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thesis entitled

A TIME-COURSE STUDY OF INTERACTIONS BETWEEN
VITAMIN A AND 'POLYBROMINATED BIPHENYLS IN RATS

presented by

R. Wasito

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Pathology

 

Major professor
Dr. Stuart D. Sleight
Date August; 8, ISBB

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A TIME-COURSE STUDY OF INTERACTIONS BETWEEN
VITAMIN A AND POLYBROMINATED BIPHENYLS IN RATS

BY

R. Wasito

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1984

ABSTRACT

A TIME-COURSE STUDYOF INTERACTIONS BETWEEN
VITAMIN A AND POLYBROMINATED BIPHENYLS IN RATS

BY

R. Wasito

Seventy-two young male rats were allotted to 4 groups
of 18 each. Diets, either vitamin A-deficient or supple-
mented were fed for 42 days and contained 0 or 100 mg of a
commercial mixture of polybrominated biphenyls (PBB) for the
last 28 days of the experiment. .Adverse clinical signs were
not observed. PBB significantly depressed weight gain in
rats fed a vitamin A-deficient diet. Hepatic lesions due to
PBB toxicosis were not diminished by vitamin A supplementa-
tion. The vitamin A-deficient diet containing PBB caused
depletion of retinol concentrations in the serum and
produced abnormal vitamin A metabolism in the liver as
manifested by the appearance of significant amounts of
retinyl acetate. Hyperplasia of the bile ducts and squamous
metaplasia of the thyroid and salivary glands occurred in

vitamin A-deficient rats given PBB.

Dedicated With Love To

Rr. Hastari Wuryastuti, my wife
My mother
My brother and his wife, my sisters and their husbands,
and their children:
Ria, Bagus, Nila, Windri, Galih, Imok, Denta, Uuk and Bayu

You

And in Memory of my Father: R. Mohammad Ichram

ii

ACKNOWLEDGEMENTS

The author is grateful to Dr; S.D. Sleight, my major
adviser, for his dedication and love during my course of
study.

Drs. HJL Stowe and RJL Jensen, my graduate committee,
for their thoughtful suggestions for the thesis.

The government of the Republic of Indonesia and the
Rockefeller Foundation for their financial support.

Drs. Margit S. Rezabek, Darlene Dixon, Mark G. Evans,
Robert E. Galbraith and many colleagues for their valuable
assistance during the course of the experiment.

Irene Brett, Anne Goatley, Mae K. Sunderlin and Frances
M. Whipple for their assistance in the laboratory work, and
Cheryl Assaff for typing this thesis. The warmth and
friendship shown to me by personnel in the histopathology,
clinical nutrition laboratories and the Department of
Pathology, Michigan State University is a particularly
pleasant memory.

My deepest appreciation to my wife, for her assistance,
and almost one year, she tolerated my preoccupation and my
absence with an affectionate understanding and continuing
love during my study in the United States which will always

be remembered.

TABLE OF CONTENTS

LIST OF TABLES O O O O O O O O O O O O O O 0

LIST OF FIGURES O O 0 O O O O O O O O O O 0

INTRODUCTION 0 O O O O O O O O O O O O O O 0

LITERATURE REVIEW 0 O O O O O O O O O O O C

Vitamin A . . . . . . . . .

Introduction . . . . . . . .
MetabOIj-Sm O O O O O O I O 0
Pathology of Vitamin A Defic'

Vitamin A Toxicity
Polybrominated Biphenyls . . .
Introduction . . . . .
Metabolism . . . . . .
Pathology . . . . . .

Interaction Between Vitamin A and Xenobiotics/

Carcinogens . . . . . . . . . . .

Influence of Xenobiotics/Carcinogens on

Vitamin A . . . . . . . .

Influence of Vitamin A on Xenobiotics/

Carcinogens . . . . . . .

Page
. vi

vii

o
mhfi

NO\O\O\I

Hi4

. 14
. 14

. 15

Comparison Between the Effects of Polybrominated

Biphenyls and Vitamin A Deficiency

MATERIALS AND METHODS . . . . . . . . . . .

RESULTS

Experimental Design . . . . . . . .

Feeding Practices . . . . . . . . .

Laboratory Investigation Procedures
Collection and Processing of
Vitamin A Analysis . . . . .
Statistical Evaluation . . .

Clinical Signs . . . . . . . . . . .
Body Weight . . . . . . . . . . . .
Organ Weights . . . . . . . . . . .
Laboratory Investigation . . . . . .
Serum Vitamin A . . . . . .
Liver Vitamin A . . . . . .

iv

0 l7

19
21
21
21
22
24

. 25
. 25
. 27
. 27
. 27
. 34

DISCUSSION

SUMMARY

REFERENCES

VITA

Pathology . . . . . .

Gross Lesions
Histopathology

Page

Table

10

LIST OF TABLES

Experimental design . . . . . . . . . . .
Organ to body weight ratios at 7 days . .
Organ to body weight ratios at 14 days .
Organ to body weight ratios at 28 days .
Sera vitamin A concentrations at 7 days .
Sera vitamin A concentrations at 14 days
Sera vitamin A concentrations at 28 days
Liver vitamin A concentrations at 7 days
Liver vitamin A concentrations at 14 days

Liver vitamin A concentrations at 28 days

vi

Page
20
28
29
30
31
32
33
35
36

37

LIST OF FIGURES

Figure Page

1 Mean body weights of rats fed diets contain-
ing 0 or 100 mg of PBB/kg and either defi-
cient in vitamin A or supplemented with
30,000 10 retinyl acetate/kg .. . .. . .. . 26

2 Photomicrograph of a liver from a rat fed a
vitamin A-deficient diet .. . .. . .. . .. 41
3 Photomicrograph of a liver from a rat fed a

vitamin A-deficient diet and necropsied after
7 days of dietary treatment with 100 mg of
PBB/kg O O O O O O O O O O O O O O O O O O O O 41

4 Photomicrograph of a liver from a rat fed a
diet-supplemented with 30,000 10 retinyl
acetate/kg after 28 days of dietary treatment
with 100 mg of PBB/kg . . . . . . . . . . . . 43

5 Photomicrograph of an extraparenchymal bile
duct from a rat fed a diet supplemented with
30,000 IU retinyl acetate/kg after 28 days of
dietary treatment with 100 mg of PBB/kg . . . 43

6 Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient
diet after 14 days of dietary treatment with
100 mg of PBB/kg .. . .. . . .. . . .. . . 45

7 Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient
diet after 28 days of dietary treatment with
100 mg of PBB/kg . . . . . . . . . . . . . . . 45

8 Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient
diet after 28 days of dietary treatment with
100 mg of PBB/kg . . . . . . . . . . . . . . . 47

9 Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient
diet after 28 days of dietary treatment with
100 mg of PBB/kg . . . . . . . . . . . . . . . 47

vii

Figure

10

11

Page

Photomicrograph of a thyroid gland from a rat
fed a vitamin A-deficient diet after 28 days
of dietary treatment with 100 mg of PBB/kg . . 49

Photomicrograph of a salivary gland from a

rat fed a vitamin A-deficient diet after 28

days of dietary treatment with 100 mg of

PBB/kg . . . . . . . . . . . . . . . . . . . . 49

viii

INTRODUCTION

The realization that nutritional imbalances and
deficiencies are still major problems in human and animal
health has resulted in numerous studies on the role of diet
on effects of environmental contaminants (xenobiotics) and
carcinogens. Polyhalogenated aromatic hydrocarbons, such as
polybrominated biphenyls (PBB), polychlorinated biphenyls
(PCB), dibenzofurans, dibenzo-p-dioxin and many others, are
cause for concern because their widespread distribution in
the environment can result in human and animal exposure.
The interaction of nutritional factors, such as vitamin A,
and environmental contaminants, such as PBB, is of current
research interest and may have important ramifications for.
human and animal health.

Vitamin A deficiency has a global distribution and is
still a serious public health problem in some countries
(WHO, 1967; Srikantia, 1982). A number of experiments
suggest that vitamin A deficiency is related to the adverse
effects of certain xenobiotics such as PCB (Innami gt 31.,
1974) and tetrachlorodibenzo-p-dioxin (TCDD) (Thunberg 23
_l,, 1983). Previous work in this laboratory by Darjono
(1982) demonstrated that the vitamin A nutritional status

has important effects upon the toxicity of PBB. He

1

2
evaluated the interaction between vitamin A status and PBB
toxicity in two experiments. He found that clinical signs
of vitamin A deficiency and mortality occurred earlier in
rats fed vitamin A-deficient diets containing 100 mg of
PBB/kg than in those fed a vitamin A-deficient diet without
PBB. Vitamin A supplementation prevented decreased weight
gain and decreased thymic weight associated with PBB
toxicosis (Darjono, 1982). Massive enlargement of the
common bile duct occurred in rats fed vitamin A-deficient
diets containing 100 mg of PBB/kg. Histologically, this
lesion consisted of extensive hyperplasia. Serum vitamin A
concentrations in rats fed vitamin A-deficient diets were
also lowered by PBB as compared to rats fed a diet .
containing PBB and supplemented with vitamin A. The
interaction between vitamin A deficiency and PBB toxicosis
also affected vitamin A metabolism in the liver as
manifested by the appearance of significant amounts of
retinyl acetate in the vitamin A profile. Darjonoks
experiments were not designed to study the pathogenesis of
the lesions. The interaction of vitamin A and PBB toxicosis
was also evaluated in a pilot experiment by Wasito and
Galbraith (1983, unpublished data). Decreased weight gain,
low serum vitamin A concentration, enlargement of the liver,
swollen and vacuolated hepatocytes, hyperplasia of the bile
duct, keratinization of the ultimobranchial follicles in the

thyroid gland and squamous metaplasia of the salivary gland

3
were observed only after prolonged feeding of a vitamin A-
deficient diet containing 100 ppm PBB.

We therefore decided to do a time-course study over a
4-week period so as to better assess the interaction between
vitamin A status and PBB. The first objective of this study
was to evaluate and compare serum and hepatic vitamin A
contents in young rats as affected by the interaction
between dietary levels of vitamin A and PBB. The second
objective was to evaluate the pathogenesis of the previously
described histologic lesions which were due to the combined
effects of vitamin A deficiency and PBB with emphasis on
those suggestive of vitamin A deficiency. The third
objective was to further characterize whether or not the

vitamin A nutritional status affected the toxicity of PBB.

1-4

if)

LITERATURE REVIEW

Vitamin A

Introduction

 

Vitamin A is present in the diet either as preformed
vitamin A (retinol and its esters) or as its precursor (B-
carotene) (Davis, 1978; GOpalan and Rao, 1979). Structur-
ally, the vitamin A molecule consists of a hydrophobic head
(B-ionone ring), a conjugated iSOprenoid side chain and a
polar terminal group. The conjugated isoprenoid chain is a
double bond system which is isomerized by light, enzymes or
heat to change the shape of the molecule. The polar
terminal group can be enzymatically or chemically converted
to retinyl palmitate, retinal and retinoic acid (Zile and
Cullum, 1983).

Vitamin A is necessary for normal growth and differen-
tiation of epithelial cells and bone (Wolbach, 1954).
Retinol, retinyl esters, retinal, and retinoic acid are also
capable of supporting these functions (Zile and Cullum,
1983).

Vitamin A is also important in reproduction because it
is known to affect spermatogenesis, oogenesis and placental,
fetal and embryonal deve10pment. Retinol, retinyl esters or

retinal are necessary for these functions. Lotthammer

4

5
(1979) reported that cattle fed B-carotene deficient rations
had delayed ovulation and increased in the incidence of
follicular and luteal cysts. In another study, however,
these effects were not demonstrated (Folman gt al,, 1979).
Retinoic acid is considered as biochemically inactive
insofar as the reproductive functions of vitamin A are
concerned (Thompson 33 al,, 1964). However, an in yitgg
study indicated that retinoic acid activated cell cleavage
in the early stage of spermatogenesis and stimulated
cellular differentiation in mouse cryptorchid testes (Haneji
gt 31., 1983).

Vitamin A is also responsible for vision and color
perception. The active form of vitamin A for this function
is retinal derived from retinyl esters or retinol (Wald,
1960). 'Vitamin A-deficient animals often have xerophthalmia
because of keratinization of corneal and conjunctival
epithelial cells, and squamous metaplasia and atrophy of
lacrimal glands (Wolbach, 1954).

Vitamin A values are given in International Units (IU)
of vitamin A. The IU values can be converted to micrograms
by applying the following factors: 1 IU of vitamin A = 0.3
ug retinol; 0.6 ug B-carotene; and 1.2 ug other provitamin A
carotenoids with vitamin A activity (W110, 1967). In rats,
the minimal requirement for maintaining weight gains,
optimal longevity and some hepatic vitamin A storage is
about 2,000 IU per kg of diet (Anonymous, National Academy

Sciences, 1972).

Metabolism

 

As already mentioned, the major sources of vitamin A in
the diet are B-carotene and retinyl esters. B-carotene is
converted to vitamin A primarily in the intestinal mucosa.
The biosynthetic process involves two soluble enzymes. 8-
carotene is cleaved at the central double bond by B-carotene
15-15'-dioxygenase to yield two molecules of retinaldehyde.
Retinaldehyde is then reduced to retinol by retinaldehyde
reductase (Goodman and Olson, 1969). Dietary retinyl esters
are hydrolyzed in the intestinal lumen and the resulting
retinol is then absorbed into the mucosal cells. Retinol
that is absorbed or newly synthesized in the mucosal cells
is reesterified with long chain saturated fatty acids. The
retinyl esters formed are then absorbed into the body in
association with lymph chylo microns. During chylomicron
metabolism, virtually all of the retinyl esters remaining
with the chylomicron remnant are removed from the circula-
tion by the liver (Blomhoff _e_1:_ _a_l., 1982). After uptake of
the chylomicron-associated retinyl esters, hydrolysis and
reesterification occur in the liver. The retinyl esters
formed, mainly retinyl palmitate, are stored in association
with lipid droplets in the liver (Goodman, 1980).

Transport of vitamin A from the liver to the tissue is
in the form of the free alcohol retinol. In the liver,
hydrolysis of retinyl esters is responsible for formation of
retinol prior to the mobilization of hepatic vitamin A. The

retinol formed is bound to a specific plasma binding protein

7
(retinol binding protein or RBP) (Rask _e_t_ 31., 1980). The
concentration of RBP in plasma is regulated by vitamin A
status. Thus, in: vitamin A deficiency, RBP molecules are
not secreted from the liver. In plasma, RBP forms a complex
with thyroxin binding prealbumin. The RBP molecules
interact with a cell membrane receptor on epithelial cells
and transfer vitamin A across the membrane into the cells
(Heller, 1975; Rask and Peterson, 1976). The uptake of
vitamin A by the cells causes a reduction of the affinity of
RBP for thyroxin binding prealbumin. The RBP molecules
which are not able to interact with the thyroxin binding
prealbumin are then excreted in the urine (Rask _e_t 31.,
1980) .

In the cell, vitamin A is bound to a cellular receptor
protein. Retinol, retinal and retinoic acid are specific-
ally bound to cytosolic-binding protein (Liau 3E 31., 1981).

Retinoic acid is transported by serum albumin (Smith e_t_:_
31., 1973). It is rapidly metabolized to more polar
compounds which are excreted in the urine and bile. The
major biliary metabolite of retinoic acid is identified as

retinol-B-glucuronide (Goodman, 1980).

Pathology 31 Vitamin _A_ Deficiency and Vitamin _A Toxicity

 

 

Gross lesions and clinical signs: In the rat,

 

manifestations of vitamin A deficiency include xerophthal-
mia, ataxia, paresis, humped posture, rough coat and slight

focal alopecia (Klein-Szanto _e_t; 31., 1980).

8

Vitamin A toxicity affects the skin (dryness and
scaling), the hair (dryness and loss), the optic disc
(papilledema), the liver (cirrhosis), bone (malformation)
and arteries, heart and kidney (calcification) (Grey _e_t; 31.,
1965; Strebel _e__t_:_ 31., 1969; Russell _e_t: 31., 1974; Davis,
1978).

Histopathology: As previously mentioned, vitamin A is

 

necessary for the normal differentiation of epithelial cells
in many tissues (Wolbach, 1954). A deficiency of vitamin A
results in squamous metaplasia in which a squamous
keratinizing type of epithelium replaces the normal form of
epithelium of salivary glands, trachea, bronchi, testes,
kidney, urinary bladder, cornea, conjunctiva, meibomian
glands, pancreas and skin. Klein-Szanto _e_t 31. (1980)
noticed that in vitamin A-deficient rats, males deve10ped
squamous metaplasia of the trachea and salivary glands
earlier than females. Sialoadenitis, especially of the
submaxillary gland was always present. On the other hand,
squamous metaplasia of the renal pelvis developed earlier in
females.

Others have found that edema and atrophy of testes
(Wolbach and Howe, 1925), squamous metaplasia of the bile
ducts (Beaver, 1961) and keratinization of the ultimo-
branchial follicles of the thyroid glands (Krupp, 1972) were

related to vitamin A deficiency in rats.

Polybrominated Biphenyls

Introduction

 

Polybrominated biphenyls (PBB) were manufactured for
use as flame retardants in industrial processes. The
production of PBB by Michigan Chemical Corporation as a
flame retardant under the trade name Firemaster (FM) began
in 1970 and ceased in 1975.

An analysis of FM indicated that it consisted of 2%
tetrabromobiphenyl, 10.6% pentabromobiphenyl, 62.8%
hexabromobiphenyl, 13.8% heptabromobiphenyl and 11.4% other
biphenyls (Kay, 1977). The mixture causes a mixed-type
induction of liver microsomal drug metabolizing enzymes
since it induces phenobarbital (PB)-type and 3- _
methylcholanthrene (MC)-type microsomal enzymes (Dent _e_t
_a_1_., 1976). Apparently the toxicity of FM is mostly
attributed to its congeners with MC-type induction
capability. In addition, mono ortho-substituted congeners
in PM, such as 2,4,5,3',4',5'-heyxabromobipheny1 (HBB)
(Dannan _e_t; 31., 1978), 2,4,5,3',4'-pentabromobiphenyl
(Dannan 33 _a_l., 1982a) and 2,3,4,5,3',4'-HBB (Dannan _e_t-131.,
1982b) were reported to be MC-type inducers and were
considered toxic. Congener 2,2',4,4',5,5'-HBB comprises
approximately 50% of the mixture, is a PB-type inducer, and
is relatively nontoxic (Akoso 31; a_1., 1982b). Another PB-
type inducer, 2,4,5,2',5'-pentabromobipheny1 is also not

toxic (Dannan _e_t_ 31., 1982b).

10

The PM mixture containing PBB begins to melt at 72° C
and decomposes at 300 to 400° C. It is insoluble in water
and highly soluble in organic solvents (fat soluble). It
has a low vapor pressure. PBB can be degraded by
ultraviolet radiation (Ruzo and Zabik, 1975).

In 1973 and 1974, livestock feed was contaminated by PM
at a facility in Michigan. Clinical signs described in
dairy cattle fed PBB-contaminated feed included decreased
milk production, anorexia, alOpecia and abnormal growth
(Jackson and Halbert, 1974). Subsequently, other farm
animals were also found to be contaminated by ingestion of
PBB-contaminated feed (Damstra 31; 31., 1982). Some
investigators associated skin rashes, joint swelling,
hematologic changes, immunologic defects, increased
susceptibility to respiratory infection and neurologic
symptoms to exposure of peOple to PBB (Bekesi and Holland,
1978; Valciukas, 1978; Weil e_t__a_1_., 1981). However, the
levels of PBB in serum or adipose tissue were not well
correlated with reported signs of PBB toxicity in man (Brown

33 31., 1981; Weil 3:5 31., 1981).

Metabolism

 

Results of animal studies indicate that PBB concentrate
in hepatic and adipose tissues are poorly metabolized and
very slowly excreted. Thus, the potential exists for
bioaccumulation and long term effects of PBB exposure (Dent

3t; 31., 1976; DiCarlo 3t 31., 1978).

11
As already mentioned, PBB toxicosis is mostly attributed to
congeners with MC-type induction capability. The mechanism
of toxicity of PBB is unknown. However, it is suggested
that an initial event is the stereospecific reversible
binding of a congener to a cytosolic polypeptide receptor
molecule called the Ah receptor (Poland 31': 31., 1976). The
congener-receptor binding is required for aryl hydrocarbon
hydroxylase (AHH) induction (Robertson 31331., 1983). The
PBB receptor complex is then translocated to the nucleus
resulting in the synthesis of liver microsomal drug
metabolizing enzymes (P-450) (Nebert 3E 31., 1982) .

The various forms of P-450 represent a family of
hemoproteins which are monooxygenases that possess catalytic
activity towards many substrates. Monooxygenases are
enzymes that insert one atom of atmospheric oxygen into
their substrates. Accordingly, to perform this
monooxygenation, the P-450 receives two electrons from
cofactors NADPH or NADH. These electrons are received one
at a time via reductases (flavoproteins). It is not known
whether the P-450 molecules are arranged in rosettes around
reductase molecules or the reductase molecules move among P-
450 molecules. In most organisms, the electron chains are
embedded in the endoplasmic reticulum, inner mitochondrial
membrane and the nuclear envelope. Therefore, monooxygenase
activities require'the integrity of an electron flow between
the cofactor NADPH or NADH and the oxygenated form of P-450.

After passage of the electrons to P-450, one atom of

12

atmospheric oxygen is transferred to the substrate at the P-
450 enzyme-active site. This process involves activation of
iron of heme. The other atom of oxygen is found in cellular
water. It is believed that oxygenation occurs at or within
the surface of the microsomal (lipoidal) membrane. The
resulting more polar metabolites are water soluble and are
then excreted in the urine or bile (Nebert _e_t_ 31., 1982) .

PBB are lipophilic and have long half-lives in body
tissues. In the rat, the half-life of PBB is 23.1 weeks in
serum and 69.3 weeks in fat (Miceli and Marks, 1981).
Although PBB are poorly metabolized, excretion of PBB is
primarily via the bile into the feces. Milk and eggs are
the major routes of excretion in lactating mammals and egg-
laying birds, respectively (Matthews _e_t_ 31., 1977).
Transplacental transfer of PBB was demonstrated in rats and
cows and detrimental effects in young suckling on PBB-
contaminated mothers have been reported (Rickert 3_t__a_l_.,
1978; Willett and Durst, 1978; Moore 31'; 31., 1978; Detering

33 _a_l_., 1975).

Pathology

 

Gross lesions: Hepatomegaly, thymic involution and

 

loss of weight were consistent features noted in PBB-treated
rats and mice (Sleight and Sanger, 1976; Gupta and Moore,
1979). Kimbrough 3331. (1978) and Kimbrough 3331. (1981)
observed neOplastic nodules in the livers of rats fed FM FF-
1 at high doses. Tumor promoting effects of PBB in rats

were also reported by Jensen 3!; fl: (1982) who found

13

increased numbers of y-glutamyl transpeptidase (GGT)
positive enzyme-altered foci and neoplastic nodules.
Thyroid enlargement was noted in rats (Sleight 3’3 31., 1978)
and in sows and their pigs (Werner and Sleight, 1981).
Darjono 3t 31. (1983) reported that rats fed a vitamin A-
deficient diet containing 100 ppm PBB had extensive
enlargement of the common bile ducts.

Histopathology: In the liver, severe hypertrophy and
vacuolation of hepatocytes in rats fed 100 ppm or 500 ppm of
PBB for 30 or 60 days were noticed by Sleight and Sanger
(1976). Similar histologic features were also observed in
the liver of sows and their pigs (Werner and Sleight, 1981).
Hepatic necrosis (McCormack _e_t-31., 1980) and intracyto-
plasmic inclusions within hepatocytes (Akoso, 1981) were
occasionally observed in rats as a result of exposure to
PBB. Large acidophilic hepatocytes with enlarged nuclei and
multiple nucleoli were described by Jensen _e_t 31. (1982) in
rats in a study of the tumor promoting activity by PBB.
Darjono g 31. (1983) observed hyperplasia of the extra-
parenchymal bile ducts in rats after prolonged feeding of a
vitamin A-deficient diet containing 10 or 100 ppm PBB.

In the thyroid gland, Sleight 3E 31. (1978),
Mangkoewidjojo (1979) and Akoso _ei 31. (1982a) observed
hypertr0phy and hyperplasia of the follicular cells as well
as depletion of follicular colloid.

In the thymus, thymic cortical atrophy and loss of

demarcation between the cortical and the medullary regions

14
were observed in rats fed FM FF-l at high doses (Gupta and
Moore, 1979) and in rats given 2,3,4,5,3',4'-HBB (Dannan 31;
31., 1982b) .

Interaction Between Vitamin A
and Xenobiotics/Carcinogens

 

Influence 31 Xenobiotics/Carcinogens 33 Vitamin 3

 

Research by Goerner (1938) with rabbits revealed that
intraperitoneal exposure to dibenzanthracene caused a marked
decrease of hepatic vitamin A storage. Decrease of hepatic
vitamin A also occurred in rats following exposure to
methylcholanthrene (MC) (Bauman 33 31., 1942) and
benzo(a)pyrene (BaP) (Carruthers, 1942). These results
suggest that the depletion of vitamin A by the carcinogenic
polycyclic aromatic hydrocarbons may be an important factor
in cancer. Certain xenobiotics such as polychlorinated
biphenyls (PCB) (Kato 3E 31., 1978), phenobarbital (Backes
$31., 1979), tetrachlorodiphenyldioxine (TCDD) (Thunberg
313 31., 1980), ethanol (Sato and Lieber, 1982) and
polybrominated biphenyls (PBB) (Mangkoewidjojo, 1979; Akoso
_e_t_ 31., 1982a; Darjono, 1982) have also been reported to
lower hepatic vitamin A storage. Kato e_t 31. (1978)
speculated that PCB and DDT caused decreases in vitamin A
levels because these substances induce synthesis of
microsomal enzymes that may lead to the metabolism of

vitamin A. This hypothesis may be supported by observations

that liver microsomal enzymes affect the hydroxylation of

IIa

15

lipid-soluble drugs (Brodie g 31., 1958), steroids
(Talalay, 1965) and fatty acids (Das 33 _a_l_., 1968).
Aflatoxin Bl was also reported to lower liver and serum
vitamin A levels (Newberne and Suphakarn, 1977).
Chikaraishi and Suzue (1977) also reported that aflatoxin
may inhibit the conversion of B-carotene to vitamin A as

well as enhance the depletion of vitamin A.

Influence 33 Vitamin A 33 Xenobiotics/Carcinogens

 

Innami 33 31. (1974) observed that rats fed a 0.1% PCB
diet supplemented with 3,400 IU of vitamin A/100 g of the
PCB diet for 6 weeks grew better than those fed only a 0.1%
PCB diet. Rats fed a vitamin A-deficient diet with 0.1% PCB
had a significant growth retardation compared to those given
only a 0.1% PCB diet. In the thymus, vitamin A was reported
to have a protective effect against thymic depletion in rats
fed 100 ppm PBB (Darjono _e_t_ 31., 1983).

Moon 33 fl (1983) reported that several retinoids:
axerophthene, retinyl acetate (RAc), retinyl methyl ether
(RME), N-(4-hydroxyphenyl)retinamide, possess mammary
anticarcinogenic activity when given chronically in the diet
to experimental animals. Sporn 33 31. (1977) demonstrated
that lB-cis-retinoic acid inhibited the development of
bladder cancers in rats caused by direct instillation of N-
methyl-N-nitrosurea (MNU) into the bladder. Other investi-
gators have indicated a protective effect of vitamin A
against 3-methylcholanthrene (MC)-induced lung tumors in

rats (Cone and Nettesheim, 1973) and a decrease in

l6

benzo(a)pyrene (BaP)-induced lung tumors in hamsters
(Saffiotti 33 31., 1967).

Wattenberg (1983) reported that retinoids act by
suppressing the expression of neoplasia by cells previously
exposed to doses of a carcinogenic agent. However, the
mechanisms by which retinoids inhibit carcinogenesis are
still unclear. Retinoic acid is thought to act on tissues
by binding to a specific intracellular protein or retinoic
acid receptor (RAR). There are several reports to support
this hypothesis. In one study, 3 human breast carcinomas
and 1 lung carcinoma were shown to contain RAR whereas the
specimens of the adjacent normal tissues did not contain any
detectable RAR (Ong 33 31., 1975). Significantly higher
levels of RAR were reported in cervical, endometrial and
ovarian cancers (Palan and Seymour, 1980) and in the
transitional cell type of bladder tumors in humans (Fagg 33
g” 1982) .

On the other hand, an E1 33.333 study by Jetten (1983)
indicated that retinoids stimulated sarcoma growth factor
(SGF) and 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced
growth of normal rat kidney fibroblast cells NRK 536-3 (SA
6) in soft agar. These results are in contrast to the
observation by Todaro 33 31. (1978) who demonstrated that
retinoids block phenotypic cell transformation produced by
SGF using a different subclone NRK 536-7. Retinoids were
reported to inhibit the cell proliferation of an SV40-

transformed line. However, proliferation of two cell lines

 

s

6‘"

.6.“

‘4.
‘5

‘9
A

0.; ‘.

‘\

.u‘

5.!

17

that were transformed by a Kirsten and Moloney strain of
murine sarcoma virus and produced growth factor into culture
medium was remarkably stimulated by retinoids (Hiragun 33
31., 1983). Hennings _331. (1982) and Chauvenet and Paque
(1982) suggested a potential danger of retinoid therapy when
performed on growth factor producing neoplasms. This
oncogenic effect of retinoids might explain the retinoid-
induced tumor enhancement in hamster cheek pouches initiated
with dimethylbenzanthracene (Levij and Polliack, 1968) and
in the lungs of hamsters initiated with BaP (Smith 33 31.,
1975) .

Comparison Between the Effects 33
Polybrominated Biphenyls and Vitamin 3 Deficiency

 

 

There are several similarities between the effects of
polybrominated biphenyls (PBB) and related compounds and
vitamin A deficiency. For example, PBB toxicosis and
vitamin A deficiency are known to cause a wasting-type
syndrome (Wolbach, 1925; Hayes, 1971; Sleight and Sanger,
1976; Gupta and Moore, 1979).

The thymic involution observed in rats fed FM FF-l at
high doses (Gupta and Moore, 1979) is similar to that
reported to occur in vitamin A-deficient rats (Wolbach,
1925). A similar loss of cortical thymocytes was also
described in tetrachlorodibenzo-p-dioxin (TCDD) toxicosis

(Kociba 33 31., 1979) .

l8

McConnell 33 31. (1979) reported keratinization and
squamous metaplasia of the Meibomian glands in rhesus
monkeys highly contaminated with polychlorinated biphenyls
(PCB). These epithelial changes were similar to those
reported for vitamin A-deficient animals (Wolbach, 1954).

There is evidence that both exposure to PBB and vitamin
A deficiency may play a role in carcinogenesis. Kimbrough
3_t 31. (1978), Kimbrough _e_t 31. (1981) and Jensen _e_t_ _a3.
(1982) reported that PBB are hepatocarcinogenfl:in.rats.
Epidemiological studies have suggested that vita min A
deficiency in humans is associated with cancer of the
stomach (Abels 33 31., 1941) and cancer of the respiratory

tract (Basu 33 31., 1976).

MATERIALS AND METHODS

Experimental Design

Seventy-two male Sprague-Dawley ratsa, weighing
approximately 60 g at 21 days old were used. During 2 days
of acclimation, the rats were fed a regular commercial
pelleted dietb and tap water ad libitum.

The rats were randomly allotted to 4 groups of 18 each.
Rats in these groups were fed diets either deficient in
vitamin A (groups A and B) or containing 30,000 IU retinyl
acetate/kg (groups C and D) for 14 days. At this time (day
0), rats in their respective groups were given either 0
(groups A and C) or 100 mg of PBBC/kg of diet (groups B and
D) for 28 days. The experimental design is illustrated in
Table 1.

The rats were housed 6 to a cage in stainless wire-
top, plastic cages and the bedding was changed once a week.
Room temperature was maintained at ZIJF’C. Lights were

controlled automatically to allow 12 hours of darkness.

 

aCharles River Breeding Laboratories, Inc., Portage,
Michigan.

bTeklad, A Harlan Sprague Dawley, Inc. Co., Winfield,
Iowa.

CFiremaster BP—6, Michigan Chemical Co., St. Louis,
Michigan.

19

20

.ooumoflcca who no ooaaax ouo3 msoum some CH mum“ xflmm

 

o e a mx\mmm as OOH + mx\a.=H ooo.om mx\a SH ooo.om

 

 

 

 

 

 

 

Q
a e a mx\< SH ooo.om mx\< DH ooo.om o
o o o mx\mmm ma OOH + « baofioammo a ucoaoamoo m
o m we < acoHOHwoo m ucoHonoo <
mm «a 5 mm : o sac . o man on «a- was asouo
Axon. :oaumcfleume human
.mx\oumuoom
ahcfluou DH ooo.om nufi3 coucoeoammzm no 4 :fiemufi> ca ucofloamoo nonufio ouoz
scans mx\mmm no me ooa no o magnamucoo muofio new ones wumm .cmamoc Hmncoefiuomxm .H oflnme

21

Feeding Practices

The basal diet for the rats was a commercial vitamin A
deficient dietd. Diets were supplemented with vitamin A by
adding 30,000 IU retinyl acetatee/kg feed to the basal diet.

Cottonseed oil was used as the vehicle for mixing
vitamin A and PBB in the feed. Diets were mixed at the
beginning of the experiment and were refrigerated.

Feed was provided in porcelain containers with
stainless steel tOps. Drinking water was available ad
libitum in inverted bottles with rubber stoppers and
stainless steel sipper tubes. Fresh water was provided
every other day.

Rats were observed daily for clinical signs. Feed
consumption was recorded every other day and body weights

were recorded weekly.

Laboratory Investigation Procedures

 

Collection and Processing 33 Tissues

 

Final body weights were recorded prior to the necrOpsy
and rats were killed with ether anesthesia. A blood sample
was obtained from the heart while the rat was anesthetized.
Serum was removed after coagulation and centrifugation and

was stored at ~25° C until chemical analyses were done.

 

dTeklad Mills, Madison, Wisconsin.

eRoche Chemical Division, Hoffman-La Roche, Inc.,
Nutley, New Jersey.

22

A necr0psy was performed immediately after the animal
was killed. All tissues were examined grossly and the liver
and thymus were weighed with a tOploading balancef at time
of necropsy; The thyroid glands were weighed on an
analytical balance9 after being fixed with 10% neutral
buffered formalin for 24 hours.

Tissues for histologic examination were preserved in
10% neutral buffered formalin. Tissues collected included
eyelid, eye, salivary gland, thyroid, trachea, brain,
thymus, heart, lung, liver, spleen, adrenal gland, kidney,
stomach, intestine, pancreas, urinary bladder and testes.
Liver tissue for vitamin A analysis was wrapped with
aluminum foil and stored at ~25? C until analyses were done.

Formalin-fixed tissues were trimmed, processed in an

Autotechniconh

and embedded in paraffin. The tissues were
then cut with a microtome at 5 u and stained with

hematoxylin and eosin.

Vitamin 3 Analysis
The determination of vitamin A in the serum and liver
tissue was done in the Clinical Nutrition Laboratory,

Department of Large Animal Surgery and Medicine, Michigan

 

fMettler Series P, Model 163 (Readability: 0.01 g),
Mettler Instrument Corp., Highstown, New Jersey.

gModel H-lS (Readability: 0.0001 g), Mettler
Instrument Corp., Highstown, New Jersey.

hHistomatic, Model 165, Fisher Scientific Co.,
Pittsburg, Pennsylvania.

23

State University. Vitamin A was quantitated by the method
established by Stowe (1982) as a modification of the high
performance liquid chromatography procedure described by
Dennison and Kirk (1977).

In preparation for vitamin A quantitation in the serum,
1 ml of serum was mixed with 1 m1 absolute ethanol and
vortexed for 5 seconds to form a suspension of the
denaturated protein. Two milliliters of hexane (68 to 690 C
boiling point) were added to the mixture which was then
vortexed for 1 minute and centrifuged at 3000 rpm for 10
minutes. The hexane supernatant was removed and passed
through a 0.45 micron Millipore filteri in a Swinny-type
filter holder. One hundred microliters of aliquot were
injected into the chromatographic systemj. Separation was
isocratic in a Microporasil columnj (30 x 3.9 cm long) with
a 60:40 mixture of degassed hexane and chloroform and the
mixture was pumped through the HPLC unit at 2.5 ml/minute at
1000 psi. Forms of vitamin A were detected with a

k set at 330 and 470 nm for excitation and

Spectrofluorometer
emission wavelengths, respectively, and equipped with a 35
ul flow cell.
Liver vitamin A concentrations on a dry weight basis
were determined as follows. To determine dry weight of
iMillipore Corp., Bedford, Massachusetts.

JWaters Associated, Inc., Milford, Massachusetts.

kAminco Bowman (J4-8961) Spectrofluorometer, American
Instrument Company, Silver Spring, Maryland.

24

liver, 2 grams of liver tissue were placed in an aluminum
pan and dried in an oven at 56° C. The dried liver tissues
were weighed after 24 hours in the oven. For the quantita-
tion of the vitamin A level, 1 gram of liver tissue was
placed in a large tube and distilled water was added up to 5
ml. The mixture was then homogenized1 and from this
homogenate, 0.5 ml was pipetted into a disposable test tube.
An equal amount of absolute ethanol was added and then the
mixture was vortexed for 5 seconds. For the extraction, 5
ml hexane was used which was then vortexed for 5 minutes and
centrifuged at 3000 rpm for 10 minutes. The hexane super-
natant was removed and the subsequent steps were as

described for serum samples.

Statistical Evaluation

For organ to body weight ratios and weight gain, the
data were analyzed using analysis of variance and Student
'Newmaaneul's test. For vitamin A analysis, the data for
groups A and B or groups C and D were compared by the
Student's t-test. The differences were considered

significant at the level of p<0.05.

 

1Polytron Homogenizer, Kinematica GmBH, Switzerland.

RESULTS

Clinica1 Signs

There were no adverse clinical signs of either vitamin
A deficiency or PBB toxicosis observed in any of the rats
during this experiment. Daily feed intake was not altered

throughout the study.

Body Weight

 

Body weights increased steadily from the start of the
experiment until the experiment was terminated. At the end
of the experiment, the body weights of rats fed a diet
deficient in vitamin A and containing 100 mg of PBB/kg
(group B) were the lowest (273.3 g) when compared to rats
fed a vitamin A deficient diet (group A; 296.6 g)lor to rats
fed a diet supplemented with 30,000 10 retinyl acetate and
containing 0 (group C; 324.7 g) or 100 mg (group D; 314.1 g)
of PBB/kg (Fig. 1).

On day 7, the weight gain of rats in group B was
significantly different from groups C and D (p<0.05), but
not from group A. On day 14, 21 or 28, the weight gain of
rats in group B was significantly different from those in

groups A, C and D (p<0.05).

25

Grams

26

350

I
300 / 3
= ’
I A
”x
250 _ /
/’ ‘
200
9
4”
150 I! 1’
/ I 1
100 _ + PBB
----o DEFICIENTA
50 ——o DEFICIENT A+ 100 mg of PBB/kg
——-0 30,000 lU A
——a 30,000 IU A+ 100 mg of PBB/kg

 

 

-14 -7 O 7 14 21 28
Day

Figure 1. Mean body weights of rats fed diets containing 0 or

1 00 mg of PBB/kg and either deficient in vitamin A
or supplemented with 30,000 IU retinyl acetate/ kg.

27

Organ Weights

 

Organ to body weight ratios are listed in Tables 2, 3
and 4. Administration of PBB caused significant increases
(p<0.05) in the liver to body weight ratios at 7, 14 and 28
days (groups B and D). Thymic weight to body weight ratios
were not significantly different from each other. Thyroid
weight to body weight ratios were significantly increased in
groups B and D on day 7 (p<0.05) as compared to groups A and

C but not in group B on day 14 and not in group D on day 28.

Laboratory Investigation

Serum Vitamin 3

 

Data relating to serum vitamin A concentrations are
tabulated (Tables 5, 6 and 7). After 7, 14 or 28 days of
dietary treatment with 100 mg of PBB/kg, there were no
significant effects on the total vitamin A concentrations in
the sera of rats fed a diet supplemented with 30,000 IU
retinyl acetate/kg (group D). Total vitamin A
concentrations in the sera were still maintained in rats
given 100 mg PBB/kg in a vitamin A-deficient diet for 7 or
14 days (group B). By day 28, PBB caused significant
decreases of the total vitamin A concentrations in the sera
of rats in this group. Decreases were caused mainly by

significant lowering of retinol concentrations (p<0.05).

28

.uonuo some sown uconommao hflucmowmacmflm uozo

.U use d mmDoum scum .mo.ovm. ucmuommao hauCMUAMHcmHmn

.Q can m mmzoum eoum Amo.ovmv acouomuwo haucmoawflcoamm

.Qm H some on commoumxo mum mama

.0 hop :0 a can m mmsoum :H mumu mo muoflc ou cocoa mmms

.1

 

 

 

 

 

 

 

.0
Two Hmucoafiuomxo ou Hoaum name a; a can 0 museum :3.” upon mo muofio ou coccm < cunEmuE/g
be.aus.mfl omo.onmm.o nm.oum.m . ooa ooo.om e o
am.HH~.m omo.onos.o «H.oua.m o ooo.om m o
no.mua.ma omo.ousm.o am.ouo.e ooa o o m
am.ous.m omo.oumm.c m~.oum.m o o m a
ism m coa\me. Asa m ooa\m. 13m m OOH\m. lmx\ms. 1mx\=H. moan asouo
odoumca msewna uo>HA mmm 4 cflemufl> we
as cmucoeoammsm .oz
mofiumu unmflo3 >603 ou :mmuo coflumofiwfiooe uofio
.mwmn h an weapon unmfim3 >oon ou ammuo .m manna

9
2

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.Q can 0 .< mmsoum sown acouowufio waucmonHcmHm uozo

.Q msoum sown Amo.ovm. acouommfio maucmofimacmfimc

.umsuo some souw unoHoMMAc aflucmoawwcmflm uozo

.3 new a masons scum Am0.0va. ucmuouufie saucmoauacmnmn

.Q can m mmsoum sown .mo.ovmv ucououmwo afiucmofiuficmfimm

.mm H come on commoumxo mum mama

.o amp :0 a cam m masoum ca mumu uo muoflc ou cocoa mmma

fl

 

 

 

 

 

 

 

. .o
Two Hmucoefluomxo ou newum mamc «A a new 0 mmsoum ca mumu mo muowc ou cocoa 4 :HEmuHP.
MH.HH~.HH oOO.OHOm.O am.OHO.O OOH OOO.Om O O
33.3Hm.m uOO.OHOm.O mm.Oum.m O OOO.Om O o
mO.HHO.O o~O.O.+.OO.O n~.OHm.O OOH O O m
em.HHm.s omO.OHOm.O ad.Oum.m . O O O a
12m m OOH\OEO 13m O OOH\OO 12m m OOH\O. .mx\me. .mx\OHO mum“ asouo
caoumna unease uo>aq mmm 4 caemufi> no
«a cmucoeoammsm .oz
moflumu unmaoz anon ou cmmno GOAumofluHcoe nowa
.mmoc vH um moflumu ucmfims wcon ou cmmuo .m manna

 

I n~\fl-v—~

u~ an

-«-

b I..\ N

-~\v-a.~3 \fluvauad

DU

- n.‘ 5.

RAM

30

.U can m .< mmsoum souu acoHMMMHo xHucmonwcmHm uozm

.0 cum a museum scum HO0.0vaO acmumOmHO HHuamoHHHcmHmO

.m muons some .O0.0va. ucmumOOHO sHbcmoHOHcmHmm

.uonuo coco scum ucoHoMMHU haucmoHMHcmHm uozo

.O can a masoum some AOO.Ova. unmumOOHO HHucmoHOHcmHmo

.0 new m mmsoum eouw .mo.ovm. acououwflc mHucmoHMHcmHmn

omflzm

.om H name no commoumxo mum mama

.o amp co m can m mmsoum CH mumu mo muoflo 0» cocoa mmm«

.1

 

 

 

 

 

.O
ammo HmucoEHHomxo on HOHHQ mamo «H Q cum 0 mmsoum ca mumu mo muoHo ou cmccm a CHEmuS/e
O.OH.HHO.O OOO.OHmm.O oO.OHO.O OOH OOO.Om O a
OH.HHO.O OOO.O.m~.O nH.O+O.~ O OOO.Om O o
OO.HHO.O OHO.OHOH.O oO.OHO.O . OOH O O m
mOOHOO ONO.OH-.O am.OH~.m O O O O
izm O OOHHOEO 13m m OOH\OO 13m m OOH\O. .mx\ms. .mx\OH. moan asouu
oHouxna moshca uo>wq mmm < :HEmuw> mo
«3 cmucoeonmsm .oz
moflumn unmfloz woos ou ammuo GOODMUHMHGOE umfia

.mmmc am an moHumH unmflmz anon

cu cmmHO .v OHQMB

31

.o mac :0 a cam m mmsoum ca

.0 msoum Scum ucOHOMMHc hHuCMOHMHcmHm uozn

.< msoum scum ucouoHMHc hHDCMOHMHcmHm uo2m

.om H same no commoumxo mum mama

mumu mo mpoflc cu oocom mmm«

k.

 

 

 

 

 

 

 

.O
amp HMHGOEHHomxo ou HOHHQ name «A a 0cm 0 mmsouv cw mumu mo madame ou coccm < GHEmuH?‘
nO.OmHHO.O~O nO.~HHHm.OOm O n~.OaHO.OO OOH OO0.0m O o

O.OHHHO.HOH O.OOHHO.HO~ O m.HOHO.OO O OO0.0m O o
OO.OOHHH.HOH OO.OmHHO.OOm m.OHH.m OH.HHHO.OH OOH O O m
m.HOHH.OOO O.OHHO.OOH O.O~HH.O~ O.HHHH.O~ O O O a
Hmuoa Hocfiuom oumuoo< oumuHEHmm .mx\msv Amx\DH. mumu msouo
HmaHuom Hachmm mmm < :HemuH> Ho
*« « .Hmmsm .oz
AHE\m:. d :Hemufl> asuom coHumonHooe uoHQ
.mhmc h um mcoHumuucoocoo < :HEmuH> mumm .m oHnma

32

.o amp :0 Q cam m museum :H mumu mo muoac ou ooocm mmm«

.U msoum scum #:0HOMMH0 haucmoHMHcmHm uozn

.4 msoum scum acouomch aHucmoHMHcmHm uozm

.Qm H come no commoumxo mum mama

fi

 

 

 

 

 

 

.O
amp HmucOEHHomxo on HOHHQ mmmo OH O can 0 mmsoum ca mumu mo muowo ou coco—O 4 cflemufwe
nO.OOHHm.OOO nH.OOHHO.OOO O.OHHO.~H nO.OHHO.Om OOH OOO.Om O a

O.OOHHH.OHO O.OOHH.OOH O.OHm.O O.O~HO.OO O OOO.Om O o
OO.OOHO.O~O OO.OOHO.mOO ~.HNHO.O OH.~mHm.Om OOH O O m
O.HOuO.O~O m.HOHO.~OO «.mHHH.O O.OHHO.OH O O O 4
Hmuoe Hocfiuom demu004 oumuHEHmm Amx\mev Amx\aav mpmn msouw
HmcHuom HacHuom mmm 4 :HsmuH> mo
44 « .Hmmsm .oz
AHE\mc. 4 :HEmuH> Esuom coHDMOHMHUOE HOOD
.mwmc 4H um mcofiumugcoocoo 4 GHEmHO> whom .o mHnt

33

.0 macho eoum ucoquMHc wHucmOHMHcmHm Oozo

.4 Ozoum some AO0.0vaO ucmnmumHO HHOOOOHOHOOHOO
.o Osoum some AOO.OvOO bcmummuHO HHbcmonHamHmn
.4 msoum scum acoHOMMHc wHucmowmwcmHm uozm

.Qm H cmos mm commoumxm mum mumn

.o amp co Q can m museum :H mpmu mo muoHo ou cocoa mmm««

 

 

 

 

 

 

.O
xmc Hmucoefluomxo ou HoHum mxmc 4H m can 0 mmsoum CH mumu mo muowc ou cocoa 4 CHEOOHPO
OO.OOHHO.OOO OO.OOHHO.OOO O.HHHO.OO OO.OHHO.OH OOH OOO.Om O O

~.OOHO.OHO O.mOHm.~OO 0.0~H0.0~ O.OHHO.NO O OO0.0m O o
oH.OmHO.OO um.OOHO.OO O m~.¢HHm.OO OOH O O m
0.00Hm.Hmm 0.00H0.00H O 0.0HH0.00 O O O 4
Hmuoe HOCHuom oumuoo4 oumuHEHmm Amx\wE.. .mx\DHV wumu msouu
HmaHuom HmcHuom mmm 4 :HemuH> Ho
«4 « .Hmmsm .oz
AHE\m:. 4 GHEMUH> asumm coflumOHMHcOE UOHQ

 

 

.mhoo mm um mcofiumuucoocoo 4 :OEmuH> muom .n manna

34

Liver Vitamin 3

Data for liver vitamin A concentrations are listed in
Tables 8, 9 and 10. PBB decreased hepatic vitamin A
concentrations in rats fed diets either deficient in vitamin
A (group B) or supplemented with retinyl acetate (group D).
In rats in group B at 7 or 14 days and in rats in group D at
7, 14 or 28 days, there were significant decreases of the
vitamin A concentrations in the livers (p<0.05). By day 28,
rats in group B had a significant increase in the vitamin A
concentration in the liver (p<0.05). Although the hepatic
vitamin A concentration (pg/g) in rats in groups B and D
were decreased at 7, 14 or 28 days, calculation of the data
on a mg/whole liver wet weight basis indicated that the
decrease was only significant in rats in group D at 14 or 28
days (p<0.05). By day 28, the hepatic vitamin A (mg/whole
liver) in group B was significantly higher (p<0.05) than in

group A.

Pathology

 

Gross Lesions

 

Gross lesions in rats given PBB were observed at all
time points and were mainly in the liver and thyroid glands.
Vitamin A content of the diet did not influence the
appearance of gross lesions.

31333: At 7, 14 or 28 days, livers of rats treated

with 100 mg of PBB/kg of diet (groups B and D) were

35

.0 QDOHm E0um ucoHOHNHU >Hucmowwwcmwm 302

U

.4 mucum 80uw ucmuowwHo xHucmoHuHcmHm uozo

.o macho scum HO0.0vOO OcmumOOHO HHOOOUHOHOOHOO

.4 accuo scum Hmo.ovmv acouommwc >HucmonHcmHmm

.om H come mm commmumxo one come

.o amp co a can m mascum cH mumH mo muowc OD compo mma««

.o >mo HancoEHuomxo ou HoHum mamo 4H 0 can 0 mascum cw mumu mo muowo o» cocoa 4 cHEmuH>«

 

 

 

 

 

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O.HHH.O H.OOOHO.O~OH O.~HHO.OO O ~.OOOHO.OOOH O OOO.Om O o
UHHH.OHHOH.O OO.OHN.OH OO.OHO.O O.OHH.O OO.OHO.OH OOH O O O
OOO.OHOOH.O O.OH~.OH ~.~HH.O O m.OH~.Om O O O a
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HMUOB COMUMHHCOUCOU HOCmuwm Oumumvd OUMUMEHQQ «immm 4 CMEMUM> MO
chwuom HacHuom mecoEonmsm HonEsz
4 cHEmuH> um>ma coflumonwcoE umHo
.mxmc h um mcoHumHucoocoo 4 cHEmuH> uo>Ha .m mHnme

36

.o xmc Hmucoewuomxm

.U accum Ecuu ucwuwmuwo wHucmonHcmHm uoz

D

.4 macaw Eouu acmuouch >HucmonHcmHm uozo

.U macaw Ecum Hmo.ovmv Demuomufio hHucmonHcmHmn

.O OOOHO scum AOO.OvO. acmumOOHO HHOOOOHOHOOHOO

.om H come we commoumxo mum come

.0 amp co m can m museum CH mumu mo muowp cu cocoa mmm««

cu HoHum wasp «H a can 0 mascum cH mumu mo mumHo ou cocoa 4 CHEOHH>«

 

 

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O.OHO.HH O.OOOHO.OOOH H.HH~.OH O O.OOnHO.OOOH O OOO.OH O O
UHOO.OH~HH.O OO.~HO.O om.OH~.O m.mHO.H mO.OHO.H OOH O O O
HOO.OHOOH.O O.OHO.OH m.OHO.O O O.nH~.OH O O O a
iuo>HH\meO Hm\mnv HO\OOO Hm\mzv Hm\mnv me\msv me\OHO mumu OOOHO
Hmuoe cowumuucmocou Hocwumm mumuoo4 mumuHEHmm «cmmm 4 CHEmuw> mo
HacHuom Hacwumm omucoEonmsm Honesz

 

4 CHEmuH> uo>Ha

 

coHumonHooE umHo

 

.mmmp «H um mcoHumHucoocoo 4 cHEmuH> Ho>Hq

.O OHOOO

7
3

.o xmo Hmucwewummxo ou Howum mxwo «H a

.4 macaw Eon Hmo.ovmv ucmHOMLHQ >HucmonHcmHmo

.U msoum Eoum Hmo.ovmv ucmumumHn mHucmoHuHcmHmn

.4 meon EoHu uncummuwc mHucmonHcmHm uozm

.om H come no pommmumxm one name

.0 >m© no.0 can m mascum CH mum» mo muowo Cu cocoa mmm«e

can u mmsoum cw mumu mo muoHo cu coupe 4 cHEmuH>«

 

 

 

 

 

OOOHTHH OOONNHOOOO OOOHTO O OOONNHNOOO OOH OOO.OH O O
O.OHO.ON O.OOOHOOOH O.~H0.0H O O.OOOHO.OOON O OOO.OO O o
UOHOOHOHHO oOOHHO O oH.OHO.O OO.OHO.O OOH O O O
OOO.OHONO.O O.HHO.~ O 0.0HH.H H.HH~.H O O O O
HOO>HH\OEO HO\OOO HO\OOO .O\m:. HO\OOO me\msv ‘ me\OHO mum“ OOoOo
HMUOB COquHuCOOr—OU HOCMUO“ 0UNUOU¢ wumquHmm {immm C CwEmuwxr NO
HacHumm chwumm cmucmeHmmsm HonEsz
4 cHEmuH> Ho>ma .coHumonflooE uoHQ
.m>m© mm um mcoHumuucoocoo 4 CHEmuH> uw>wq .oH oHnme

38
enlarged, had a whitish mottling and a slight yellow
discoloration.
Thyroid: At 7, 14 or 28 days, dietary treatment with
100 mg of PBB/kg caused the thyroid glands to appear
slightly larger than those that had dietary treatments

without PBB and have a deep brown discoloration.

Histopatholggy

 

31y33: Livers of rats fed diets either deficient in
vitamin A (group A) or supplemented with 30,000 10 retinyl
acetate/kg (group C) were normal (Fig. 2). By days 7, 14 or
28, the addition of 100 mg of PBB/kg to groups B and D
caused enlarged hepatocytes and cytoplasmic vacuolation
(Fig. 3). Multifocal areas of necrosis with lymphocyte
accumulation occurred in 1 rat in group B at 7 days and in 1
rat in group D at 28 days (Fig. 4).

Bile duct: PBB did not cause any changes in the bile

 

ducts of rats in group D (Fig. 5). However, at 14 and 28
days, the bile ducts of rats fed a vitamin A-deficient diet
with 100 mg of PBB/kg (group B) had hyperplasia of the
epithelial surfaces and pyknotic nuclei. At 14 days, 3 of 6
rats in group B had slight hyperplasia of the bile ducts
(Fig. 6), while at 28 days, there was mild (4 of 6 rats),
moderate (1 of 6 rats) or marked (1 of 6 rats) hyperplasia
of the bile ducts (Fig. 7, 8 and 9). There were no bile
duct lesions observed in this group at 7 days. Only 1 of 6

rats in group A at 28 days had mild hyperplasia of the bile

39

duct, while no histologic lesions of the bile ducts were
observed in any rats before this time.

Thyroid: Lesions in the thyroid gland were observed
only in rats in group B at 28 days. The lesion was
characterized by early evidence of squamous metaplasia of
the ultimobranchial follicles (Fig. 10). Rats from the
other groups did not have any changes in the thyroid.

Salivary gland: As with the thyroid, histologic

 

lesions of the salivary gland were observed only in rats in
group B at 28 days. There was early evidence of squamous
metaplasia of larger ducts (Fig. 11). There were no
histologic lesions observed in any of the rats in the other
groups.

Lesions were not seen in histologic sections from
eyelid, eye, trachea, brain, thymus, heart, lung, spleen,
adrenal gland, kidney, stomach, intestine, pancreas, urinary

bladder and testes.

40

Figure 2. Photomicrograph of a liver from a rat fed a
vitamin A-deficient diets Notice the normal lobular pattern
with hepatic cords radiating from the central vein toward

the portal triads, compact cytoplasm of hepatocytes and
prominent sinusoids. (H & E stain, 450x.)

Figure 3. Photomicrograph of a liver from a rat fed a
vitamin A-deficient diet and necropsied after 7 days of
dietary treatment with 100 mg of PBB/kg. Notice swollen
hepatocytes and numerous cytoplasmic vacuoles of various

sizes in the central and midzonal areas. (H & E stain,
450x.)

41

 

Figure 3

42

Figure 4. Photomicrograph of a liver from a rat fed a
diet supplemented with 30,000 IU retinyl acetate/kg after 28
days of dietary treatment with 100 mg of PBB/kg. Notice an
area of necrosis with accumulation of lymphocytes. (H & E
stain, 450x.)

Figure 5. Photomicrograph of an extraparenchymal bile
duct from a rat fed a diet supplemented with 30,000 IU
retinyl acetate/kg after 28 days of dietary treatment with
100 mg of PBB/kg. Notice the normal columnar epithelial
cells lining the duct. (H & E stain, 450x.)

43

 

4

Figure

 

Figure 5

44

Figure 6. Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient diet after 14 days
of dietary treatment with 100 mg of PBB/kg. Notice the

slight epithelial hyperplasia with pyknotic nuclei. (H & E
stain, 450x.)

Figure 7. Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient diet after 28 days
of dietary treatment with 100 mg of PBB/kg to illustrate
mild epithelial hyperplasia with pyknotic nuclei. Notice

hyperplasia of the epithelial cells as compared to Figure 6.
(H & E stain, 450x.)

45

 

Figure 6

 

Figure 7

46

Figure 8. Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient diet after 28 days
of dietary treatment with 100 mg of PBB/kg to illustrate
moderate epithelial hyperplasia with pyknotic nuclei.

Notice hyperplasia of the epithelial cells as compared to
‘Figures 6 and 7. (H & E stain, 450x.)

Figure 9. Photomicrograph of an extraparenchymal bile
duct from a rat fed a vitamin A-deficient diet after 28 days
of dietary treatment with 100 mg of PBB/kg to illustrate
marked epithelial hyperplasia with pyknotic nuclei. Notice
the papillary projections of the hyperplastic epithelial
cells into the lumen of the duct. (H & E stain, 450x.)

47

 

Figure 9

1_______-IIIlIIIIIIIIIIIIII'I'IIIALLLL,

48

Figure 10. Photomicrograph of a thyroid gland from a
rat fed a vitamin A-deficient diet after 28 days of dietary
treatment with 100 mg of PBB/kg to illustrate early evidence
of squamous metaplasia, Notice keratinization of the
ultimobranchial follicles. (H & E stain, 576x.)

Figure 11. Photomicrograph of a salivary gland from a
rat fed a vitamin A-deficient diet after 28 days of dietary
treatment with 100 mg of PBB/kg to illustrate early evidence
of squamous metaplasia. Notice squamous type of epithelial
cells lining portions of the duct instead of the usual
cuboidal or columnar type of epithelial cells. (H & E
stain, 576x.)

49

 

Figure 10

 

Figure 11

DISCUSSION

In most research on the effects of toxic chemicals in
laboratory animals, the animals are fed a diet adequate in
vitamin A. The results of many experiments have shown that
vitamin A status has an important influence on the toxic
effects of environmental contaminants (Innami 33 31., 1974;
Darjono, 1982; Thunberg 33 31,, 1983). In the studies noted
above, dietary modifications were made when the experiments
were started. ‘Vitamin A deficiency is still a serious
veterinary and public health problem (WHO, 1967; Srikantia,
1982). Therefore, people or animals are often depleted in
vitamin A when exposed to chemicals such as PBB or related
compounds. In the present study, an attempt was made to
simulate natural exposure of animals to chemicals such as
PBB when they are marginally deficient in vitamin A. This
was done by feeding a vitamin A-deficient diet for 14 days
prior to the administration of PBB.

Although administration of PBB accelerated the
depletion of total vitamin A in the sera in rats fed a
vitamin A-deficient diet (group B), neither weight loss nor
clinical manifestations of vitamin A deficiency was observed
during the time frame of the study. The lowest body weight
was observed in rats in group B as compared to the other

groups. By day 14 and thereafter, the weight gain of rats
50

51
in group B was significantly lower than for the other groups
(p<0.05). It is possible that the combined effect of a
vitamin A-deficient diet and PBB adversely affected the
weight gain. 'Vitamin A deficiency impairs animals' ability
to utilize food, especially protein (Hayes, 1971). PBB
could cause defective absorption of nutrients from the gut
based on the fact that tetrachlorodibenzo-p-dioxin (TCDD)
was reported to impair active intestinal absorption of
glucose and leucine in male Sprague-Dawley rats (Ball and
Chhabra, 1981).

The gross and histologic effects of PBB on the liver
were similar to those previously reported (Sleight and
Sanger, 1976) and were not diminished by vitamin A
supplementation. PBB increased the liver weight to body
weight ratios (p<0.05) at 7, 14 or 28 days. Histologically,
there were enlarged hepatocytes and cytoplasmic vacuoles.
In general, a similar manifestation was seen in the thyroid
weight to body weight ratios. Thyroid weight was increased
in rats in groups B and D. Decreases in thymic weight to
body weight ratios and histopathological changes in the
thymus were not observed in this study. Darjono (1982)
demonstrated that rats fed a vitamin A-deficient diet with
100 ppm PBB had the lowest thymic weight when compared to
those fed diets containing either adequate or supplemented
vitamin A and 100 ppm PBB. In Darjonods study, however,
diets were fed until clinical signs of vitamin A deficiency

and death losses occurred in rats fed vitamin A-deficient

52
diets containing 100 ppm PBB. It is likely that in his
experiments, thymic involution was due mainly to vitamin A
deficiency and PBB simply accelerated depletion in vitamin
A.

In the present study, rats were fed diets either
vitamin A-deficient or supplemented 14 days prior to
experimental day 0. In rats fed a vitamin A-deficient diet
(group A), the hepatic vitamin A concentrations decreased
with time (Tables 8, 9 and 10). By day 28, when the hepatic
vitamin A concentration was markedly depleted (2.3 ug/g),
the vitamin A concentration in the serum was 231.3 ng/ml as
compared to the serum vitamin A concentrations of 408.1
ng/ml on day 7 or 528.0 ng/ml on day 14. Therefore, serum
vitamin A was still maintained during the period that liver
vitamin A stores became severely depleted. Although hepatic
vitamin A stores were nearly depleted, only 1 rat in group A
had histologic lesions suggestive of vitamin A deficiency at
28 days.

In rats, a vitamin A-deficient diet should be
supplemented with approximately 2000 IU vitamin A/kg to
attain satisfactory weight gain, Optimal longevity and
hepatic vitamin A storage (Anonymous, National Academy of
Sciences, 1972). Supplementation of the vitamin A-deficient
diet with 30,000 IU retinyl acetate/kg (group C) markedly
increased the hepatic vitamin A concentrations (from 37.2
ug/g to 1724.9 ug/g at 7 days, 19.8 ug/g to 1503.0 ug/g at

14 days and 2.3 ug/g to 2853.0 ug/g at 28 days). Hepatic

53

vitamin A concentrations were much higher than those
reported by Darjono (1982). He reported a hepatic vitamin A
concentration of 97.9 ug/g in rats fed a diet supplemented
with 30,000 IU retinyl palmitate/kg for 66 days. The total
serum vitamin A concentrations varied between the specified
times: 7, 14 or 28 days. By day 7, the concentration of
the serum vitamin A in group C (361.0 ng/ml) was nearly the
same as in group A (408.1 ng/ml). However, by days 14 and
28, group C had higher serum vitamin A concentrations (836.1
ng/ml and 614.3 ng/ml, respectively) than group A (528.0
ng/ml and 231.3 ng/ml, respectively). Serum vitamin A
concentrations in group C at 14 or 28 days were also higher
than those reported by Darjono (1982). He reported a serum
vitamin A concentration of 456.0 ng/ml in rats fed a diet
supplemented with 30,000 IU retinyl palmitate/kg for 66
days. Supplementation of the vitamin A-deficient diet with
retinyl acetate was apparently more effective in maintaining
vitamin A concentrations when compared to dietary
supplementation with retinyl palmitate. It is possible that
the retinyl acetate used in this experiment was more stable
than the retinyl palmitate used by Darjono. It is also
possible that retinyl acetate is more easily absorbed when
compared to retinyl palmitate. Darjono's.rats were somewhat
older but this would still not account for any major
differences in vitamin A storage or utilization.

PBB caused significant decreases in total liver vitamin

A (mg/whole liver) only in the vitamin A-supplemented rats

54
at 14 and 28 days. Chemicals such as PBB commonly increase
liver size. Values calculated on a ug/g basis may not be an
accurate indication of total liver vitamin A. For example,
rats in certain groups had a decreased concentration of
hepatic vitamin A, but their total hepatic vitamin A was no
lower than in the control livers. Therefore, in evaluating
hepatic vitamin A associated with exposure to PBB, the total
hepatic vitamin A (mg/whole liver) must be considered in
addition to the concentration per unit of weight.

In the present study, the decrease of the hepatic
vitamin A concentrations was due to the decrease of retinyl
palmitate concentrations. The retinyl palmitate
concentrations were nearly totally depleted in rats fed a
vitamin A-deficient diet (group A). Addition of PBB to
diets of rats fed a vitamin A-deficient diet (group B)
resulted in the appearance of significant amounts of retinyl
acetate in the livers (from 0.0 to 3.1 ug/g at 7 days, 0.0
to 7.5 ug/g at 14 days and 1.1 to 7.8 ug/g at 28 days).
Similar effects were observed by Darjono (1982). As a
result, by day 28, the total hepatic vitamin A (mg/whole
liver) in group B was significantly higher (p<0.05) than in
group A. Retinyl acetate was not detected in rats fed a
diet supplemented with vitamin A and containing PBB (group
D). In the normal rat liver, there is primarily retinol and
retinyl palmitate which concentrates mainly in the fat
storing cells. Ninety-five per cent of the total vitamin A

is stored in the liver, predominantly in the form of retinyl

55
palmitate (De Leeuw 33 31., 1983). Since the source of
retinol for peripheral target tissues is from the liver via
hydrolysis of retinyl palmitate, it is possible that an
increased turnover of hepatic retinyl palmitate may explain
the mechanism of PBB-induced vitamin A deficiency. PBB may
alter reesterification of retinyl esters in the liver and
lead to an accumulation of retinyl acetate. The effects of
PBB on vitamin A metabolite concentrations could occur at
either the intestinal or hepatic levels. More likely, the
action of PBB on hepatocytes causes an increased production
of the esterification enzyme which favors retinyl acetate
production. Simultaneously, there could also be a
depressing effect upon hepatocytes which favor the enzyme
associated with formation of retinyl palmitate. Whether or
not the accumulation of retinyl acetate in the liver is
harmful to hepatocytes is unknown. Additional research,
however, is needed to elucidate the importance of retinyl
acetate in PBB exposure.

PBB accelerated depletion of serum vitamin A
concentrations in rats in group B to the extent that
histologic lesions of vitamin A deficiency develOped before
noticeable clinical signs of vitamin A deficiency occurred.
Rats in group B (3 of 6 rats at 14 days and all rats at 28
days) had histologic lesions suggestive or typical for
vitamin A deficiency. The histologic lesions consisted of
early hyperplasia of the bile ducts and early evidence of

squamous metaplasia of the ultimobranchial follicles in the

56

thyroid gland and of larger ducts of the salivary gland.
Since rats from the other groups had no lesions in the bile
ducts, thyroid or salivary glands, the lesions in rats in
group B are likely related to the interaction between PBB
toxicosis and vitamin A deficiency.

The results are very important to those in human and
animal health because they provide further evidence of the
ability of PBB or related compounds to accelerate the
depletion of vitamin A. Therefore, such chemicals can
induce histologic lesions and clinical signs suggestive of
vitamin A deficiency. These effects could be more rapid and

severe in vitamin A-deficient animals or people.

SUMMARY

Seventy-two male Sprague-Dawley rats, weighing
approximately 60 g at 21 days old were allotted to 4 groups
of 18 each. Two weeks prior to the beginning of the
experiment, rats in 2 groups (A and B) were fed a vitamin A-
deficient diet whereas the remaining 2 groups (C and D) were
fed a diet supplemented with 30,000 IU retinyl acetate/kg.
Starting on day 0, rats were given either 0 (groups A and C)
or 100 mg of PBB/kg of diet (groups B and D). Six rats in
each group were killed at days 7, 14 and 28, respectively.

There were no clinical signs of either PBB toxicosis or
vitamin A deficiency observed during the experiment. Feed
intake was relatively normal. However, rats fed a vitamin
A-deficient diet containing 100 mg of PBB/kg had the lowest
body weight when the experiment was terminated. Weight gain
was significantly depressed in this group.

Liver weights were greatly increased in rats fed a diet
containing 100 mg of PBB/kg (groups B and D). Histo-
logically, there were enlarged hepatocytes and cytoplasmic
vacuolation. The gross and histologic effects of PBB on the
liver were not diminished by vitamin A supplementation.
Thyroid weight was increased in rats in groups B and D as

compared to rats in groups A and C.

57

58
Thymic weight was not affected by PBB toxicosis or by
vitamin A deficiency in the present study.

Three of six rats fed a vitamin A-deficient diet
containing PBB for 14 days had slight hyperplasia of the
extraparenchymal bile ducts. By 28 days, the vitamin A-
deficient diet containing PBB induced mild to marked
hyperplasia of the epithelial cells lining the extraparen-
chymal bile ducts. Evidence of early squamous metaplasia
was seen in the ultimobranchial follicles of the thyroid
gland and the larger ducts of the salivary gland.

Analysis of the serum and hepatic vitamin A indicated
that rats fed a vitamin A-deficient diet containing PBB
(group B) had marked depletion in the retinol concentration
in the serum on day 28 and in the retinyl palmitate
concentration in the liver on days 14 and 28. Group B also
had an abnormal vitamin A profile in the liver manifested by
the appearance of significant amounts of retinyl acetate on
days 7, 14 or 28 days. This disturbance of vitamin A
metabolism in the liver may be related to an alteration in
the reesterification of retinyl palmitate.

This experiment has shown that dietary levels of
vitamin A have an important influence on effects of exposure
to PBB. The combined effect of a high dose of PBB and low
vitamin A in the diet greatly accelerated the depletion of
serum and hepatic vitamin A concentrations and therefore
induced squamous metaplasia of the thyroid and salivary

glands and hyperplasia of the bile ducts.

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VITA

VITA

The author was born in Tuban, East Java, Indonesia, on
June 20, 1952. He graduated from the Faculty of Veterinary
Medicine, Gadjah Mada University in January, 1978, and was
appointed as teaching assistant at the Pathology Department
in the same university following graduation.

He got a scholarship from the Ministry of Education and
Culture for Indonesia called "Bakat dan Prestasi" from 1975
through 1978 and was also awarded the "Lawton Award" from
Dr. Kirkpatrick Lawton, Michigan State University in 1975.
He was the outstanding student in his class as a
professional in 1977 and as a veterinarian in 1978.

He was awarded a fellowship by the Rockefeller
Foundation and was admitted as a graduate student in the
Department of Pathology, Michigan State University in the
Fall of 1982. He received a Master of Science degree in
1984.

The author is happily married to Dr. Rr. Hastari

Wuryastuti.

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