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This is to certify that the

thesis entitled

Effect of Acute Polybrominated Biphenyl
Treatment or Orchidectomy on Hypothalamic
and Hepatic Androgen Metabolism

presented by

Donald Leroy Nilke

has been accepted towards fulfillment

of the requirements for
Master of d . Pharmacology and
egree 1n
Science Toxicology

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Major professor

 

 

 

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EFFECT OF ACUTE POLYBROHINATED BIPHENYL
TREAIMENT OR ORCHIDECTOMY ON HYPOTHALAMIC

AND HEPATIC ANDROGEN METABOLISM

BY

Donald Leroy Wilke

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Pharmacology

1982

c§/&cmae7

ABSTRACT
EFFECT OF POLYBROMINATED BIPHENYL

TREATMENT OR ORCHIDECTOMY ON HYPOTHALAMIC
AND HEPATIC ANDROGEN METABOLISM

BY
Donald L. Wilke

Reproductive effects seen in PBB exposed animals may be
related to increased steroid metabolism. To determine
whether PBB effects on reproduction also involved the
hypothalamus, hypothalamic androgen aromatization (HA) was
measured. Rats were treated with a single dose of
Firemaster BP-6 (150 mg/kg i.p.), assigned to a control
group, or orchidectomized at 21 days of age. HA activity
was measured in adult rats by incubation of hypothalamic
homogenate with 3H-androstenedione, and the 3H-estrone
produced was measured by liquid scintillation counting
following purification by two successive HPLC steps. in
gitgg incubations of testosterone with liver microsomes
confirmed PBB induction of steroid metabolism. HA activity
in control males was approximately 3 times greater than the
activity in control females. Prepubertal castration caused
a decrease in HA activity in male rats but not to levels
seen in female rats. PBB treatment resulted in a trend

toward decreased aromatization in male and female rats.

TABLE OF CONTENTS

List Of TableSo O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O iv
List Of Figures. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O v
--TEXT PAGES--

INTRODUCTION — General Introduction to Thesis.... 1-17

Polybrominated Biphenyls as Reproductive
TOXicantSOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Reproductive Consequences of End-Organ
Metabolism of Testosterone.......... 6

Experimental Hypothesis and Objectives.... 14

SECTION I — Assay Development.................... 18-36

INTRODUCTION...OOOOOOOOOOOOOOOOOOOOOOOOOOOO 18
METHODS
Development of HPLC Systems........... 21

Preliminary Studies with Placental
Microsomes........................ 24

Purification of Tritiated

Androstenedione................... 25
Incubation of Hypothalamic Tissue..... 28
Method Validation..................... 30

RESULTS AND DISCUSSION..................... 32

SECTION II - Effects of Orchidectomy or PBB
Treatment on Hepatic Testosterone Metabolism..... 37-50

INTRODUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 37

ii

METHODS
Animals and Treatment................. 40

Preparation of Liver Microsomes....... 40

Determination of In Vitro Testosterone

MetabOIismOOOOOOOOOOOOOOOOOOOOOOOO 41
High Performance Liquid Chromatography 42
Statistical Analysis.................. 42
RESULTS
Sex Differences in Testosterone
oxj-dationOOOOOOOOOOOOOOOOOOOOOOOOO 43
Effect of PBS Treatment............... 43
Effect of Castration on Testosterone
MetabOIismOOOOOOOOOOOOOOOOOOOOOOOO 46
DISCUSSIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 48

SECTION III - Effect of Orchidectomy or Acute PBB
Treatment on Hypothalamic Aromatase Activity..... 51-61

INTRODUCTION............................... 51
METHODS
Animals and Treatment................. 54
Assay of Hypothalamic Aromatase....... 54
Statistical Analysis.................. 55
RESULTS.................................... 57
DISCUSSION................................. 60

SUMARY AND CONCLUSIONSOOOOOOOOOOOOOOOOOOOOOOOOOO 62-64

LITERATURE CITED...oooooooooooooooooooooooooooooo 65-73

iii

LIST or TABLES

TABLE

1 Purity of Estrone Produced by Aromatization
Following Reverse Phase Chromatography with
35% THFOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

2 Products of oxidative testosterone metabol-

ism in Vitro by livers of orchidectomized,
control, and P88 treated rats..............

Effect of orchidectomy or PBB treatment at

21 days on the hypothalamic aromatase
activity in adult male and female rats.....

iv

PAGE

34

45

59

LIST OF FIGURES

FIGURE PAGE
1 Formation of active metabolites of

testosteroneOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 8
2 Intermediates formed in the aromatization

Of androstenedioneOOOOOOOOOOOOOOOOOOOOOOOOO ll
3 Interactions between the hypothalamus/

pituitary, gonadal steroid synthesis, and

hepatic steroid metabolism................. 15
4 Chromatograms of reference steroids........ 22
5 Radiochromatogram of productslgf incubation

of placental microsomes with C-testo-

steroneOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 26
6 Radiochromatogram of fractions collected

from system B chromatography of zero-time
blank and hypothalamic homogenate

inCUbationooooooooooooooooooooooooooooooooo 33
7 Time and protein dependence of incubation

of hypothalamic homogenate................. 36
8 Products of oxidative testosterone metabol-

ism in vitro by livers of orchidectomized,

control, and P83 treated rats.............. 44
9 Total testosterone metabolism in vitro by

liver microsomes from orchidectomized,

control, and P38 treated rats.............. 47
10 Summary of hypothalamic aromatase assay.... 56
11 Effect of orchidectomy or PBB treatment at

21 days on the hypothalamic aromatase

activity in adult male and female rats..... 58

INTRODUCTION

General Introduction to Thesis

INTRODUCTION

Polybrominated Biphenyls as Reproductive Toxicants
Michigan's polybrominated biphenyl (PBB) contamina-
tion problem began during 1973 with the accidental
incorporation of a commercial PBB mixture into cattle
feed. Accidental shipment of 'Firemaster BP-6' from the
St. Louis, Michigan plant of Michigan Chemical Corporation
resulted in the incorporation of polybrominated biphenyls
into at least three lots of cattle feed produced by the
Michigan Farm Bureau. Estimates of PBS concentration in
the contaminated feed have ranged as high as 13,500 ppm
(Kay, 1977). Feed consumption by cattle, and the
subsequent distribution of contaminated meat and dairy
products throughout the state have resulted in the
exposure of most of Michigan's population to P38.
Toxicological studies have determined that the number of
Michigan residents with detectable PBB levels ranges from
as low as 43% in the upper peninsula to as high as 96% in
the lower peninsula (Eyster, 1976). Following the
discovery of the PBS contamination in 1973, scientific and
anecdotal reports of adverse human and animal health

1

2
effects of P835 began to occur.

The study of the P38 contamination has been
complicated by the fact that PBBs, like the chemically and
toxicologically related PCBs (polychlorinated biphenyls),
are not produced or distributed as single compounds, but
rather as complex mixtures of polyhalogenated biphenyl
compounds. In addition, other non-biphenyl constituents
such as polybromonapthalenes or polybromodibenzofurans may
be present (Dent, 1978), or may be formed during cooking
or processing of PBS contaminated foods (O'Keefe, 1978).
Firemaster BP-6 is a mixture of at least 18 FEB congeners
of varying degrees of bromination (DiCarlo et al, 1978).
The major congeners present in Firemaster BP-6 are
2,4,5,2',4',5'—hexabromobiphenyl (approximately 60%) and
2,3,4,5,2',4',5'heptabromobiphenyl (ll - 27%) (Dent,
1978). Tetra-, penta-, octa-, and higher bromobiphenyls
are also present (Harris et al, 1978).

The effects of polybromobiphenyls on human and animal
health have been the subject of some controversy. Uncon-
firmed reports of non-specific toxicosis and malaise in
both exposed humans and cattle have been reported in the
popular press, and in the scientific literature. High
dose feeding of P333 to cattle have resulted in anorexia,
diarrhea, salivation and lacrimation, fetal death,
”general depression", skin, hoof, and kidney lesions, and
elevated SGOT, SGPT, and BUN (Durst et a1, 1978; Roble et

al, 1978). Anecdotal reports of P83 toxicosis in humans

3

have listed symptomatology including numbness, nausea,
weight loss, anorexia, hepatitis, fainting, headache,
fatigue, irritability, depression, acne, and a variety of
other symptoms (DiCarlo et a1, 1978). Michigan Department
of Public Health studies found ”...no consistent pattern
of illness or symptoms..." (DiCarlo et al, 1978), but
subsequent human studies have identified at least a few
health effects probably linked to PBS ingestion. Toxico-
logical studies of PBS exposed Michigan residents have
revealed immuno-deficiencies in both humoral and cell-
mediated responses (Bekesi, 1979), and increased incidence
of halogenacne among exposed individuals (DiCarlo et al,
1978). Other studies have shown a general impairment of
good health among exposed children (Barr, 1978), and a
variety of clinical abnormalities in liver function
(DiCarlo et a1, 1978) in exposed adults. The clinical
picture is a complicated one, however, and some investi-
gators still maintain that there exists '... no relation-
ship between PBB levels and physical or laboratory find-
ings...[exposed individuals have]...few objective findings
and reactive depression may be responsible for the high
prevalence of constitutional symptoms.” (Stross, 1981).

PBBs are known potent inducers of hepatic and extra-
hepatic mixed-function oxidase (MFO) enzyme metabolism
(McCormack et a1, 1978; Dent, 1978; Kluwe et a1, 1978;
Goldstein et a1, 1979; Dent et a1, 1978; Robertson et a1,

1981; Dent et a1, 1976; Babish and Stowesand, 1977). The

4

FEB mixture, Firemaster BP-6 is an established ”mixed"
inducer of hepatic mixed function oxidases, with an effect
similar to the simultaneous administration of both pheno-
barbital (PB), a P-450 inducer, and 3-methy1cholanthrene
(3-MC), a P-448 inducer. Treatment with PCB mixtures
results in a pattern of MFO induction virtually identical
to that seen with combined phenobarbital and 3-MC treat-
ment (Ryan et a1, 1977). Microsomal proteins produced by
PBB treatment, however, have been shown to differ slightly
from those produced by concurrent phenobarbital and 3-MC
treatment by SDS-PAGE electrophoresis (Dent, 1978).

Treatment of animals with proven inducers of MFOs
such as phenobarbital or PCBs has led to increased micro-
somal metabolism of steroid hormones (Nowicki and Normal,
1972; Orberg and Kihlstrom, 1973; Orberg and Lundberg,
1974; Derr, 1978; Sanders et al, 1974). This increased
steroid metabolism may be responsible in part for the
alterations in reproductive functions seen in PCB treated
animals. In rodent systems, PCBs have produced uterine
atrophy, reductions in plasma progesterone concentration,
lengthened estrous cycles, and additional reproductive
effects (Jonsson et a1, 1976; Kimbrough et a1, 1978;).
Recent reports in the literature have shown that admin-
istration of phenobarbital during development results in
permanent reproductive deficits (Gupta et a1,1980; Gupta
et a1, 1982) including permanent reductions in circulating

testosterone levels.

5

The MFO inducing properties of PB and PCBs are shared
by PBBs. Several endocrine-related alterations observed
in animals treated with PBBs suggest that these compounds
modify endogenous steroid sex hormone metabolism to the
extent that reproductive function is diminished. Female
rats exposed perinatally to PBBs had delayed vaginal open-
ing (Harris et al, 1978) and displayed lengthened estrous
cycles (Johnson et a1, 1980). Rhesus monkeys also display-
ed lengthened menstrual cycles which were associated with
flattened and lengthened serum progesterone peaks
(Lambrecht et a1, 1978). Rhesus monkeys exposed to PBBs
in the diet had hypoactive seminiferous tubules (Allen et
a1, 1978) and young bulls fed PBB had testicular atrophy
and reduced spermatogenesis (Jackson and Halbert, 1974).

PBBs have been shown to attenuate the biological
response to a variety of steroid hormones by increasing
their microsomal metabolism. A marked reduction in the
time of progesterone anesthesia in rats was noted concur-
rent with PBB induction of progesterone lGa-hydroxylation
(McCormack et al, 1979; Arneric et al, 1980). The utero-
tropic action of exogenously administered estradiol and
estrone was decreased by PBB treatment, and the increase
in cytosolic estrogen receptors normally seen with

estradiol administration was blunted (Bonhaus et a1,1981).

6

Male reproductive dysfunction has also been found
with PBB administration. PBB treatment has resulted in
decreased seminal vesicle and prostate weights (Johnston
et al, 1980), an effect which may have been mediated by
increased hepatic testosterone metabolism. PBBs have been
shown to increase oxidative testosterone metabolism in
33359 in an age and sex dependant manner (Newton et al,
1982), and to decrease Sa-reduction of testosterone in
both male and female rats. Decreased efficacy of reproduc—
tive steroid hormones due to increased metabolism is one
possible mechanism for the reproductive deficits seen in
PBB treated animals. If increased metabolism of circulat-
ing steroids, particularly androgens, occurred during the
critical period of sexual differentiation (see below) the
reproductive deficits seen with PBB and with other MFO
inducers could be permanent, due to effects on brain dif-
ferentiation or decreases in cytosolic androgen receptors

in peripheral reproductive tissues.

Reproductive Consequences of End-Organ Metabolism of
Testosterone

The effects of reproductive hormones such as
testosterone fall into one of two catagories:

1. Direct effects of testosterone on reproduction in

normal or castrated adult animals, and

7

2. Effects of testosterone resulting from perinatal
sexual differentiation or "imprinting" of target organs
such as brain and secondary sex structures.

A number of effects seen in adult animals require
perinatal sexual differentiation or "imprinting" by
testosterone (or the absence of testosterone in the
female). Testosterone imprinting often requires the
metabolic activation of the androgen. Generally, target
organ metabolism of testosterone forms one of two active
species: Sa-dihydrotestosterone or estradiol (see Figure
l). Sa-Reduction of testosterone to form Sa-dihydro-
testosterone (DHT) is an essential step required by some
tissues such as the external genitalia before masculin-
ization occurs. Even in tissues such as the Wolffian
ducts, which are affected directly by testosterone, DBT
exhibits greater potency (Bardin and Catterall, 1981).
Aromatization, on the other hand, is required for the
differentiation of the mammalian brain to establish
appropriate male behavior patterns (MacLusky and
Naftolin, 1981). Aromatization also plays a role in
androgen action in some muscle tissue, notably the levator
ani muscle (Bardin and Catterall, 1981).

Morphological differentiation relies on the presence
of testosterone during the critical period of sexual
differentiation (days 17 to 28 post-conception in the

rat). Fetal testis, under the control of the fetal

o
Testosterone Au-Androstenedione

 

       

' H
So-Dihydro- l78—Estradiol Estrone
testosterone

 

30-Androstane-
diol

FIGURE 1. Formation of active metabolites of testosterone.

9
pituitary and/or the placenta (Wilson, 1981) secrete at
least two organizing hormones needed for male differen-
tiation. The first hormone is known as Mullerian
Regression Factor (MRF), and is a peptide hormone of
approximately 70,000 MW. MRF causes the atrophy and
regression of the female structures known as the Mullerian
Ducts. The second hormone required for sexual differen—
tiation is testosterone. In the absence of MRF and
testosterone the fetus develops as a morphological female.
Unlike MRF, testosterone is a steroid which must undergo
metabolism in at least some target tissues for sexual
differentiation to be complete. Defects in testosterone
synthesis result in a condition known as female
pseudo-hermaphroditism, a condition characterized by
undescended testes, absence of male external genitalia,
and feminine psychosocial orientation. Defects in
Sa-reduction of testosterone, while indistinguishable
externally from testosterone deficiency, prevent
development of external genitalia and the urogenital
sinus, although normal development of internal male
structures (Wolffian ducts) is allowed. In addition,
Sa-reductase deficiency allows central male diffentiation
to proceed normally via the aromatase pathway resulting in
male sexual behavior patterns in rodents, and allows
normal male behavior to develop in humans (MacLusky and

Naftolin, 1981; Ehrhardt and Meyer-Bahlburg, 1981).

10

Brain sexual differentiation depends on the
production of estrogens (17B—estradiol and estrone) from
perinatal circulating androgens (testosterone and
androstenedione). The aromatization reaction which
results in the production of estrogens from androgens
occurs in the brain regions affected (Naftolin et a1,
1975; Naftolin et al, 1972; Selmanoff et al, 1977) and is
believed to follow the same biochemical pathway seen in
placental microsomes (see Figure 2) (Selmanoff et al,
1977). Briefly, the aromatizable androgen is hydroxylated
at the l9-position, then further oxidized at the 19-carbon
resulting in the formation of the gem-diol in equilibrium
with the l9-oxo-androgen (Thompson and Siiteri, 1974;
Braselton et a1, 1974). The last hydroxylation reaction
is the rate limiting step in aromatization, and results in
the formation of ZB-hydroxy-l9-oxo-androstenedione,
followed immediately by the loss of the #19 carbon, the
spontaneous collapse of the A ring into an aromatic
configuration, and the reduction of the 3-keto group to a
3-hydroxy group (Goto and Fishman, 1977). The formation
of these hydroxylated intermediates requires the presence
of NADPH and molecular oxygen.

Central effects which rely upon aromatization of
androgens to estrogens include the following: gonadotropin
secretion (Worgul et al, 1981), prolactin secretion,

learning, circadian rhythmns (MacLusky and Naftolin,

 

11

AROIA'I'IZA'I'IOM

 

io-hydroxy-

 

   

FIGURE 2. Intermediates formed in the aromatization of
androstenedione.

12
1981), reproductive behaviors (Clemens and Pomerantz,
1982; Whalen and Olson, 1978), agression and play, and
regulation of hepatic drug and steroid metabolism
(Gustafsson et a1, 1981). There is an extensive
literature on the role which aromatization plays in adult
as well as in perinatal brain (for list of references see
MacLusky and Naftolin, 1981; Naftolin et al, 1975; and
McEwen, 1982). In adult brain, aromatization regulates
feedback control of gonadotropin secretion (Naftolin et a1
1975, Worgul et al, 1981), and plays a role in the
behavioral response to circulating or administered
androgens (Clemens and Pomerantz, 1982; Whalen and Olson,
1978; Christensen and Clemens, 1975).

Aromatization of androgen to estrogen also results in
differentiation of the neonatal brain. During the
critical period of sexual differentiation, the neonatal
brain is still plastic, and may be organized as a male
brain by the administration of either estradiol or
aromatizable androgen (MacLusky and Naftolin, 1981).
Administration of Sa-reduced androgens alone is
insufficient to cause masculinization of the neonatal
brain, although Sa—reduction of testosterone may play some
role in brain differentiation (Martini, 1982). A wide
variety of experimental evidence confirms the important
role of androgen aromatization for brain sexual

differentiation. Perinatal castration results in

 

13
feminization of behavior and gonadotropin secretion
patterns in male rats (Naftolin et al, 1975; McEwen, 1982;
Whalen and Olson, 1981), testosterone or estradiol
administration to either castrate male or female rats
results in masculinization (Poplow and Ward, 1977; Whalen
and DeBold, 1974), and administration of the estrogen
antagonist MER-25 or the aromatization inhibitor f

androsta-l,4,6-triene-3,l7 dione results in feminization

 

of male fetuses (Naftolin et at, 1975; Clemens and

If

Pomerantz, 1982; Whalen and Olson, 1981; Gladue and
Clemens, 1980; Clemens and Gladue, 1978; Christensen and
Clemens, 1975).

Aromatase activity in both male and female fetal
brain has been shown to peak during the critical period of
sexual differentiation (George and Ojeda, 1982), and
testosterone titers in male rats also peak during the same
time period (Orth et a1, 1981; Weisz and Ward, 1980).

Some researchers have shown that the developing brain is
exquisitely sensitive to even a short—term drop in
circulating testosterone titer (Ward and Weisz, 1980), and
that androgen deprivation during the period of brain
development will result in demasculinization and
feminization of adult behavior (Ward, 1972).

Regulation of hepatic drug and steroid metabolism
lies within the sexually differentiated region of the

hypothalamus (Gustafsson, et a1 1981). Influences which

14
feminize behavior also feminize hepatic steroid metabolic
patterns. Control of hepatic steroid metabolism has been
shown to reside in the pre-optic area of the hypothalamus
(Gustafsson et al, 1981) the area with the maximal
concentration of brain aromatase (Selmanoff et a1, 1977),
and the region of brain which is visibly sexually
dimorphic (Gorski et a1, 1978). A number of effects of
steroids on hepatic metabolism have been shown to be
mediated through the hypothalmic-pituitary axis, and to be
dependant on brain aromatization of steroids (Einarsson et
a1, 1973; Gustafsson et a1, 1981; Bardin and Catterall,
1981). The hypothalmic-pituitary axis, therefore, not only
regulates reproductive physiology directly, but may
further modulate reproductive events through control of

hepatic steroid metabolism (Figure 3).

Experimental Hypothesis and Objectives

 

PBBs are potent inducers of mixed function oxidase
enzyme systems, enzymes which are also responsible for the
metabolism of endogenous steroid hormones. If PBBs
decrease circulating steroid levels during the critical
period of sexual development, impaired central
differentiation may occur, with corresponding defects in
central regulation of reproductive function. PBBs may

produce some of their reproductive effects through

15

Testosterone

\

Estradiol

l

     

Pituitary

FSH
LII

Testes

AFTER GUSTAFFSON, ET.AL. ENVIR. HLTH. PERSPEC. VOL 38

FIGURE 3. Interactions between the hypothalamus/pituitary,

gonadal steroid synthesis, and hepatic steroid
metabolism.

 

16
alterations in central hypothalamic- pituitary control.

One indicator of the degree of brain differentiation
is the activity of the hypothalamic aromatase enzyme.
Research has established that females possess much lower
levels of hyothalmic aromatase in adulthood than do males
(Weisz and Gibbs, 1974), and treatments altering the
neonatal or prepubertal endocrine environment might be
expected to perturb the normal development of the
aromatizing enzyme system. The hypothalamic aromatase,
then, may be used as one indicator of reproductive
function.

In order to use the hypothalamic aromatase as an
indicator of central nervous system neuroendocrine
function, an improved aromatase assay was needed.
Previously available assay techniques are either too time
consuming or insufficiently accurate (see Naftolin et a1
1975; George and Ojeda, 1982).

In order to test our hypothesis that PBB induction of
hepatic MFOs may alter central reproductive control
through effects on hypothalamic aromatase the following
experimental objectives were defined:

1. Develop a sensitive and reproducible assay for the
determination of hypothalmic aromatase.

2. Determine normal levels of hypothalamic aromatase
in male, female, and castrate male rats for use in

experimental design of further studies on effects of P383

 

17
on central differentiation.
3. Determine the effects of pre-pubertal castration
and PBB treatment on hypothalamic androgen aromatization.
4. Confirm the effects of polybrominated biphenyls on

hepatic testosterone metabolism.

 

F-

SECTION I

Assay Development

INTRODUCTION

In order to accurately determine the activity of the
aromatase enzyme system in the hypothalamus, it was
necessary to develop an improved assay. Although
aromatizing activity is central to the normal control of
reproduction, hypothalamic aromatase is active at very low
levels, and radioactive tracer methods are necessary for
its detection. In general, previous assays for aromatase

have relied on one of two methods: production of 3H O

2
from the aromatization of 2-3H-androstenedione (Thompson
and Siiteri, 1974), or isolation and purification of
3H-estrogens by solvent partition, thin layer chromato-
graphy, derivatization, and reverse isotope dilution
followed by recrystallization to constant specific
activity (Dessi-Fulgheri and Lupo, 1982; Selmanoff et.al.,
1977; Naftolin et.al., 1972). Assays relying on the
production of 3H20 do not always agree quantitatively
with assays which detect directly the production of
tritiated estrogen products (George and Ojeda, 1982). In

addition, assays relying on the production of tritiated

18

19
water have been used primarily in tissues with higher
aromatase activity than that reported in adult
hypothalamus, and these assays may yield inaccurate
results in the presence of non-aromatase enzymes capable
of releasing tritium from 2-3H-androgens (Hersey et.al.,
1981). Although methods involving derivatization and
multiple recrystallizations are more accurate, these
techniques are time consuming and recoveries must be
calculated using dual isotope techniques. These
disadvantages rendered previously available techniques
unsuitable, and high performance liquid chromatography
(HPLC) seemed to hold the greatest promise of providing a
rapid, sensitive and reproducible method. HPLC makes
possible the determination of recovery by U.V. absorbance
of reference standards added to the incubation extract,
and offers the possibility of determining the production
of several compounds in one chromatographic step. A valid
enzyme assay would require the following conditions to be
met: a) Incubation conditions which give time and protein
dependant aromatization of the androgenic substrate, b)
HPLC conditions which allow the rapid separation of the
estrogenic products of aromatization, and c) proof of the
radiochemical purity of the estrogens isolated by HPLC.
Incubation conditions for placental and hypothalamic
aromatase have been well described in the literature

(Naftolin et.al., 1975; Selmanoff et.al., 1977; Ryan,

20
1959), but existing methods for determination of the
reaction products are not suitable for detailed studies of
the enzyme. The inital objective was therefore the

development of a suitable HPLC system.

METHODS

Development of HPLC Systems

HPLC methods were developed on a Waters Assoc.
chromatographic system consisting of a M-6000 pump, a
model 440 U.V. absorbance detector set at 254nm, and a
Waters 760 data module. Reference steroids were purchased
from Steraloids Inc. (Wilton, N.H.), and dissolved in
methanol for chromatography.

A variety of solvents were tested to determine which
would adequately separate testosterone, androstenedione,
estrone, and l7B-estradiol. Two solvent systems were
developed for the assay, and two additional systems were
used to confirm the purity of the peaks collected during
the assay (Figure 4):

System A: acetonitrile/water (70:30), 1.0 ml/min,

Waters 10p radial-Pak C-18 cartridge;

System B: THF/water (35:65), 2.5 ml/min, Waters

lOu radial-Pak C-18 cartridge;

System C: acetonitrile/THF/water (55:5:40),

1.0 ml/min, Altex 5p Ultrasphere ODS

column (15 cm length);

21

22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Q)
2 L. m
m U L
'53 L11 1 1 1 1 1 irng 1 1 ka 1 1 1
O 1
.8 "GE,
< Her, C D
>
3 "Biz
‘1
E
2 MeE2 E1""2
UL..-
[.1 1 1 1 1 1 l 1 11J. 1 1 1 1 1 1
.2. 4 3 12 g' 4 a 12 I6—

Time (min)

FIGURE 4. Chromatograms of reference steroids.
A) acetonitrile/water (70:30), C-18 column
B) THE/water (35:65), C-l8 column
C) acetonitrile/THF/water (55:5:40), C-18
D) cyclohexane/n-octanol (95:5), normal phase

E =estrone, E =17B-estradiol, 1£§=
androstenedio e, MeE =estrone-3-methyl ether,
MeE2=17B-estradiol-3-methyl ether.

23
System D: cyclohexane/n-octanol (95:5), 2.0 ml/min.
Waters 10u radial-Pak normal phase;

System A allowed the rapid separation of the products
of aromatization (estrone and l7B-estradiol) from the
tritiated androstenedione precursor. Since the estrogens
elute before the androgen (Figure 4a), the chance of
product contamination by the highly radioactive substrate
was minimized.

System B, using THF and water, exhibited an entirely
different pattern of solvent selectivity. Chromatograms
of reference steroids (Figure 4b) show that the estrogens
eluted behind testosterone and androstenedione. Because
of the differences in selectivity exhibited by
acetonitrile and THE, it is unlikely that a compound
co-eluting with one of the estrogens on system A would
also co-elute on system B. Therefore, preliminary
chromatography of a sample on system A with collection of
the estrogen fractions, followed by a second
chromatographic purification of the collected fraction on
system B, should yield estrogen peaks of high purity.

Systems C and D (Figure 4c and 4d) were developed to
separate the 3-methyl ethers of estrone and estradiol from
androstenedione and the parent estrogens. These systems
were used to confirm the purity of the estrone peak

collected during chromatography on system B.

24

Preliminary Studies With Placental Microsomes

Placental microsomes were used as a source of
aromatase for the initial experiments, since placenta has
an active, well characterized aromatizing enzyme system
(see Ryan, 1959; Braselton et.al., 1974). A healthy,
fullterm human placenta was obtained from Sparrow Hospital
(Lansing MI), and placental microsomes were prepared
according to the method of Ryan (Ryan, 1959). The
placenta was minced, the vascular tissue dissected away,
and segments of the placenta were placed into cold (4°
C) 0.05M phosphate buffer and homogenized in a Waring
blender. The placental homogenate was centrifuged at
10,000 x g for 20 minutes, and the supernatant was drawn
off, placed into ultacentrifuge tubes, and centrifuged at
105,000 x g for 60 minutes. The microsomal pellet was
resuspended in 200mM sucrose/ 0.05M phosphate buffer at a
dilution such that 1 ml of microsomes was equivalent to 10
g of tissue. The microsomes were then stored at -70° C
until used.

Placental microsomes equivalent to 1 g of tissue were
incubated in 0.05M phosphate buffer (pH=7.0) at 37° C
for one hour in a total volume of 1 ml containing 40uM
l4C-testosterone (25uCi/umole), and an NADPH
regenerating system consisting of 2.4 uM NADP, 11.2
uM glucose-6-phosphate, and 0.4 U glucose-6-phosphate
dehydrogenase. Following incubation for one hour, the

reaction was terminated by the addition of 5 ml of

 

25
extracted twice with methylene chloride. The combined
methylene chloride extracts were evaporated to dryness

under N and partitioned between 1.0N NaOH and toluene.

2:
The NaOH layer was neutralized by the addition of 5N HCl,
and extracted twice with methylene chloride. The
methylene chloride extract was evaporated to dryness under
N2, and dissolved in 0.5 m1 of methanol for
chromatography.

Chromatography of the placental incubation extracts
resulted in radiochromatograms showing good separation of
the 14C-estrogens produced from the 14C-testosterone
precursor (Figure 5a and 5b). Both system A and system B
show the estogens adequately separated from testosterone,

and measurable but minimal 17B-oxidation of estradiol to

es trone .

Purification of Tritiated Androstenedione

 

Following the successful incubation of placental

. . 14
microsomes with

C-testosterone, hypothalamic incuba-
tions under the same conditions were attempted using
(1,2,6,7)-3H-androst-4-ene-3,17-dione (90 Ci/mmole) as
the precursor. These initial experiments with hypothala-
mic tissue failed to yield tritiated estrogens above those
seen as ”background" in incubation blanks. Because of the
high background in the substrate, two purification steps,

corresponding to chromatography of the 3H-androstene-

dione on system A and then on system B were used to purify

 

26

2500 +
IA l E2
2000 ;

 

 

 

 

 

 

 

 

 

   

1500
DPM per
fraction
1000
500
0 IO. 0 O O
0 3 6 9 12 15 18 21 24 27 30
fraction 0
2500 + “ ‘
E
; B T 2
2000 ; fl
1500 ;
DPM per
fraction
1000
500 E
1
9 12 15 18 21 24 27 30
fraction 0
FIGURE 5.

Radiochromatogram of produiss of incubation of
placental microsomes with C-testosterone.

A) chromatography on system A

B) chromatography on system B

E =estrone4 E =17B-estradiol, T=testo-
s erone, A =a§drostenedione.

27
the substrate before incubation. Repeated hypothalamic
incubations still failed to yield tritiated estrone,
although incubations of placental microsomes did produce
the expected estrogens.

Multiple rechromatographies of the 3H-androstene-
dione using systems similar to HPLC systems A and B
disclosed the presence of androsta-l,4,6-triene-3,17-dione
present as an impurity. The synthesis of the tritiated
androstenedione by New England Nuclear, the supplier, used
androsta-l,4,6-triene-3,l7-dione as the starting material.
Since the 1,4,6-triene has been shown to be a potent
aromatase inhibitor (Schwartzel and Brodie, 1973), its
presence was suspected of inhibiting the hypothalamic
aromatase. The purification steps employed initially
(chromatography on system A followed by chromatography on
system B) had failed to remove the 1,4,6-triene because
the compound co-eluted with androstenedione on both
systems.

An improved purification technique was therefore
developed which used chromatography on system B to remove
estrogens present as impurities, followed by chromato-
graphy of the androstenedione peak on a system with 40%
acetonitrile as the eluent. Chromatography of the
androstenedione with 40% acetonitrile removed all traces

of 1,4,6—triene present.

28

Incubation of Hypothalamic Tissue

Adult male Sprague-Dawley rats were killed by
decapitation, and the entire brain removed and frozen on
dry ice. Anterior hypothalamus containing the median
pre-optic area was removed using a dissection similar to
that reported by Naftolin (Naftolin et.al., 1972). The
median pre-optic area has been shown to contain greater
than 2/3 of the brain aromatase (Selmanoff et.al., 1977).
The dissected hypothalamic tissue was homogenized in 0.05M
phosphate buffer (pH=7.0), and microgram quantities of
estradiol and estrone were added to allow easy calculation
of recovery following incubation. The use of cold estrone
and estradiol in the incubation was suggested by Naftolin
(Naftolin et.al., 1975) as a technique to prevent further
metabolism of 3H-estrogens produced by aromatiation. In
this case, the estrogen ”trap" also functioned to allow
easy calculation of recovery. This technique was possible
because estrogens do not “product inhibit" the aromatase
enzyme (Naftolin et.al., 1975; Schwarzel and Brodie,
1973).

Each assay tube contained hypothalamic homogenate
(5 — 10 anterior hypothalami), 200nM 3H-androstenedione
(90 Ci/mmole) and an NADPH regenerating system consisting
of 2.4uM NADP, 12uM glucose-G-phosphate and 0.4 U
glucose-6-phosphate dehydrogenase in a total volume of 0.5

ml. The reaction was started by the addition of the

29
3H-androstenedione and the assay tubes were incubated
with agitation for 3 hours at 37° C. The reaction was
terminated by the addition of 5 ml of methylene chloride.
Blanks were prepared by the addition of methylene chloride
at zero time, or by incubation of boiled cerebral cortex
homogenates for 3 hours.

Each incubation tube was extracted twice with 5 ml of
methylene chloride, the combined extracts were dryed over
anhydrous NaZSO4 and evaporated to dryness under N2.
Each sample was dissolved in 0.5 m1 of methanol/ water
(90:10), and extracted with 1.0 ml of hexane to remove
non-polar lipids. The methanol/water layer containing the
estrogens was evaporated to dryness and redissolved in 50
pl of methanol prior to chromatography.

The samples in methanol were chromatographed on HPLC
system A, and the estrone and estradiol fractions
collected for rechromatography. The estrone peak was
again collected during chromatography on system B, and the
3H-estrone present was quantified by liquid scintil-
lation counting in a Packard Tri-Carb, (Model 460 C).
Recovery of estrone was calculated from U.V. absorbance

during chromatography on system B.

30
Method Validation

Several test incubations were performed in order to
verify the purity of the estrone peak collected and
counted following chromatography on system B. The first
incubation was carried out in 6 tubes, each containing a
different amount of placental microsomes. After incuba-
tion as described above, the estrogenic products in each
tube were subjected to chromatography using system A, then
split into two aliquots. The first aliquot was chromato-
graphed on system B, and the specific activity of the
estrone collected was determined. The second aliquot was
run on system B, the estrone peak collected, and then
methylated by the method of Brown (Brown, 1955). The
3-methyl ether of estrone was then chromatographed on
system C, and the specific activity of the estrone methyl
ether compared to the specific activity of the estrone
obtained during chromatography on system B.

Peak purity was also checked for incubations contain-
ing homogenates of hypothalamic tissue. Three tubes, each
with 10 hypothalami, were incubated as described above.
Peak purity of the estrone produced following chromato-
graphy on system B was determined as described above for
placental microsomes, however following methylation the
specific activity of the estrone methyl ether produced was
checked on both system C and system D. In addition, time

and protein dependancy of the hypothalamic incubations

31
were verified using pooled homogenate equivalent to two to

eight hypothalami, and for times up to 3 hours.

RESULTS AND DISCUSSION

Chromatography with system A was useful in separating
the estrone produced from the highly radioactive
3H-androstenedione substrate. Since the estrogens
eluted first on system A, the chance of the androstene-
dione ”tailing into" the estrogen peaks was eliminated.
The second chromatographic step with system B then
separated the estrogens from each other, and from any
residual androstenedione which might remain.

Estrone recovered following chromatography with
system B averaged 70 - 80 % of the estrone added to the
incubation tubes. Figure 6 shows a radiochromatogram
(HPLC system B) of the estrone produced by incubation of
3H-androstenedione with hypothalmic tissue in comparison
with that seen in a blank incubation. Since hypothalamic
tissue has been shown to contain little 17B-dehydrogenase
(George and Ojeda, 1982), and since only trace amounts of
estradiol were observed following incubation of
hypothalmic homogenates (Figure 6), the estradiol peak was
not further characterized, and was not used in the
calculation of hypothalamic aromatizing activity.

Chromatography on system B produced estrone with an

32

DPl/frsctlon

FIGURE 6.

33

 

 

 

1500 -» .
l blank
IOOO -
500 -—
i i T
5 IO IS 20
1500-'-

1000 '*

 

500 '”

 

 

 

5 IO 15 20
tractlon t

Radiochromatogram of fractions collected from
system B chromatography of zero-time blank and
hypothalamic homogenate incubation. Fractions
collected every 30 seconds beginning at 4
minutes. Cross-hatch pattern corresponds to
extrone peak with U.V. detector (280 nm).

34

.wmocm enouumw Hocfimfiuo no on no manna on» no wwucaooamo «

 

 

 

 

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35
average purity of 95% or greater (Table 1). This was
shown by collection of the estrone fractions from system
B, methylation, and rechromatography on system C or D.
Since the criterion for purity using recrystallization is
normally a 5% or smaller change in isotope ratio, the
purity of estrone produced by this method is comparable to
that produced by repeated recrystallization.

Both time and protein dependancy were initially
determined using placental microsomes (data not shown),
and confirmed using hypothalmic incubations. This
incubation technique showed linear time dependancy to 3
hours, and was protein dependant with hypothalamic protein
equivalent to 2 to 8 hypothalami (Figure 7).

These results indicate that hypothalamic aromatase
may be measured following separation of estrogens on HPLC
system A and isolation and quantification of estrone on
HPLC system B. Whereas previous methods have offered
recoveries of around 50% (Weisz and Gibbs, 1974), this
method offers recoveries of 70 - 80%, and is more rapidly
performed than previous techniques which have relied on
solvent partition, thin-layer chromatography,
derivatization, and reverse isotope dilution with multiple
recrystallizations. This method offers isolation of
products of high (95% or greater), proven purity and it is
sensitive enough to allow the detection of less than 10
femtomoles of tritiated estrogen produced by

aromatization.

36

5000
A
B
4000 /. 120°
:1

3000

2000
,9’
O

l000 / 300 I

DPfl/ “be

\O

b
0PM/Iolr
\

 

 

j v
n 1 ( 2 3 I» 4 1 1
no hours) Hypothalamic

Protein equivalents

FIGURE 7. Time (A) and protein dependence (B) of
incubation of hypothalamic homogenates.

SECTION II
Effects of Orchidectomy or PBB

Treatment on Hepatic Testosterone Metabolism

INTRODUCTION

Hepatic oxidative metabolism of testosterone acts to
produce physiologically less active forms of this
androgen. These compounds then readily undergo phase II
metabolism and are excreted. Increases in sex steroid
metabolism may be expected to result in impaired
reproductive function if homeostatic mechanisms are unable
to compensate. Polybrominated biphenyls are known potent
inducers of hepatic oxidative metabolism (Dent, 1978; Dent
et.al., 1976; McCormack et.al., 1978), and have been shown
to specifically induce the hepatic mixed-function oxidase
system responsible for oxidative metabolism of steroids
(Nowicki and Norman, 1972; Arneric et.al. 1980; Newton
et.al., 1982). In vivo studies have shown that PBBs
reduce the efficacy of exogenously administered steroids.
PBBs decrease the uterotropic action of exogenously
administered estradiol (Bonhaus et.al., 1981), and reduce
the time of progesterone induced anaesthesia in rats
(McCormack et.al., 1979).

PBBs have produced a variety of endocrine and
reproductive effects which may be linked to increased
microsomal metabolism of sex steroids. PBB fed bulls have

37

38
shown testicular atrophy and decreased spermatogenesis
(Jackson and Halbert, 1974; Kimbrough et.al., 1978). Male
rats treated perinatally with PBBs showed decreased
prostate and seminal vesicle weights, and female rats
displayed lengthened estrous cycles (Johnson et al, 1980).
In addition, the hypertrophic response of rat seminal
vesicle to exogenous testosterone was decreased (McCormack
et al, 1979).

Polybrominated biphenyls are “mixed” inducers of
hepatic microsomal enzymes, having the inducing properties
of both P-450 inducers such as phenobarbital, and P-448
inducers such as 3-methylcholanthrene. Although the
individual PBB congeners have been shown to possess either
P-450 or P-448 inducing activity, the PBB mixture,
Firemaster BP-6, contains congeners which induce both
P-450 and P-448 (Dent, 1978).

PBB induction of hepatic testosterone metabolism has
been shown to follow a pattern which is dependent on both
the age and the sex of the animal treated (Newton et al,
1982). The sex difference in MFO induction may be related
to perinatal imprinting of steroid metabolism which occurs
under the influence of circulating testosterone (Skett and
Gustafsson, 1979; Einarsson et a1 1973; Gustafsson et al,
1981). Since circulating steroid levels also affect
hepatic steroid metabolism directly, changing steroid

levels during sexual maturation of the animals may also

39
play a role in the age difference in response to PBBs.
The objective of this study was to examine the
effects of acute PBB treatment on hepatic testosterone
metabolism, and to compare male and female testosterone
metabolic patterns with those seen in PBB induced and

orchidectomized animals.

METHODS

Animals and Treatment
Weaned, 21 day old male and female Sprague-Dawley
rats were obtained from Spartan Farms (Haslett, MI). The
animals were randomly assigned to one of five treatment
groups according to sex. The treatment groups were as
follows:
1. control male;
2. control female;
3. PBB treated males, given 150 mg/kg of Firemaster
BP-6 (i.p. in peanut oil) at 22 days of age;
4. PBB treated females, 150 mg/kg Firemaster BP-6
(i.p. in peanut oil) at 22 days of age;
5. males castrated at 22 days of age.
Each treatment group contained 15 to 17 animals. The
animals were maintained on a 12 hour in phase lightzdark
cycle, with water and food (Wayne Lab Blox) available ad

libitum until sacrifice at 75 days of age.

Preparation of Liver Microsomes
Animals were killed by decapitation, and the livers
immediately removed into ice-cold 1.15% KCl. The livers

40

41
were then weighed, and homogenized in 4 volumes of 66mM
Tris buffer using a glass and teflon Potter-Elvehjem
homogenizer. The homogenate was centrifuged at 10,000 x g
for 20 minutes, the supernatant drawn off and
recentrifuged at 105,000 x g for 60 minutes. Following
ultracentrifugation, the microsomal pellet was
re-suspended in 66mM Tris/ 200mM sucrose buffer and frozen
at -70° C until used. Microsomal protein concentra-
tions were determined with a modification of the Lowry
method (Lowry et.al., 1951) using bovine serum albumin as

the standard.

Determination of In Vitro Testosterone Metabolism

Hepatic oxidative testosterone metabolism was
measured using the method of Newton (Newton et.al., 1982).
Each incubation tube contained 0.8 mg of microsomal
protein, 115 umole testosterone, and an NADPH regenerating
system consisting of 0.3 umole NAD, 0.7 umole NADP,
5.8 umole glucose-6-phosphate and l U of glucose-6-
phosphate dehydrogenase in a total volume of 1.06 ml.
After preincubation for 5 minutes the reaction was started
.by the addition of testosterone. The incubation mixtures
were maintained at 37° C for 30 minutes with agitation.

Reactions were terminated by the addition of 5 ml of
chloroform/diethyl ether (3:1). Each incubation tube was

extracted 3 times with 5 m1 of the chloroform/ether

42
mixture, and the extract dryed by passage over anhydrous
Na2804. The dryed extracts were evaporated to dryness
under N2, resuspended in 1.0 ml ethanol, and filtered

prior to chromatography.

High Performance Liquid Chromatography

The oxidative metabolites of testosterone were
quantified by the method of Newton (Newton et.al., 1982).
The testosterone metabolites 7a-hydroxytestosterone,
16a-hydroxytestosterone, 6B-hydroxytestosterone, and
androstenedione were separated and quantified by HPLC.
The chromatography system consisted of a Waters M-6000
pump, 441 detector (254 nm), 720 system controller, Waters
Intelligent Sample Programmer (WISP), and Waters 760 data
module. The column used was an Altex 5p Ultrasphere
ODS column, with 24% THF as the eluent at a flow rate of
1.0 ml/min. Metabolites were quantified by external
standard quantitation using known reference steroids

(Steraloids, Wilton N.H.) as standards.

Statistical Analysis

Metabolite concentrations were determined by
integration of peak areas using the Waters 760 data
module. Data were analyzed using a completely random
analysis of variance (a=0.05) for each metabolite,
followed by Duncan's New Multiple Range Test (a=0.05) for

comparison of treatment means.

RESULTS

Sex differences in testosterone oxidation

Control male rats showed greater 16a-hydroxylation,
6B-hydroxylation, and 17B-oxidation than did control
females (Figure 8b, 8c, 8d and Table 2). Control females,
however, had increased production of 7a-hydroxytestoster-

one when compared with control male animals (Figure 8a).

Effect of PBB treatment

 

PBB treatment increased the production of 7a-hydroxy-
testosterone in microsomes from both male and female rats,
and increased the production of lGa-hydroxytestosterone
and 6B-hydroxytestosterone in male but not in female rats
(Figure 8a, 8b, 8c and Table 2). PBB treated males
exhibited 7a-hydroxytestosterone levels comparable to
those seen in control females. Androstenedione production
was unaffected by PBB treatment in male rats, but was

increased by PBB treatment in female rats (Figure 8d).

43

LE MG PRUT MIN

NH

PRflT/HIH

_.
J

LETT

NH

5 FPUT HUI

lWMLEsfl'

B FPOTdflH

LET)

HM

FIGURE 8 .

44

HEF’F’iT I C TESTOSTERONE METHE'

 

 

 

 

 

 

 

 

 

 

 

 

       

   

 

 

 

 

 

 

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EHPEPIMENTHL GROUP

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3 com me PBB ma cas

Products of oxidative testosterone metabolism
in vitro by livers of orchidectomized,
control, or PBB treated rats.

A) 7a-hydroxytestosterone

B) 16a-hydroxytestosterone

C) 68-hydroxytestosterone

D) androstenedione

CON MA=control male, PBB MA=PBB treated males,
CAS MA=castrate males, CON FE=control females,
PBB FE= PBB treated females.

45

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46

Effect of castration on testosterone metabolism

The effect of castration was to reduce total
testosterone metabolism to levels seen in normal female
rats (Figure 9 and Table 2). Although castration failed
to change the level of 7a-hydroxylation when compared to
male controls, orchidectomized males produced levels of
l6a-hydroxy- testosterone, 6B-hydroxytestosterone, and
androstenedione similar to those seen in the females

tested (control and PBB treated females).

HHDLEEMB PRDTKNIH

FIGURE 9.

47

HEPHTIC TESTOSTERONE METRE:

 

 

 

 

 

 

 

 

 

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Total testosterone metabolism in vitro by
liver microsomes from orchidectomized, control,
or PBB treated rats.

CON MA=control male, PBB MA=PBB treated males,
CAS MA=castrate males, CON FE=control females,
PBB FE=PBB treated females.

DISCUSSION

A summary of total testosterone metabolism (Figure 9)
reveals that in general, a single dose of PBB (150 mg/kg)
was sufficient to induce testosterone metabolism for at
least 55 days following treatment in both male and female
rats. Castrate males showed total testosterone metabolism
similar to that seen in control females, indicating that
testosterone supports its own metabolism.

Since differences in metabolism were first linked to
the sex of animals (Nicholas and Barron, 1932),
experiments which attempted to define the role of the sex
steroids in modulating metabolism have been numerous.
Neonatal castration of male rats altered hepatic
metabolism to a pattern resembling that seen in female
animals, although adult castration had variable effects on
the pattern of drug metabolism (Einarsson et al., 1973).
Castration before 4 weeks of age has been shown to result
in feminization of hepatic metabolism (Kato, 1974). The
present experiment clearly demonstrated a castration
induced feminization of 16a- and GB- hydroxylation and
androstenedione production, while 7a-hydroxylation was
unaffected.

48

49

PBB treatment resulted in induction of total
testosterone metabolism. Male animals showed induction of
the P-4SO linked enzymes 16a- and GB- hydroxylase, while
female animals exhibited marked increases in 7a-hydroxy1a-
tion. Testosterone lGa-hydroxylase activity is sexually
differentiated by circulating androgens during neonatal
development, whereas 63-hydroxy1ase activity is under
direct androgenic control (Einarsson et a1, 1973).
Testosterone 7a-hydroxylase is regulated by non-hormonal
factors in adulthood, and shows both higher basal activity
as well as greater inducibility in female rats (Einarsson
et al, 1973).

Newton et a1 (1982) have previously demonstrated that
chronic PBB treatment results in induction of hepatic
steroid metabolism. The results of this study are in
general agreement with those reported by Newton, although
some differences in the exact pattern of enzyme induction
were seen. These differences are attributable to the
differences between the single dose treatment used in this
study, the chronic dietary intake used by Newton, and age
differences at time of treatment and sacrifice.

From the above results, it is apparent that even a
single dose of PBB is capable of marked induction of
testosterone metabolism. Whether the increased metabolism
of steroids is related to reproductive effects associated

with PBB treatment is of current interest. Decreased

50
testosterone levels during the perinatal period of sexual
differentiation could lead to permanent reproductive
deficits. Gupta et al (1982) have already shown that
perinatal treatment with phenobarbital, a short-term
inducer of steroid metabolism, is capable of eliciting a
permanent decrease in circulating testosterone levels.
Increased testosterone metabolism during the critical
period of brain differentiation could permanently alter
reproductive function in both male and female animals.
PBB, a potent inducer of mixed function oxidases, should
therefore be tested for effects on brain differentiation

and neuroendocrine modulation of reproductive function.

SECTION I I I
Effect of Orchidectomy or Acute PBB

Treatment on Hypothalamic Aromatase Activity

INTRODUCTION

The aromatization of androgens to estrogens in the
central nervous system is a key step in the sexual
differentiation of the brain, and the consequent sex
differences seen in adult sexual behavior and physiology.
In man (Naftolin et al, 1971) as in the rat (Naftolin et
al, 1975; Weisz and Gibbs, 1974), circulating androgens
from the fetal testis are aromatized into estrogens in the
anterior hypothalamus, and direct the organization of the
brain to the male pattern. Any interruption of this
process either through reduction in circulating
testosterone titers (Orth et al, 1981; Ward and Weisz,
1980), or blockade of the aromatase pathway in the
hypothalamus (Gorski et al, 1977; Whalen and Olson, 1981),
results in feminization of adult physiology and behavior
(Ward, 1972; Gorski et al, 1978; Ward and Weisz, 1980).

Aromatization of testosterone and androstenedione to
their corresponding estrogens, l7B-estradiol and estrone,
also plays a key role in the regulation of adult
reproductive physiology and behavior. Worgul et a1 (1981)
have shown that in the dog, as in the rat, central
aromatization is responsible for regulation of LH

51

52
secretion in the male. Other investigators have
conjectured that it is androgen aromatization rather than
circulating estrogens that modulate hypothalamo-pituitary
feedback mechanisms in the female (Selmanoff et al, 1977).

Because of the key role played by hypothalamic
aromatase in sexual differentiation of the brain and in
adult neuroendocrinology, any alteration in this enzyme
system could have far reaching reproductive consequences.
PBBs have been shown to be potent inducers of the hepatic
mixed-function oxidases which metabolize circulating
steroids (Dent, 1978; Newton et al, 1982). Induction of
MFOs may reduce circulating steroid levels, and thus alter
hypothalamic aromatization, consequently affecting
reproductive performance.

Although the presence of aromatase has been verified
in the brain tissue of a variety of species (Naftlin et
al, 1975; Clemens and Pomarantz, 1982; Sholl et al, 1982;
Worgul et al, 1981; Reddy et a1, 1973; Callard et al,
1978), little is known about its specific interactions
with normal and perturbed endocrine systems. Early
research demonstrated that rabbit aromatase could be
influenced by castration or hormone treatment in adult
animals (Reddy et a1, 1973). The rabbit data is
interesting, but since the rabbit is a reflex ovulator,
the data have little predictive value in rat or human

systems.

' ‘
IJJ

Because PBBs may alter the fetal endocrine environ-
ment by induction of steroid metabolism, PBBs could affect
the hypothalamic aromatase system. Since PBBs have known
reproductive effects, some of which may be mediated
through central pathways, it is important to examine the
hypothesized interaction of P883 with hypothalmic
aromatase. The following experiments were performed to
determine the effect of PBB treatment on hypothalamic
aromatase, and compare the effect with that produced by

orchidectomy.

METHODS

Animals and Treatment

Weaned, 21 day old male and female Sprague-Dawley
rats were assigned to PBB treated, control, or
orchidectomized groups as described previously. Animals
were maintained on a 12 hour light cycle with food and

water available ad libitum until sacrifice at 75 days of

 

age.

Assay of hypothalamic aromatase

 

At 75 days of age, animals were killed by
decapitation and the brain was immediately removed onto
dry ice. The brain was stored for 24 hours at -70° C
until assayed for hypothalamic aromatase activity.
(1,2,6,7)-3H-Androst-4-ene-3,l7-dione (New England
Nuclear, Boston MA), was purified by two successive HPLC
procedures as previously described (section I) to insure
removal of androsta-l,4,6-triene-3,l7 dione and any other
radiochemical contaminants.

On the day of the assay, the frozen brains were
dissected on a cold-plate by the method of Naftolin et a1
(1972), and the anterior hypothalamus containing the

54

55
median pre-optic area was removed into ice cold 0.05M
phosphate buffer. Five or six hypothalami were pooled in
each assay tube, and homogenized between teflon and glass.
An NADPH regenerating system and additional buffer (pH=7)
were added to make a total volume of 0.5 ml as described
in section I, and the reaction was initiated by the
addition of lOuCi of (1,2,6,7)-3H-androstenedione (90
Ci/mmole). The reaction mixtures were incubated at 37°
C for 3 hours, and the reaction terminated by the addition
of 5 ml of methylene chloride. Samples were prepared for
HPLC and subjected to chromatography on HPLC system A
followed by chromatography on HPLC system B as described
in section I (see Figure 10). Tritiated estrone was
collected during chromatography on system B, and
radioactivity was determined by liquid scintillation
counting using a Packard Tri—Carb (Model 460 C). Estrone
recovery was determined by U.V. absorbance, and the

production of tritiated estrone per brain was calculated.

Statistical Analysis

 

Data were analyzed by a completely random analysis of
variance (a=0.05), and differences between treatment
groups in mean estrone production per brain were
determined using Duncan's New Multiple Range Test

(a=0.05).

56

DIAGRAM 0F¥METHOD

T1 SSUG Incubotes

extraction with
methylene chloride

Extract

partition between
90% MeOH/hexone

 

ill *

Steroid froctlon Llolglgroction
HPLC
70% acetonitrile

.sterolds Neutrol Isteroids

HPLC
35% THF

esgone * estfiol

{:Fmethylotion
<:>rechromotogroohy

 

Phenoll

 

)ootionol

FIGURE 10. Summary of hypothalamic aromatase assay.
Incubation conditions described in text.

RESULTS

Figure 11 summarizes the effects of sex, castration
and PBB treatment on hypothalamic aromatase activity.
Both control and PBB treated female brains had aromatase
levels significantly lower than those displayed by any of
the male brains (Table 3). PBB treated males exhibited a
trend toward decreased aromatase, but the activity did not
differ significantly from that seen in control males. PBB
treatment also did not significantly affect aromatase in
female brains. Castration on day 21 significantly
decreased hypothalamic aromatase when compared to control
male brains, although castration did not lower aromatase

to the levels seen in female brains.

57

58

 

    

I r

 

I'-

'3!

3"
l

u”

 

. L."
IS:

 

DPH UF EIHERHIH

 

   

‘IIII’I'III

 

131311 1111 FEB 1111 E11: 1111 1311-1 FE PBB FE
HPERIMEHTHL GROUP
I BEHIH HEIIIMHTHSE

  

FIGURE 11. Effect of orchidectomy or PBB treatment at 21
days on the hypothalamic aromatase activity in
adult male and female rats.

CON MA=control male, PBB MA=PBB treated males,
CAS MA=castrate males, CON FE=control females,
PBB FE=PBB treated females.

59

TABLE 3
Effect of orchidectomy or PBB treatment
at 21 days on the hypothalamic
aromatase activity in adult male and
female rats.

 

brain aromatase

 

group DPM El/ brain
control male 676 :TSO (3)a
p313 male 525 i 57 131""D
castrate male 407 i 69 (3)b
control female 187 :y53 (3)c
pea female 100 :t 8 (31°

 

expressed as mean i s. e. m. (n)

a,b,c letters denote values which do
not differ significantly from other
values designated with the same letter.

DISCUSSION

These experiments demonstrate for the first time that
pre-pubertal castration can affect hypothalamic aromatase
activity in rats. A previous report by Reddy et a1 (1973)
described increased aromatase in rabbits following adult
castration. Since rabbits are reflex ovulators,
hypothalamo-pituitary control of reproduction may be
expected to differ substantially from that seen in rats or
humans. The castration induced decrease in hypothalamic
aromatase reported here may indicate that some parameters
of central neuroendocrine differentiation are still
plastic after the classical period of brain differentia-
tion which ends at day 5 after birth (MacLusky and
Naftolin, 1981).

Although castration at day 22 resulted in a
significant decrease in aromatase activity, PBB treatment
had no significant effect. The trend toward decreased
aromatase seen in PBB treated male rats, although not
statistically significant, was in the same direction as
that seen in castrate animals, and may reflect the effect
of decreased steroid levels expected with PBB. It must be
kept in mind that these are preliminary experiments,

60

61
performed with few replicates (n=3). Future experiments
with larger numbers of replicates should be more
definitive.

Because of the effect on hypothalamic aromatase seen
with castration, it is important to determine if perinatal
PBB treatment will show the same effect; i.e. feminization
of hypothalamic aromatase activity in the male brain.
Because of the important role that aromatase plays in
differentiation (McEwen et al 1980; Naftolin et a1, 1975;
Whalen and Olson, 1978; Clemens and Pomerantz, 1982),
regulation of adult sexual behavior (Popolow and Ward,
1976; Whalen and Rezek, 1977), and regulation of
gonadotropin secretion (Selmanoff et al, 1977; Naftolin et
al, 1975; Worgul et al, 1981) it is critical to determine
if aromatase activity is affected by reproductive
toxicants such as PBB and phenobarbital (McCormack et a1,
1979; Johnson et al, 1980; Gupta et al, 1982).

Perinatal PBB treatment may be expected to lower
available steroid levels during brain differentiation, and
thus impair the development of the hypothalamic aromatase
system. Further experiments utilizing neonatal castration
and PBB treatment will help determine the role, if any,
that central effects play in the reproductive alterations
seen following treatment by mixed-function oxidase

inducers.

SUMMARY AN D CONCLUS IONS

SUMMARY AND CONCLUSIONS

Exposure to PBBs results in induction of hepatic
steroid metabolism. This may account for the reproductive
effects associated with PBB exposure. In addition to
direct effects on reproductive organs, decreases in
circulating androgen titers may affect reproduction by
altering central neuroendocrine control mechanisms. In
addition, PBBs may exert central effects directly on the
hypothalamus to alter aspects of reproductive physiology.
In order to determine if PBB effects on reproduction
involved the hypothalamus, a new, sensitive assay for
hypothalamic aromatase was developed. Hypothalamic
aromatase activity was measured in orchidectomized
animals, and in control and PBB treated male and female
rats. PBB induction of hepatic steroid metabolism was
confirmed by in vitro incubations of testosterone with
hepatic microsomes.

The assay of hypothalamic aromatase employed dual
HPLC chromatographies and liquid scintillation counting to
isolate and quantify the estrone produced by aromatization
of androstenedione by hypothalamic homogenates in vitro.
This technique offered good recoveries of estrone (70 -

62

63
80%), and was sensitive enough to detect the production of
less that 10 femtomoles of tritiated estrogen. The
estrone isolated by the HPLC technique was of very high
(greater than 95%) purity. Recoveries were calculated
using U.V. absorbance of carrier estrogens added during
incubation. The assay was a substantial improvement over
existing methods, and was easily and rapidly performed.

Incubation of hepatic microsomes with testosterone
has confirmed that a single dose of PBBs given at the time
of weaning (22 day old rats), is sufficient to induce
hepatic MFO activity in adulthood. PBBs increased overall
testosterone metabolism in both male and female rats,
whereas orchidectomy of 22 day old males decreased in
vitro testosterone oxidation to levels comparable to those
seen in female rats. Production of 7a-hydroxy-
testosterone was increased in both male and female rats by
PBB treatment, but lGa-hydroxytestosterone and 6B-hydroxy
testosterone production was elevated only in male rats.
Castration did not alter 7a-hydroxytestosterone production
but did decrease the production of l6a- and 6B-hydroxy-
testosterone to female levels.

Female rats had hypothalamic aromatase activities
substantially below those seen in male animals. PBB
treated males exhibited a trend toward decreased aromatase
activity, but the activity did not differ significantly
from that seen in control males. Castration on day 22

significantly decreased hypothalamic aromatase when

64
compared to control male brains, but did not lower
aromatase levels to those seen in female brains.

These experiments demonstrated that hypothalamic
aromatase may be affected by manipulation of the endocrine
environment in weanling rats. Sexual differentiation of
the hypothalamus is exquisitely sensitive to even small
alterations in circulating steroid levels (Ward and Weisz,
1980), and endocrine manipulation during neonatal life may
result in a deficit of hypothalamic development leading to
permanent reproductive effects. PBBs may increase steroid
metabolism during sexual differentiation, and thus produce
lifelong defects in reproductive physiology. Further
experiments are needed to determine whether the presence
of PBBs during neonatal life is capable of permanently

altering central neuroendocrine control of reproduction.

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