella
typhimurium. I. Activation through conjugation with
glutathione 1g vitro. Chem. Biol. Interactions 20, 1-16.

 

 

Reed, D., and Beatty, P. (1980). Biosynthesis and
regulation of glutathione: Toxicological implications.
In Reviews 1g Biochemical Toxicology (E. Hodgson, J. Bend
and R. Philipot, eds.). Pp. 213-241, Elsevier/North
Holland, New York.

 

Render, J.A., Aust, S.D., and Sleight, S.D. (1982). Acute
pathologic effects of 3,3',4,4',5,5'-hexabromobiphenyl in
rats: Comparison of its effects with Firemaster BP-6 and
2,2',4,4',5,5'-hexabromobiphenyl. tggigglt ABEL
Pharmacol. 62, 428-444.

 

Rifkind, AHB., Firpo, Jr., A., and Alonso, ELR. (1984).
Coordinate induction of cytochrome P-448 mediated mixed
function oxidases and histopathologic changes produced
acutely in chick embryo liver by polychlorinated biphenyl
congeners. Toxicol. Appl. Pharmacol. 72, 343-354.

 

Ringer, RJL (1978). PBB fed to immature chickens: Its
effects on organ weights and function and on the
cardiovascular system. Environ. Health Perspect. 23,
247-255.

 

Robertson, L.W., Andres, J.L., and Safe, S.H. (1983).
Toxicity of 3,3',4,4'- and 2,2',5,5'-tetrabromobiphenyl:
Correlation of activity with aryl hydrocarbon hydroxylase
induction and lack of protection by antioxidants. g1
Toxicol. Environ. Health 11, 81-91.

 

133

Robertson, L.W., Parkinson, A., Campbell, M.A., and Safe, 8.
(1982). Polybrominated biphenyls as aryl hydrocarbon
hydroxylase inducers: Structure-activity correlations.
Chem.-Biol. Interactions 42, 53-66.

Rous, P., and Kidd, CLG. (1941). Conditional neoplasms and
subthreshold neoplastic states: A study of the tar
tumors of rabbits. g1 Exp. Med. 73, 365-390.

 

Rutenberg, A.M., Kim, H., Fischbein, J.W., Hanker, J}S.,
Wasserkrug, H.LH, and Seligman, A.M. (1969).
Histochemical and ultrastructural demonstration of gamma-
glutamyl transpeptidase activity. 11 Histochegt
Cytochem. 17, 517-526.

 

 

Sato, R., Nishibayashi, H., and Ito, A. (1969).
Characterization of two hemoproteins of liver microsomes.
In Microsomes and Drug Oxidations “LR. Gillette, AJL
Conney, G.J. Cosmides, R.W. Estabrook, J.R. Fouts and
G.J. Mannering, eds.). Pp. 111-128, Academic Press, New
York.

Scherer, E. (1981). Use of a programmable pocket
calculator for the quantitation of precancerous foci.
Carcinogenesis 2, 805-807.

 

Schulte-Hermann, R., Ohde, G., Schuppler, J., and Timmerman-
Trosiener, I. (1981). Enhanced proliferation of
putative preneoplastic cells in the rat liver following
treatment with the tumor promoters phenobarbital,
hexachlorocyclohexane, steroid compounds, and nafenopin.
Cancer Res. 41, 2556-2562.

 

Scribner, JJL, and Sflss,IL (1978L. Tumor initiation and
promotion. In International Review gt Experimental
Pathology (G.W. Richter and M.A. Epstein, edsJ. Vbl.
18, pp. 137-198, Academic Press, New York.

 

 

 

Selikoff, I.J., and Anderson, H.A. (1979). A survey of the
general population of Michigan for health effects of PBB
exposure. Submitted to the Department of Health.

Sladek, N.E., and Mannering, G.J. (1966). Evidence for a
new P-450 hemoprotein in hepatic microsomes from
methylcholanthrene-treated rats. Biochem. Biophys. Res.
Commun. 24, 668-674.

 

Sleight, S.D., Mangkoewidjojo, S., Akoso, B.T., and Sanger,
\LL. (1978). Polybrominated biphenyl toxicosis in rats
fed an iodine-deficient, iodine-adequate, or iodine-
excess diet. Environ. Health Perspect. 23, 341-346.

 

 

134

Sleight, S.D., and Sanger, V.L. (1976). Pathologic
features of polybrominated biphenyl toxicosis in the rat
and guinea pig. _J_._ Am. Vet. Med. Assoc. 169, 1231-1235.

Smith, R.L. (1973). The Excretory Function gt At1g.
Chapman and Hall, London.

 

 

 

Snoke, J.Eu. and Bloch, K. (1952). Formation and
utilization of y-glutamylcysteine in glutathione
synthesis. _J_._Biol. Chem. 199, 407-414.

 

Steel, R.G.D., and Torrie, J.H. (1980). Principles and
Procedures gt Statistics. A Biomedical Approach (C.
Napier and J.W. Maisel, eds.). McGraw-Hill, New York.

 

 

 

 

Stott, W.T., Reitz, R.H., Schumann, A.M., and Watanabe, P.G.
(1981). Genetic and nongenetic events in neoplasm. Fd.
Cosmet. Toxicol. 19, 567-576.

 

Strobel, H.W., Lu, ANY.H.,?Heidema, J., and Coon, M.J.
(1970). Phosphatidylcholine requirement in the enzymatic
reduction of hemoprotein P-450 and in fatty acid,
hydrocarbon, and drug hydroxylation. Biol. Chem: 245,
4851-4856.

 

Stross, JZK., Smoklery I.A., Isbister, J., and‘Wilcox, K.R.
(1981). The human health effects of exposure to
polybrominated biphenyls. Toxicol. Appl. Pharmacol. 58,
145-150.

 

Sundstrdm, G., Hutzinger, O., and Safe, 8. (1976).
Identification of 2,2',4,4',5,5'-hexabromobiphenyl as the
major component of flame retardant Firemaster BP-6.
Chemosphere 1, 11-14.

 

Surak, J.G., and Bradley, R.L. (1976). Transport of
organochlorine chemicals across cell membranes. Environ.
Res. 11, 343-352.

Sweeney, CLD., Jones, K;G., Cole, F.M., Basford, D., and
Krestynski, F. (1979). Iron deficiency prevents liver
toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Science
204, 332-335.

Thor,IL, Moldéus,jR” and Orrenius, S. (1979). Metabolic
activation and hepatotoxicity effect of cysteine, n-
acetylcysteine, and methionine on glutathione
biosynthesis and bromobenzene toxicity in isolated rat
hepatocytes. Arch. Biochem. Biophys. 192, 405-413.

 

135

Thunberg,'Lu Ahlborg, UAL, Hakansson,IL, Krantz,(L, and
Monier, M. (1980L. Effects of 2,3,7,8-
tetrachlorodibenzo-p-dioxin on the hepatic storage of
retinol in rats with different dietary supplies of
vitamin A (retinol). Arch. Toxicol. 45, 273-285.

Trosko, J;E., Dawson, 8., and Chang, C.-C. (1981). PBB
inhibits metabolic cooperation.in1Chinese hampster V79
cells in culture by various polybrominated biphenyl (PBB)
congeners. Carcinogenesis 3, 181-186.

 

Tsushimoto, G., Trosko, J.E., Change, C.-C., and Aust, S.D.
(1982). Inhibition of metabolic c00peration in Chinese
hampster V79 cells in culture by various polybrominated
biphenyl (PBB) congeners. Carcinogenesis 3, 181-186.

Tuey, DAL, and Matthews, HJL (1980). Distribution and
excretion of 2,2',4,4',5,5'-hexabromobiphenyl in rats and
man: Pharmacokinetic model predictions. Toxicol. Appl.
Pharmacol. 53, 420-431.

 

 

van Miller, J3P., Hsa, I.C., and Allen, J}R. (1975).
Distribution and metabolism <mf H-2,5,2U5'-
tetrachlorobiphenyl in rats. Proc. Soc. Exg Biol. Med.
148, 682-687.

 

Vermilion, J3L., Ballou, ELP., Massey, V., and Coon, M.J.
(1981). Separate roles for FMN and FAD in catalysis by
liver microsomal NADPH-cytochrome P-450 reductase. 9;
Biol. Chem. 256, 266-277.

 

Vermilion, J.L., and Coon, M.J. (1978). Purified liver
microsomal NADPH-cytochrome P-450 reductase. Spectral
characterization of oxidation-reduction states. g; Biol.
Chem. 253, 2694-2704.

Vos, CLG., and van Genderon, H. (1973). Toxicological
aspects of immunosuppression. In Pesticides and the
Environment. A Continuing Controversy (W.B. Diechmann,
ed.). Symposia Specialists, Florida, pp. 527-546.

 

 

Watanabe, K” and Williams, GAL, (1978L. Enhancement of
rat hepatocellular-altered foci by the liver promoter
phenobarbital: Evidence that foci are precursors of
neoplasms and that the promoter acts on carcinogen-
induced lesions. g1 Natl. Cancer Inst. 61, 1311-1314.

 

 

Waxman, D.J., and Walsh, C. (1982). Phenobarbital-induced
rat liver cytochrome P-450 purification and
characterization of two cflosely related isozymic forms.
g1 Biol. Chem. 257, 10446-10457.

 

136

Welton, AWE”, and Aust, S.D. (1974). Multiplicity of
cytochrome P-450 hemoproteins in rat liver microsomes.
Biochem. BiOphys. Res. Comm. 56, 898-906.

Werner, P.R., and Sleight, S.D. (1981). Toxicosis in sows
and their pigs caused by feeding diets containing
polybrominated biphenyls to sows during pregnancy and
lactation. Amer. g1 Vet. Res. 42, 183-188.

 

White, I.NJL (1976). The role of liver glutathione in the
acute toxicity of retrorsine to rats. Chem.-Biol.

 

Interactions 13, 333-342.

Whitlock, Jru, J3P., and Galeazzi, D.R. (1984). 2,3,7,8-
Tetrachlorodibenzo-p-dioxin receptors in wild type and
variant mouse hepatoma cells. L Biol. Chem. 256, 980-
985.

 

Wilkinson, CLF., and Brattsten, IuB. (1972-73). Microsomal
drug metabolizing enzymes in insects. In Drug Metabolism
Reviegg (F.J. DiCarlo, ed.). Vol. 1, pp. 153-227, Marcel
Dekker, New York.

 

Williams, G.M. (1980). The pathogenesis of rat liver
cancer caused by chemical carcinogens. Biochim. Biophys.

 

ACta 605, 187-189.

Yasukochi, Y., and Masters, BJLS. (1976). Some properties
of a detergent-solubolized NADPH-cytochrome c (cytochrome
P-450) reductase purified by biospecific affinity
chromatography. g1 Biol. Chem. 251, 5337-5344.

 

Yoshida, K., Sakurada, T., Kitanka, H., Fukazawa, H., Kaise,
N., Kaise, K., Yamamoto, M., Saito, 8., and Yoshinaga, K.
(1980). A solid-phase radioimmunoassay for free
thyroxine in serum compared with equilibrium dialysis
method. Tohoku g1 Exp: Med. 132, 375-383.

 

Zampaglione, N.D., Jollow, J., Mitchell, J.R., StripP: B.,
Hamrick, M., and Gillette, J.R. (1973). Role of
detoxifying enzymes in bromobenzene-induced liver
necrosis. g1 Pharmacol. Exp. Ther. 187, 218-227.

 

 

VITA

The author was born in Newark, New Jersey on July 10,
1958. She received her primary and secondary education in
the public and private school systems of East Orange, New
Jersey. She received the Degree of Doctor of Veterinary
Medicine from the School of‘Veterinary Medicine, Tuskegee
Institute, Tuskegee Institute, Alabama.

In June of 1982 she began a M.S. program as a minority
fellow in the Department of Pathology at Michigan State
University. In 1984 she was admitted into the
M111t:iciis;cj.p].ir1a1:y D<DC‘tC>rEil Iflrc>g1:a.m i.n
Pathology/Environmental Toxicology. In 1984 the author
became a N.I.H. Pathotoxicology Fellow.

In 1985 the author was admitted into a 2-year
Postdoctoral Training Program in Pathotoxicology and
Laboratory Animal Medicine at Rockefeller University, New

York, New York.

137

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