Citation
A genetic approach to the regulation of the constitutive forms of cytochrome P-450

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Title:
A genetic approach to the regulation of the constitutive forms of cytochrome P-450
Added title page title:
Cytochrome P-450
Creator:
Hawke, Roy Lee, 1951-
Publication Date:
Language:
English
Physical Description:
viii, 156 leaves : ill., graphs ; 29 cm.

Subjects

Subjects / Keywords:
Cytochromes ( jstor )
Female animals ( jstor )
Liver ( jstor )
Metabolites ( jstor )
Microsomes ( jstor )
Propionates ( jstor )
Rats ( jstor )
Sex linked differences ( jstor )
Steroids ( jstor )
Testosterone ( jstor )
Cytochrome P-450 Enzyme System ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF ( mesh )
Microsomes, Liver ( mesh )
Pharmacology and Therapeutics thesis Ph.D ( mesh )
Testosterone -- metabolism ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 147-154.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Roy Lee Hawke.

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University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
023215290 ( ALEPH )
10187564 ( OCLC )
ACB2408 ( NOTIS )

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Full Text













A GENETIC APPROACH TO THE REGULATION OF THE
CONSTITUTIVE FORMS OF CYTOCHROME P-450





By



ROY LEE HAWKE


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1983




A GENETIC APPROACH TO THE REGULATION OF THE
CONSTITUTIVE FORMS OF CYTOCHROME P-450
By
ROY LEE HAWKE
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1983


TO LYNNE TWIEST
This dissertation is dedicated to my wife, Lynne Twiest, for her
support, patience, and sacrifice which were instrumental in the
attainment of this degree, and to my daughters Dawne and Heather who I
regret have had to make their own special sacrifices during the many
years of my education.


LIBRARY BINDING CO., INC., St.
ACKNOWLEDGEMENTS
I would like to extend sincere thanks and appreciation to my
dissertation committee members, and to all of the faculty members and
support staff of the Department of Pharmacology who have helped me in
innumerable ways over the last four years.
I wish to especially acknowledge the heroic efforts of both Debbie
Baylor in the typing of these manuscripts as well as other "rush" jobs
which were often thrust upon her, and Jeff Jones who was forever asked
to make slides on a moment's notice or less.
I would like to acknowledge the expert technical assistance
provided by Lynn Raynor over the course of these studies. Her endless
effort in keeping the laboratory functioning smoothly has made things
easier on us all. I am truly grateful for the support and friendship
she has extended toward me over the years. The meticulous efforts of
my sister, Becky, who spent her summers working in our lab, are also
gratefully acknowledged.
Finally, I wish to express my gratitude to Allen Neims whose
greatest accomplishment was not in making this mere dissertation
possible, but rather instilling in me a genuine fascination for science
and an excitement for research which I hope to carry with me until the
end of my career. He allowed our student-teacher relationship to
continuously evolve such that I now have the confidence to think out
loud. I will sorely miss our collaboration and the many hours of
discussion spent on topics ranging from Gators to P-450.


PREFACE
This dissertation is composed of five chapters representing
different aspects of my doctoral research. Each chapter has been
written in a standard manuscript style with an introduction, a
materials and methods section, results and discussion. Some
unavoidable redundancy will, therefore, be encountered in the
introduction and discussion of each chapter as a consequence of this
style of presentation.
IV


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
PREFACE iv
ABSTRACT vi i
INTRODUCTION 1
CHAPTER ONE HETEROGENEITY OF CYTOCHROME P-450 IN THE
SITE-SPECIFIC HYDROXYLATIONS OF STEROIDS
BY AKR/J MOUSE LIVER MICROSOMES 5
Introduction 5
Materials and Methods 8
Results 13
Discussion 31
CHAPTER TWO THE INFLUENCE OF STRAIN AND SEX ON SITE-
SPECIFIC TESTOSTERONE HYDROXYLASE
ACTIVITIES OF MOUSE HEPATIC MICROSOMES 37
Introduction 37
Materials and Methods 40
Results 43
Discussion 58
CHAPTER THREE DEVELOPMENT OF SITE-SPECIFIC TESTOSTERONE
HYDROXYLASES IN HEPATIC MICROSOMES FROM
AKR/J AND BALB/cJ MICE 64
Introduction 64
Materials and Methods 67
Results 70
Discussion 81
CHAPTER FOUR GENETIC REGULATION OF HEPATIC MICROSOMAL
TESTOSTERONE 15a- and 16a-HYDR0XYLASE
ACTIVITIES IN FEMALE AKR/J AND BALB/cJ
MICE. 85
Introduction 85
Materials and Methods 88
Results 92
Discussion 109
v


TABLE OF CONTENTSContinued
PAGE
CHAPTER FIVE ENDOCRINE FACTORS IN THE REGULATION OF
HEPATIC MICROSOMAL TESTOSTERONE 15a- AND
16a-HYDROXYLASE ACTIVITIES IN AKR/J AND
BALB/cJ MICE 113
Introduction 113
Materials and Methods 117
Results 122
Discussion 139
GENERAL CONCLUSIONS 144
REFERENCES 147
BIOGRAPHICAL SKETCH 155
vi


Abstract of Dissertation Presented to t'ne Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
A GENETIC APPROACH TO THE REGULATION OF THE CONSTITUTIVE
FORMS OF CYTOCHROME P-450
By
Roy Lee Hawke
August, 1983
Chairman: Allen H. Neims
Major Department: Pharmacology and Therapeutics
The genetic, hormonal, and developmental regulation of various
forms of the cytochromes P-450 in untreated isogenic strains of mice
has been examined by virtue of the remarkable position specificity
demonstrated by these constitutive isozymes in the hydroxyl at ion of
testosterone. Careful kinetic analysis has established optimal assay
conditions for the measurement of hydroxylase activity in mouse hepatic
microsomes, and a new radiochromatographic procedure for the separation
and quantitation of six monohydroxylated products of testosterone is
described.
Detailed statistical analysis of results from studies on the
effects of sex, strain, and postnatal development on the rate of
hydroxylation at the 6a, 6b, 7a, 15a, 15b and 16a positions of
testosterone provided evidence for both heterogeneity and polymorphism
in the cytochromes P-450 catalyzing these hydroxylations and/or in
their modes of regulation. The most interesting findings from these


studies related to the 15a and 16a hydroxylations. With respect to
testosterone 16a-hydroxylase activity, 1) rates were 2- to 3-fold
higher in microsomes from males than females of all eight strains
studied; 2) prominent strain differences were detected only between
female mice; and 3) postnatal development was more rapid than the
postnatal increase in cytochrome P-450 content and the development of
other hydroxylase activities. With respect to testosterone
15a-hydroxylase activity, 1) sexual dimorphism was strain-dependent in
that rates in females were 2- to 3-fold higher than rates in males in
only four of eight strains; 2) strain differences detected between
males were independent of those found between females; and 3) a 10-day
lag in postnatal development, which was observed for this activity
only, was independent of sex but not of strain.
Analysis of genetic crosses and endocrine studies revealed that
1) 16a-hydroxyl ase activity was reversibly inducible by androgens, and
a single genetic locus regulates the basal level of activity in female
mice; 2) the strain-specific sexual dimorphism of 15a-hydroxyl ase
activity is controlled by a single genetic locus which probably is
expressed in differences between strains in responsiveness to sex
hormones; and 3) the induction of 15a-hydroxylase activity by estradiol
benzoate was independent of strain while suppression of this activity
by androgen was strain-dependent.
vi i i


INTRODUCTION
The cytochrome P-450 monoxygenase system is involved in the
biosynthesis and/or catabolism of many endogenous compounds including
cholesterol, bile acids, corticosteroids, prostaglandins, and sex
steroids. The same enzyme system participates in the metabolic
oxidation of a variety of xenobiotics such as drugs, polycyclic
aromatic hydrocarbons, food additives, pesticides, and industrial
pollutants; it thus plays an important role in xenobiotic
detoxification and elimination. Where oxidative metabolism leads to
the generation of highly reactive electrophilic metabolites, such as
epoxides or N-hydroxides, the "detoxification" reactions can result in
carcinogenic, mutagenic or teratogenic metabolites. The broad
substrate and product specificity of the cytochrome P-450 oxidizing
system reflects the presence of several forms of the enzyme. Each of
these forms of cytochrome P-450 may be more or less species-specific.
Each form is likely to have more or less distinctive substrate and
product specificity and inducer and inhibitor sensitivity, and to the
under more or less distinctive control genetically, developmentally,
and distributionally. Therefore, the P-450 monoxygenase system
combines great breadth and specificity with regard to species, sex,
age, tissue, etc. Whether or not the various isozymes represent
distinct gene products and the extent to which post-translational
modification, allosteric modifiers, lipid environment (these enzymes
1


2
are membrane-bound), etc. modulate the subtrate and/or product
specificity are not resolved.
Although the existence of multiple isozymes of cytochrome P-450 in
mammalian liver is now well established, surprising uncertainty remains
with regard to the extent of heterogeneity and the physical, chemical,
and biological basis for multiplicity especially in the constitutive
forms characteristic of the untreated animal. These forms are of
particular interest because of the possibilitity that their regulation
and functions may differ from those of the inducible isozymes. The
constitutive isozymes represent the bulk of the cytochromes P-450
present in the untreated liver, are immunologically distinct from the
induced forms, and possess greater specificity towards endogenous
substrates. Although under most circumstances constitutive isozymes
account for a large proportion of hepatic xenobiotic metabolism,
surprisingly little is known about the degree of overlap in catalytic
properties between isozymes, the mechanisms by which these forms are
independently regulated by physiological and/or environmental factors,
and the extent of allelic polymorphism in the isozymes. Clearly such
issues are of great significance for understanding and predicting
xenobiotic interactions and human individuality in the response to
drugs.
This dissertation represents a study on the genetic, hormonal and
developmental regulation of various constitutive forms of the
cytochrome P-450 isoyzmes that contribute to the site-specific
monohydroxylations of testosterone in hepatic microsomes prepared from
untreated isogenic strains of mice. Several substrates, including


3
testosterone and other endogenous steroids, have been used earlier with
success to probe the heterogeneity of the cytochromes P-450 by virtue
of the remarkable regio- and positional selectivity exhibited by
certain isozymes towards these substrates. Indeed, changes in the
relative specificity for site-specific hydroxylations as a function of
species, age, sex and other factors have provided much of the initial
evidence for the existence of multiple forms of cytochrome P-450. Our
application of a common methodology in the comparative study of hepatic
microsomal steroid hydroxylations in male and female mice of several
isogenic strains provides us with valuable insight into the modes of
regulation and polymorphic expression of these constitutive isozymes of
cytochrome P-450. This study also serves as a useful probe of the
catalytic specificity and heterogeneity of those constitutive forms of
the enzyme active toward testosterone.
It is crucial to the rationale of this dissertation that the
investigation involve isogenic animals. Although heterogeneity in the
constitutive cytochromes P-450 probably reflects the presence of a
finite number of isozymic forms of this enzyme, the degree of
complexity could be exaggerated by the many allelic differences that
randombred or outbred animals can be expected to display. The use of
inbred strains prevents intrastrain allelic variations and provides a
homogeneous background for selected studies of the heterogeneity of
cytochrome P-450 by way of interstrain comparisons.
This thesis is composed of five chapters, the first of which
describes a convenient one-dimensional TLC-radiochromatographic
procedure for the separation and quantitation of six monohydroxylated


4
products of testosterone metabolism and the development of optimal
conditions for the measurement of hydroxylase activity in mouse hepatic
microsomes. Chapters two and three deal with the effects of sex,
strain, and postnatal development on the rates of hydroxylation at the
6a, 63, 7a, 15a, 153, and 16a positions of testosterone. Finally,
chapters four and five represent detailed studies on the genetic and
endocrine regulation of testosterone hydroxylase activities, primarily
at the 15a and 16a positions, in two selected strains of mice. A final
discussion serves to integrate conclusions reached in each chapter.
Each chapter has been prepared for submission as an independent
manuscript for publication.


CHAPTER ONE
HETEROGENEITY OF CYTOCHROME P-450 IN THE SITE-SPECIFIC HYDROXYLATIONS
OF STEROIDS BY AKR/J MOUSE LIVER MICROSOMES
Introduction
The concept of isozymic multiplicity has been used often to
explain the broad substrate and product specificities of the hepatic
microsomal monoxygenase system and its distinctive responses to various
inhibitors and inducers (1,2). Direct evidence for heterogeneity in
the cytochromes P-450 of mammalian liver is abundant. Several forms of
the enzyme have been resolved (3,4), and various isozymes have been
differentiated by subunit molecular weight, immunological properties,
catalytic properties, peptide maps, partial amino acid sequences, and
even nucleic acid hybridization (5,6).
Despite this progress, surprising uncertainty remains with regard
to the extent of heterogeneity, the degree of overlap in catalytic
properties between forms, and the mode of biological regulation of
individual forms and/or catalytic activities (7). In this situation,
where one does not know exactly how many forms there are or how they
relate to one another, unrecognized polymorphism would introduce
serious and unneeded complexity. Recent reports add credibility to
this concern (8,9) and underscore the importance of conducting selected
experiments with isogenic animals. Our lack of knowledge is especially
apparent in the case of the "constitutive" forms of hepatic cytochrome
5


6
P-450, here defined as those present in untreated animals. Notably,
only a small fraction of the cytochromes P-450 found in untreated rat
liver crossreacts with antibodies raised to the isozymes induced by
phenobarbital, polycyclic aromatic hydrocarbons, or pregnenalone 16a-
carbonitn1e (10,11). These constitutive isozymes are of particular
interest because of the possibility that their substrate specificity,
function and regulation may differ from those of the inducible
isozymes.
Several substrates which yield multiple products, including
biphenyl (12), warfarin (13), and benzo[a]pyrene (14), have been used
successfully to probe the heterogeneity of the cytochromes P-450 by
virtue of the remarkable regio- and positional stereoselectivity
exhibited by certain isozymes toward these substrates. Testosterone,
an endogenous compound, has also served in this regard (15,16,17).
With rat liver microsomes, the relative specificity for hydroxylation
at the 7a, 16a and 60 positions changes with age (18,19), sex (20,21),
neonatal imprinting (20,22) and exposure to xenobiotics (23,24,25).
Some sex and strain differences in the pattern of hydroxylation of
testosterone have also been observed with mouse liver microsomes (26).
Our current work focuses on the site-specific monohydroxyl at ion of
steroids, primarily testosterone, by hepatic microsomes from untreated,
inbred mice. We have found that the usual assay methods developed for
studies of testosterone metabolism in the rat are inappropriate in mice
because the array of metabolic products is different. This report
deals with 1) the development of a reasonably rapid and economical one
dimensional TLC procedure suited to our purpose; 2) the development of


7
optimal assay conditions for the measurement of hydroxylase activity in
mouse microsomes; 3) the finding of substantial sexual dimorphism in
selective hydroxylations of testosterone by liver microsomes from AKR/J
mice; 4) the differential effects of potassium phosphate buffer on
selective hydroxylations of testosterone by preparations from these
animals; and 5) comparative experiments with progesterone as
substrate.


8
Materials and Methods
Chemicals. [4-^C]Testosterone (56.1 Ci/mol) and
[4-^C]-progesterone (57.2 Ci/mol) were purchased from New England
Nuclear, Waltham, Massachusetts. Radiochemical purities exceeded 93%
according to the TLC procedure described below. 63-, 7a-, 16a-, and
163-Hydroxytestosterone and 63-, 16a-, and 21-hydroxyprogesterone were
purchased from Steraloids, Inc., Wilton, New Hampshire. 23- and
153-Hydroxytestosterone were gifts from G.D. Searle and Co., Chicago,
Illinois. 15a-Hydroxytestosterone was kindly provided by the Upjohn
Company, Kalamazoo, Michigan, and 15a-hydroxyprogesterone was a gift
from Dr. S. Solomon, McGill University, Montreal, Canada.
6a-Hydroxytestosterone and additional samples of several of the
compounds listed above were provided by Dr. D. N. Kirk from the Medical
Research Council Steroid Reference Collection.
Testosterone and progesterone, which were purified by
recrystallization twice from acetone/hexane and ethanol/water,
respectively, and NADPH were purchased from Sigma Chemicals, St. Louis,
Missouri. All other chemicals ware purchased from the Fisher
Scientific Company, Pittsburg, Pennsylvania, and were of the highest
quality available.
Animals. Male and female AKR/J mice (8 weeks old, 20-25 g) were
purchased from Jackson Laboratories, Bar Harbor, Maine. Mice of the
same sex were housed in groups of up to six animals on corncob Sani-cel
bedding under a controlled environment for at least three weeks before
use. Smoking and the use of insecticides were prohibited, and 12-hour


9
1ight/12-hour dark cycles were maintained. Animals were provided
Purina Rodent Laboratory Chow and tap water ad libitum.
Preparation of microsomes. Mice were killed by decapitation
between 8 am and 10 am, and livers were excised and homogenized in ice-
cold 250 nil potassium phosphate buffer, pH 7.4, containing 150 mM
potassium chloride (5 ml/g of liver). Microsomes were prepared by
differential centrifugation (27) and were resuspended in 100 mM
potassium phosphate buffer, pH 7.4, to give a final suspension
containing approximately 10 mg microsomal protein per ml. The protein
content of microsomal suspensions was determined by the method of Lowry
et al. (28) with bovine serum albumin as standard.
Enzyme assays. Cytochrome P-450 content was measured by the
method of Omura and Sato (29) after microsomal suspensions were first
diluted ten-fold in 100 mM phosphate buffer, pH 7.4, containing 20%
glycerol (v/v) and 0.2% Emulgen 911 (v/v). Absorbance at 450 nm was
measured in relation to the steady baseline between 480-500 nm.
Virtually identical results were obtained without Emulgen as long as
careful baseline corrections were included.
The microsomal biotransformation of testosterone was assayed under
conditions similar to those described by Shiverick and Neims (30).
Reaction mixtures routinely contained 100 mM potassium phosphate
buffer, pH 7.4, 10 mM MgCl2, 1*0 mM NADPH, 131 yM [14C]steroid,
testosterone or progesterone (0.5 yCi) added in 10 yl of ethanol, and
0.4-0.5 mg of microsomal protein (0.2-0.3 mg for progesterone
incubations) in a final volume of 1.0 ml. The enzymatic reaction was
initiated by the addition of NADPH and allowed to proceed for 10 min at


10
37 C before termination by the addition of 5 ml of methylene chloride.
Controls routinely lacked microsomes, but zero-time and
heat-inactivated samples gave similar results.
Separation and assay of testosterone metabolites. Each incubation
mixture was shaken with methylene chloride for 30 min before collection
of the organic phase, which was then evaporated to dryness at room
temperature under a stream of nitrogen. The residue, which contained
98% of the added radioisotope, was dissolved in 50 pi of ethyl acetate.
Aliquots (25 pi) of the extract and relevant standards were applied to
scored, methanol-washed, 20 x 20 cm, silica gel 60 [r254''Precoa1:ec'>
plastic-backed thin layer chromatography plates, 0.2 mm thick (E.
Merck, Darmstadt, Germany). The plates were developed three times in
ether/benzene/methanol, 55/44/0.5 (v/v) in unlined tanks. Optimal
separation of the radiolabeled metabolites of testosterone was achieved
at 17 and an ambient humidity between 30 and 60%, conditions which
were associated with a development time of about 100 min per run. Even
at suboptimal conditions, the desired resolution could be achieved by
increasing the fraction of methanol in the developing solvent. Rarely,
a fourth development was needed to separate 15a-hydroxytestosterone
from a small zone of radioactivity just below it. Progesterone
metabolites were resolved by the same TLC system except that the plates
were developed only twice.
Radioactive zones on the TLC plates were located by autoradio
graphy with SB-5 X-ray film (Eastman Kodak Co., Rochester, N.Y.).
After the plates were first covered by a single layer of Scotch 810
transparent tape, each zone was cut out and counted directly in 10 ml


11
of Liquiscint (National Diagnostics, Somerville, N.J.) in a Beckman LS
7000 liquid scintillation counter with external quench correction
(Beckman Instruments, Inc., Fullerton, CA); recovery of applied to
the plate was greater than 95%. The Scotch tape, which was found not
to affect counting efficiency, was quite helpful in avoiding chipping
of the gel during cutting.
Metabolites were identified by comigration with unlabeled
reference standards in five different solvent systems. The standards
were visualized by fluorescence quenching. Identification and the
radiochemical purity of each putative hydroxytestosterone metabolite
was further assessed by acetylation. For these experiments, radio
active zones were cut and eluted into ethyl acetate containing 40-50 ug
of the unlabeled standard. Sufficient numbers of each zone were
combined to yield about 15,000 cpm. Samples were evaporated to dryness
under nitrogen and incubated in 100 pi of pyridine and 100 yl of acetic
anhydride in foil-covered, tightly capped test tubes. After 24 hours,
the samples were dried and chromatographed as described above except
that the plates were developed only once. More than 85% of radiolabel
in each metabolite zone was recovered during TLC of the acetylated
derivatives. The Rf values of the radioactive areas were compared to
both internal and external acetylated reference standards.
Identification of progesterone metabolites was based only on
comigration with reference standards on the TLC system described
above.
Calculations and statistical analyses. The amount of each of the
various hydroxysteroid metabolites was computed from the fraction of


12
radiolabel in a particular zone relative to the total radioactivity
applied to that lane of the TLC plate. Specific activities of steroid
"hydroxylases" are expressed as nmoles of product formed per min per mg
microsomal protein. Molecular activities are defined here as units of
hydroxylase activity per nmole of cytochrome P-450. The data were
analyzed by the unpaired Student's t-test. A P-value of less than 0.05
was taken as significant. Unless otherwise indicated, each value is
expressed as a mean and standard error.


13
Results
Identification and TLC of testosterone metabolites. The
incubation of [^C]testosterone with NADPH, oxygen and hepatic
microsomes from AKR/J male mice resulted in the formation of a large
number of metabolites. This is seen in Figure 1 (left panel) which
depicts the reaction products resolved by a 2-dimensional TLC procedure
similar to that reported by Ford et al_. (26). For routine analysis, we
developed a more rapid and economical one-dimensional TLC system. The
latter system was designed especially to separate and quantitate the
15a- and 16a-hydroxytestosterone metabolites because pilot experiments
had revealed these metabolites to be most significant for our purposes.
An autoradiogram of the one-dimensional chromatogram (Figure 1, right
panel) shows that twelve distinct zones of radioactivity were resolved.
Comparison of the one- and two-dimensional chromatograms revealed that
zones 1 and 2 (which comigrated with the 15a- and 16a-hydroxytesto-
sterone reference standards, respectively) as well as zones 4, 5, 6 and
7, were essentially devoid of obvious contaminants.
The relative mobilities of the metabolites contained in zones 1
through 8 and a set of hydroxytestosterone standards are presented in
Table 1. The metabolites were identified tentatively by comparison
with these external standards, as well as by comigration with internal
standards on one- and two-dimensional TLC. The presence of these
metabolites had been established by GC-mass spectroscopy (31).
Testosterone is in zone 11, and the metabolite in zone 5 is not yet
identified.


14
ZONE
4
Figure 1. Autoradiograms of two-dimensional (left panel) and
one-dimensional (right panel) chromatograms' of
[14C]testosterone metabolites. AKR/J male liver
microsomes were incubated with [^(^testosterone under
the conditions described in the text. Testosterone and its
metabolities were extracted and then spotted onto silica gel
F-254 plates. Two-dimensional chromatography was conducted
by first developing the plates horizontally two times with
chloroform/ethanol (92:8) and then developing the plates
vertically three times with ether/benzene/methanol
(55:44:0.5). The one-dimensional plates were developed
three times with the latter solvent system. The major
radiolabeled zones on the one-dimensional plate were
numbered arbitrarily with lowest numbers closest to the
origin.


TABLE 1
CHROMATOGRAPHIC COMPARISON OF RADIOLABELED TESTOSTERONE METABOLITES
AND THEIR ACETYLATED DERIVATIVES WITH HYDROXYTESTOSTERONE STANDARDS
AND THEIR DIACETYL DERIVATIVES.
Hydroxytestosterone
Standards
Metabolite
Zone^-
Mobil it.y
Std 14C
Rf^
Acetylated Derivatives
Std 14c
15a
1
0.07
0.07
0.64
0.64
16a
2
0.09
0.09
0.68
0.68
7a
3
0.12
0.12
0.65
0.66
0.54
153
4
0.16
0.15
0.62
0.62

0.34

5

0.21
0.73
6a
6
0.25
0.23
0.75
0.75
63
7
0.36
0.35
0.72
0.72
23
8
0.46
0.45
0.81
0.82
163
8
0.46
0.45
0.59
0.59

0.50
Testosterone
11
0.74
0.73


TABLE l--extended.
-^[^C]Testosterone, 131 yM, was incubated with hepatic microsomes from male AKR/J mice
under conditions described in text. Radiolabeled metabolites were resolved into zones as
defined in Figure 1.
^One-dimensional TLC was performed according to the text and Figure 1. Standards (Std)
were located by fluorescence quenching; metabolites were located by radioautography.
In all cases, standards admixed with radiolabeled metabolites comigrated with the
metabolite in question. Since the plates were developed 3 times, mobilities are
expressed relative to the solvent front after the third run.
^Standards and radiolabeled metabolites were acetylated as described in the text.
Standard (Std) and metabolite (^C) derivatives were located by fluorescence
quenching and radioautography, respectively. When standards were admixed with
metabolites before acetylation, the derivatized standard comigrated with the acetylated
metabolite in question. Acetylated metabolites were resolved by a single development in
the one-dimensional TLC system described in the text.


17
Further identification, as well as assessment of the radio
chemical purity, of the metabolites contained in zones 1-8 was
accomplished by acetylation of standards and the labeled material in
each zone. After acetylation, essentially all of the radioactivity in
zones 1 and 2 migrated with Rf's indistinguishable from the diacetyl
derivatives of 15a- and 16a-hydroxytestosterone, respectively (Table
1). As suggested by the results depicted in Figure 1, zones 3 and 8
contained more than one labeled compound. About 60% of the label in
zone 3 comigrated with the diacetyl 7a-hydroxytestosterone standard.
Zone 8 contained substantial quantities of both 23- and
163-hydroxytestosterone, 55% and 28%, respectively, as well as an
unidentified product. Derivatization confirmed that the radiolabel in
zones 4, 6 and 7 was composed almost entirely of 153-, 6a-, and
63-hydroxytestosterone, respectively, although a minor (approximately
20%) contaminant was found along with 153-hydroxytestosterone in zone
4. The stabilities of individual diacetylated metabolites (or
standards) were not determined, and lability could have contributed to
some of the apparent contamination.
The [^C]testosterone metabolites produced by AKR/J male
hepatic rnicrosomes were also identified by their comigration with
reference standards in 4 other TLC systems used in earlier studies of
the biotransformation of testosterone by rodent liver rnicrosomes (Table
2). Careful examination of Table 2 reveals that none of these solvent
systems resolved all of the metabolites in question. Nonetheless, the
sum of these results supports our identification of metabolites.


TABLE 2
COMPARISON OF THE MOBILITIES OF RADIOLABELED TESTOSTERONE
METABOLITES FORMED IN THE PRESENCE OF HEPATIC MICROSOMES FROM
MALE AKR/J MICE WITH HYDROXYTESTOSTERONE STANDARDS ON FOUR
TLC SYSTEMS.
Rf
Testosterone Solvent System^.
Metabolite
/£ B C D
Std
14C
Std
14C
Std
14C
Std
14C
15a-Hydroxy
0.26
0.26
0.39
0.39
0.27
0.28
0.04
0.04
16a-Hydroxy
0.30
0.30
0.46
0.46
0.34
0.35
0.05
0.05
7a-Hydroxy
0.43
0.43
0.53
0.51
0.41
0.42
0.07
0.08
153-Hydroxy
0.43
0.43
0.46
0.47
0.39
0.39
0.09
0.09
Zone 5

0.57

0.56

0.48
0.12
6a-Hydroxy
0.60
0.61
0.63
0.60
0.50
0.48
0.14
0.12
63-Hydroxy
0.64
0.67
0.67
0.64
0.57
0.55
0.19
0.17
Testosterone
0.88
0.89
0.86
0.86
0.86
0.85
0.60
0.60


TABLE 2extended.
1TLC was conducted under conditions described in the text except for solvents:
A, methylene chloride/acetone (9/5, v/v); B, chioroform/ethyl acetate/ethanol
(40/10/7); C, chloroform/ethanol (9/1); and D, chioroform/ether (7/3). Plates were
developed once with solvents B, C and D, but twice with A. Standards (Std) were
located by fluorescence quenching, and metabolites (^C) were located by
radio- autography. In all cases, standards admixed with radiolabeled metabolites
comigrated with the zone in question.
hs ince plates were developed twice with solvent A, mobilities (not Rf's) are
expressed relative to the solvent front after the second run.


20
Characteristics of the enzymatic reaction. Typically, 20-25% of
the substrate, testosterone (initial concentration, 131 yM), was
consumed in 10 min upon incubation with 0.4-0.5 mg microsomal protein
The metabolites in zones 1 (15a-hydroxytestosterone) through 7
(63-hydroxytestosterone) accounted for more than 65% of the total
metabolism. 60-Hydroxytestosterone was the major metabolite formed in
the presence of both male and female liver microsomes. Each of these
metabolites was produced more rapidly in the presence of microsomes
than any other subcellular fraction.
Rates of formation of each of these hydroxytestosterone
metabolites were proportional to microsomal protein concentration
(Figure 2). Figure 3 depicts the relationship between time and the
production of metabolites. Rates of formation of 15a- and
16a-hydroxytestosterone were constant for 15 min. The rates of
formation of 7a-, 150-, 6a- and 60-hydroxytestosterones were constant
for at least 10 min. After 15 min of incubation, the rates of
formation of all products, especially 60- and 150-hydroxytestosterone,
decreased significantly.
The metabolism of testosterone by hepatic microsomes from male and
female mice. Figure 4 compares the relative rates of formation of six
hydroxytestosterone metabolites in the presence of microsomes prepared
from male and female AKR/J mice. A female predominance was observed in
hydroxylase activity at the 6a, 60, 150 and especially the 15a position
of testosterone. Hydroxylation at 7a was not affected by sex, and
hydroxylation at the 16a position of testosterone was uniquely
predominant in males. Table 3 compares the apparent Km and Vmax
for


21
Figure 2. Effects of protein concentration on the rates of formation of
various hydroxytestosterone metabolites. The standard
reaction mixture, which was incubated for 10 minutes at
37C, contained 131pM [^C]-testosterone, 100 mM phosphate
and NADPH as described in the text. The microsomes were
prepared from male AKR/J liver.


22
Figure 3. Effects of time of incubation on the formation of various
hydroxytestosterone metabolites. The standard reaction
mixture, which was incubated at 37C, contained 0.4 mg
microsomal protein prepared from male AKR/J liver, 131 pM
[^C]-testosterone, 100 mM phosphate and NADPH as
described in the text.


TESTOSTERONE HYDROXYLASE ACTIVITY
(nmoles/min/mg protein) EXCLUDING 6/9
23
HYDROXYTESTOSTERONE METABOLITES
0.7
0.5
0.3
0.1
15a I6a 7a
X
*
T
15/9 6a 6/S
I
j.
t4.0
3.0
2.0
1.0
cfy cdy cfj cfg cfg
Figure 4. Rates of formation of six hydroxytestosterone metabolites by
microsomes prepared from male and female AKR/J mice. [^C]-
testosterone was incubated with microsomes under standard
incubation conditions described in the text. Note different
scale for 60-hydroxytestosterone. N=5 and indicates
P<0.01 for male-female comparison.
6/3 HYDROXYLASE ACTIVITY
(nmoles /min/mg protein)


TABLE 3
APPARENT KINETIC CONSTANTS FOR HEPATIC MICROSOMAL TESTOSTERONE
HYDROXYLASES IN MALE AND FEMALE AKR/J MICE
Hydroxytestosterone
Metabolite
Apparent Kinetic
Constants ( SE)
Male
Female
Ktn
(pM)
(nmol/mg^lO min)
Km
(pM)
(nmol/mg^lO min)
15a
4.2 ( 1.2)
1.43 (0.19)
3.7 ( 0.4)
5.15* (0.54)
16a
1.4 ( 0.2)
4.19 (0.43)
4.9 ( 1.7)
1.22* (0.12)
7a
2.7 ( 1.0)
0.81 (0.09)
5.6 ( 0.5)
0.86 (0.07)
15s
45.9 ( 7.7)
1.50 (0.18)
41.3 ( 7.6)
2.40* (0.33)
6a
5.1 ( 0.6)
2.20 (0.23)
6.1 ( 0.2)
3.92* (0.36)
63
60.9 (11.3)
29.20 (2.89)
80.1 (10.6)
53.11* (7.14)


TABLE 3extended.
IHepatic microsomes from male and female mice were incubated with 6 different concentrations
of [^^testosterone (5-500 yM) of varying specific radioactivity (total of 0.17-1.5
yCi/incubation) under conditions defined in the text except that incubation times were
adjusted to assure measurement of initial velocities at each substrate concentration,
n = 3.
-^max 1S expressed as nmoles of product formed per mg of microsomal protein per 10 minutes,
indicates P < 0.05 with regard to male/female comparisons.


26
production of each metabolite in the presence of male and female
microsomes. Lineweaver-Burk plots for each metabolite were essentially
linear with preparations from either sex. There were no significant
differences between male and female mice in the apparent Km values
for any of the six testosterone hydroxylations. For the 6a, 7a, 15a
and 16a hydroxylations, the apparent Kms for testosterone were low
(1.4 to 6.1 pt-1) in comparison to those for 63 and 153 hydroxyl ations
(41 to 80 pM).
Maximal velocity for 15a hydroxylation was more than 3-fold
higher with female than male preparations, while Vmax for 16a
hydroxylation of testosterone was more than 3-fold higher with male
than female microsomes. Both VRiax's and relative rates for 6a,
63 and 153 hydroxylations were significantly higher in female
preparations, but differences in each case were less than 2-fold.
7a-Hydroxylation was the only activity studied that was not affected by
sex, although only 60% of the radiolabel in its zone is
7a-hydroxytestosterone. Admixture of liver microsomes from male and
female mice in 4 different proportions yielded additive activities for
production of the 15a-, 16a- and other hydroxytestosterones (data not
shown). Therefore, we find no evidence for the presence of a
reversible, non-stoichiometric activator or inhibitor for any of the
hydroxylations. The mixing experiments also decrease the probability
of product inhibition as an explanation for the sexual dimorphism.
The content of cytochrome P-450 in hepatic microsomes was not
dependent on sex (inale and female, 0.816 and 0.760 nmoles P-450/mg
microsomal protein, respectively). Thus the sexually dimorphic


27
hydroxylase activities persist even when results are expressed in terms
of molecular activity. Indeed, we found less day-to-day variation in
molecular activities than in specific activities.
The effects of potassium phosphate concentration on testosterone
hydroxyl ation. All forementioned experiments were conducted in 100 mM
phosphate buffer. Figure 5 presents the results of experiments in
which the relative rates of hydroxylation of testosterone by male and
female microsomes were studied at 30, 100 and 330 mM phosphate, all at
pH 7.4. The effects of changing potassium phosphate concentration were
strongly dependent on the position of hydroxylation, and in the case of
16a and 15a hydroxylations, on gender as well. An 11-fold increase in
phosphate concentration resulted in substantial inhibition (75 to 90%)
of 15a and 7a hydroxylation, little or no effect on 15b and
6a-hydroxylation, and a modest stimulation of 6b and 16a (male only)
hydroxylation. Male 15a hydroxylation was relatively insensitive to
changes in potassium phosphate concentration between 30 and 100 mM, but
female activity decreased 2-fold.
Progesterone. A limited number of experiments were conducted with
progesterone as substrate in order to gain some insight into the
substrate specificity of the hydroxylases under investigation. Results
with hepatic microsomes from male and female AKR/J mice are presented
in Table 4. Progesterone was actively metabolized, and 15a-, 16a-, 6b-
and 21-hydroxyprogesterone were identified as important products. Like
testosterone, 6b hydroxylation of progesterone was quantitatively most
important and exhibited a small but significant female predominance.
The female predominance seen in 15a hydroxylation with testosterone was


28
even more striking with progesterone in that a 5 to 6-fold female/male
ratio was observed. The male predominance in 16a hydroxylation with
testosterone (3-fold) was not seen with progesterone.


% ACTIVITY % ACTIVITY
29
HYDROXYTESTOSTERONE METABOLITES
POTASSIUM PHOSPHATE BUFFER (mM)
Figure 5. Effects of potassium phosphate buffer concentration on the
rates of formation of different hydroxytestosterone
metabolites in the presence of hepatic microsomes from male
and female AKR/J mice. Livers from 2 mice were pooled for
preparation of microsomes. Incubation conditions are
described in the text; the initial [^C]-testosterone
concentration was 131 pM, but the concentration of potassium
phosphate, pH 7.4, was varied as indicated. All hydroxylase
activities are expressed in relation to activity at 100 mM
phosphate (defined as 100%) since that concentration is used
in the standard assay, n = 3. and + indicate PC0.05 for
30-100 mM and 100-330 mM comparisons, respectively.


TABLE 4
RATES OF FORMATION OF HYDROXYPROGESTERONE METABOLITES IN THE
PRESENCE OF HEPATIC
MICROSOMES FROM
MALE AMD FEMALE AKR/J
MICE
Hydroxyprogesterone
Metabolite
Rates of Formationi
(nmoles/mq protein/min)
Ratio of
Activities
9
d
9/£
15a
1.23
0.22*
5.59
16a
0.72
0.75
0.96
6b
4.43
3.45**
1.29
21
0.39
0.29
1.35
a.[14c]-Progesterone, 131 pM, was incubated for 10 minutes with
microsomes under conditions described in the text; phosphate
concentration was 100 mM. Metabolites were resolved by TLC and
assayed radiometrically.
indicates P < 0.005 (male/female comparison).
**Indicates P < 0.025 (male/female comparison).


31
Discussion
These results initiate a set of detailed studies with highly
inbred mice on the heterogeneity, substrate specificity and regulation
of the constitutive forms of cytochrome P-450. The overall project is
designed to control for possible allelic polymorphism in the P-450's
and later, to take advantage of potential strain differences. Our
initial goal was to establish a reasonably rapid and economical system
for assay of the major monohydroxylated metabolites of testosterone.
We have developed a one-dimensional radiochromatographic technique for
the assay of several hydroxytestosterone metabolites produced by mouse
liver microsomes. The method has better resolving power than other
reported TLC systems and is more rapid, less tedious and/or less
expensive than the two-dimensional TLC or the paper chromatographic
procedures that have recently been used in studies with mice (26,32).
We have focused on six hydroxytestosterone metabolites which
together comprise more than two-thirds of total products. Metabolites
were identified by comigration with reference standards in five
different solvent systems; identifications were confirmed by study of
diacetyl derivatives. While our method is good for assay of the 15a-,
16a-, 6a- and 63-hydroxytestosterone metabolites, 7a- and
153-hydroxytestosterone account for only about 60% and 80% of the
labeled metabolites in zones 3 and 4, respectively. We are especially
cautious in the interpretation of 7a-hydroxylase activity because of
the susceptibility of this metabolite to spontaneous dehydration to the
4,6-diene both before and during TLC (31).


32
Large discrepancies exist in the reported rates of 16a, 7a and 63
hydroxylation of testosterone in the presence of hepatic rnicrosomes
from various strains of mice (32-35). These discrepancies, which also
include differences in the rank order of the different hydroxylase
activities, could reflect allelic polymorphism between isogenic strains
of mice, but at least some of the discrepancy seems to be
methodological; it results from certain features peculiar to murine
hepatic steroid metabolism. Ford et^ ad_. (31) have reported the
two-dimensional TLC separation of several of the metabolites of
testosterone formed by mouse liver rnicrosomes. At least two of these
metabolites, 15a- and 150-hydroxytestosterone, have not been reported
after incubation of testosterone with rat or rabbit rnicrosomes. Mouse
liver rnicrosomes also possess little if any 4-ene reductase activity
(31). Yet many earlier experiments with mice have been conducted
employing solvent systems (Table 2) and assay conditions optimized for
studies with rat or rabbit.
We have examined the rates of formation of each metabolite as a
function of various assay conditions. Subcellular fractionation
revealed that the hydroxylases in question were concentrated in the
microsomal fraction and that varying the NADPH concentration 2-fold did
not influence results. As discussed below, because the concentration
of potassium phosphate buffer had differen- tial effects on
hydroxylation at different positions, we elected to maintain phosphate
at a concentration nearly that found in liver cytosol (ca. 70 mM).
Under our standard incubation conditions, testosterone concentration
was at least 20 times greater than the Km's for the formation of all


33
products except 63- and 153- hydroxytestosterone. This assured
catalytic rates near Vmax for most products, and rates that were at
least 60% Vmax for all products. While the amount of each product
formed was proportional to both time of incubation and microsomal
protein concentration, our standard incubation conditions remain just
inside the limits of linearity for the formation of 63- and
153-hydroxytestosterone, perhaps reflecting the higher Km's for
these hydrcxylations and/or differences between P-450 isozymes.
There was little variability between AKR/J mice of the same sex
with regard to rates of formation of six different hydroxytesto-
sterones; standard deviations approximated 15%. The rates of 15a, 153,
6a and 63 hydroxylation of testosterone were higher in female than male
AKR/J microsomes with 15a-hydroxylation exhibiting the greatest degree
of sexual dimorphism (3 to 4-fold female predominance) despite the lack
of sexual dimorphism in P-450 content. Hydroxylation at 7a was not
sexually dimorphic, and the rate of formation of 16a-hydroxytesto-
testosterone was 3 to 4-fold faster in male than female microsomes.
Sex-dependent differences in steroid and drug metabolism in rats have
been well documented (36,37), and recent evidence suggests that these
differences reflect the presence of sex-dependent forms of hepatic
microsomal cytochrome P-450 (38,39). Reports of sex differences in
drug metabolism in mice are inconsistent, perhaps reflecting
strain dependence and/or a smaller quantitative difference between the
sexes in mice (40,41). The sexual dimorphisms we find for 15a and 16a
hydroxylation of testosterone are larger than typically seen with other


34
substrates in mice, perhaps because it is an endogenous substrate.
Sex differences in the selective hydroxylations of testosterone were
also exhibited by A/J and 129/J (but not the C57BL/6J) mice (26).
Studies of testosterone metabolism in reconstituted P-450 systems
from animals induced with different xenobiotics have suggested that
hydroxylations at the 6p, 7a and 16a positions are catalyzed primarily
by different forms of cytochrome P-450 (42,43,44). This concept has
been extended recently to untreated animals (45,46). By virtue of the
broad substrate and/or product specificity exhibited by certain forms
of cytochrome P-450, it is likely that hydroxylase activity at a given
position on the steroid nucleus reflects the activity of more than one
monoxygenase present in hepatic microsomes. Recently, Thomas et al_.
(47) have established the existence of more than one testosterone 16a-
hydroxylase in rat liver microsomes. They found a large proportion of
testosterone 16a hydroxylation in microsomes from untreated rats to be
immunologically distinct from that due to cytochrome P-450^. Our
finding of a male predominance (3- to 4-fold) in 16a-hydroxylase
activity toward testosterone but not progesterone suggests that the
steroid 16a-hydroxylase activity of mouse hepatic microsomes may also
be heterogeneous. Based on analogous experiments, a similar
interpretation was proposed by Kremers et al_. (48) for the rat. The
differential effects of potassium phosphate concentration also support
this conclusion. Male but not female testosterone 16a-hydroxylase
activity was stimulated by high potassium phosphate concentrations.
Our findings probably reflect a different distribution of P-450's
in male and female AKR/J liver, with each P-450 having more or less


35
discrete specificity with regard to the selective hydroxylation of
testosterone. Nonetheless, it seems quite probable that any given
"hydroxylase" (e.g. testosterone 16a-hydroxylase) is the result of
activities imparted by more than one enzyme. Certainly P-450's with
the functional propensity to direct most hydroxylation of testosterone
to the 15a or 16a positions can be distinguished from each other and
from other testosterone hydroxylases by comparison of sexes. To what
extent male and female forms of the P-450's differ qualitatively and/or
quantitatively cannot be surmised at this time. Kinetic experiments
reveal that the sex differences in AKR/J mice were not due to
differences in apparent Km's between sexes. Furthermore, mixing
experiments did not suggest a gender-dependent activator or inhibitor.
Our results could indicate differences in the concentrations of certain
forms of cytochrome P-450 between males and females.
It is not known whether the female predominance in 15a, and to a
lesser extent 153, 6a and 63 hydroxylation reflects quantitative or
qualitative differences in one or several P-450 isozymes.
Interestingly, the female predominance in 15a hydroxylation observed
with testosterone is even more striking with progesterone. The greater
sex difference results from higher rates of 15a hydroxylation toward
progesterone than testosterone with female microsomes; male
15a-hydroxylase activities are similar with both substrates. This
result suggests qualitative differences between the process of
15a hydroxylation in males and females and indicates that steroid
15a-hydroxylase is also a heterogeneous enzyme. This conclusion is
also supported by the effects of potassium phosphate. Rates for 15a


36
hydroxylation of testosterone in females were found to be 2-fold higher
at 30 mM potassium phosphate than at 100 mM while little difference was
observed in 15a-hydroxylase activity in males at these two
concentrations. This interpretation is more convincing when one
considers that the effects of phosphate concentration on other
hydroxylations (except 16a) are the same with male and female
preparations. More detailed experiments of the stimulatory and
inhibitory effects of potassium phosphate (cf. 49) are in progress.


CHAPTER TWO
THE INFLUENCE OF STRAIN AND SEX ON SITE-SPECIFIC TESTOSTERONE
HYDROXYLASE ACTIVITIES OF MOUSE HEPATIC MICROSOMES
Introduction
The isolation of a number of inducible cytochrome P-450 isozymes
from liver microsomes of rat (43,50) and rabbit (51,52) as distinct
proteins with different chemical and physical properties (3,4) has
established the multiplicity of mammalian hepatic microsomal cytochrome
P-450. Recently, attention has also been focused on the various forms
of cytochrome P-450 from untreated rats (4,6) and rabbits (45,53),
partly because immunological techniques have revealed a lack of
cross-reactivity between various inducible forms and the bulk of these
"constitutive" isozymes (10,11,47). The molecular basis for
heterogeneity within the constitutive forms is a particularly difficult
problem because each isozyme likely occurs in relatively small amounts.
Nonetheless, these forms are of special interest because of the
possibility that their physiological function(s) and regulation may be
quite different from those of the inducible isozymes. These isozymes
likely have greater specificity than the inducible forms toward
endogenous substrates like steroids, and their regulation involves
factors such as neonatal and pubertal sexual differentiation (38,39).
The extent of heterogeneity in both inducible and constitutive
isozymes of P-450 could be exaggerated in studies of randombred or
outbred animals by the many allelic differences that such animals are
37


38
likely to display. Johnson et_ al_. (8) have recently described
differences in progesterone 21-hydroxylase activity and form 1 P-450
between individual New Zealand White rabbits. Moreover, Vlasuk et
a]_.(9) have reported that Holtzman and Long-Evans rats, and even
different colonies within those strains, could be distinguished by the
characteristics of their phenobarbital-induced cytochromes P-450.
While it is not clear whether this degree of complexity reflects the
presence of genetically distinct P-450 apoproteins or is the result of
postranslational modification(s) of one or more gene products, it is
clear that the heterogeneity of cytochrome P-450 and its regulation
represent a complex problem involving both genetic and environmental
influences.
Several substrates, including testosterone and other endogenous
steroids, have been used successfully to probe the heterogeneity of the
cytochromes P-450 by virtue of the remarkable regio- and positional
selectivity exhibited by certain isozymes. We reasoned that a detailed
comparative study of hepatic microsomal steroid hydroxylations in male
and female animals of several isogenic mouse strains could provide
valuable insight into the catalytic specificity, regulation and
polymorphic expression of several forms of cytochrome P-450 in
untreated animals. Strain differences in steroid hydroxylation have
been reported for many species (54,55,56) and in some instances the
patterns of inheritance have been worked out (57,58). In regard to the
hydroxylation of testosterone by mouse liver microsomes, large
discrepanices exist between reported rates for 16a, 7a, 6ft and other
hydroxylations in studies by various investigators (26,32,33). These


39
differences could reflect a large degree of polymorphism among isogenic
mouse strains, but methodologic differences between laboratories
probably have contributed (59). In this study, the use of inbred
strains controls for intrastrain allelic variations and a common
methodology applies in all experiments. These factors allow more
meaningful interstrain and gender comparisons.


40
Materials and Methods
Materials. Testosterone [4-l^C] (56.1 yCi/ymol) was
purchased from New England Nuclear, Waltham, Ma. The radiochemical
purity was greater than 98% as established by TEC on silica gel
F^54 plates with the solvent system ether/benzene/methanol,
55:44:0.5 (v/v). Testosterone was obtained from Sigma Chemical Co.,
St. Louis, Mo., and was recrystallized twice from acetone/hexane.
Reference steroid standards were obtained through sources described
previously (59).
Precoated (0.2 mm) silica gel 60 F254 TLC plates (E. Merck,
Darmstadt, Germany) were purchased from Scientific Products, Brunswick,
N.J. SB-5 X-ray film for autoradiography was obtained from the Eastman
Kodak Co., Rochester, N.Y., and Liquiscint liquid scintillation
cocktail was obtained from National Diagnostics, Somerville, N.J.
NADPH was purchased from Sigma Chemicals, St. Louis, Mo. All other
materials were purchased from the Fisher Scientific Co., Pittsburgh,
Pa., and were of the highest quality commercially available.
Animals. Mice of 8 strains were obtained at the age of 6-8
weeks (20-25 g) from Jackson Laboratories (Bar Harbor, Me). Mice of
the same sex were housed 4-6 per cage on corncob Sani-cel bedding. The
animals were maintained on a 12 hr light/12 hr dark photoperiod and
were allowed both food (Purina Rodent Chow) and water ad libitum. All
mice were 11-14 weeks of age at the time of study.
Preparation of microsomes. Given the possibility of circannual
variation in testosterone hydroxylase activity (26), all animals were
studied between September and December. Male and female mice of a


41
given strain were killed by decapitation on the same day between 8 and
10 am. Livers were excised immediately, and gall bladders were
carefully removed. The livers were then rinsed, and each gram of liver
was homogenized with a motor-driven glass-Teflon homogenizer in 5 ml of
ice-cold 250 nil potassium phosphate buffer, pH 7.4, containing 150 mM
potassium chloride. Microsomes were prepared by differential
centrifugation. Briefly, the homogenate was centrifuged at 10,000 g
for 15 min, and the supernatant fraction was recentrifuged for 1 hr at
110,000 g. Microsomal pellets from 1 g of liver were resuspended in 2
ml of 100 mM potassium phosphate, pH 7.4, and assayed immediately.
Microsomal suspensions contained approximately 8-10 mg protein per ml
as determined by the method of Lowry et aj_. (28). Cytochrome P-450
content was determined by the method of Omura and Sato (29) after
microsomal suspensions were diluted 10-fold in 100 mM phosphate, pH
7.4, containing 20% glycerol (v/v) and 0.2% Emulgen 911.
Radiometric assay of testosterone hydroxylase activities. The
rates of formation of 6a-, 63-, 7a-, 15a-, 15$-, and
16a-hydroxytestosterone were determined by the radiometric assay we
described earlier (59). Briefly, 0.4-0.5 mg microsomal protein were
incubated at 37 C in a shaking water bath with l^C-enriched
testosterone (131 nmol containing 0.5 yCi; final concentration, 131 yM)
added in 10 yl ethanol; 1.0 ymol NADPH (final concentration, 1.0 mM);
10.0 ymol MgCl2 (final concentration 10.0 mM); and potassium
phosphate buffer, pH 7.4 (final concentration, 100 mM) in a final
volume of 1.0 ml. Reactions were started by the addition of NADPH and
stopped 10 min later by the addition of 5 ml of ice-cold methylene


42
chloride. Each sample was then transferred to a 10-ml Teflon-lined
screw cap test tube and shaken vigorously for 30 min. Phases were
separated by centrifugation, and the organic phase was collected and
evaporated to dryness at room temperature under a stream of nitrogen.
Residues were resuspended in 50 yl ethyl acetate and resolved by
TLC. Routinely, plastic-backed silica gel TLC plates were developed
3-times with ether/benzene/methanol (55:44:0.5, v/v). The testosterone
metabolites formed in the presence of hepatic microsomes from at least
one male and one female from each of the 8 strains were also resolved
by two-dimensional TLC; silica gel plates (20 x 20 cm) were developed
in the first dimension with chioroform/ethanol (92:8) and in the second
dimension 3-times with the solvent system described above. All
chromatograms were subjected to radioautography, and radiolabeled zones
were cut out and quantitated by scintillation spectrophotometry.
Statistics. Two-way analysis of variance was conducted for each
hydroxylase activity in order to test for the influence of sex, strain,
and the interaction between sex and strain on enzymatic activity (60).
Where the interaction between sex and strain was found to be
significant, Duncan's multiple comparison procedure was used to compare
the 8 strains at both levels of sex, and also to compare the two sexes
at each level of strain (61).


43
Results
The data collected in this study are reasonably straightforward;
the rates of formation of six different monohydroxytestosterone
metabolites in the presence of hepatic microsomes from males and
females of eight strains of mice were determined. Mean values for the
various testosterone hydroxylase activities for five animals of each
sex and strain are presented in Tables 5 (specific activity or
catalytic activity/mg microsomal protein) and 6 (apparent molecular
activity or catalytic activity/nmole P-450). Standard deviations for
the 16 groups of animals ranged from 10 to 30 percent of the respective
means.
Earlier experiments (59) with hepatic microsomes from male and
female AKR/J mice afford background for assessment of the results
presented in Tables 5 and 6. 1) The six metabolites under scrutiny
comprised more than 65 percent of the total biotransformation products
of testosterone. 2) The formation of each metabolite was proportional
to time of incubation and microsomal protein concentration under the
conditions of assay. 3) The initial testosterone concentration in
these experiments was 131 yM; the apparent K^'s for production of
each metabolite were less than 7 yM except for 15f>-hydroxytestosterone
(50 yM) and 60-hydroxytestosterone (80 yM). In no case was more than
30 percent of the testosterone consumed during incubation.
Even a casual examination of the results depicted in Tables 5 and
6 revealed certain striking findings. 1) The rates of hydroxylation of
testosterone at six positions were quite similar in the various strains
of animals, especially among males. For example, 63-hydroxylase


TABLE 5
SPECIFIC ACTIVITIES OF TESTOSTERONE HYDROXYLASES: RATES OF FORMATION
OF VARIOUS METABOLITES IN THE PRESENCE OF HEPATIC MICROSOMES
FROM MALES AND FEMALES OF EIGHT STRAINS OF MICE.
Testosterone Specific Activity^
Metabolite (nmoles product/10 inin/mg microsomal protein)
BALB/cJ
C3H/HeJ
C57BL/6J
SWR/J
DBA/2J
A/J
129/J
AKR/J
MALE
I 5a-OH
1.68
1.45
1.63
1.11
1.20
1.33
1.60
1.37
1 6a-OH
4.80
4.77
4.20
3.79
4.07
4.33
3.06
3.87
7a-OH
1.15
1.75
1.64
1.56
1.94
0.93
1.65
1.15
15(3-OH
1.09
0.65
1.45
1.46
0.86
1.43
1.34
1.19
6a-OH
2.73
1.45
3.45
3.05
2.04
2.57
2.89
2.90
6(3 -OH
18.56
21.70
18.49
32.09
20.41
26.32
23.66
27.58


TABLE 5--extended.
FEMALE
15a-0H
1.96
1.18
1.30
1.40
2.39
2.78
3.36
4.64
16a-0H
2.34
1.39
1.39
1.67
2.31
1.87
0.93
1.15
7 cx-OH
1.06
2.02
2.02
2.27
2.68
0.82
2.52
1.06
150-OH
1.00
0.76
1.01
1.71
1.18
1.28
1.52
1.94
6a-0H
2.39
1.64
3.21
4.04
3.04
2.48
4.17
3.78
6B-0H
18.72
23.38
14.25
30.27
23.63
22.69
27.08
32.29
An=5 for each sex and
strain. See
text for
statistical
analyses.


TARLE 6
APPARENT MOLECULAR ACTIVITIES OF TESTOSTERONE HYDROXYLASES:
RATES OF
FORMATION
MICROSOMES
OF VARIOUS
FROM MALES
METABOLITES
AND FEMALES
IN THE PRESENCE OF
OF EIGHT STRAINS
HEPATIC
OF MICE.
Testosterone
Metabol ite
(nmoles
Apparent Molecular
product/10 min/nmole
Activity^ b
cytochrome P-450-
)
BALB/cJ
C3H/HeJ
C57BL/6J
SWR/U DBA/2J
A/J
129/J
AKR/J
MALE
15a-OH
2.34
1.81
2.54
1.14
1.94
1.95
2.88
1.67
16a-OH
6.71
6.08
6.50
3.89
6.67
6.35
6.01
4.79
7a-OH
1.60
2.22
2.52
1.59
3.21
1.36
2.81
1.43
1 5(3 -OH
1.54
0.84
2.26
1.49
1.41
2.09
2.43
1.45
6a-OH
3.87
1.87
5.29
3.11
3.41
3.75
5.26
3.56
6(3 -OH
25.93
27.71
28.64
32.73
33.52
38.35
41.17
33.28
CTi


TABLE 6--extended.
FEMALE
15a-0H
3.10
1.86
2.74
1.62
3.37
4.86
6.06
6.19
16a-0H
3.76
2.18
2.94
1.93
3.29
3.24
1.66
1.53
7a-0H
1.68
3.18
4.31
2.61
3.82
1.41
4.60
1.41
153-OH
1.60
1.18
2.15
1.98
1.65
2.22
2.72
2.59
6a-0H
3.39
2.57
7.19
4.67
4.36
4.28
7.53
5.07
63-OH
29.86
36.65
30.33
34.98
33.16
38.43
47.42
43.35
^N=5 for each
strain and
sex. See
text for
statistical
analyses.
Cytochrome P-450 was measured spectrophotometrically; see text for details.


48
activity was about 10-fold higher than other activities in ail strains.
2) The rate of 16a hydroxylation proceeded about 2 to 3-fold faster in
male than female preparations of all strains. 3) The rate of most
other hydroxylations, especially 15a hydroxylation, proceeded more
rapidly in female than male microsomes, but the effect was
strain-specific. 4) For most comparisons, mean specific activities and
apparent molecular activities lead to similar conclusions because mean
P-450 content per mg microsomal protein varied little between strains
or sexes. Of the eight strains studied, only C3H/HeJ and C57BL/6J
demonstrated a consistent sex difference in hepatic cytochrome P-450
content. In these two strains P-450 content was 20-30% greater in male
than female microsomes.
Results of a two-way analysis of variance for five hydroxylase
activities in 80 animals (5 males and 5 females for 8 strains) are
presented in Table 7 in terms of values. 7a-Hydroxylation is
omitted from detailed analysis because only about 60% of radiolabel in
its TLC zone was 7a-hydroxytestosterone (59). "Total" represents
the percent of the total variability for a given hydroxylase activity
that can be accounted for by the sum of the effects of sex, strain and
the sex-strain interaction. Unusually high values were obtained for
15a- and 16a-hydroxylase activities in that 85-90% of all individual
variability in these activities could be accounted for by these
variables. By comparison, only 50% of the variability in
63-hydroxylase activity could be attributed to the same factors.
Except for 63-hydroxyl at ion, the effect of sex, strain and the
interaction between sex and strain were all significant determinants of


TABLE 7
ANALYSIS OF VARIANCE (ANOVA) BETWEEN SEX, STRAIN, AND THE
INTERACTION BETWEEN SEX AND STRAIN AND THE SELECTIVE
HYOROXYLATION OF TESTOSTERONE.
Position of
Hydroxylation
R^ Values
Sex
Strain
Sex + Strain
Total
Total
15a
0.8194
0.3054
0.3109
0.8357
0.8679
16a
0.7770
0.1015
0.0272
0.9057
0.9370
156
0.0217
0.5149
0.1776
0.7142
0.7276
6a
0.0666
0.5372
0.1150
0.7188
0.7496
63
0.0010*
0.4784
0.0527*
0.5322
0.5656
^N=80 for specific activity at each position of hydroxylation since 5
males and 5 females were studied for each of 8 strains.
-ANOVA of apparent molecular activities instead of specific
activities.
:k
Failed test for significance. All other values are significant
(P < 0.05).


50
the rates of position-specific monohydroxyl at ion of testosterone (P <
0.05). For 16a hydroxylation, sex was the most important determinant.
For 15a hydroxylation, the interaction between sex and strain (i.e.,
the effect of one factor depends upon the level of the other factor)
was most important. In the other cases, strain was the most important
determinant.
Male and female comparisons of testosterone hydroxylase activities
for each strain are presented in Table 8. Sex ratios are given only
when the differences were statistically signficant. Statistical
analysis of either specific activities or apparent molecular activities
results in similar interpretations except in a few cases where the
differences between the sexes was 40% or less. As indicated in Tables
5 and 6, the rate of 16a hydroxylation was 2-3 times higher in the male
preparations of all eight strains studied. In contrast, female
activity was greater for most other hydroxylase activities, but
differences were strain-specific. The most prominent strain-specific
sex difference was observed for 15a-hydroxylase activity. In the
AKR/J, A/J, 129/J and DBA/2J strains, 15a-hydroxylation proceeded 2-3
times faster in female than male microsomes while in the other four
strains no significant sex difference was observed. Other experiments
(see below) have revealed that 15a-hydroxylase activity is not
dependent on phase of the estrus cycle.
In order to assess the statistic significance of differences
between strains with regard to rate of site-specific testosterone
hydroxylation, the activities of male and female microsomes were
analyzed separately by Duncan's multiple comparison procedure


TABLE 8
MALE:FEMALE COMPARISON OF APPARENT MOLECULAR ACTIVITIES
IN THE SELECTIVE HYDROXYLATION OF TESTOSTERONE
Position of
Hydroxylation Male/Female (M/F) or Female/Male (F/M) Ratic£
DBA/2J
SWR/J
129/J
AKR/J
BALB/cJ
A/J
C3H/HeJ
C57BL/6J
16a
M/F
2.0
2.0
3.6
3.1
1.8
2.0
2.8
2.2
15a
F/M
1.7
*
2.1
3.7

2.5
*
*
153
F/M
*+
1.3+
*
1.8
*
*
* *+
6a
F/M
*+
1.5
1.4
1.4
*
*
* 1.4+
Based on results presented in Table 6.
*Failed test for significance (a level = .05; Duncan's Multiple Range Test).
+Test for significance differs when analysis is based on ratios of specific activity
instead of apparent turnover number.


52
(Figure 6). Comparisons were done on apparent molecular activities
instead of specific activities for reasons enumerated below. Many
differences between the strains (a level = 0.05) were observed in the
hydroxylase activity at each of four sites on testosterone.
Hydroxylation at 7 a was omitted from consideration for the reasons
described above; 63 hydroxylation was not analyzed because it did not
reveal a significant dependence on strain (Table 7). Those strains
with hydroxylase activities that viere statistically indistinguishable
are joined by lines in Figure 6. Such groupings between males were
distinct from female groups, and female mice generally exhibited a
greater degree of polymorphism for most hydroxylase activities. This
sex-strain interaction was most pronounced in the rate of 15a
hydroxylation (Figure 6). For example, while male activities of the
C3H/HeJ, AKR/J and SWR/J strains were net distinguishable, female
15a-hydroxylase activity of the AKR/J strain was 3- to 4-fold greater
than that of the C3H/HeJ and SUR/J strains. Male 15a-hydroxyl ase
activity varied only 1- to 2-fold among the eight strains of mice.
Although interstrain variation in the other hydroxylations was small, a
2 to 3-fold strain difference could be found depending on the sex,
strain and hydroxylase activity in question. No obvious correlations
indicative of groups of hydroxylase activities were detected.
Possible interstrain relationships in the relative pattern of
testosterone hydroxylation at all six positions were explored by
expressing each hydroxylase activity as a percentage of the total
hydroxylase activity (6a + 63 + 7a + 15a + 153 + 16a) observed in a
strain. The overall patterns of hydroxylase activity were nearly


TESTOSTERONE HYDROXYLASE ACTIVITY (nmoles/IOmln/nmole P-450)
53
Figure 6. A comparison of strains v/ith regard to apparent molecular
activities for the 15a-, 16a-, 6a- and 15p-hydroxyl at ions of
testosterone. Males and females were analyzed separately by
the Duncan's multiple comparison procedure. The strains
linked by a vertical solid line were indistinguishable
statistically at the a level of 0.05. Abbreviations: A/J,
AJ; AKR/J, AK; BALB/cJ, BL; C3H/HeJ, C3; C57RL/6J, B6;
DBA/2J, D2; SHR/J, SW; and 129/J, 1.1.


54
identical among the different inbred male mice (Figure 7) although
small differences v/ere found for each hydroxyl at ion. In all strains
the rank order of hydroxylase activity was 63 > 16a > 6a > 7a 2 15a >_
153. In contrast, female patterns of testosterone hydroxylation were
quite different in different strains (Figure 8). Only the C57BL/60,
DBA/2J, C3H/HeJ and SWR/J strains had similar rank orders of
hydroxylase activity. Qualitative variations from this pattern were
seen for certain hydroxylase activities in the other four strains.
Changes in the pattern of hydroxylation at the 15a and 16a positions
were observed for the 129/J strain, and at the 15a, 16a and 7a
positions for the AKR/J and A/J strains. A change in the rank order of
only 7a-hydroxylase activity was noted for the BALR/cJ strain. This
difference did not reflect the contaminant that chromatographs with
7a-hydroxytestosterone because the unknown metabolite was found to
remain in equal proportion to the 7a-hydroxytestosterone metabolite in
both sexes of all eight strains as evidenced by two dimensional TLC.
Indeed, two-dimensional TLC did not reveal any previously unidentified
major strain-specific metabolites in either males or females which
might have been undetected by routine one-dimensional TLC or which
might have interfered with quantitation of the six hydroxylase
activities of interest.
Testosterone hydroxylase specific activities and cytochrome P-450
content of untreated animals were found to fluctuate from month to
month by as much as 50% during the course of these and subsequent
experiments. This variation in microsomal activities of untreated
animals has been observed by others (41,62). Ford et al_. (26) have


RELATIVE RATE OF FORMATION
55
SITES OF TESTOSTERONE HYDROXYLATION
I
Figure 7. Relative rates of formation of 6a-, 63-, 7a-, 15a-, 153- and
16a-hydroxytestosterone (expressed as a fraction of the rate
of formation of these 6 metabolites) in the presence of
hepatic microsomes from males of 8 strains of mice. Note
the 10-fold difference in the scale for 63 hydroxyl ation.


RELATIVE RATE OF FORMATION
56
SITES OF TESTOSTERONE HYDROXYLATION
Figure 8. Relative rates of formation of 6a-, 63-, 7a-, 15a-, 153- and
16a-hydroxytestosterone (expressed as a fraction of the rate
of formation of these 6 metabolites) in the presence of
hepatic microsomes from females of 8 strains of mice. Note
the in-fold difference in the scale for 63 hydroxylation.


57
reported a circannual variation in total hepatic hydroxylase activity
of mice towards testosterone; total testosterone hydroxylase specific
activity was reported to be 3-4 times higher in December than in April.
We did not observe a circannual phenomenon, and we are able to
compensate for the variability noted above by expressing results as
apparent molecular activities, defined here as nmoles of product formed
per min per nmole cytochrome P-450. ANOVA indicated that in virtually
all cases unaccountable variability in data obtained with 80 animals is
decreased with use of apparent molecular activities in comparison to
specific activities.


58
Discussion
This study in inbred nice of the relationship between strain, sex
and the sex-strain interaction and site-specific monohydroxylations of
testosterone provides evidence for both heterogeneity and polymorphism
in the isozymes of hepatic microsomal cytochrome P-450 and/or their
modes of regulation. With the exception of 63 hydroxyl ation, a
surprisingly large portion (73-93%, Table 7) of individual variability
can be accounted for by strain, sex and/or the sex-strain interaction.
The significance of strain or the sex-strain interaction as
determinants of catalytic activity in individual animals indicates
genetic polymorphism in the 6a, 7a, 15a, 153 and 16a hydroxyl ations of
testosterone. Discrete phenotypic classes were not distinguished
because of the limited magnitude of the interstrain variations in
hydroxylase activities.
Because of the prominent interaction between sex and strain, more
detailed evaluation of polymorphism necessitated separate consideration
of males and females (Figure 6). Despite the striking conservation
among microsomal preparations from males of pattern and rank order of
hydroxylase activities (Figure 7), there are statistically significant
differences between strains in four activities (6a, 15a, 153, 16a).
Catalytic activities toward selected positions on the testosterone
molecule vary by about two-fold, and usually not in parallel with
changes in other hydroxylases in the same strain. These results bear
not only on polymorphism, but also suggest extensive isozymic
heterogeneity. We do not have sufficient information to indicate
whether the polymorphisms we have observed reflect differences in


59
isozyme-proteins or genetic alterations at any of several possible
levels of regulation. Both possibilities have precedent in that Dieter
and Johnson (56) have described differences between steroid
hydroxylases isolated from different strains of rabbits whereas the
regulatory Ah locus described by Mebert and colleagues is widely
appreciated (63).
Polymorphism among preparations from female mice is more striking,
especially with regard to 15a-hydroxylase activity. In only four of
the eight strains were female 15a-hydroxylase activities significantly
higher than that in males of the same strain. This is reflected by the
prominent sex-strain interaction as a determinant of activity. There
is no apparent correlation between this polymorphism and the Ah locus
since two strains which exhibit this dimorphism, A/J and AKR/J, are
responsive and non-responsive to polycyclic aromatic hydrocarbons,
respectively (64). Figures 6 and 8 also reveal several other
statistically significant differences in various hydroxylase activities
among preparations from the various female mice. The differences are
greater than those seen between males, yet once again parallelisms in
groups of strains or activities are not apparent. On the basis of the
data presented in Figures 7 and 8, one might conclude that testosterone
hydroxylase activity is highly conserved between inbred strains of mice
and female factors, presumably endocrine, are capable of perturbing
this homeostasis. Subsequent studies revealed the independence of
results from estrus (unpublished observations). Moreover, other
experiments in castrated males (unpublished) accentuated polymorphism.


60
It is now our opinion that endogenous testosterone actually blunts an
underlying polymorphism that is expressed more fully in the female.
The best evidence for involvement of multiple isozymes derives
from the influence of sex on the various hydroxylase activities. We
clearly can distinguish independence of 16a-hydroxyl ase and
15a-hydroxylase activities from each other and from the other
activities. Several other experiments also indicate substantial
heterogeneity in the constitutive P-450 isozymes in mouse liver that
contribute to testosterone hydroxyl at ion. These include the
differential effects of phosphate concentration (59) and endocrine
manipulations (65). Indeed, based on the approach developed by Kremers
et al_. (48) in rats, we have already presented evidence (59) for the
contribution of more than one isozyme to both 16a- and 15a-hydroxylase
activities in mice. Several other reports bear on the relationship
between our results and constitutive and inducible cytochrome P-450
isozymes of mice. Counarin hydroxylation exhibits a 5 to 10-fold
polymorphism in both untreated and phenobarbital treated mice (66), yet
this polymorphism is not reflected in the metabolism of a large number
of other xenobiotics or in the 63, 7a or 16a hydroxylation of
testosterone. To date five or six different forms of liver microsomal
P-450 from mice induced with different xenobiotics have been purified
(3,67). Two of these isozymes, A2 and C2 prepared from
phenobarbital-treated R6D2F;j/J mice, catalyze the 7a and 63
hydroxylation of testosterone, respectively (42). Additional physical
evidence for multiple forms of cytochrome P-450 in the mouse has been
provided by the high performance liquid chromatographic elution


61
profiles of solubilized hepatic P-450's in untreated phenobarbital and
fl-naphthoflavone treated mice (68). To our knowledge, a constitutive
form of cytochrome P-450 has yet to be purified from mice.
Sex-dependent differences in steroid and drug metabolism by the
hepatic microsomal monoxygenase system have been well characterized in
the rat (36,37,69,70). Reports of such differences in mice are sparse
and often inconsistent. Until now, most of the P-450 associated sex
differences reported for mice have been strain-dependent and show a
small female predominance (40,71,72). Our results for the 6a, 6g, 7a,
15a and 15p hydroxylations of testosterone support these observations.
Strain differences in the female predominance of 15a-hydroxylase
activity are substantial and offer the opportunity to study the genetic
constraints on the sexual expression of different forms of P-450.
Preliminary studies (73) suggest that the regulation of this activity
is similar to that described previously (74,75,76) for the
15-hydroxylase system of female rats active toward 5a-androstane-3a,
170-diol 3,17-disulfate. The only significant sex difference we found
to be strain-independent was 16a hydroxylation, which occurred 2 to 3
times faster in male preparations from all eight strains. This finding
of male predominance is distinctive for mouse hepatic P-450
monoxygenase activities and may be characteristic of mice in general.
Steroid hydroxylations have been studied most extensively in the rat,
and 16a hydroxylation is higher in males with several steroid
substrates (48,77). Hepatic 16a hydroxylation of testosterone in the
rat has been shown to be catalyzed by a sex-dependent form of
cytochrome P-450 that is neonatally imprinted (38,78). This activity


62
is distinct from rat phenobarbital -induced 16a-hydroxyl ase activity
(22, 47). We have previously shown (59) in the AKR/J strain that these
sex differences in 15a- and 15a-hydroxylase activities are not
associated with differences in apparent Km values for testosterone.
One observation seems to speak against the rather extensive
heterogeneity implied in the foregoing discussion, namely, the finding
that the rate of each hydroxylation parallels a two-fold time-to-time
variation in the P-450 content of hepatic microsomes. This, of course,
had prompted us to analyze most results in terms of catalytic activity
per nmole P-450 (apparent molecular activity). Only if these
hydroxylations are catalyzed either by one or two forms comprising the
bulk of the constitutive P-450, or by several forms under identical
regulation with the bulk of constitutive P-450, (or that we and others
introduce an unknown methodologic factor that influences recovery of
all cytochromes P-450) could we anticipate all hydroxylase activities
to be equally affected. All of these remain valid possibilities;
recent observations (Singh, Hawke and Neims, unpublished results) with
mice treated with polylC to induce interferon yielded results that bear
on the question. In this circumstance, P-450 content decreased about
50% and all hydroxylase activities were affected nearly in parallel.
It is our opinion that the circannual variation in total testosterone
hydroxylase activity reported by Ford et_ a]_. (26) reflects a related
phenomenon although no reference to P-450 content was made.
In summary, we suppose that several isozymes of P-450 are involved
in the six hydroxylations of testosterone we studied. Each isozyme
probably has more or less discrete substrate (or position) specificity


63
and total hydroxylase activity at each position reflects the
contribution of more than one isozyme. Vie further presume that each
isozyme in a given strain of mice is under more or less discrete
regulation (e.g. by sex), yet capable of parallel changes with the bulk
of constitutive cytochrome P-450. Finally, we have also presented
evidence of polymorphism in several of the isozymes, with respect to
enzyme-protein and/or its regulation. It follows that the strain
differences detected in this study may be indicative of a much greater
degree of genetic polymorphism that would be apparent in studies of the
isolated forms of cytochrome P-450.


CHAPTER THREE
DEVELOPMENT OF SITE-SPECIFIC TESTOSTERONE HYDROXYLASES IN HEPATIC
MICROSOMES FROM AKR/J AND BALB/cJ MICE
Introduction
The cytochrome P-450 monoxygenase system is involved in the
biotransformation of a diverse array of compounds, from drugs and other
xenobiotics to endogenous substrates such as cholesterol, sex steroids,
fatty acids and prostaglandins (79,80). Originally, cytochrome P-450
was thought to be a single, catalytically nonspecific enzyme. There is
now substantial physical, catalytic and immunological evidence that
cytochrome P-450 consists of a family of hemoprotein isozymes each with
different and more or less overlapping catalytic specificities
(3,4,67). Most of this progress in establishing cytochrome P-450
heterogeneity has dealt with the inducible P-450 isozymes. Some recent
reports have focused on forms of cytochrome P-450 from untreated rats
(45; and rabbits (45,53), partly because immunological techniques have
revealed a lack of cross-reactivity between various inducible forms and
the bulk of these constitutive isozymes (10,11,47)
The extent of, and the molecular basis for, heterogeneity within
the constitutive forms of P-450 remain largely unkown because each
isozyme likely occurs in relatively small amounts in hepatic
microsomes. Nonetheless, these forms are of particular interest
because of the possibility that their physiological function(s) and
64


65
regulations may be quite different from those of the inducible
isozymes. The isozymes probably have greater specificity than the
inducible forms toward endogenous substrates such as steroids.
We are studying the constitutive forms of cytochrome P-450 in
untreated animals by examining and comparing the position specific
monohydroxylations of testosterone in isogenic strains of mice. The
use of inbred strains has already provided valuable information on the
genetic regulation, structure and temporal expression of the inducible
isozymes associated with the Ah cluster (63). Identification of
genetic polymorphisms in the various testosterone hydroxylations have
enabled us to explore some of the genetic and hormonal controls
applicable to the constitutive isozymes (73,81). These results in turn
have been useful probes of the multiplicity and specificity of certain
constitutive forms of hepatic cytochromes P-450 active toward
testosterone.
Differences in the developmental patterns of monoxygenase
activities have also provided insight into the heterogeneity and
regulation of P-450. While many groups of different enzymes can be
shown to share similar developmental patterns (82,83), the maturation
of different hepatic monoxygenase activities has been observed to vary
with substrate (84), sex (85), strain (86) and xenobiotic induction
(87). While comparison of the developmental patterns for several
testosterone hydroxylase activities should provide insight into the
heterogeneity of the constitutive forms of cytochrome P-450, comparison
of patterns between two strains of mice may provide even more valuable
information on genetic control of temporal expression. Developmental


66
control of the induction of specific forms of hepatic microsomal
cytochrome P-450 by temporal genes has been proposed in a number of
species (83,89). The existence of temporal genes which modify the
expression of lysosomal and enzymes during development has been
suggested by Paigen and colleagues (82,90).
In this study we describe position specificity in the maturation
of testosterone hydroxyl at ion and report a strain difference only in
the development of 15a-hydroxyl ase activity. The latter finding
suggests the existence of temporal genes in control of the expression
of certain constitutive cytochrome P-450 activities.


67
Materials and Methods
Testosterone [4-l^C] (56.1 yCi/ymol) was purchased from New
England Nuclear, Waltham, Ma. The radiochemical purity was greater
than 98% as established by TLC on silica gel F254 plates with the
solvent system ether/benzene/methanol, 55:44:0.5 (v/v). Testosterone
was obtained from Sigma Chemical Co., St. Louis, Mo., and was
recrystallized twice from acetone/hexane. Reference steroids were
obtained from sources described previously (59). Precoated (0.2 mm)
silica gel 60 F254 TLC plates (E. Merck, Darmstadt, Germany) were
purchased from Scientific Products, Brunswick, N.J., and SB-5 X-ray
film for autoradiography was obtained from Eastman Kodak Co.,
Rochester, N.Y. Liquiscint liquid scintillation mixture was obtained
from National Diagnostics, Somerville, N.J.; Emulgen 911 was purchased
from the Kao-Atlas Co., Tokyo, Japan. NADPH, D-glucose-6-phosphate and
EDTA were purchased from Sigma Chemicals, St. Louis, Mo. All other
materials and reagents were purchased from the Fisher Scientific Co.,
Pittsburg, Pa., and were of the highest quality commercially
available.
Animals. Timed-pregnant AKR/J and BALB/cJ mice were received
from Jackson Laboratories, Bar Harbor, Me., between the 13 and 15th
days of gestation. Pregnancy had been determined by the observation of
a vaginal plug the day after mating and by gross observation or
palpation after the tenth day of gestation (91). After spontaneous
delivery, females and their litters were housed in separate cages on
corncob Sani-cel bedding. The animals were maintained on a 12 hr
light/12 hr dark photoperiod and allowed both food (Purina Rodent Chow)
and water ad 1ibitum. Mice were weaned at 21 days of age and housed


68
4-6 per cage thereafter according to sex. At designated ages at least
3 male and female litter mate pairs were randomly selected from
separate litters for study. The liver of each animal was investigated
individually except where indicated.
Preparation of microsomes. Mice were killed by decapitation
between 8 and 10 a.m., and livers were excised and rinsed immediately.
All preparative steps were performed at 4 C. Liver homogenates from
28, 35, 49 and 77 day-old animals were prepared from minced tissue in
ice-cold 150 mM potassium chloride-250 mM potassium phosphate, pH 7.4,
as described previously. Microsomes were prepared by differential
centrifugation (27). Homogenates from younger mice were prepared by
identical procedures except that 150-170 mg of liver from 2 day-old
animals (3 pooled livers) or 300-350 mg of liver from 10 or 21 day-old
mice (2 pooled livers) were admixed with a fixed volume (5 ml) of
potassium chloride-phosphate buffer. Microsomal pellets were
resuspended in 100 mM phosphate buffer, pH 7.4, to yield a final
suspension containing approximately 15-20 mg microsomal protein per ml.
Protein content of microsomal suspensions was determined by the method
of Lowry et al_. (28).
Enzyme assays. Cytochrome P-450 content was measured by the
method of Omura and Sato (29) with a Beckman 5260 Spectrophotometer
after microsomal suspensions or whole liver homogenates were diluted
five-fold in 100 mM phosphate buffer, pH 7.4, containing 20% glycerol
and 0.2% Emulgen 911. Peak heights were measured in relation to the
steady baseline observed between 480-500 nm. G1ucose-6-phosphatase


69
activity was measured by the method described by Baginski et al_. (92)
using 0.8-1.0 mg microsomal protein per incubation.
Radiometric assay for hydroxytestosterone metabolites. The
rates of hydroxylation of testosterone at the 6a, 6g, 15a, 15g and 16a
positions were determined by the radioassay procedure we described
previously (59). Briefly, 0.4-0.5 mg of microsomal protein were
incubated at 37 C in a shaking water bath with NADPH (1.0 ymol),
MgCl2 (10.0 ymol), potassium phosphate buffer (final concentration,
0.1 M, pH 7.4) and [^C] enriched testosterone (0.131 ymol
including at least 0.5 yCi in 10 yl ethanol) in a final volume of 1.00
ml. Measurement of activities in preparations from 2, 10 and 21 day
old animals required 1.0-1.5 mg of microsomal protein per incubation.
Reactions were initiated by the addition of NADPH and terminated after
10 min by the addition of 5 ml of ice-cold CH2C12- Each sample was
then transferred to a 10 ml screw-top Teflon-lined test tube, shaken
vigorously for 30 min, and centrifuged for 15 min to separate phases.
The organic phase was removed and evaporated to dryness at room
temperature under a stream of nitrogen. Residues were resuspended in
50 yl ethyl acetate, and the radiolabelled metabolites of testosterone
were separated by TLC on plastic-backed silica gel plates developed
three times with ether/benzene/methanol, 55:44:0.5 (v/v).
Statistical analysis. Statistical significance of differences
between sexes was calculated by Student's t-test; P < 0.05 was
considered significant. All values are expressed as mean and standard
error.


70
Results
Postnatal development of hepatic microsomal cytochrome P-450.
As depicted in Figure 9, both male and female AKR/J mice experienced a
substantial increase in cytochrome P-450 concentration in microsomes
between days 1 and 21 of age (0.16 to 0.91 nmol/mg of protein).
Microsomal cytochrome P-450 concentration increased only slightly more
between days 21 and 77. Adult values were 5-6 fold higher than those
at age 2 days regardless of sex.
The recovery of total cytochrome P-450 and another microsomal
enzyme, glucose-6-phosphatase, was measured in preparations from 10 and
77 day old mice of each sex to determine if recovery of microsomes
varied with age. At both ages, 55-62% of homogenate
glucose-6-phosphatase activity was recovered in the microsomal
preparation. The recovery of cytochrome P-450 in the microsomal pellet
was 55-60% and 70-75% of the homogenate content from 10 and 77 day old
animals, respectively. Since the yield of microsomal protein per gram
of liver changed little with postnatal maturation (range at all ages,
20.7-32.7 mg/gram liver), the pattern of P-450 maturation depicted in
Figure 9 would be the same had one expressed cytochrome P-450
concentration in terms of liver weight instead of microsomal protein.
Postnatal development of microsomal testosterone hydroxylase
activities. The developmental patterns of both testosterone 16a- and
15a-hydroxylase activities are illustrated in Figures 10 and 11. In
2-day-old female mice the specific activity of testosterone
16a-hydroxylase (Fig. 10) was about 12% of the adult value. This
activity increased 6 to 7-fold in both male and female mice during the


nmole P-450/mg PROTEIN
71
Figure 9. Cytochrome P-450 content in hepatic microsomes from AKR/J
male and female mice of varying age. Each value represents
the mean of 3 determinations.


SPECIFIC ACTIVITY (
72
Figure 10. Testosterone 16a-hydroxylase activity in hepatic microsomes
from male ( ) and female ( o ) AKR/J mice of varying age.
Each value represents the mean of 3 determinations.


73
3 weeks after birth. After 21 days of age female 16a-hydroxylase
activity tended to plateau while activity in males continued to
increase such that there was a statistically significant difference
between sexes by day 28. By day 77, a 3-fold male predominance in
16a-testosterone hydroxylase had been attained.
In contrast, during the first 10 days after birth when 16a- and
all other hydroxylase activities were increasing (see below), the
specific activity of testosterone 15a-hydroxylase activity in AKR/J
mice remained unchanged (Figure 11). This 10-day lag in the postnatal
maturation of 15a-hydroxyl ase activity was independent of sex and in
sharp contrast to the 3-fold increase in cytochrome P-450 concentration
that occurred over the same interval. It follows, of course, that
15a-hydroxylase activity per nmole cytochrome P-450 (apparent molecular
activity) actually decreased substantially between days 2 and 10 of
age. Between 10 and 35 days of age, 15a-hydroxylase activity increased
ten-fold, an interval during which cytochrome P-450 concentration only
doubled. Male 15a-hydroxylase activity remained relatively constant
after 35 days of age but activity in females increased steadily such
that a 3-fold female predominance was manifest at 77 days of age.
We have already reported that testosterone 6a-, 60-, 150-, and
especially, 15a- and 16a-hydroxylase activities exhibited sexual
dimorphism in adult AKR/J mice (59). In the present study, no
significant gender differences were observed before day 28, and most
were not apparent until after the 35th day of age. The developmental
curves for 63-, 6a-, and 150-hydroxylase activities in male and female
AKR/J liver are presented in Figures 12 and 13, respectively. In order


SPECIFIC ACTIVITY (
74
Figure 11. Testosterone 15a-hydroxylase activity in hepatic microsomes
from male ( ) and female ( o ) AKR/J mice of varying age.
Each value represents the mean of 3 determinations.


RELATIVE SPECIFIC ACTIVITY
75
AGE (days)
Figure 12. Testosterone 6a-( ), 53-( ), and 153-( A ) hydroxylase
activities in hepatic microsomes from male AKR/J mice of
varying ages. Cytochrome P-450 content (nmoles per mg
microsomal protein) is indicated (....) for comparison.
Specific activities were scaled to converge at age 35 days
for the purpose of prepubertal and postpubertal
comparisons. The relative specific activity of 1.0 at age
35 days is equivalent to 0.387, 3.654 and 0.184 nmoles
product per min per mg microsomal for 6a-, 63- and 153-
hydroxylases, respectively. Each value represents the mean
of 3 determinations.


RELATIVE SPECIFIC ACTIVITY
76
Figure 13. Testosterone 6ct-( ), 6f$-( o ), and 153-( a ) hydroxylase
activities in hepatic microsomes from female AKR/J mice of
varying ages. Cytochrome P-450 content (nmoles per mg
microsomal protein) is indicated (....) for comparison.
Specific activities were scaled to converge at age 35 days
for the purpose of prepubertal and postpubertal
comparisons. The relative specific activity of 1.0 at age
35 days is equivalent to 0.445, 3.930 and 0.223 nmoles
product per min per mg microsomal for 6a-, 63- and 153-
hydroxylases, respectively. Each value represents the mean
of 3 determinations.


77
to distinguish relative rates of maturation before and after puberty,
the scales for each hydroxylase activity have been set such that all
curves converge to a common point at age 35 days. Results for total
cytochrome P-450 are included for clarity of comparison. Male and
female 6a-, 63- and 153-hydroxyl ase activities gradually increased
ten-fold between days 2 and 35 of age, in parallel to the five-fold
increase seen in cytochrome P-450 concentration. After puberty, 6a-
and 153-hydroxylase activities in female preparations increased to
values greater than the correspondng levels in male microsomes.
Postnatal maturation of testosterone hydroxylase activities in
BALB/cJ mice. We have previously reported that, unlike the situation
in AKR/J mice, no female predominance in any testosterone hydroxylase
activity can be detected in the BALB/cJ strain (81). Therefore, it was
interesting to compare the development of hydroxylase activities in
female BALB/cJ mice with the AKR/J. Cytochrome P-450 concentration in
microsomal preparations from BALB/cJ mice was approximately 2-fold
higher than in AKR/J on days 2 and 10 of age (Table 9). To facilitate
comparison, hydroxylase activities are presented in Table 9 in terms of
apparent molecular activities or hydroxylase activity per nmole of
cytochrome P-450. The specific activity for any hydroxylation can be
computed by multiplying apparent molecular activity by age-specific
P-450 concentration which is also listed in Table 9 for convenience.
Developmental curves for 6a-, 63-, 163- and 16a-hydroxylase activity in
BALB/cJ were similar to those seen in the AKR/J strain. The striking
similarity in rates of maturation of these activities between the two
strains between age 2 and 10 days is revealed in Table 10, which


78
presents results as ratios of apparent molecular activities at the two
ages in each strain. These ratios of apparent molecular activities
between day 10 and day 2 should approximate 1 if cytochrome P-450 and
hydroxylase activity mature in parallel. 16a-Hydroxylase activities
increased 1.6 times faster than P-450 in both strains, whereas other
hydroxylases increase at rates within 30% of that of the increase in
P-450. The maturation of 15a-hydroxylase activity in the two strains
is quite different. As noted above, in AKR/J mice there was no
increase in 15a-hydroxylase activity between days 2 and 10; indeed,
specific activity decreased by 9% during this interval. This of course
is consistent with the low (0.33) ratio of apparent molecular
activities at days 10 and 2. During the same age interval, the
specific activity of 15a-hydroxylase in the BALB/cJ increased by 84%,
nearly in parallel with the increase in P-450.
In BALB/cJ females, which do not exhibit the adult female
predominance in most hydroxylase activities that is characteristic of
AKR/J mice, apparent molecular activities decreased between days 10 and
77 in all mice as P-450 content continued to increase to adult levels
(Table 9).


79
TABLE 9
DEVELOPMENT OF HYDROXYLASE ACTIVITIES IN TWO MOUSE STRAINS IN
RELATION TO THE DEVELOPMENT OF CYTOCHROME P-450
Position of Molecular activities or P-450 content3
Hydroxyl at ion
AKR/J
BALB/cJ
DAY 2
DAY 10
DAY 77
DAY 2
DAY 10
DAY 77
15a
1.57
0.52
6.29
1.08
0.85
1.80
16a
1.76
2.85
2.08
2.78
4.53
3.32
150
1.27
1.10
2.71
1.69
1.29
1.11
6a
2.17
2.60
5.31
1.92
2.47
2.37
60
25.70
27.60
37.22
23.51
24.17
23.90
Cytochrome P-450
0.16
0.44
1.15
0.33
0.77
1.10
aHydroxylase activities are expressed as nmoles product formed per 10
inin per nmole cytochrome P-450. Cytochrome P-450 contents are expressed
in terms of nmoles P-450 per mg microsomal protein. All values represent
the mean of 3 determinations. All standard deviations were 10 to 20
percent of respective means.


80
TABLE 10
MATURATION OF EACH HYDROXYLASE MOLECULAR ACTIVITY
BETWEEN DAYS 2 and 10 OF AGE IN AKR/J AND
BALB/cJ MICE
Ratio of Molecular Activities2
Position of Hydroxylation Day 10/Da,y 2
AKR/J BALB/cJ
15a
0.33
0.83
16a
1.62
1.63
153
0.87
0.76
6a
1.20
1.29
6(5
1.07
1.03
aThe ratios of hydroxylase activities (expressed per
nmole P-450; see Table 9) at ages 10 and 2 days have
been computed for 5 activities.


81
Discussion
Independent patterns of maturation of the 7a, 60 and 16a
hydroxylation of testosterone by rat liver microsomes were first
observed by Jacobson and Kuntzman (93). Recently, differences in the
developmental pattern of several monoxygenase activities in untreated
C57BL/6N male mice have been submitted as evidence for the involvement
of distinct isozymes in the various activities (84). We have
previously studied the effects of sex and strain on the site-specific
hydroxylations of testosterone in inbred mice (81) and now extend the
experiments to include development to gain further insight into the
heterogeneity and regulation of the P-450 isozymes.
Our results provide developmental evidence for at least three
functional forms of cytochrome P-450 involved in the specific
hydroxylations of testosterone, namely, one involved in 6a, 6$ and 15g
hydroxylation, another in 16a hydroxylation, and a third in 15a
hydroxylation. The observation that 6a, 60 and 150 hydroxylase
activities cannot be distinguished developmentally suggests either
coordinate development of distinct P-450 isozymes or involvement of a
single form of cytochrome P-450 with broad specificity toward the 6a,
63 and 15g and perhaps other positions of testosterone. The fact that
all three activities show female predominance in adult AKR/J but not
8ALB/cJ mice is consistent with both explanations. The concept of
coordinate development of three different enzymes has already been
established in the case Of lysosomal acid hydroxylases in mice (82,90),
and could well apply to 6a, 60 and 150 hydroxylase activities since


82
recent biochemical and strain studies suggest some independence between
these activities (59,81).
The existence of a distinct P-450 isozyme with activity
especially for 16a-hydroxylation of testosterone is suggested by the
more rapid development of this activity relative to the postnatal
increase in cytochrome P-450 content and the other hydroxylase
activities, and by the fact that testosterone 16a hydroxylation is the
only activity to experience a three-fold postpubertal increase in male
mice only. Both characteristics apply to both AKR/J and BALB/cJ
animals. In contrast, 16a-hydroxylase activity exhibits little or no
increase until after the fourth week of age in rats (69). In these
animals 16a-hydroxylase activity is androgen-dependent in rats and
directed toward several steroid substrates (20,48,77). The distinctive
postnatal and postpubertal patterns of 16a hydroxylation may well
reflect regulation of two different isozymes bearing 16a-hydroxylase
activity. We have already presented evidence for multiplicity in mouse
16a-hydroxyl ase (59); moreover, in the rat phenobarbital-induced and
androgen-imprinted 16a-hydroxylases have been solubilized and resolved
(22,38,47).
The development of testosterone 15a-hydroxylase activity also
reveals postnatal and postpubertal patterns of maturation that are
easily distinguished from those of P-450 and other hydroxylases, but in
this case the distinctions are strain-specific. In AKR/0 mice
15a-hydroxylase activity did not increase during the first ten days
after birth, and there was a three-fold increase in only female
activity after puberty. Neither situation applies to development of


83
15a-hydroxylase activity in BALB/cJ mice. As with 16a-hydroxylase
activity, we may be dealing with more than one 15a-hydroxylase and
strain-specificity in their regulation. The threefold rise in
testosterone 15a-hydroxylase activity in female AKR/J mice after
puberty may signify the emergence of a sex-dependent female form of
cytochrome P-450 which mainly catalyzes 15a hydroxylation with some
activity toward the 6a, 63 and 153 positions of testosterone. Indeed,
Gustafsson and Ingelman-Sundberg have described a 15-hydroxyl ase in
female rat hepatic microsomes active on steroids and steroid sulfate
conjugates (74,75,76), the activity of v/hich has been associated with a
52,000 dal ton protein found only in females (94). One of the most
intriguing questions raised by our results is whether or not this
postpubertal (or sexually dimorphic) difference between strains relates
to the differences among strains in the maturation of 15a-hydroxylase
activity during the first ten days after birth. There is no obvious
reason to link the capacity for expression of an adult female activity
or protein, with the delayed expression of 15a-hydroxylase activity in
young male and female pups; yet both are characteristics of AKR/J but
not BALB/cJ animals.
The difference in rates of maturation of 15a-hydroxylase
activity between days 2 and 10 of age in AKR/J (-9%) and BALB/cJ (+84%)
are perhaps best interpreted in terms of temporal genes. Paigen (95)
has suggested that genetic elements, called temporal genes, determine
the developmental program for individual proteins, and strain
differences have been described (90,96). The responsiveness of
drug-metabolizing enzymes to specific hormones has been shown to change


84
during development (97,98), and there is evidence in mouse, rat, and
rabbit (88,89) for temporal control of the induction of forms of
cytochrome P-450 associated with the Ah locus. It will be interesting
in future experiments to explore the potential relationship between
polymorphisms we have observed in the early neonatal developmental
patterns and sexual dimorphism in the adult.


CHAPTER FOUR
GENETIC REGULATION OF HEPATIC MICROSOMAL TESTOSTERONE 15a- AND
16a-HYDR0XYLASE ACTIVITES IN FEMALE AKR/J AND BALB/cJ MICE
Introduction
The cytochrome P-450 monoxygenase system is involved in the
biotransformation of a diverse array of compounds including drugs,
carcinogens, pesticides, steroids and fatty acids (79,80). The
existence of different isozymes of cytochrome P-450 in mammalian liver
is now well established with the isolation and purification of a number
of forms which vary in subunit molecular weight, immunological and
catalytic properties, and amino acid sequences (3,4) from rat
(43,46,50), rabbit (45,51-53) and mouse (42,67). But the extent of
cytochrome P-450 heterogeneity, the degree of overlap in substrate
specificity between isozymes and the mechanisms by which these isozymes
are independently regulated by physiological and enviromental factors,
are not well understood.
Individual animals and people can be quite heterogeneous with
respect to both environmental exposures and genetic factors known to
influence drug metabolism. Sometimes it is the combination of
environmental and genetic factors that influence monoxygenase
activities. Genetic differences in sensitivity to induction of P-450
isozymes by polycyclic aromatic hydrocarbons has been attributed to
polymorphism in the Ah locus or cluster by Nebert and Jensen (63).
85


86
Indeed, the induction of specific forms of cytochrome P-450 by various
inducers has allowed some progress into the molecular biology of the
monoxygenase system as evidenced by recent nucleic acid hybridization
experiments (5,6). Less is understood about the constitutive forms of
cytochrome P-450, here defined as those isozymes present in untreated
animals. Immunological techniques have already revealed a lack of
structural homology between the major inducible forms and the bulk of
the constitutive P-450 isozymes in hepatic microsomes of untreated
rats (10,11,47). It seems likely that these constitutive isozymes will
differ from the inducible P-450's with regard to function(s) and mode
of biological regulation. Studies into the molecular biology of the
constitutive isozymes would be greatly facilitated by identification of
genetic polymorphisms in the regulation and/or structure of these
forms.
While several examples of genetic polymorphism of drug oxidation
in man have been described (e.g. 99-102), the mechanism underlying
these polymorphisms remains unknown. Preliminary studies suggest that
the impairment in debrisoquine 4-hydroxyl at ion resides at the level of
the microsomal monoxygenase system (103). The recent demonstration of
polymorphism of debrisoquine oxidation in various rat strains has
allowed more detailed biochemical studies (104,105). Vlasuk et al_. (9)
have found inter- and intra-strain polymorphism among four molecular
forms of closely related phenobarbital-induced cytochromes P-450 from
rats. Oieter and Johnson (56) have attributed a strain difference in
the 63 hydroxylation of progesterone by highly purified preparations of
P-450 from untreated rabbits to the presence or absence of one of two


87
subforms. Progress has likely been slowed by the happenstance that
oxidative metabolism of any drug represents the cumulative action of
more than a single form of cytochrome P-450. Polymorphism in a single
isozyme might then be masked in the background of other contributing
isozymes except in instances where oxidation is predominantly catalyzed
by an isozyme with great substrate specificity. The coumarin
hydroxylase locus (106) remains the only monoxygenase polymorphic
allele to be mapped in the mouse.
Constitutive forms of P-450 may be more specific in the oxidation
of endogenous substrates (45,46,107) than inducible forms. It follows
that polymorphism associated with alteration in the structure or
regulation of constitutive cytochromes P-450 might be more easily
detected with endogenous substrates than with xenobiotics. Indeed,
strain differences in steroid hydroxyl ation have been reported for many
species (86,54,55,56). While some patterns of inheritance have been
worked out (57,58), in most instances the exact nature of these
polymorphisms is not yet known.
The study of the regiospecific hydroxylations of testosterone in
isogenic mouse strains promises to provide valuable information on the
genetic and hormonal factors regulating the predominant forms of
cytochrome P-450 in hepatic microsomes from untreated animals. We have
previously reported genetic and sexual polymorphisms in the
testosterone 15a- and 16a-hydroxylase activities of mice (59,81). Our
present studies were undertaken to elucidate the genetic and endocrine
basis of the sex and strain differences in testosterone hydroxylation
at these two positions in inbred mice.


88
Materials and Methods
Material s. Testosterone [4-^C] (56.1 yCi/pmol) was purchased
from New England Nuclear, Waltham, Ma. The radiochemical purity was
greater than 98% as established by TLC on silica gel F254 plates with
the solvent system ether/benzene/methanol, 55:44:0.5 (v/v).
Testosterone was obtained from Sigma Chemical Co., St. Louis, Mo. and
was recrystallized twice from acetone/hexane. Reference steroids were
obtained through sources previously described (59). Precoated (0.2 mm)
silica gel 60 F254 TLC plates (E. Merck, Darmstadt, Germany) were
purchased from Scientific Products, Brunswick, N.J., SB-5 X-ray film
for autoradiography was obtained from the Eastman Kodak Co., Rochester,
N.Y., and Liquiscint liquid scintillation mixture was obtained from
National Diagnostics, Somerville, N.J. Emulgen 911 was purchased from
the Kao-Atlas Co., Tokyo, Japan. NADPH and testosterone propionate
were purchased from Sigma Chemical Co. All other materials and
reagents were purchased from the Fisher Scientific Co., Pittsburgh,
Pa., and were of the highest quality commercially available.
Animals. Male and female AKR/J, BALB/cJ and BALB/cJ x AKR/J FI
hybrid mice (CAKF^/J; 6-8 weeks old, 20-25 g) were purchased from
Jackson Laboratories, Bar Harbor, Maine. F2 and backcross animals were
bred at the University of Florida's Animal Resources Facility. Mice of
the same sex were housed in groups of six or less per cage on corncob
Sani-cel bedding in an animal room with a controlled environment.
Smoking and the use of insecticides were prohibited, and a 12-hour
1ight/12-hour dark cycle was maintained. Animals were fed Purina
Rodent Laboratory Chow and tap water ad 1ibitum. Mice were maintained


89
under these conditions for at least three weeks before study at the age
of 77 days. Orchiectomies, ovariectomies and sham operations were
routinely performed under methoxyflurane anesthesia at the age of 56
days. Adequacy of the operative procedures was confirmed by inspection
at the time of death. Where indicated, intact, sham-operated or
gonadectomized mice received daily subcutaneous injections of 100 yg of
testosterone propionate in 0.2 ml of sesame seed oil for seven days.
Control animals received vehicle only. Mice were killed 24 hours after
the last injection.
Preparation of microsomes. Mice were killed by decapitation
between 8 am and 10 am, and livers were excised immediately and
carefully separated from gall bladders. The livers were then rinsed
and homogenized in 5 ml/gram liver of ice-cold 150 rrM potassium
chloride 250 mM potassium phosphate, pH 7.4, with a motor-driven
glass-Teflon homogenizer. Microsomes were prepared by differential
centrifugation (27). Rriefly, the homogenate was centrifuged at 10,000
xg for 15 min, and the resulting supernatant was centrifuged for 1 hr
at 110,000 xg. Microsomal pellets from 1 gram of liver were
resuspended in 2 ml of 100 mM phosphate, pH 7.4, and were used
immediately. Microsomal suspensions contained approximately 8-10 mg
microsomal protein per ml as determined by the method of Lowry et al.
(28). Cytochrome P-450 content was determined by the method of Omura
and Sato (29) with a Beckman 5260 Spectrophotometer after microsomal
suspensions were diluted ten-fold in 100 mM phosphate, pH 7.4,
containing 20% glycerol and 0.2% Emulgen 911. Peak heights at 450 nm
were measured in relation to the steady baseline at 480 to 500 nm.


90
Radiometric assay for determination of testosterone hydroxylase
activities. Testosterone hydroxylase activities at the 6a, 63, 15a,
15$ and 16a positions viere determined by a radioassay procedure
described previously (59). Briefly, 0.4-0.5 mg of microsomal protein
viere incubated at 37 C in a shaking water bath with NADPH (1.0 ymol),
MgClo (10.0 ynol), potassium phosphate (final concentration, 0.1 M, pH
7.4) and [-^C]-enriched testosterone (0.131 nmol, 0.5 yCi in 10 yl
ethanol) all in a final volume of 1.00 ml. Reactions were initiated by
the addition of NADPH and terminated 10 min later by the addition of 5
ml of ice-cold methylene chloride. Each sample was then transferred to
a 10 ml Teflon-lined screw-capped test tube, shaken vigorously for 30
min and centrifuged to separate phases. The organic phase was removed
and evaporated to dryness at room temperature under nitrogen. Residues
were resuspended in 50 yl ethyl acetate and the radiolabelled
metabolites of testosterone were separated by TLC on plastic-backed
silica gel plates developed three times in ether/benzene/methanol,
55:44:0.5 (v/v).
Calculation and statistical evaluation of results. Testosterone
hydroxylase activities were expressed as apparent molecular activities
or nmoles of product formed per min per nmole of cytochrome P-450
assuming that 1 nmole [^C] hydroxytestosterone was derived from 1
nmole of [^C] testosterone. This was done to minimize the impact of
variation in cytochrome P-450 content (81). Me do not know whether the
relatively small day-to-day variation in P-450 content/ng microsomal
protein is biological or methodologic. Expression of results in terms
of specific activity or product formed per mg microsomal protein


91
increased intrastrain variation a small amount but did not change basic
conclusions. The fluctuation in cytochrome P-450 content is not due to
induction by environmental exposure to polycyclic aromatic hydrocarbons
since the AKR/J strain is non-responsive (64). Statistical analysis of
the data was performed according to Student's t-test; P < 0.05 was
taken as significant. All values represent the mean of five
determinations and are expressed with standard errors.
The number of genes involved in determining quantitative
differences in testosterone hydroxylase activity between the RALB/cJ
and AKR/J mouse strains was estimated by the method of Falconer (108),
where N, the number of independent loci involved, is given by the
following relationship:
_ 1/8 (mean parent 1 mean parent 2)^
(F2 variance FI variance)


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A GENETIC APPROACH TO THE REGULATION OF THE
CONSTITUTIVE FORMS OF CYTOCHROME P-450
By
ROY LEE HAWKE
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1983

TO LYNNE TWIEST
This dissertation is dedicated to my wife, Lynne Twiest, for her
support, patience, and sacrifice which were instrumental in the
attainment of this degree, and to my daughters Dawne and Heather who I
regret have had to make their own special sacrifices during the many
years of my education.

ACKNOWLEDGEMENTS
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I would like to extend sincere thanks and appreciation to my
dissertation committee members, and to all of the faculty members and
support staff of the Department of Pharmacology who have helped me in
innumerable ways over the last four years.
I wish to especially acknowledge the heroic efforts of both Debbie
Baylor in the typing of these manuscripts as well as other "rush" jobs
which were often thrust upon her, and Jeff Jones who was forever asked
to make slides on a moment's notice or less.
I would like to acknowledge the expert technical assistance
provided by Lynn Raynor over the course of these studies. Her endless
effort in keeping the laboratory functioning smoothly has made things
easier on us all. I am truly grateful for the support and friendship
she has extended toward me over the years. The meticulous efforts of
my sister, Becky, who spent her summers working in our lab, are also
gratefully acknowledged.
Finally, I wish to express my gratitude to Allen Neims whose
greatest accomplishment was not in making this mere dissertation
possible, but rather instilling in me a genuine fascination for science
and an excitement for research which I hope to carry with me until the
end of my career. He allowed our student-teacher relationship to
continuously evolve such that I now have the confidence to think out
loud. I will sorely miss our collaboration and the many hours of
discussion spent on topics ranging from Gators to P-450.

PREFACE
This dissertation is composed of five chapters representing
different aspects of my doctoral research. Each chapter has been
written in a standard manuscript style with an introduction, a
materials and methods section, results and discussion. Some
unavoidable redundancy will, therefore, be encountered in the
introduction and discussion of each chapter as a consequence of this
style of presentation.
IV

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
PREFACE iv
ABSTRACT vi i
INTRODUCTION 1
CHAPTER ONE HETEROGENEITY OF CYTOCHROME P-450 IN THE
SITE-SPECIFIC HYDROXYLATIONS OF STEROIDS
BY AKR/J MOUSE LIVER MICROSOMES 5
Introduction 5
Materials and Methods 8
Results 13
Discussion 31
CHAPTER TWO THE INFLUENCE OF STRAIN AND SEX ON SITE-
SPECIFIC TESTOSTERONE HYDROXYLASE
ACTIVITIES OF MOUSE HEPATIC MICROSOMES 37
Introduction 37
Materials and Methods 40
Results 43
Discussion 58
CHAPTER THREE DEVELOPMENT OF SITE-SPECIFIC TESTOSTERONE
HYDROXYLASES IN HEPATIC MICROSOMES FROM
AKR/J AND BALB/cJ MICE 64
Introduction 64
Materials and Methods 67
Results 70
Discussion 81
CHAPTER FOUR GENETIC REGULATION OF HEPATIC MICROSOMAL
TESTOSTERONE 15a- and 16a-HYDR0XYLASE
ACTIVITIES IN FEMALE AKR/J AND BALB/cJ
MICE. 85
Introduction 85
Materials and Methods 88
Results 92
Discussion 109
v

TABLE OF CONTENTS—Continued
PAGE
CHAPTER FIVE ENDOCRINE FACTORS IN THE REGULATION OF
HEPATIC MICROSOMAL TESTOSTERONE 15a- AND
16a-HYDROXYLASE ACTIVITIES IN AKR/J AND
BALB/cJ MICE 113
Introduction 113
Materials and Methods 117
Results 122
Discussion 139
GENERAL CONCLUSIONS 144
REFERENCES 147
BIOGRAPHICAL SKETCH 155
vi

Abstract of Dissertation Presented to t'ne Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
A GENETIC APPROACH TO THE REGULATION OF THE CONSTITUTIVE
FORMS OF CYTOCHROME P-450
By
Roy Lee Hawke
August, 1983
Chairman: Allen H. Neims
Major Department: Pharmacology and Therapeutics
The genetic, hormonal, and developmental regulation of various
forms of the cytochromes P-450 in untreated isogenic strains of mice
has been examined by virtue of the remarkable position specificity
demonstrated by these constitutive isozymes in the hydroxyl at ion of
testosterone. Careful kinetic analysis has established optimal assay
conditions for the measurement of hydroxylase activity in mouse hepatic
microsomes, and a new radiochromatographic procedure for the separation
and quantitation of six monohydroxylated products of testosterone is
described.
Detailed statistical analysis of results from studies on the
effects of sex, strain, and postnatal development on the rate of
hydroxylation at the 6a, 6b, 7a, 15a, 15b and 16a positions of
testosterone provided evidence for both heterogeneity and polymorphism
in the cytochromes P-450 catalyzing these hydroxylations and/or in
their modes of regulation. The most interesting findings from these
VI1

studies related to the 15a and 16a hydroxyl ations. With respect to
testosterone 16a-hydroxylase activity, 1) rates were 2- to 3-fold
higher in microsomes from males than females of all eight strains
studied; 2) prominent strain differences were detected only between
female mice; and 3) postnatal development was more rapid than the
postnatal increase in cytochrome P-450 content and the development of
other hydroxylase activities. With respect to testosterone
15a-hydroxylase activity, 1) sexual dimorphism was strain-dependent in
that rates in females were 2- to 3-fold higher than rates in males in
only four of eight strains; 2) strain differences detected between
males were independent of those found between females; and 3) a 10-day
lag in postnatal development, which was observed for this activity
only, was independent of sex but not of strain.
Analysis of genetic crosses and endocrine studies revealed that
1) 16a-hydroxylase activity was reversibly inducible by androgens, and
a single genetic locus regulates the basal level of activity in female
mice; 2) the strain-specific sexual dimorphism of 15a-hydroxyl ase
activity is controlled by a single genetic locus which probably is
expressed in differences between strains in responsiveness to sex
hormones; and 3) the induction of 15a-hydroxylase activity by estradiol
benzoate was independent of strain while suppression of this activity
by androgen was strain-dependent.
vi i i

INTRODUCTION
The cytochrome P-450 monoxygenase system is involved in the
biosynthesis and/or catabolism of many endogenous compounds including
cholesterol, bile acids, corticosteroids, prostaglandins, and sex
steroids. The same enzyme system participates in the metabolic
oxidation of a variety of xenobiotics such as drugs, polycyclic
aromatic hydrocarbons, food additives, pesticides, and industrial
pollutants; it thus plays an important role in xenobiotic
detoxification and elimination. Where oxidative metabolism leads to
the generation of highly reactive electrophilic metabolites, such as
epoxides or N-hydroxides, the "detoxification" reactions can result in
carcinogenic, mutagenic or teratogenic metabolites. The broad
substrate and product specificity of the cytochrome P-450 oxidizing
system reflects the presence of several forms of the enzyme. Each of
these forms of cytochrome P-450 may be more or less species-specific.
Each form is likely to have more or less distinctive substrate and
product specificity and inducer and inhibitor sensitivity, and to the
under more or less distinctive control genetically, developmentally,
and distributionally. Therefore, the P-450 monoxygenase system
combines great breadth and specificity with regard to species, sex,
age, tissue, etc. Whether or not the various isozymes represent
distinct gene products and the extent to which post-translational
modification, allosteric modifiers, lipid environment (these enzymes
1

2
are membrane-bound), etc. modulate the subtrate and/or product
specificity are not resolved.
Although the existence of multiple isozymes of cytochrome P-450 in
mammalian liver is now well established, surprising uncertainty remains
with regard to the extent of heterogeneity and the physical, chemical,
and biological basis for multiplicity especially in the constitutive
forms characteristic of the untreated animal. These forms are of
particular interest because of the possibilitity that their regulation
and functions may differ from those of the inducible isozymes. The
constitutive isozymes represent the bulk of the cytochromes P-450
present in the untreated liver, are immunologically distinct from the
induced forms, and possess greater specificity towards endogenous
substrates. Although under most circumstances constitutive isozymes
account for a large proportion of hepatic xenobiotic metabolism,
surprisingly little is known about the degree of overlap in catalytic
properties between isozymes, the mechanisms by which these forms are
independently regulated by physiological and/or environmental factors,
and the extent of allelic polymorphism in the isozymes. Clearly such
issues are of great significance for understanding and predicting
xenobiotic interactions and human individuality in the response to
drugs.
This dissertation represents a study on the genetic, hormonal and
developmental regulation of various constitutive forms of the
cytochrome P-450 isoyzmes that contribute to the site-specific
monohydroxylations of testosterone in hepatic microsomes prepared from
untreated isogenic strains of mice. Several substrates, including

3
testosterone and other endogenous steroids, have been used earlier with
success to probe the heterogeneity of the cytochromes P-450 by virtue
of the remarkable regio- and positional selectivity exhibited by
certain isozymes towards these substrates. Indeed, changes in the
relative specificity for site-specific hydroxylations as a function of
species, age, sex and other factors have provided much of the initial
evidence for the existence of multiple forms of cytochrome P-450. Our
application of a common methodology in the comparative study of hepatic
microsomal steroid hydroxylations in male and female mice of several
isogenic strains provides us with valuable insight into the modes of
regulation and polymorphic expression of these constitutive isozymes of
cytochrome P-450. This study also serves as a useful probe of the
catalytic specificity and heterogeneity of those constitutive forms of
the enzyme active toward testosterone.
It is crucial to the rationale of this dissertation that the
investigation involve isogenic animals. Although heterogeneity in the
constitutive cytochromes P-450 probably reflects the presence of a
finite number of isozymic forms of this enzyme, the degree of
complexity could be exaggerated by the many allelic differences that
randombred or outbred animals can be expected to display. The use of
inbred strains prevents intrastrain allelic variations and provides a
homogeneous background for selected studies of the heterogeneity of
cytochrome P-450 by way of interstrain comparisons.
This thesis is composed of five chapters, the first of which
describes a convenient one-dimensional TLC-radiochromatographic
procedure for the separation and quantitation of six monohydroxylated

4
products of testosterone metabolism and the development of optimal
conditions for the measurement of hydroxylase activity in mouse hepatic
microsomes. Chapters two and three deal with the effects of sex,
strain, and postnatal development on the rates of hydroxylation at the
6a, 63, 7a, 15a, 15^, and 16a positions of testosterone. Finally,
chapters four and five represent detailed studies on the genetic and
endocrine regulation of testosterone hydroxylase activities, primarily
at the 15a and 16a positions, in two selected strains of mice. A final
discussion serves to integrate conclusions reached in each chapter.
Each chapter has been prepared for submission as an independent
manuscript for publication.

CHAPTER ONE
HETEROGENEITY OF CYTOCHROME P-450 IN THE SITE-SPECIFIC HYDROXYLATIONS
OF STEROIDS BY AKR/J MOUSE LIVER MICROSOMES
Introduction
The concept of isozymic multiplicity has been used often to
explain the broad substrate and product specificities of the hepatic
microsomal monoxygenase system and its distinctive responses to various
inhibitors and inducers (1,2). Direct evidence for heterogeneity in
the cytochromes P-450 of mammalian liver is abundant. Several forms of
the enzyme have been resolved (3,4), and various isozymes have been
differentiated by subunit molecular weight, immunological properties,
catalytic properties, peptide maps, partial amino acid sequences, and
even nucleic acid hybridization (5,6).
Despite this progress, surprising uncertainty remains with regard
to the extent of heterogeneity, the degree of overlap in catalytic
properties between forms, and the mode of biological regulation of
individual forms and/or catalytic activities (7). In this situation,
where one does not know exactly how many forms there are or how they
relate to one another, unrecognized polymorphism would introduce
serious and unneeded complexity. Recent reports add credibility to
this concern (8,9) and underscore the importance of conducting selected
experiments with isogenic animals. Our lack of knowledge is especially
apparent in the case of the "constitutive" forms of hepatic cytochrome
5

6
P-450, here defined as those present in untreated animals. Notably,
only a small fraction of the cytochromes P-450 found in untreated rat
liver crossreacts with antibodies raised to the isozymes induced by
phenobarbital, polycyclic aromatic hydrocarbons, or pregnenalone 16a-
carbonitn1e (10,11). These constitutive isozymes are of particular
interest because of the possibility that their substrate specificity,
function and regulation may differ from those of the inducible
isozymes.
Several substrates which yield multiple products, including
biphenyl (12), warfarin (13), and benzo[a]pyrene (14), have been used
successfully to probe the heterogeneity of the cytochromes P-450 by
virtue of the remarkable regio- and positional stereoselectivity
exhibited by certain isozymes toward these substrates. Testosterone,
an endogenous compound, has also served in this regard (15,16,17).
With rat liver microsomes, the relative specificity for hydroxylation
at the 7a, 16a and 6b positions changes with age (18,19), sex (20,21),
neonatal imprinting (20,22) and exposure to xenobiotics (23,24,25).
Some sex and strain differences in the pattern of hydroxylation of
testosterone have also been observed with mouse liver microsomes (26).
Our current work focuses on the site-specific monohydroxyl at ion of
steroids, primarily testosterone, by hepatic microsomes from untreated,
inbred mice. We have found that the usual assay methods developed for
studies of testosterone metabolism in the rat are inappropriate in mice
because the array of metabolic products is different. This report
deals with 1) the development of a reasonably rapid and economical one¬
dimensional TLC procedure suited to our purpose; 2) the development of

7
optimal assay conditions for the measurement of hydroxylase activity in
mouse microsomes; 3) the finding of substantial sexual dimorphism in
selective hydroxylations of testosterone by liver microsomes from AKR/J
mice; 4) the differential effects of potassium phosphate buffer on
selective hydroxylations of testosterone by preparations from these
animals; and 5) comparative experiments with progesterone as
substrate.

8
Materials and Methods
Chemicals. [4-^CjTestosterone (56.1 Ci/mol) and
[4-^C]-progesterone (57.2 Ci/mol) were purchased from New England
Nuclear, Waltham, Massachusetts. Radiochemical purities exceeded 93%
according to the TLC procedure described below. 63-, 7a-, 16a-, and
163-Hydroxytestosterone and 63-, 16a-, and 21-hydroxyprogesterone were
purchased from Steraloids, Inc., Wilton, New Hampshire. 23- and
153-Hydroxytestosterone were gifts from G.D. Searle and Co., Chicago,
Illinois. 15a-Hydroxytestosterone was kindly provided by the Upjohn
Company, Kalamazoo, Michigan, and 15a-hydroxyprogesterone was a gift
from Dr. S. Solomon, McGill University, Montreal, Canada.
6a-Hydroxytestosterone and additional samples of several of the
compounds listed above were provided by Dr. D. N. Kirk from the Medical
Research Council Steroid Reference Collection.
Testosterone and progesterone, which were purified by
recrystallization twice from acetone/hexane and ethanol/water,
respectively, and NADPH were purchased from Sigma Chemicals, St. Louis,
Missouri. All other chemicals ware purchased from the Fisher
Scientific Company, Pittsburg, Pennsylvania, and were of the highest
quality available.
Animals. Male and female AKR/J mice (8 weeks old, 20-25 g) were
purchased from Jackson Laboratories, Bar Harbor, Maine. Mice of the
same sex were housed in groups of up to six animals on corncob Sani-cel
bedding under a controlled environment for at least three weeks before
use. Smoking and the use of insecticides were prohibited, and 12-hour

9
1ight/12-hour dark cycles were maintained. Animals were provided
Purina Rodent Laboratory Chow and tap water ad libitum.
Preparation of microsomes. Mice were killed by decapitation
between 8 am and 10 am, and livers were excised and homogenized in ice-
cold 250 nil potassium phosphate buffer, pH 7.4, containing 150 mM
potassium chloride (5 ml/g of liver). Microsomes were prepared by
differential centrifugation (27) and were resuspended in 100 mM
potassium phosphate buffer, pH 7.4, to give a final suspension
containing approximately 10 mg microsomal protein per ml. The protein
content of microsomal suspensions was determined by the method of Lowry
et al. (28) with bovine serum albumin as standard.
Enzyme assays. Cytochrome P-450 content was measured by the
method of Omura and Sato (29) after microsomal suspensions were first
diluted ten-fold in 100 mM phosphate buffer, pH 7.4, containing 20%
glycerol (v/v) and 0.2% Emulgen 911 (v/v). Absorbance at 450 nm was
measured in relation to the steady baseline between 480-500 nm.
Virtually identical results were obtained without Emulgen as long as
careful baseline corrections were included.
The microsomal biotransformation of testosterone was assayed under
conditions similar to those described by Shiverick and Neims (30).
Reaction mixtures routinely contained 100 mM potassium phosphate
buffer, pH 7.4, 10 mM MgCl2, 1.0 mM NADPH, 131 pM [14C]steroid,
testosterone or progesterone (0.5 pCi) added in 10 pi of ethanol, and
0.4-0.5 mg of microsomal protein (0.2-0.3 mg for progesterone
incubations) in a final volume of 1.0 ml. The enzymatic reaction was
initiated by the addition of NADPH and allowed to proceed for 10 min at

10
37° C before termination by the addition of 5 ml of methylene chloride.
Controls routinely lacked microsomes, but zero-time and
heat-inactivated samples gave similar results.
Separation and assay of testosterone metabolites. Each incubation
mixture was shaken with methylene chloride for 30 min before collection
of the organic phase, which was then evaporated to dryness at room
temperature under a stream of nitrogen. The residue, which contained
98% of the added radioisotope, was dissolved in 50 pi of ethyl acetate.
Aliquots (25 pi) of the extract and relevant standards were applied to
scored, methanol-washed, 20 x 20 cm, silica gel 60 [r254''Precoa1:ec'>
plastic-backed thin layer chromatography plates, 0.2 mm thick (E.
Merck, Darmstadt, Germany). The plates were developed three times in
ether/benzene/methanol, 55/44/0.5 (v/v) in unlined tanks. Optimal
separation of the radiolabeled metabolites of testosterone was achieved
at 17° and an ambient humidity between 30 and 60%, conditions which
were associated with a development time of about 100 min per run. Even
at suboptimal conditions, the desired resolution could be achieved by
increasing the fraction of methanol in the developing solvent. Rarely,
a fourth development was needed to separate 15a-hydroxytestosterone
from a small zone of radioactivity just below it. Progesterone
metabolites were resolved by the same TLC system except that the plates
were developed only twice.
Radioactive zones on the TLC plates were located by autoradio¬
graphy with SB-5 X-ray film (Eastman Kodak Co., Rochester, N.Y.).
After the plates were first covered by a single layer of Scotch 810
transparent tape, each zone was cut out and counted directly in 10 ml

11
of Liquiscint (National Diagnostics, Somerville, N.J.) in a Beckman LS
7000 liquid scintillation counter with external quench correction
(Beckman Instruments, Inc., Fullerton, CA); recovery of applied to
the plate was greater than 95%. The Scotch tape, which was found not
to affect counting efficiency, was quite helpful in avoiding chipping
of the gel during cutting.
Metabolites were identified by comigration with unlabeled
reference standards in five different solvent systems. The standards
were visualized by fluorescence quenching. Identification and the
radiochemical purity of each putative hydroxytestosterone metabolite
was further assessed by acetylation. For these experiments, radio¬
active zones were cut and eluted into ethyl acetate containing 40-50 ug
of the unlabeled standard. Sufficient numbers of each zone were
combined to yield about 15,000 cpm. Samples were evaporated to dryness
under nitrogen and incubated in 100 pi of pyridine and 100 yl of acetic
anhydride in foil-covered, tightly capped test tubes. After 24 hours,
the samples were dried and chromatographed as described above except
that the plates were developed only once. More than 85% of radiolabel
in each metabolite zone was recovered during TLC of the acetylated
derivatives. The Rf values of the radioactive areas were compared to
both internal and external acetylated reference standards.
Identification of progesterone metabolites was based only on
comigration with reference standards on the TLC system described
above.
Calculations and statistical analyses. The amount of each of the
various hydroxysteroid metabolites was computed from the fraction of

12
radiolabel in a particular zone relative to the total radioactivity
applied to that lane of the TLC plate. Specific activities of steroid
"hydroxylases" are expressed as nmoles of product formed per min per mg
microsomal protein. Molecular activities are defined here as units of
hydroxylase activity per nmole of cytochrome P-450. The data were
analyzed by the unpaired Student's t-test. A P-value of less than 0.05
was taken as significant. Unless otherwise indicated, each value is
expressed as a mean and standard error.

13
Results
Identification and TLC of testosterone metabolites. The
incubation of [^C]testosterone with NADPH, oxygen and hepatic
microsomes from AKR/J male mice resulted in the formation of a large
number of metabolites. This is seen in Figure 1 (left panel) which
depicts the reaction products resolved by a 2-dimensional TLC procedure
similar to that reported by Ford et a]_. (26). For routine analysis, we
developed a more rapid and economical one-dimensional TLC system. The
latter system was designed especially to separate and quantitate the
15a- and 16a-hydroxytestosterone metabolites because pilot experiments
had revealed these metabolites to be most significant for our purposes.
An autoradiogram of the one-dimensional chromatogram (Figure 1, right
panel) shows that twelve distinct zones of radioactivity were resolved.
Comparison of the one- and two-dimensional chromatograms revealed that
zones 1 and 2 (which comigrated with the 15a- and 16a-hydroxytesto-
sterone reference standards, respectively) as well as zones 4, 5, 6 and
7, were essentially devoid of obvious contaminants.
The relative mobilities of the metabolites contained in zones 1
through 8 and a set of hydroxytestosterone standards are presented in
Table 1. The metabolites were identified tentatively by comparison
with these external standards, as well as by comigration with internal
standards on one- and two-dimensional TLC. The presence of these
metabolites had been established by GC-mass spectroscopy (31).
Testosterone is in zone 11, and the metabolite in zone 5 is not yet
identified.

14
ZONE
&
Figure 1. Autoradiograms of two-dimensional (left panel) and
one-dimensional (right panel) chromatograms'of
[14C]testosterone metabolites. AKR/J male liver
microsomes were incubated with [^(^testosterone under
the conditions described in the text. Testosterone and its
metabolities were extracted and then spotted onto silica gel
F-254 plates. Two-dimensional chromatography was conducted
by first developing the plates horizontally two times with
chloroform/ethanol (92:8) and then developing the plates
vertically three times with ether/benzene/methanol
(55:44:0.5). The one-dimensional plates were developed
three times with the latter solvent system. The major
radiolabeled zones on the one-dimensional plate were
numbered arbitrarily with lowest numbers closest to the
origin.

TABLE 1
CHROMATOGRAPHIC COMPARISON OF RADIOLABELED TESTOSTERONE METABOLITES
AND THEIR ACETYLATED DERIVATIVES WITH HYDROXYTESTOSTERONE STANDARDS
AND THEIR DIACETYL DERIVATIVES.
Hydroxytestosterone
Standards
Metabolite
Zone^-
Mobil it.y—
Std 14C
Rf£
Acetylated Derivatives
Std 14c
15a
1
0.07
0.07
0.64
0.64
16a
2
0.09
0.09
0.68
0.68
7a
3
0.12
0.12
0.65
0.66
0.54
153
4
0.16
0.15
0.62
0.62
—
0.34
—
5
—
0.21
0.73
6a
6
0.25
0.23
0.75
0.75
63
7
0.36
0.35
0.72
0.72
23
8
0.46
0.45
0.81
0.82
163
8
0.46
0.45
0.59
0.59
—
0.50
Testosterone
11
0.74
0.73

TABLE l--extended.
-£[^C]Testosterone, 131 yM, was incubated with hepatic microsomes from male AKR/J mice
under conditions described in text. Radiolabeled metabolites were resolved into zones as
defined in Figure 1.
^One-dimensional TLC was performed according to the text and Figure 1. Standards (Std)
were located by fluorescence quenching; metabolites were located by radioautography.
In all cases, standards admixed with radiolabeled metabolites comigrated with the
metabolite in question. Since the plates were developed 3 times, mobilities are
expressed relative to the solvent front after the third run.
^Standards and radiolabeled metabolites were acetylated as described in the text.
Standard (Std) and metabolite (^C) derivatives were located by fluorescence
quenching and radioautography, respectively. When standards were admixed with
metabolites before acetylation, the derivatized standard comigrated with the acetylated
metabolite in question. Acetylated metabolites were resolved by a single development in
the one-dimensional TLC system described in the text.

17
Further identification, as well as assessment of the radio¬
chemical purity, of the metabolites contained in zones 1-8 was
accomplished by acetylation of standards and the labeled material in
each zone. After acetylation, essentially all of the radioactivity in
zones 1 and 2 migrated with Rf's indistinguishable from the diacetyl
derivatives of 15a- and 16a-hydroxytestosterone, respectively (Table
1). As suggested by the results depicted in Figure 1, zones 3 and 8
contained more than one labeled compound. About 60% of the label in
zone 3 comigrated with the diacetyl 7a-hydroxytestosterone standard.
Zone 8 contained substantial quantities of both 23- and
163-hydroxytestosterone, 55% and 28%, respectively, as well as an
unidentified product. Derivatization confirmed that the radiolabel in
zones 4, 6 and 7 was composed almost entirely of 153-, 6a-, and
63-hydroxytestosterone, respectively, although a minor (approximately
20%) contaminant was found along with 153-hydroxytestosterone in zone
4. The stabilities of individual diacetylated metabolites (or
standards) were not determined, and lability could have contributed to
some of the apparent contamination.
The [^C]testosterone metabolites produced by AKR/J male
hepatic rnicrosomes were also identified by their comigration with
reference standards in 4 other TLC systems used in earlier studies of
the biotransformation of testosterone by rodent liver rnicrosomes (Table
2). Careful examination of Table 2 reveals that none of these solvent
systems resolved all of the metabolites in question. Nonetheless, the
sum of these results supports our identification of metabolites.

TABLE 2
COMPARISON OF THE MOBILITIES OF RADIOLABELED TESTOSTERONE
METABOLITES FORMED IN THE PRESENCE OF HEPATIC MICROSOMES FROM
MALE AKR/J MICE WITH HYDROXYTESTOSTERONE STANDARDS ON FOUR
TLC SYSTEMS.
Rf
Testosterone Solvent System^.
Metabolite
A^ B C D
Std
14C
Std
14C
Std
14C
Std
14C
15a-Hydroxy
0.26
0.26
0.39
0.39
0.27
0.28
0.04
0.04
16a-Hydroxy
0.30
0.30
0.46
0.46
0.34
0.35
0.05
0.05
7a-Hydroxy
0.43
0.43
0.53
0.51
0.41
0.42
0.07
0.08
153-Hydroxy
0.43
0.43
0.46
0.47
0.39
0.39
0.09
0.09
Zone 5
—
0.57
—
0.56
—
0.48
0.12
6a-Hydroxy
0.60
0.61
0.63
0.60
0.50
0.48
0.14
0.12
63-Hydroxy
0.64
0.67
0.67
0.64
0.57
0.55
0.19
0.17
Testosterone
0.88
0.89
0.86
0.86
0.86
0.85
0.60
0.60

TABLE 2—extended.
1TLC was conducted under conditions described in the text except for solvents:
A, methylene chloride/acetone (9/5, v/v); B, chioroform/ethyl acetate/ethanol
(40/10/7); C, chloroform/ethanol (9/1); and D, chloroform/ether (7/3). Plates were
developed once with solvents B, C and D, but twice with A. Standards (Std) were
located by fluorescence quenching, and metabolites (^C) were located by
radio- autography. In all cases, standards admixed with radiolabeled metabolites
comigrated with the zone in question.
hs ince plates were developed twice with solvent A, mobilities (not Rf's) are
expressed relative to the solvent front after the second run.

20
Characteristics of the enzymatic reaction. Typically, 20-25% of
the substrate, testosterone (initial concentration, 131 yM), was
consumed in 10 min upon incubation with 0.4-0.5 mg microsomal protein
The metabolites in zones 1 (15a-hydroxytestosterone) through 7
(63-hydroxytestosterone) accounted for more than 65% of the total
metabolism. 60-Hydroxytestosterone was the major metabolite formed in
the presence of both male and female liver microsomes. Each of these
metabolites was produced more rapidly in the presence of microsomes
than any other subcellular fraction.
Rates of formation of each of these hydroxytestosterone
metabolites were proportional to microsomal protein concentration
(Figure 2). Figure 3 depicts the relationship between time and the
production of metabolites. Rates of formation of 15a- and
16a-hydroxytestosterone were constant for 15 min. The rates of
formation of 7a-, 150-, 6a- and 60-hydroxytestosterones were constant
for at least 10 min. After 15 min of incubation, the rates of
formation of all products, especially 60- and 150-hydroxytestosterone,
decreased significantly.
The metabolism of testosterone by hepatic microsomes from male and
female mice. Figure 4 compares the relative rates of formation of six
hydroxytestosterone metabolites in the presence of microsomes prepared
from male and female AKR/J mice. A female predominance was observed in
hydroxylase activity at the 6a, 60, 150 and especially the 15a position
of testosterone. Hydroxylation at 7a was not affected by sex, and
hydroxylation at the 16a position of testosterone was uniquely
predominant in males. Table 3 compares the apparent Km and Vmax
for

21
Figure 2. Effects of protein concentration on the rates of formation of
various hydroxytestosterone metabolites. The standard
reaction mixture, which was incubated for 10 minutes at
37°C, contained 131pM [14C]-testosterone, 100 mM phosphate
and NADPH as described in the text. The microsomes were
prepared from male AKR/J liver.

22
Figure 3. Effects of time of incubation on the formation of various
hydroxytestosterone metabolites. The standard reaction
mixture, which was incubated at 37°C, contained 0.4 mg
microsomal protein prepared from male AKR/J liver, 131 pM
[^C]-testosterone, 100 mM phosphate and NADPH as
described in the text.

TESTOSTERONE HYDROXYLASE ACTIVITY
(nmoles/min/mg protein) EXCLUDING 6/9
23
HYDROXYTESTOSTERONE METABOLITES
0.7
0.5
0.3
0.1
15a
X
16a
*
T
7a
15/9 6 a
X
6/S
I
cfg cftj> cfc¡> cfc¡> cf cf<¡>
t4.0
3.0
2.0
1.0
Figure 4. Rates of formation of six hydroxytestosterone metabolites by
microsomes prepared from male and female AKR/J mice. [â„¢C]-
testosterone was incubated with microsomes under standard
incubation conditions described in the text. Note different
scale for 60-hydroxytestosterone. N=5 and * indicates
P<0.01 for male-female comparison.
6/3 HYDROXYLASE ACTIVITY
(nmoles /min/mg protein)

TABLE 3
APPARENT KINETIC CONSTANTS FOR HEPATIC MICROSOMAL TESTOSTERONE
HYDROXYLASES IN MALE AND FEMALE AKR/J MICE
Hydroxytestosterone
Metabolite
Apparent Kinetic
Constants (± SE)—
Male
Female
Km
(pM)
(nmol/mg^lO min)
Km
(pM)
(nmol/mg^lO min)
15a
4.2 ( 1.2)
1.43 (0.19)
3.7 ( 0.4)
5.15* (0.54)
16a
1.4 ( 0.2)
4.19 (0.43)
4.9 ( 1.7)
1.22* (0.12)
7a
2.7 ( 1.0)
0.81 (0.09)
5.6 ( 0.5)
0.86 (0.07)
CO.
IT)
i-H
45.9 ( 7.7)
1.50 (0.18)
41.3 ( 7.6)
2.40* (0.33)
6a
5.1 ( 0.6)
2.20 (0.23)
6.1 ( 0.2)
3.92* (0.36)
63
60.9 (11.3)
29.20 (2.89)
80.1 (10.6)
53.11* (7.14)

TABLE 3—extended.
IHepatic microsomes from male and female mice were incubated with 6 different concentrations
of [^^testosterone (5-500 yM) of varying specific radioactivity (total of 0.17-1.5
yCi/incubation) under conditions defined in the text except that incubation times were
adjusted to assure measurement of initial velocities at each substrate concentration,
n = 3.
-^max 1S expressed as nmoles of product formed per mg of microsomal protein per 10 minutes,
indicates P < 0.05 with regard to male/female comparisons.

26
production of each metabolite in the presence of male and female
rnicrosoines. Lineweaver-Burk plots for each metabolite were essentially
linear with preparations from either sex. There viere no significant
differences between male and female mice in the apparent Km values
for any of the six testosterone hydroxylations. For the 6a, 7a, 15a
and 16a hydroxylations, the apparent Km's for testosterone were low
(1.4 to 6.1 pM) in comparison to those for 6g and 15g hydroxylations
(41 to 80 pM).
Maximal velocity for 15a hydroxylation was more than 3-fold
higher with female than male preparations, while Vmax for 16a
hydroxylation of testosterone was more than 3-fold higher with male
than female microsomes. Both Vmax's and relative rates for 6a,
65 and 15g hydroxylations were significantly higher in female
preparations, but differences in each case were less than 2-fold.
7a-Hydroxylation was the only activity studied that was not affected by
sex, although only 60% of the radiolabel in its zone is
7a-hydroxytestosterone. Admixture of liver microsomes from male and
female mice in 4 different proportions yielded additive activities for
production of the 15a-, 16a- and other hydroxytestosterones (data not
shown). Therefore, we find no evidence for the presence of a
reversible, non-stoichiometric activator or inhibitor for any of the
hydroxylations. The mixing experiments also decrease the probability
of product inhibition as an explanation for the sexual dimorphism.
The content of cytochrome P-450 in hepatic microsomes was not
dependent on sex (male and female, 0.816 and 0.760 nmoles P-450/mg
microsomal protein, respectively). Thus the sexually dimorphic

27
hydroxylase activities persist even when results are expressed in terms
of molecular activity. Indeed, we found less day-to-day variation in
molecular activities than in specific activities.
The effects of potassium phosphate concentration on testosterone
hydroxyl ation. All forementioned experiments were conducted in 100 mM
phosphate buffer. Figure 5 presents the results of experiments in
which the relative rates of hydroxylation of testosterone by male and
female microsomes were studied at 30, 100 and 330 mM phosphate, all at
pH 7.4. The effects of changing potassium phosphate concentration were
strongly dependent on the position of hydroxylation, and in the case of
16a and 15a hydroxylations, on gender as well. An 11-fold increase in
phosphate concentration resulted in substantial inhibition (75 to 90%)
of 15a and 7a hydroxylation, little or no effect on 15b and
6a-hydroxylation, and a modest stimulation of 6b and 16a (male only)
hydroxylation. Male 15a hydroxylation was relatively insensitive to
changes in potassium phosphate concentration between 30 and 100 mM, but
female activity decreased 2-fold.
Progesterone. A limited number of experiments were conducted with
progesterone as substrate in order to gain some insight into the
substrate specificity of the hydroxylases under investigation. Results
with hepatic microsomes from male and female AKR/J mice are presented
in Table 4. Progesterone was actively metabolized, and 15a-, 16a-, 6b-
and 21-hydroxyprogesterone were identified as important products. Like
testosterone, 6b hydroxylation of progesterone was quantitatively most
important and exhibited a small but significant female predominance.
The female predominance seen in 15a hydroxylation with testosterone was

28
even more striking with progesterone in that a 5 to 6-fold female/male
ratio was observed. The male predominance in 16a hydroxylation with
testosterone (3-fold) was not seen with progesterone.

% ACTIVITY % ACTIVITY
29
30 100 330
Figure 5. Effects of potassium phosphate buffer concentration on the
rates of formation of different hydroxytestosterone
metabolites in the presence of hepatic microsomes from male
and female AKR/J mice. Livers from 2 mice were pooled for
preparation of microsomes. Incubation conditions are
described in the text; the initial [^C]-testosterone
concentration was 131 pM, but the concentration of potassium
phosphate, pH 7.4, was varied as indicated. All hydroxylase
activities are expressed in relation to activity at 100 mM
phosphate (defined as 100%) since that concentration is used
in the standard assay, n = 3. * and + indicate PC0.05 for
30-100 mM and 100-330 mM comparisons, respectively.

TABLE 4
RATES OF FORMATION OF HYDROXYPROGESTERONE METABOLITES IN THE
PRESENCE OF HEPATIC
MICROSOMES FROM
MALE AMD FEMALE AKR/J
MICE
Hydroxyprogesterone
Metabolite
Rates of Formationi
(nmoles/mq protein/min)
Ratio of
Activities
9
d
9/£
15a
1.23
0.22*
5.59
16a
0.72
0.75
0.96
6b
4.43
3.45**
1.29
21
0.39
0.29
1.35
a.[14c]-Progesterone, 131 pM, was incubated for 10 minutes with
microsomes under conditions described in the text; phosphate
concentration was 100 mM. Metabolites were resolved by TLC and
assayed radiometrically.
indicates P < 0.005 (male/female comparison).
**Indicates P < 0.025 (male/female comparison).

31
Discussion
These results initiate a set of detailed studies with highly
inbred mice on the heterogeneity, substrate specificity and regulation
of the constitutive forms of cytochrome P-450. The overall project is
designed to control for possible allelic polymorphism in the P-450's
and later, to take advantage of potential strain differences. Our
initial goal was to establish a reasonably rapid and economical system
for assay of the major monohydroxylated metabolites of testosterone.
We have developed a one-dimensional radiochromatographic technique for
the assay of several hydroxytestosterone metabolites produced by mouse
liver microsomes. The method has better resolving power than other
reported TLC systems and is more rapid, less tedious and/or less
expensive than the two-dimensional TLC or the paper chromatographic
procedures that have recently been used in studies with mice (26,32).
We have focused on six hydroxytestosterone metabolites which
together comprise more than two-thirds of total products. Metabolites
were identified by comigration with reference standards in five
different solvent systems; identifications were confirmed by study of
diacetyl derivatives. While our method is good for assay of the 15a-,
16a-, 6a- and 63-hydroxytestosterone metabolites, 7a- and
153-hydroxytestosterone account for only about 60% and 80% of the
labeled metabolites in zones 3 and 4, respectively. We are especially
cautious in the interpretation of 7a-hydroxylase activity because of
the susceptibility of this metabolite to spontaneous dehydration to the
4,6-diene both before and during TLC (31).

32
Large discrepancies exist in the reported rates of 16a, 7a and 63
hydroxylation of testosterone in the presence of hepatic rnicrosomes
from various strains of mice (32-35). These discrepancies, which also
include differences in the rank order of the different hydroxylase
activities, could reflect allelic polymorphism between isogenic strains
of mice, but at least some of the discrepancy seems to be
methodological; it results from certain features peculiar to murine
hepatic steroid metabolism. Ford et^ ad_. (31) have reported the
two-dimensional TLC separation of several of the metabolites of
testosterone formed by mouse liver rnicrosomes. At least two of these
metabolites, 15a- and 150-hydroxytestosterone, have not been reported
after incubation of testosterone with rat or rabbit rnicrosomes. Mouse
liver rnicrosomes also possess little if any 4-ene reductase activity
(31). Yet many earlier experiments with mice have been conducted
employing solvent systems (Table 2) and assay conditions optimized for
studies with rat or rabbit.
We have examined the rates of formation of each metabolite as a
function of various assay conditions. Subcellular fractionation
revealed that the hydroxylases in question were concentrated in the
microsomal fraction and that varying the NADPH concentration 2-fold did
not influence results. As discussed below, because the concentration
of potassium phosphate buffer had difieren- tial effects on
hydroxylation at different positions, we elected to maintain phosphate
at a concentration nearly that found in liver cytosol (ca. 70 mM).
Under our standard incubation conditions, testosterone concentration
was at least 20 times greater than the Km's for the formation of all

33
products except 63- and 153- hydroxytestosterone. This assured
catalytic rates near Vmax for most products, and rates that were at
least 60% Vmax for all products. While the amount of each product
formed was proportional to both time of incubation and microsomal
protein concentration, our standard incubation conditions remain just
inside the limits of linearity for the formation of 63- and
153-hydroxytestosterone, perhaps reflecting the higher Km's for
these hydrcxylations and/or differences between P-450 isozymes.
There was little variability between AKR/J mice of the same sex
with regard to rates of formation of six different hydroxytesto-
sterones; standard deviations approximated 15%. The rates of 15a, 153,
6a and 63 hydroxylation of testosterone were higher in female than male
AKR/J microsomes with 15a-hydroxylation exhibiting the greatest degree
of sexual dimorphism (3 to 4-fold female predominance) despite the lack
of sexual dimorphism in P-450 content. Hydroxylation at 7a was not
sexually dimorphic, and the rate of formation of 16a-hydroxytesto-
testosterone was 3 to 4-fold faster in male than female microsomes.
Sex-dependent differences in steroid and drug metabolism in rats have
been well documented (36,37), and recent evidence suggests that these
differences reflect the presence of sex-dependent forms of hepatic
microsomal cytochrome P-450 (38,39). Reports of sex differences in
drug metabolism in mice are inconsistent, perhaps reflecting
strain dependence and/or a smaller quantitative difference between the
sexes in mice (40,41). The sexual dimorphisms we find for 15a and 16a
hydroxylation of testosterone are larger than typically seen with other

34
substrates in mice, perhaps because it is an endogenous substrate.
Sex differences in the selective hydroxylations of testosterone were
also exhibited by A/J and 129/J (but not the C57BL/6J) mice (26).
Studies of testosterone metabolism in reconstituted P-450 systems
from animals induced with different xenobiotics have suggested that
hydroxylations at the 6p, 7a and 16a positions are catalyzed primarily
by different forms of cytochrome P-450 (42,43,44). This concept has
been extended recently to untreated animals (45,46). By virtue of the
broad substrate and/or product specificity exhibited by certain forms
of cytochrome P-450, it is likely that hydroxylase activity at a given
position on the steroid nucleus reflects the activity of more than one
monoxygenase present in hepatic microsomes. Recently, Thomas et al_.
(47) have established the existence of more than one testosterone 16a-
hydroxylase in rat liver microsomes. They found a large proportion of
testosterone 16a hydroxylation in microsomes from untreated rats to be
immunologically distinct from that due to cytochrome P-450^. Our
finding of a male predominance (3- to 4-fold) in 16a-hydroxylase
activity toward testosterone but not progesterone suggests that the
steroid 16a-hydroxylase activity of mouse hepatic microsomes may also
be heterogeneous. Based on analogous experiments, a similar
interpretation was proposed by Kremers et al_. (48) for the rat. The
differential effects of potassium phosphate concentration also support
this conclusion. Male but not female testosterone 16a-hydroxylase
activity was stimulated by high potassium phosphate concentrations.
Our findings probably reflect a different distribution of P-450's
in male and female AKR/J liver, with each P-450 having more or less

35
discrete specificity with regard to the selective hydroxylation of
testosterone. Nonetheless, it seems quite probable that any given
"hydroxylase" (e.g. testosterone 16a-hydroxylase) is the result of
activities imparted by more than one enzyme. Certainly P-450's with
the functional propensity to direct most hydroxylation of testosterone
to the 15a or 16a positions can be distinguished from each other and
from other testosterone hydroxylases by comparison of sexes. To what
extent male and female forms of the P-450's differ qualitatively and/or
quantitatively cannot be surmised at this time. Kinetic experiments
reveal that the sex differences in AKR/J mice were not due to
differences in apparent Km's between sexes. Furthermore, mixing
experiments did not suggest a gender-dependent activator or inhibitor.
Our results could indicate differences in the concentrations of certain
forms of cytochrome P-450 between males and females.
It is not known whether the female predominance in 15a, and to a
lesser extent 153, 6a and 63 hydroxylation reflects quantitative or
qualitative differences in one or several P-450 isozymes.
Interestingly, the female predominance in 15a hydroxylation observed
with testosterone is even more striking with progesterone. The greater
sex difference results from higher rates of 15a hydroxylation toward
progesterone than testosterone with female microsomes; male
15a-hydroxylase activities are similar with both substrates. This
result suggests qualitative differences between the process of
15a hydroxylation in males and females and indicates that steroid
15a-hydroxylase is also a heterogeneous enzyme. This conclusion is
also supported by the effects of potassium phosphate. Rates for 15a

36
hydroxylation of testosterone in females were found to be 2-fold higher
at 30 mM potassium phosphate than at 100 mM while little difference was
observed in 15a-hydroxylase activity in males at these two
concentrations. This interpretation is more convincing when one
considers that the effects of phosphate concentration on other
hydroxylations (except 16a) are the same with male and female
preparations. More detailed experiments of the stimulatory and
inhibitory effects of potassium phosphate (cf. 49) are in progress.

CHAPTER TWO
THE INFLUENCE OF STRAIN AND SEX ON SITE-SPECIFIC TESTOSTERONE
HYDROXYLASE ACTIVITIES OF MOUSE HEPATIC MICROSOMES
Introduction
The isolation of a number of inducible cytochrome P-450 isozymes
from liver microsomes of rat (43,50) and rabbit (51,52) as distinct
proteins with different chemical and physical properties (3,4) has
established the multiplicity of mammalian hepatic microsomal cytochrome
P-450. Recently, attention has also been focused on the various forms
of cytochrome P-450 from untreated rats (4,6) and rabbits (45,53),
partly because immunological techniques have revealed a lack of
cross-reactivity between various inducible forms and the bulk of these
"constitutive" isozymes (10,11,47). The molecular basis for
heterogeneity within the constitutive forms is a particularly difficult
problem because each isozyme likely occurs in relatively small amounts.
Nonetheless, these forms are of special interest because of the
possibility that their physiological function(s) and regulation may be
quite different from those of the inducible isozymes. These isozymes
likely have greater specificity than the inducible forms toward
endogenous substrates like steroids, and their regulation involves
factors such as neonatal and pubertal sexual differentiation (38,39).
The extent of heterogeneity in both inducible and constitutive
isozymes of P-450 could be exaggerated in studies of randombred or
outbred animals by the many allelic differences that such animals are
37

38
likely to display. Johnson et_ al_. (8) have recently described
differences in progesterone 21-hydroxylase activity and form 1 P-450
between individual New Zealand White rabbits. Moreover, Vlasuk et
a]_.(9) have reported that Holtzman and Long-Evans rats, and even
different colonies within those strains, could be distinguished by the
characteristics of their phenobarbital-induced cytochromes P-450.
While it is not clear whether this degree of complexity reflects the
presence of genetically distinct P-450 apoproteins or is the result of
postranslational modification(s) of one or more gene products, it is
clear that the heterogeneity of cytochrome P-450 and its regulation
represent a complex problem involving both genetic and environmental
influences.
Several substrates, including testosterone and other endogenous
steroids, have been used successfully to probe the heterogeneity of the
cytochromes P-450 by virtue of the remarkable regio- and positional
selectivity exhibited by certain isozymes. We reasoned that a detailed
comparative study of hepatic microsomal steroid hydroxylations in male
and female animals of several isogenic mouse strains could provide
valuable insight into the catalytic specificity, regulation and
polymorphic expression of several forms of cytochrome P-450 in
untreated animals. Strain differences in steroid hydroxylation have
been reported for many species (54,55,56) and in some instances the
patterns of inheritance have been worked out (57,58). In regard to the
hydroxylation of testosterone by mouse liver microsomes, large
discrepanices exist between reported rates for 16a, 7a, 6ft and other
hydroxylations in studies by various investigators (26,32,33). These

39
differences could reflect a large degree of polymorphism among isogenic
mouse strains, but methodologic differences between laboratories
probably have contributed (59). In this study, the use of inbred
strains controls for intrastrain allelic variations and a common
methodology applies in all experiments. These factors allow more
meaningful interstrain and gender comparisons.

40
Materials and Methods
Materials. Testosterone [4-l^C] (56.1 yCi/ymol) was
purchased from New England Nuclear, Waltham, Ma. The radiochemical
purity was greater than 98% as established by TEC on silica gel
F^54 plates with the solvent system ether/benzene/methanol,
55:44:0.5 (v/v). Testosterone was obtained from Sigma Chemical Co.,
St. Louis, Mo., and was recrystallized twice from acetone/hexane.
Reference steroid standards were obtained through sources described
previously (59).
Precoated (0.2 mm) silica gel 60 F254 TLC plates (E. Merck,
Darmstadt, Germany) were purchased from Scientific Products, Brunswick,
N.J. SB-5 X-ray film for autoradiography was obtained from the Eastman
Kodak Co., Rochester, N.Y., and Liquiscint liquid scintillation
cocktail was obtained from National Diagnostics, Somerville, N.J.
NADPH was purchased from Sigma Chemicals, St. Louis, Mo. All other
materials were purchased from the Fisher Scientific Co., Pittsburgh,
Pa., and were of the highest quality commercially available.
Animals. Mice of 8 strains were obtained at the age of 6-8
weeks (20-25 g) from Jackson Laboratories (Bar Harbor, Me). Mice of
the same sex were housed 4-6 per cage on corncob Sani-cel bedding. The
animals were maintained on a 12 hr light/12 hr dark photoperiod and
were allowed both food (Purina Rodent Chow) and water ad libitum. All
mice were 11-14 weeks of age at the time of study.
Preparation of microsomes. Given the possibility of circannual
variation in testosterone hydroxylase activity (26), all animals were
studied between September and December. Male and female mice of a

41
given strain were killed by decapitation on the same day between 8 and
10 am. Livers were excised immediately, and gall bladders were
carefully removed. The livers were then rinsed, and each gram of liver
was homogenized with a motor-driven glass-Teflon homogenizer in 5 ml of
ice-cold 250 nil potassium phosphate buffer, pH 7.4, containing 150 mM
potassium chloride. Microsomes were prepared by differential
centrifugation. Briefly, the homogenate was centrifuged at 10,000 g
for 15 min, and the supernatant fraction was recentrifuged for 1 hr at
110,000 g. Microsomal pellets from 1 g of liver were resuspended in 2
ml of 100 mM potassium phosphate, pH 7.4, and assayed immediately.
Microsomal suspensions contained approximately 8-10 mg protein per ml
as determined by the method of Lowry et aj_. (28). Cytochrome P-450
content was determined by the method of Omura and Sato (29) after
microsomal suspensions were diluted 10-fold in 100 mM phosphate, pH
7.4, containing 20% glycerol (v/v) and 0.2% Emulgen 911.
Radiometric assay of testosterone hydroxylase activities. The
rates of formation of 6a-, 63-, 7a-, 15a-, 15$-, and
16a-hydroxytestosterone were determined by the radiometric assay we
described earlier (59). Briefly, 0.4-0.5 mg microsomal protein were
incubated at 37° C in a shaking water bath with l^C-enriched
testosterone (131 nmol containing 0.5 yCi; final concentration, 131 yM)
added in 10 yl ethanol; 1.0 ymol NADPH (final concentration, 1.0 mM);
10.0 ymol MgCl2 (final concentration 10.0 mM); and potassium
phosphate buffer, pH 7.4 (final concentration, 100 mM) in a final
volume of 1.0 ml. Reactions were started by the addition of NADPH and
stopped 10 min later by the addition of 5 ml of ice-cold methylene

42
chloride. Each sample was then transferred to a 10-ml Teflon-lined
screw cap test tube and shaken vigorously for 30 min. Phases were
separated by centrifugation, and the organic phase was collected and
evaporated to dryness at room temperature under a stream of nitrogen.
Residues were resuspended in 50 yl ethyl acetate and resolved by
TLC. Routinely, plastic-backed silica gel TLC plates were developed
3-times with ether/benzene/methanol (55:44:0.5, v/v). The testosterone
metabolites formed in the presence of hepatic microsomes from at least
one male and one female from each of the 8 strains were also resolved
by two-dimensional TLC; silica gel plates (20 x 20 cm) were developed
in the first dimension with chioroform/ethanol (92:8) and in the second
dimension 3-times with the solvent system described above. All
chromatograms were subjected to radioautography, and radiolabeled zones
were cut out and quantitated by scintillation spectrophotometry.
Statistics. Two-way analysis of variance was conducted for each
hydroxylase activity in order to test for the influence of sex, strain,
and the interaction between sex and strain on enzymatic activity (60).
Where the interaction between sex and strain was found to be
significant, Duncan's multiple comparison procedure was used to compare
the 8 strains at both levels of sex, and also to compare the two sexes
at each level of strain (61).

43
Results
The data collected in this study are reasonably straightforward;
the rates of formation of six different monohydroxytestosterone
metabolites in the presence of hepatic microsomes from males and
females of eight strains of mice were determined. Mean values for the
various testosterone hydroxylase activities for five animals of each
sex and strain are presented in Tables 5 (specific activity or
catalytic activity/mg microsomal protein) and 6 (apparent molecular
activity or catalytic activity/nmole P-450). Standard deviations for
the 16 groups of animals ranged from 10 to 30 percent of the respective
means.
Earlier experiments (59) with hepatic microsomes from male and
female AKR/J mice afford background for assessment of the results
presented in Tables 5 and 6. 1) The six metabolites under scrutiny
comprised more than 65 percent of the total biotransformation products
of testosterone. 2) The formation of each metabolite was proportional
to time of incubation and microsomal protein concentration under the
conditions of assay. 3) The initial testosterone concentration in
these experiments was 131 yM; the apparent K^'s for production of
each metabolite were less than 7 yM except for 15f>-hydroxytestosterone
(50 yM) and 60-hydroxytestosterone (80 yM). In no case was more than
30 percent of the testosterone consumed during incubation.
Even a casual examination of the results depicted in Tables 5 and
6 revealed certain striking findings. 1) The rates of hydroxylation of
testosterone at six positions were quite similar in the various strains
of animals, especially among males. For example, 63-hydroxylase

TABLE 5
SPECIFIC ACTIVITIES OF TESTOSTERONE HYDROXYLASES: RATES OF FORMATION
OF VARIOUS METABOLITES IN THE PRESENCE OF HEPATIC MICROSOMES
FROM MALES AND FEMALES OF EIGHT STRAINS OF MICE.
Testosterone Specific Activity^
Metabolite (nmoles product/10 inin/mg microsomal protein)
BALB/cJ
C3H/HeJ
C57BL/6J
SWR/J
DBA/2J
A/J
129/J
AKR/J
MALE
I 5a-OH
1.68
1.45
1.63
1.11
1.20
1.33
1.60
1.37
1 6a-OH
4.80
4.77
4.20
3.79
4.07
4.33
3.06
3.87
7a-OH
1.15
1.75
1.64
1.56
1.94
0.93
1.65
1.15
1 5(3-OH
1.09
0.65
1.45
1.46
0.86
1.43
1.34
1.19
6a-OH
2.73
1.45
3.45
3.05
2.04
2.57
2.89
2.90
6p -OH
18.56
21.70
18.49
32.09
20.41
26.32
23.66
27.58

TABLE 5--extended.
FEMALE
15a-0H
1.96
1.18
1.30
1.40
2.39
2.78
3.36
4.64
16a-0H
2.34
1.39
1.39
1.67
2.31
1.87
0.93
1.15
7 cx-OH
1.06
2.02
2.02
2.27
2.68
0.82
2.52
1.06
150-OH
1.00
0.76
1.01
1.71
1.18
1.28
1.52
1.94
6a-0H
2.39
1.64
3.21
4.04
3.04
2.48
4.17
3.78
60-OH
18.72
23.38
14.25
30.27
23.63
22.69
27.08
32.29
An=5 for each sex and
strain. See
text for
statistical
analyses.

TARLE 6
APPARENT MOLECULAR ACTIVITIES OF TESTOSTERONE HYDROXYLASES:
RATES OF
FORMATION
MICROSOMES
OF VARIOUS
FROM MALES
METABOLITES
AND FEMALES
IN THE PRESENCE OF
OF EIGHT STRAINS
HEPATIC
OF MICE.
Testosterone
Met a bol ite
(nmoles
Apparent Molecular
product/10 min/mnole
Activity^ b
cytochrome P-450-
)
BALB/cJ
C3H/HeJ
C57BL/6J
SWR/J DBA/2J
A/J
129/J
AKR/J
MALE
15a-OH
2.34
1.81
2.54
1.14
1.94
1.95
2.88
1.67
16a-OH
6.71
6.08
6.50
3.89
6.67
6.35
6.01
4.79
7a-OH
1.60
2.22
2.52
1.59
3.21
1.36
2.81
1.43
1 5(3 -OH
1.54
0.84
2.26
1.49
1.41
2.09
2.43
1.45
6a-OH
3.87
1.87
5.29
3.11
3.41
3.75
5.26
3.56
6(3 -OH
25.93
27.71
28.64
32.73
33.52
38.35
41.17
33.28
-p»
cr>

TABLE 6--extended.
FEMALE
15a-0H
3.10
1.86
2.74
1.62
3.37
4.86
6.06
6.19
16a-0H
3.76
2.18
2.94
1.93
3.29
3.24
1.66
1.53
7a-0H
1.68
3.18
4.31
2.61
3.82
1.41
4.60
1.41
153-OH
1.60
1.18
2.15
1.98
1.65
2.22
2.72
2.59
6a-0H
3.39
2.57
7.19
4.67
4.36
4.28
7.53
5.07
63-OH
29.86
36.65
30.33
34.98
33.16
38.43
47.42
43.35
â– ^N=5 for each
strain and
sex. See
text for
statistical
analyses.
Cytochrome P-450 was measured spectrophotometrically; see text for details.

48
activity was about 10-fold higher than other activities in ail strains.
2) The rate of 16a hydroxylation proceeded about 2 to 3-fold faster in
male than female preparations of all strains. 3) The rate of most
other hydroxylations, especially 15a hydroxylation, proceeded more
rapidly in female than male microsomes, but the effect was
strain-specific. 4) For most comparisons, mean specific activities and
apparent molecular activities lead to similar conclusions because mean
P-450 content per mg microsomal protein varied little between strains
or sexes. Of the eight strains studied, only C3H/HeJ and C57BL/6J
demonstrated a consistent sex difference in hepatic cytochrome P-450
content. In these two strains P-450 content was 20-30% greater in male
than female microsomes.
Results of a two-way analysis of variance for five hydroxylase
activities in 80 animals (5 males and 5 females for 3 strains) are
presented in Table 7 in terms of values. 7a-Hydroxylation is
omitted from detailed analysis because only about 60% of radiolabel in
its TLC zone was 7a-hydroxytestosterone (59). "Total" represents
the percent of the total variability for a given hydroxylase activity
that can be accounted for by the sum of the effects of sex, strain and
the sex-strain interaction. Unusually high values were obtained for
15a- and 16a-hydroxylase activities in that 85-90% of all individual
variability in these activities could be accounted for by these
variables. By comparison, only 50% of the variability in
63-hydroxylase activity could be attributed to the same factors.
Except for 63-hydroxyl at ion, the effect of sex, strain and the
interaction between sex and strain were all significant determinants of

TABLE 7
ANALYSIS OF VARIANCE (ANOVA) BETWEEN SEX, STRAIN, AND THE
INTERACTION BETWEEN SEX AND STRAIN AND THE SELECTIVE
HYOROXYLATION OF TESTOSTERONE.
Position of
Hydroxylation
Values—
Sex
Strain
Sex + Strain
Total
Total —
15a
0.8194
0.3054
0.3109
0.8357
0.8679
16a
0.7770
0.1015
0.0272
0.9057
0.9370
156
0.0217
0.5149
0.1776
0.7142
0.7276
6a
0.0666
0.5372
0.1150
0.7188
0.7496
63
0.0010*
0.4784
0.0527*
0.5322
0.5656
â– %I=8Q for specific activity at each position of hydroxylation since 5
males and 5 females were studied for each of 8 strains.
-ANOVA of apparent molecular activities instead of specific
activities.
:k
Failed test for significance. All other values are significant
(P < 0.05).

50
the rates of position-specific monohydroxyl at ion of testosterone (P <
0.05). For 16a hydroxylation, sex was the most important determinant.
For 15a hydroxylation, the interaction between sex and strain (i.e.,
the effect of one factor depends upon the level of the other factor)
was most important. In the other cases, strain was the most important
determinant.
Male and female comparisons of testosterone hydroxylase activities
for each strain are presented in Table 8. Sex ratios are given only
when the differences were statistically signficant. Statistical
analysis of either specific activities or apparent molecular activities
results in similar interpretations except in a few cases where the
differences between the sexes was 40% or less. As indicated in Tables
5 and 6, the rate of 16a hydroxylation was 2-3 times higher in the male
preparations of all eight strains studied. In contrast, female
activity was greater for most other hydroxylase activities, but
differences were strain-specific. The most prominent strain-specific
sex difference was observed for 15a-hydroxylase activity. In the
AKR/J, A/J, 129/J and DBA/2J strains, 15a-hydroxylation proceeded 2-3
times faster in female than male microsomes while in the other four
strains no significant sex difference was observed. Other experiments
(see below) have revealed that 15a-hydroxylase activity is not
dependent on phase of the estrus cycle.
In order to assess the statistic significance of differences
between strains with regard to rate of site-specific testosterone
hydroxylation, the activities of male and female microsomes were
analyzed separately by Duncan's multiple comparison procedure

TABLE 8
MALE:FEMALE COMPARISON OF APPARENT MOLECULAR ACTIVITIES
IN THE SELECTIVE HYDROXYLATION OF TESTOSTERONE
Position of
Hydroxylation Male/Female (M/F) or Female/Male (F/M) Ratic£
DBA/2J
SWR/J
129/J
AKR/J
BALB/cJ
A/J
C3H/HeJ
C57BL/6J
16a
M/F
2.0
2.0
3.6
3.1
1.8
2.0
2.8
2.2
15a
F/M
1.7
*
2.1
3.7
★
2.5
*
*
153
F/M
*+
1.3+
*
1.8
*
*
* *+
6a
F/M
*+
1.5
1.4
1.4
*
*
* 1.4+
¿Based on results presented in Table 6.
*Failed test for significance (a level = .05; Duncan's Multiple Range Test).
+Test for significance differs when analysis is based on ratios of specific activity
instead of apparent turnover number.

52
(Figure 6). Comparisons were done on apparent molecular activities
instead of specific activities for reasons enumerated below. Many
differences between the strains (a level = 0.05) were observed in the
hydroxylase activity at each of four sites on testosterone.
Hydroxylation at 7 a was omitted from consideration for the reasons
described above; 63 hydroxylation was not analyzed because it did not
reveal a significant dependence on strain (Table 7). Those strains
with hydroxylase activities that viere statistically indistinguishable
are joined by lines in Figure 6. Such groupings between males were
distinct from female groups, and female mice generally exhibited a
greater degree of polymorphism for most hydroxylase activities. This
sex-strain interaction was most pronounced in the rate of 15a
hydroxylation (Figure 6). For example, while male activities of the
C3H/HeJ, AKR/J and SWR/J strains were net distinguishable, female
15a-hydroxylase activity of the AKR/J strain was 3- to 4-fold greater
than that of the C3H/HeJ and SUR/J strains. Male 15a-hydroxyl ase
activity varied only 1- to 2-fold among the eight strains of mice.
Although interstrain variation in the other hydroxylations was small, a
2 to 3-fold strain difference could be found depending on the sex,
strain and hydroxylase activity in question. No obvious correlations
indicative of groups of hydroxylase activities were detected.
Possible interstrain relationships in the relative pattern of
testosterone hydroxylation at all six positions were explored by
expressing each hydroxylase activity as a percentage of the total
hydroxylase activity (6a + 63 + 7a + 15a + 153 + 16a) observed in a
strain. The overall patterns of hydroxylase activity were nearly

TESTOSTERONE HYDROXYLASE ACTIVITY (nmoles/IOmln/nmole P-450)
53
Figure 6. A comparison of strains v/ith regard to apparent molecular
activities for the 15a-, 16a-, 6a- and 15f$ -hydroxyl at ions of
testosterone. Males and females were analyzed separately by
the Duncan's multiple comparison procedure. The strains
linked by a vertical solid line were indistinguishable
statistically at the a level of 0.05. Abbreviations: A/J,
AJ; AKR/J, AK; BALB/cJ, BL; C3H/HeJ, C3; C57RL/6J, B6;
DBA/2J, D2; SWR/J, SW; and 129/J, 1.1.

54
identical among the different inbred male mice (Figure 7) although
small differences v/ere found for each hydroxyl at ion. In all strains
the rank order of hydroxylase activity was 63 > 16a > 6a > 7a 2 15a >_
153. In contrast, female patterns of testosterone hydroxylation were
quite different in different strains (Figure 8). Only the C57BL/60,
0BA/2J, C3H/HeJ and SWR/J strains had similar rank orders of
hydroxylase activity. Qualitative variations from this pattern were
seen for certain hydroxylase activities in the other four strains.
Changes in the pattern of hydroxylation at the 15a and 16a positions
were observed for the 129/J strain, and at the 15a, 16a and 7a
positions for the AKR/J and A/J strains. A change in the rank order of
only 7a-hydroxylase activity was noted for the BALR/cJ strain. This
difference did not reflect the contaminant that chromatographs with
7a-hydroxytestosterone because the unknown metabolite was found to
remain in equal proportion to the 7a-hydroxytestosterone metabolite in
both sexes of all eight strains as evidenced by two dimensional TLC.
Indeed, two-dimensional TLC did not reveal any previously unidentified
major strain-specific metabolites in either males or females which
might have been undetected by routine one-dimensional TLC or which
might have interfered with quantitation of the six hydroxylase
activities of interest.
Testosterone hydroxylase specific activities and cytochrome P-450
content of untreated animals were found to fluctuate from month to
month by as much as 50% during the course of these and subsequent
experiments. This variation in microsomal activities of untreated
animals has been observed by others (41,62). Ford et al_. (26) have

RELATIVE RATE OF FORMATION
55
SITES OF TESTOSTERONE HYDROXYLATION
I
Figure 7. Relative rates of formation of 6a-, 63-, 7a-, 15a-, 153- and
16a-hydroxytestosterone (expressed as a fraction of the rate
of formation of these 6 metabolites) in the presence of
hepatic microsomes from males of 8 strains of mice. Note
the 10-fold difference in the scale for 63 hydroxyl ation.

RELATIVE RATE OF FORMATION
56
SITES OF TESTOSTERONE HYDROXYLATION
Figure 8. Relative rates of formation of 6a-, 63-, 7a-, 15a-, 153- and
16a-hydroxytestosterone (expressed as a fraction of the rate
of formation of these 6 metabolites) in the presence of
hepatic microsomes from females of 8 strains of mice. Note
the in-fold difference in the scale for 63 hydroxylation.

57
reported a circannual variation in total hepatic hydroxylase activity
of mice towards testosterone; total testosterone hydroxylase specific
activity was reported to be 3-4 times higher in December than in April.
We did not observe a circannual phenomenon, and we are able to
compensate for the variability noted above by expressing results as
apparent molecular activities, defined here as nmoles of product formed
per min per nmole cytochrome P-450. ANOVA indicated that in virtually
all cases unaccountable variability in data obtained with 80 animals is
decreased with use of apparent molecular activities in comparison to
specific activities.

58
Discussion
This study in inbred nice of the relationship between strain, sex
and the sex-strain interaction and site-specific monohydroxylations of
testosterone provides evidence for both heterogeneity and polymorphism
in the isozymes of hepatic microsomal cytochrome P-450 and/or their
modes of regulation. With the exception of 63 hydroxyl ation, a
surprisingly large portion (73-93%, Table 7) of individual variability
can be accounted for by strain, sex and/or the sex-strain interaction.
The significance of strain or the sex-strain interaction as
determinants of catalytic activity in individual animals indicates
genetic polymorphism in the 6a, 7a, 15a, 153 and 16a hydroxylations of
testosterone. Discrete phenotypic classes were not distinguished
because of the limited magnitude of the interstrain variations in
hydroxylase activities.
Because of the prominent interaction between sex and strain, more
detailed evaluation of polymorphism necessitated separate consideration
of males and females (Figure 6). Despite the striking conservation
among microsomal preparations from males of pattern and rank order of
hydroxylase activities (Figure 7), there are statistically significant
differences between strains in four activities (6a, 15a, 153, 16a).
Catalytic activities toward selected positions on the testosterone
molecule vary by about two-fold, and usually not in parallel with
changes in other hydroxylases in the same strain. These results bear
not only on polymorphism, but also suggest extensive isozymic
heterogeneity. We do not have sufficient information to indicate
whether the polymorphisms we have observed reflect differences in

59
isozyme-proteins or genetic alterations at any of several possible
levels of regulation. Both possibilities have precedent in that Dieter
and Johnson (56) have described differences between steroid
hydroxylases isolated from different strains of rabbits whereas the
regulatory Ah locus described by Nebert and colleagues is widely
appreciated (63).
Polymorphism among preparations from female mice is more striking,
especially with regard to 15a-hydroxylase activity. In only four of
the eight strains were female 15a-hydroxylase activities significantly
higher than that in males of the same strain. This is reflected by the
prominent sex-strain interaction as a determinant of activity. There
is no apparent correlation between this polymorphism and the Ah locus
since two strains which exhibit this dimorphism, A/J and AKR/J, are
responsive and non-responsive to polycyclic aromatic hydrocarbons,
respectively (64). Figures 6 and 8 also reveal several other
statistically significant differences in various hydroxylase activities
among preparations from the various female mice. The differences are
greater than those seen between males, yet once again parallelisms in
groups of strains or activities are not apparent. On the basis of the
data presented in Figures 7 and 8, one might conclude that testosterone
hydroxylase activity is highly conserved between inbred strains of mice
and female factors, presumably endocrine, are capable of perturbing
this homeostasis. Subsequent studies revealed the independence of
results from estrus (unpublished observations). Moreover, other
experiments in castrated males (unpublished) accentuated polymorphism.

60
It is now our opinion that endogenous testosterone actually blunts an
underlying polymorphism that is expressed more fully in the female.
The best evidence for involvement of multiple isozymes derives
from the influence of sex on the various hydroxylase activities. We
clearly can distinguish independence of 16a-hydroxyl ase and
15a-hydroxylase activities from each other and from the other
activities. Several other experiments also indicate substantial
heterogeneity in the constitutive P-450 isozymes in mouse liver that
contribute to testosterone hydroxyl at ion. These include the
differential effects of phosphate concentration (59) and endocrine
manipulations (65). Indeed, based on the approach developed by Kremers
et al_. (48) in rats, we have already presented evidence (59) for the
contribution of more than one isozyme to both 16a- and 15a-hydroxylase
activities in mice. Several other reports bear on the relationship
between our results and constitutive and inducible cytochrome P-450
isozymes of mice. Counarin hydroxylation exhibits a 5 to 10-fold
polymorphism in both untreated and phenobarbital treated mice (66), yet
this polymorphism is not reflected in the metabolism of a large number
of other xenobiotics or in the 63, 7a or 16a hydroxylation of
testosterone. To date five or six different forms of liver microsomal
P-450 from mice induced with different xenobiotics have been purified
(3,67). Two of these isozymes, A2 and C2 prepared from
phenobarbital-treated R6D2F;j/J mice, catalyze the 7a and 63
hydroxylation of testosterone, respectively (42). Additional physical
evidence for multiple forms of cytochrome P-450 in the mouse has been
provided by the high performance liquid chromatographic elution

61
profiles of solubilized hepatic P-450's in untreated phenobarbital and
fl-naphthoflavone treated mice (68). To our knowledge, a constitutive
form of cytochrome P-450 has yet to be purified from mice.
Sex-dependent differences in steroid and drug metabolism by the
hepatic microsomal monoxygenase system have been well characterized in
the rat (36,37,69,70). Reports of such differences in mice are sparse
and often inconsistent. Until now, most of the P-450 associated sex
differences reported for mice have been strain-dependent and show a
small female predominance (40,71,72). Our results for the 6a, 6g, 7a,
15a and 15p hydroxylations of testosterone support these observations.
Strain differences in the female predominance of 15a-hydroxylase
activity are substantial and offer the opportunity to study the genetic
constraints on the sexual expression of different forms of P-450.
Preliminary studies (73) suggest that the regulation of this activity
is similar to that described previously (74,75,76) for the
15-hydroxylase system of female rats active toward 5a-androstane-3a,
170-diol 3,17-disulfate. The only significant sex difference we found
to be strain-independent was 16a hydroxylation, which occurred 2 to 3
times faster in male preparations from all eight strains. This finding
of male predominance is distinctive for mouse hepatic P-450
monoxygenase activities and may be characteristic of mice in general.
Steroid hydroxylations have been studied most extensively in the rat,
and 16a hydroxylation is higher in males with several steroid
substrates (48,77). Hepatic 16a hydroxylation of testosterone in the
rat has been shown to be catalyzed by a sex-dependent form of
cytochrome P-450 that is neonatally imprinted (38,78). This activity

62
is distinct from rat phenobarbital -induced 16a-hydroxylase activity
(22, 47). We have previously shown (59) in the AKR/J strain that these
sex differences in 15a- and 15a-hydroxylase activities are not
associated with differences in apparent Km values for testosterone.
One observation seems to speak against the rather extensive
heterogeneity implied in the foregoing discussion, namely, the finding
that the rate of each hydroxylation parallels a two-fold time-to-time
variation in the P-450 content of hepatic microsomes. This, of course,
had prompted us to analyze most results in terms of catalytic activity
per nmole P-450 (apparent molecular activity). Only if these
hydroxylations are catalyzed either by one or two forms comprising the
bulk of the constitutive P-450, or by several forms under identical
regulation with the bulk of constitutive P-450, (or that we and others
introduce an unknown methodologic factor that influences recovery of
all cytochromes P-450) could we anticipate all hydroxylase activities
to be equally affected. All of these remain valid possibilities;
recent observations (Singh, Hawke and Neims, unpublished results) with
mice treated with polylC to induce interferon yielded results that bear
on the question. In this circumstance, P-450 content decreased about
50% and all hydroxylase activities were affected nearly in parallel.
It is our opinion that the circannual variation in total testosterone
hydroxylase activity reported by Ford et_ a]_. (26) reflects a related
phenomenon although no reference to P-450 content was made.
In summary, we suppose that several isozymes of P-450 are involved
in the six hydroxylations of testosterone we studied. Each isozyme
probably has more or less discrete substrate (or position) specificity

63
and total hydroxylase activity at each position reflects the
contribution of more than one isozyme. Vie further presume that each
isozyme in a given strain of mice is under more or less discrete
regulation (e.g. by sex), yet capable of parallel changes with the bulk
of constitutive cytochrome P-450. Finally, we have also presented
evidence of polymorphism in several of the isozymes, with respect to
enzyme-protein and/or its regulation. It follows that the strain
differences detected in this study may be indicative of a much greater
degree of genetic polymorphism that would be apparent in studies of the
isolated forms of cytochrome P-450.

CHAPTER THREE
DEVELOPMENT OF SITE-SPECIFIC TESTOSTERONE HYDROXYLASES IN HEPATIC
MICROSOMES FROM AKR/J AND BALB/cJ MICE
Introduction
The cytochrome P-450 monoxygenase system is involved in the
biotransformation of a diverse array of compounds, from drugs and other
xenobiotics to endogenous substrates such as cholesterol, sex steroids,
fatty acids and prostaglandins (79,80). Originally, cytochrome P-450
was thought to be a single, catalytically nonspecific enzyme. There is
now substantial physical, catalytic and immunological evidence that
cytochrome P-450 consists of a family of hemoprotein isozymes each with
different and more or less overlapping catalytic specificities
(3,4,67). Most of this progress in establishing cytochrome P-450
heterogeneity has dealt with the inducible P-450 isozymes. Some recent
reports have focused on forms of cytochrome P-450 from untreated rats
(45; and rabbits (45,53), partly because immunological techniques have
revealed a lack of cross-reactivity between various inducible forms and
the bulk of these constitutive isozymes (10,11,47)
The extent of, and the molecular basis for, heterogeneity within
the constitutive forms of P-450 remain largely unkown because each
isozyme likely occurs in relatively small amounts in hepatic
microsomes. Nonetheless, these forms are of particular interest
because of the possibility that their physiological function(s) and
64

65
regulations may be quite different from those of the inducible
isozymes. The isozymes probably have greater specificity than the
inducible forms toward endogenous substrates such as steroids.
We are studying the constitutive forms of cytochrome P-450 in
untreated animals by examining and comparing the position specific
monohydroxylations of testosterone in isogenic strains of mice. The
use of inbred strains has already provided valuable information on the
genetic regulation, structure and temporal expression of the inducible
isozymes associated with the Ah cluster (63). Identification of
genetic polymorphisms in the various testosterone hydroxylations have
enabled us to explore some of the genetic and hormonal controls
applicable to the constitutive isozymes (73,81). These results in turn
have been useful probes of the multiplicity and specificity of certain
constitutive forms of hepatic cytochromes P-450 active toward
testosterone.
Differences in the developmental patterns of monoxygenase
activities have also provided insight into the heterogeneity and
regulation of P-450. While many groups of different enzymes can be
shown to share similar developmental patterns (82,83), the maturation
of different hepatic monoxygenase activities has been observed to vary
with substrate (84), sex (85), strain (86) and xenobiotic induction
(87). While comparison of the developmental patterns for several
testosterone hydroxylase activities should provide insight into the
heterogeneity of the constitutive forms of cytochrome P-450, comparison
of patterns between two strains of mice may provide even more valuable
information on genetic control of temporal expression. Developmental

66
control of the induction of specific forms of hepatic microsomal
cytochrome P-450 by temporal genes has been proposed in a number of
species (83,89). The existence of temporal genes which modify the
expression of lysosomal and enzymes during development has been
suggested by Paigen and colleagues (82,90).
In this study we describe position specificity in the maturation
of testosterone hydroxyl at ion and report a strain difference only in
the development of 15a-hydroxyl ase activity. The latter finding
suggests the existence of temporal genes in control of the expression
of certain constitutive cytochrome P-450 activities.

67
Materials and Methods
Testosterone [4-l^C] (56.1 yCi/ymol) was purchased from New
England Nuclear, Waltham, Ma. The radiochemical purity was greater
than 98% as established by TLC on silica gel F254 plates with the
solvent system ether/benzene/methanol, 55:44:0.5 (v/v). Testosterone
was obtained from Sigma Chemical Co., St. Louis, Mo., and was
recrystallized twice from acetone/hexane. Reference steroids were
obtained from sources described previously (59). Precoated (0.2 mm)
silica gel 60 F254 TLC plates (E. Merck, Darmstadt, Germany) were
purchased from Scientific Products, Brunswick, N.J., and SB-5 X-ray
film for autoradiography was obtained from Eastman Kodak Co.,
Rochester, N.Y. Liquiscint liquid scintillation mixture was obtained
from National Diagnostics, Somerville, N.J.; Emulgen 911 was purchased
from the Kao-Atlas Co., Tokyo, Japan. NADPH, D-glucose-6-phosphate and
EDTA were purchased from Sigma Chemicals, St. Louis, Mo. All other
materials and reagents were purchased from the Fisher Scientific Co.,
Pittsburg, Pa., and were of the highest quality commercially
available.
Animals. Timed-pregnant AKR/J and BALB/cJ mice were received
from Jackson Laboratories, Bar Harbor, Me., between the 13 and 15th
days of gestation. Pregnancy had been determined by the observation of
a vaginal plug the day after mating and by gross observation or
palpation after the tenth day of gestation (91). After spontaneous
delivery, females and their litters were housed in separate cages on
corncob Sani-cel bedding. The animals were maintained on a 12 hr
light/12 hr dark photoperiod and allowed both food (Purina Rodent Chow)
and water ad 1ibitum. Mice were weaned at 21 days of age and housed

68
4-6 per cage thereafter according to sex. At designated ages at least
3 male and female litter mate pairs were randomly selected from
separate litters for study. The liver of each animal was investigated
individually except where indicated.
Preparation of microsomes. Mice were killed by decapitation
between 8 and 10 a.m., and livers were excised and rinsed immediately.
All preparative steps were performed at 4° C. Liver homogenates from
28, 35, 49 and 77 day-old animals were prepared from minced tissue in
ice-cold 150 mM potassium chloride-250 mM potassium phosphate, pH 7.4,
as described previously. Microsomes were prepared by differential
centrifugation (27). Homogenates from younger mice were prepared by
identical procedures except that 150-170 mg of liver from 2 day-old
animals (3 pooled livers) or 300-350 mg of liver from 10 or 21 day-old
mice (2 pooled livers) were admixed with a fixed volume (5 ml) of
potassium chloride-phosphate buffer. Microsomal pellets were
resuspended in 100 mM phosphate buffer, pH 7.4, to yield a final
suspension containing approximately 15-20 mg microsomal protein per ml.
Protein content of microsomal suspensions was determined by the method
of Lowry et al_. (28).
Enzyme assays. Cytochrome P-450 content was measured by the
method of Omura and Sato (29) with a Beckman 5260 Spectrophotometer
after microsomal suspensions or whole liver homogenates were diluted
five-fold in 100 mM phosphate buffer, pH 7.4, containing 20% glycerol
and 0.2% Emulgen 911. Peak heights were measured in relation to the
steady baseline observed between 480-500 nm. G1ucose-6-phosphatase

69
activity was measured by the method described by Baginski et al_. (92)
using 0.8-1.0 mg microsomal protein per incubation.
Radiometric assay for hydroxytestosterone metabolites. The
rates of hydroxylation of testosterone at the 6a, 6g, 15a, 15g and 16a
positions were determined by the radioassay procedure we described
previously (59). Briefly, 0.4-0.5 mg of microsomal protein were
incubated at 37° C in a shaking water bath with NADPH (1.0 ymol),
MgCl2 (10.0 ymol), potassium phosphate buffer (final concentration,
0.1 M, pH 7.4) and [^C] enriched testosterone (0.131 ymol
including at least 0.5 yCi in 10 yl ethanol) in a final volume of 1.00
ml. Measurement of activities in preparations from 2, 10 and 21 day
old animals required 1.0-1.5 mg of microsomal protein per incubation.
Reactions were initiated by the addition of NADPH and terminated after
10 min by the addition of 5 ml of ice-cold CH2C12- Each sample was
then transferred to a 10 ml screw-top Teflon-lined test tube, shaken
vigorously for 30 min, and centrifuged for 15 min to separate phases.
The organic phase was removed and evaporated to dryness at room
temperature under a stream of nitrogen. Residues were resuspended in
50 yl ethyl acetate, and the radiolabelled metabolites of testosterone
were separated by TLC on plastic-backed silica gel plates developed
three times with ether/benzene/methanol, 55:44:0.5 (v/v).
Statistical analysis. Statistical significance of differences
between sexes was calculated by Student's t-test; P < 0.05 was
considered significant. All values are expressed as mean and standard
error.

70
Results
Postnatal development of hepatic microsomal cytochrome P-450.
As depicted in Figure 9, both male and female AKR/J mice experienced a
substantial increase in cytochrome P-450 concentration in microsomes
between days 1 and 21 of age (0.16 to 0.91 nmol/mg of protein).
Microsomal cytochrome P-450 concentration increased only slightly more
between days 21 and 77. Adult values were 5-6 fold higher than those
at age 2 days regardless of sex.
The recovery of total cytochrome P-450 and another microsomal
enzyme, glucose-6-phosphatase, was measured in preparations from 10 and
77 day old mice of each sex to determine if recovery of microsomes
varied with age. At both ages, 55-62% of homogenate
glucose-6-phosphatase activity was recovered in the microsomal
preparation. The recovery of cytochrome P-450 in the microsomal pellet
was 55-60% and 70-75% of the homogenate content from 10 and 77 day old
animals, respectively. Since the yield of microsomal protein per gram
of liver changed little with postnatal maturation (range at all ages,
20.7-32.7 mg/gram liver), the pattern of P-450 maturation depicted in
Figure 9 would be the same had one expressed cytochrome P-450
concentration in terms of liver weight instead of microsomal protein.
Postnatal development of microsomal testosterone hydroxylase
activities. The developmental patterns of both testosterone 16a- and
15a-hydroxylase activities are illustrated in Figures 10 and 11. In
2-day-old female mice the specific activity of testosterone
16a-hydroxylase (Fig. 10) was about 12% of the adult value. This
activity increased 6 to 7-fold in both male and female mice during the

nmole P-450/mg PROTEIN
71
Figure 9. Cytochrome P-450 content in hepatic microsomes from AKR/J
male and female mice of varying age. Each value represents
the mean of 3 determinations.

SPECIFIC ACTIVITY (
72
Figure 10. Testosterone 16a-hydroxylase activity in hepatic microsomes
from male ( • ) and female ( o ) AKR/J mice of varying age.
Each value represents the mean of 3 determinations.

73
3 weeks after birth. After 21 days of age female 16a-hydroxylase
activity tended to plateau while activity in males continued to
increase such that there was a statistically significant difference
between sexes by day 28. By day 77, a 3-fold male predominance in
16a-testosterone hydroxylase had been attained.
In contrast, during the first 10 days after birth when 16a- and
all other hydroxylase activities were increasing (see below), the
specific activity of testosterone 15a-hydroxylase activity in AKR/J
mice remained unchanged (Figure 11). This 10-day lag in the postnatal
maturation of 15a-hydroxyl ase activity was independent of sex and in
sharp contrast to the 3-fold increase in cytochrome P-450 concentration
that occurred over the same interval. It follows, of course, that
15a-hydroxylase activity per nmole cytochrome P-450 (apparent molecular
activity) actually decreased substantially between days 2 and 10 of
age. Between 10 and 35 days of age, 15a-hydroxylase activity increased
ten-fold, an interval during which cytochrome P-450 concentration only
doubled. Male 15a-hydroxylase activity remained relatively constant
after 35 days of age but activity in females increased steadily such
that a 3-fold female predominance was manifest at 77 days of age.
We have already reported that testosterone 6a-, 60-, 150-, and
especially, 15a- and 16a-hydroxylase activities exhibited sexual
dimorphism in adult AKR/J mice (59). In the present study, no
significant gender differences were observed before day 28, and most
were not apparent until after the 35th day of age. The developmental
curves for 63-, 6a-, and 150-hydroxylase activities in male and female
AKR/J liver are presented in Figures 12 and 13, respectively. In order

SPECIFIC ACTIVITY (
74
Figure 11. Testosterone 15a-hydroxylase activity in hepatic microsomes
from male ( • ) and female ( o ) AKR/J mice of varying age.
Each value represents the mean of 3 determinations.

RELATIVE SPECIFIC ACTIVITY
75
AGE (days)
Figure 12. Testosterone 6a-( □ ), 53-( ° ), and 153-( A ) hydroxylase
activities in hepatic microsomes from male AKR/J mice of
varying ages. Cytochrome P-450 content (nmoles per mg
microsomal protein) is indicated (....) for comparison.
Specific activities were scaled to converge at age 35 days
for the purpose of prepubertal and postpubertal
comparisons. The relative specific activity of 1.0 at age
35 days is equivalent to 0.387, 3.654 and 0.184 nmoles
product per min per mg microsomal for 6a-, 63- and 153-
hydroxylases, respectively. Each value represents the mean
of 3 determinations.

RELATIVE SPECIFIC ACTIVITY
76
Figure 13. Testosterone 6ct-( â–¡ ), 6f$-( o ), and 15p-( a ) hydroxylase
activities in hepatic microsomes from female AKR/J mice of
varying ages. Cytochrome P-450 content (nmoles per mg
microsomal protein) is indicated (....) for comparison.
Specific activities were scaled to converge at age 35 days
for the purpose of prepubertal and postpubertal
comparisons. The relative specific activity of 1.0 at age
35 days is equivalent to 0.445, 3.930 and 0.223 nmoles
product per min per mg microsomal for 6a-, 63- and 153-
hydroxylases, respectively. Each value represents the mean
of 3 determinations.

77
to distinguish relative rates of maturation before and after puberty,
the scales for each hydroxylase activity have been set such that all
curves converge to a common point at age 35 days. Results for total
cytochrome P-450 are included for clarity of comparison. Male and
female 6a-, 63- and 153-hydroxyl ase activities gradually increased
ten-fold between days 2 and 35 of age, in parallel to the five-fold
increase seen in cytochrome P-450 concentration. After puberty, 6a-
and 153-hydroxylase activities in female preparations increased to
values greater than the correspondíng levels in male microsomes.
Postnatal maturation of testosterone hydroxylase activities in
BALB/cJ mice. We have previously reported that, unlike the situation
in AKR/J mice, no female predominance in any testosterone hydroxylase
activity can be detected in the BALB/cJ strain (81). Therefore, it was
interesting to compare the development of hydroxylase activities in
female BALB/cJ mice with the AKR/J. Cytochrome P-450 concentration in
microsomal preparations from BALB/cJ mice was approximately 2-fold
higher than in AKR/J on days 2 and 10 of age (Table 9). To facilitate
comparison, hydroxylase activities are presented in Table 9 in terms of
apparent molecular activities or hydroxylase activity per nmole of
cytochrome P-450. The specific activity for any hydroxylation can be
computed by multiplying apparent molecular activity by age-specific
P-450 concentration which is also listed in Table 9 for convenience.
Developmental curves for 6a-, 63-, 163- and 16a-hydroxylase activity in
BALB/cJ were similar to those seen in the AKR/J strain. The striking
similarity in rates of maturation of these activities between the two
strains between age 2 and 10 days is revealed in Table 10, which

78
presents results as ratios of apparent molecular activities at the two
ages in each strain. These ratios of apparent molecular activities
between day 10 and day 2 should approximate 1 if cytochrome P-450 and
hydroxylase activity mature in parallel. 16a-Hydroxylase activities
increased 1.6 times faster than P-450 in both strains, whereas other
hydroxylases increase at rates within 30% of that of the increase in
P-450. The maturation of 15a-hydroxylase activity in the two strains
is quite different. As noted above, in AKR/J mice there was no
increase in 15a-hydroxylase activity between days 2 and 10; indeed,
specific activity decreased by 9% during this interval. This of course
is consistent with the low (0.33) ratio of apparent molecular
activities at days 10 and 2. During the same age interval, the
specific activity of 15a-hydroxylase in the BALB/cJ increased by 84%,
nearly in parallel with the increase in P-450.
In BALB/cJ females, which do not exhibit the adult female
predominance in most hydroxylase activities that is characteristic of
AKR/J mice, apparent molecular activities decreased between days 10 and
77 in all mice as P-450 content continued to increase to adult levels
(Table 9).

79
TABLE 9
DEVELOPMENT OF HYDROXYLASE ACTIVITIES IN TWO MOUSE STRAINS IN
RELATION TO THE DEVELOPMENT OF CYTOCHROME P-450
Position of Molecular activities or P-450 content3
Hydroxyl at ion
AKR/J
BALB/cJ
DAY 2
DAY 10
DAY 77
DAY 2
DAY 10
DAY 77
15a
1.57
0.52
6.29
1.08
0.85
1.80
16a
1.76
2.85
2.08
2.78
4.53
3.32
150
1.27
1.10
2.71
1.69
1.29
1.11
6a
2.17
2.60
5.31
1.92
2.47
2.37
60
25.70
27.60
37.22
23.51
24.17
23.90
Cytochrome P-450
0.16
0.44
1.15
0.33
0.77
1.10
aHydroxylase activities are expressed as nmoles product formed per 10
min per nmole cytochrome P-450. Cytochrome P-450 contents are expressed
in terms of nmoles P-450 per mg microsomal protein. All values represent
the mean of 3 determinations. All standard deviations were 10 to 20
percent of respective means.

80
TABLE 10
MATURATION OF EACH HYDROXYLASE MOLECULAR ACTIVITY
BETWEEN DAYS 2 and 10 OF AGE IN AKR/J AND
BALB/cJ MICE
Ratio of Molecular Activities2
Position of Hydroxylation Day 10/Da,y 2
AKR/J BALB/cJ
15a
0.33
0.83
16a
1.62
1.63
153
0.87
0.76
6a
1.20
1.29
63
1.07
1.03
aThe ratios of hydroxylase activities (expressed per
nmole P-450; see Table 9) at ages 10 and 2 days have
been computed for 5 activities.

81
Discussion
Independent patterns of maturation of the 7a, 60 and 16a
hydroxylation of testosterone by rat liver microsomes were first
observed by Jacobson and Kuntzman (93). Recently, differences in the
developmental pattern of several monoxygenase activities in untreated
C57BL/6N male mice have been submitted as evidence for the involvement
of distinct isozymes in the various activities (84). We have
previously studied the effects of sex and strain on the site-specific
hydroxylations of testosterone in inbred mice (81) and now extend the
experiments to include development to gain further insight into the
heterogeneity and regulation of the P-450 isozymes.
Our results provide developmental evidence for at least three
functional forms of cytochrome P-450 involved in the specific
hydroxylations of testosterone, namely, one involved in 6a, 6$ and 15g
hydroxylation, another in 16a hydroxylation, and a third in 15a
hydroxylation. The observation that 6a, 60 and 150 hydroxylase
activities cannot be distinguished developmentally suggests either
coordinate development of distinct P-450 isozymes or involvement of a
single form of cytochrome P-450 with broad specificity toward the 6a,
63 and 15g and perhaps other positions of testosterone. The fact that
all three activities show female predominance in adult AKR/J but not
8ALB/cJ mice is consistent with both explanations. The concept of
coordinate development of three different enzymes has already been
established in the case Of lysosomal acid hydroxylases in mice (82,90),
and could well apply to 6a, 60 and 150 hydroxylase activities since

82
recent biochemical and strain studies suggest some independence between
these activities (59,81).
The existence of a distinct P-450 isozyme with activity
especially for 16a-hydroxylation of testosterone is suggested by the
more rapid development of this activity relative to the postnatal
increase in cytochrome P-450 content and the other hydroxylase
activities, and by the fact that testosterone 16a hydroxylation is the
only activity to experience a three-fold postpubertal increase in male
mice only. Both characteristics apply to both AKR/J and BALB/cJ
animals. In contrast, 16a-hydroxylase activity exhibits little or no
increase until after the fourth week of age in rats (69). In these
animals 16a-hydroxylase activity is androgen-dependent in rats and
directed toward several steroid substrates (20,48,77). The distinctive
postnatal and postpubertal patterns of 16a hydroxylation may well
reflect regulation of two different isozymes bearing 16a-hydroxylase
activity. We have already presented evidence for multiplicity in mouse
16a-hydroxyl ase (59); moreover, in the rat phenobarbital-induced and
androgen-imprinted 16a-hydroxylases have been solubilized and resolved
(22,38,47).
The development of testosterone 15a-hydroxylase activity also
reveals postnatal and postpubertal patterns of maturation that are
easily distinguished from those of P-450 and other hydroxylases, but in
this case the distinctions are strain-specific. In AKR/0 mice
15a-hydroxylase activity did not increase during the first ten days
after birth, and there was a three-fold increase in only female
activity after puberty. Neither situation applies to development of

83
15a-hydroxylase activity in BALB/cJ mice. As with 16a-hydroxylase
activity, we may be dealing with more than one 15a-hydroxylase and
strain-specificity in their regulation. The threefold rise in
testosterone 15a-hydroxylase activity in female AKR/J mice after
puberty may signify the emergence of a sex-dependent female form of
cytochrome P-450 which mainly catalyzes 15a hydroxylation with some
activity toward the 6a, 63 and 153 positions of testosterone. Indeed,
Gustafsson and Ingelman-Sundberg have described a 15-hydroxyl ase in
female rat hepatic microsomes active on steroids and steroid sulfate
conjugates (74,75,76), the activity of v/hich has been associated with a
52,000 dal ton protein found only in females (94). One of the most
intriguing questions raised by our results is whether or not this
postpubertal (or sexually dimorphic) difference between strains relates
to the differences among strains in the maturation of 15a-hydroxylase
activity during the first ten days after birth. There is no obvious
reason to link the capacity for expression of an adult female activity
or protein, with the delayed expression of 15a-hydroxylase activity in
young male and female pups; yet both are characteristics of AKR/J but
not BALB/cJ animals.
The difference in rates of maturation of 15a-hydroxylase
activity between days 2 and 10 of age in AKR/J (-9%) and BALB/cJ (+84%)
are perhaps best interpreted in terms of temporal genes. Paigen (95)
has suggested that genetic elements, called temporal genes, determine
the developmental program for individual proteins, and strain
differences have been described (90,96). The responsiveness of
drug-metabolizing enzymes to specific hormones has been shown to change

84
during development (97,98), and there is evidence in mouse, rat, and
rabbit (88,89) for temporal control of the induction of forms of
cytochrome P-450 associated with the Ah locus. It will be interesting
in future experiments to explore the potential relationship between
polymorphisms we have observed in the early neonatal developmental
patterns and sexual dimorphism in the adult.

CHAPTER FOUR
GENETIC REGULATION OF HEPATIC MICROSOMAL TESTOSTERONE 15a- AND
16a-HYDR0XYLASE ACTIVITES IN FEMALE AKR/J AND BALB/cJ MICE
Introduction
The cytochrome P-450 monoxygenase system is involved in the
biotransformation of a diverse array of compounds including drugs,
carcinogens, pesticides, steroids and fatty acids (79,80). The
existence of different isozymes of cytochrome P-450 in mammalian liver
is now well established with the isolation and purification of a number
of forms which vary in subunit molecular weight, immunological and
catalytic properties, and amino acid sequences (3,4) from rat
(43,46,50), rabbit (45,51-53) and mouse (42,67). But the extent of
cytochrome P-450 heterogeneity, the degree of overlap in substrate
specificity between isozymes and the mechanisms by which these isozymes
are independently regulated by physiological and enviromental factors,
are not well understood.
Individual animals and people can be quite heterogeneous with
respect to both environmental exposures and genetic factors known to
influence drug metabolism. Sometimes it is the combination of
environmental and genetic factors that influence monoxygenase
activities. Genetic differences in sensitivity to induction of P-450
isozymes by polycyclic aromatic hydrocarbons has been attributed to
polymorphism in the Ah locus or cluster by Nebert and Jensen (63).
85

86
Indeed, the induction of specific forms of cytochrome P-450 by various
inducers has allowed some progress into the molecular biology of the
monoxygenase system as evidenced by recent nucleic acid hybridization
experiments (5,6). Less is understood about the constitutive forms of
cytochrome P-450, here defined as those isozymes present in untreated
animals. Immunological techniques have already revealed a lack of
structural homology between the major inducible forms and the bulk of
the constitutive P-450 isozymes in hepatic microsomes of untreated
rats (10,11,47). It seems likely that these constitutive isozymes will
differ from the inducible P-450's with regard to function(s) and mode
of biological regulation. Studies into the molecular biology of the
constitutive isozymes would be greatly facilitated by identification of
genetic polymorphisms in the regulation and/or structure of these
forms.
While several examples of genetic polymorphism of drug oxidation
in man have been described (e.g. 99-102), the mechanism underlying
these polymorphisms remains unknown. Preliminary studies suggest that
the impairment in debrisoquine 4-hydroxyl at ion resides at the level of
the microsomal monoxygenase system (103). The recent demonstration of
polymorphism of debrisoquine oxidation in various rat strains has
allowed more detailed biochemical studies (104,105). Vlasuk et_ al_. (9)
have found inter- and intra-strain polymorphism among four molecular
forms of closely related phenobarbital-induced cytochromes P-450 from
rats. Dieter and Johnson (56) have attributed a strain difference in
the 6p hydroxylation of progesterone by highly purified preparations of
P-450 from untreated rabbits to the presence or absence of one of two

37
subforms. Progress has likely been slowed by the happenstance that
oxidative metabolism of any drug represents the cumulative action of
more than a single form of cytochrome P-450. Polymorphism in a single
isozyme might then be masked in the background of other contributing
isozymes except in instances where oxidation is predominantly catalyzed
by an isozyme with great substrate specificity. The coumarin
hydroxylase locus (106) remains the only monoxygenase polymorphic
allele to be mapped in the mouse.
Constitutive forms of P-450 may be more specific in the oxidation
of endogenous substrates (45,46,107) than inducible forms. It follows
that polymorphism associated with alteration in the structure or
regulation of constitutive cytochromes P-450 might be more easily
detected with endogenous substrates than with xenobiotics. Indeed,
strain differences in steroid hydroxylation have been reported for many
species (86,54,55,56). While some patterns of inheritance have been
worked out (57,58), in most instances the exact nature of these
polymorphisms is not yet known.
The study of the regiospecific hydroxylations of testosterone in
isogenic mouse strains promises to provide valuable information on the
genetic and hormonal factors regulating the predominant forms of
cytochrome P-450 in hepatic microsomes from untreated animals. We have
previously reported genetic and sexual polymorphisms in the
testosterone 15a- and 16a-hydroxylase activities of mice (59,81). Our
present studies were undertaken to elucidate the genetic and endocrine
basis of the sex and strain differences in testosterone hydroxylation
at these two positions in inbred mice.

88
Materials and Methods
Material s. Testosterone [4-^C] (56.1 yCi/pmol) was purchased
from New England Nuclear, Waltham, Ma. The radiochemical purity was
greater than 98% as established by TLC on silica gel F254 plates with
the solvent system ether/benzene/methanol, 55:44:0.5 (v/v).
Testosterone was obtained from Sigma Chemical Co., St. Louis, Mo. and
was recrystallized twice from acetone/hexane. Reference steroids were
obtained through sources previously described (59). Precoated (0.2 mm)
silica gel 60 F254 TLC plates (E. Merck, Darmstadt, Germany) were
purchased from Scientific Products, Brunswick, N.J., SB-5 X-ray film
for autoradiography was obtained from the Eastman Kodak Co., Rochester,
N.Y., and Liquiscint liquid scintillation mixture was obtained from
National Diagnostics, Somerville, N.J. Emulgen 911 was purchased from
the Kao-Atlas Co., Tokyo, Japan. NADPH and testosterone propionate
were purchased from Sigma Chemical Co. All other materials and
reagents were purchased from the Fisher Scientific Co., Pittsburgh,
Pa., and were of the highest quality commercially available.
Animals. Male and female AKR/J, BALB/cJ and BALB/cJ x AKR/J FI
hybrid mice (CAKF^/J; 6-8 weeks old, 20-25 g) were purchased from
Jackson Laboratories, Bar Harbor, Maine. F2 and backcross animals were
bred at the University of Florida's Animal Resources Facility. Mice of
the same sex were housed in groups of six or less per cage on corncob
Sani-cel bedding in an animal room with a controlled environment.
Smoking and the use of insecticides were prohibited, and a 12-hour
1ight/12-hour dark cycle was maintained. Animals were fed Purina
Rodent Laboratory Chow and tap water ad 1ibitum. Mice were maintained

89
under these conditions for at least three weeks before study at the age
of 77 days. Orchiectomies, ovariectomies and sham operations were
routinely performed under methoxyflurane anesthesia at the age of 56
days. Adequacy of the operative procedures was confirmed by inspection
at the time of death. Where indicated, intact, sham-operated or
gonadectomized mice received daily subcutaneous injections of 100 yg of
testosterone propionate in 0.2 ml of sesame seed oil for seven days.
Control animals received vehicle only. Mice were killed 24 hours after
the last injection.
Preparation of microsomes. Mice were killed by decapitation
between 8 am and 10 am, and livers were excised immediately and
carefully separated from gall bladders. The livers were then rinsed
and homogenized in 5 ml/gram liver of ice-cold 150 rrM potassium
chloride - 250 mM potassium phosphate, pH 7.4, with a motor-driven
glass-Teflon homogenizer. Microsomes were prepared by differential
centrifugation (27). Rriefly, the homogenate was centrifuged at 10,000
xg for 15 min, and the resulting supernatant was centrifuged for 1 hr
at 110,000 xg. Microsomal pellets from 1 gram of liver were
resuspended in 2 ml of 100 mM phosphate, pH 7.4, and were used
immediately. Microsomal suspensions contained approximately 8-10 mg
microsomal protein per ml as determined by the method of Lowry et al.
(28). Cytochrome P-450 content was determined by the method of Omura
and Sato (29) with a Beckman 5260 Spectrophotometer after microsomal
suspensions were diluted ten-fold in 100 mM phosphate, pH 7.4,
containing 20% glycerol and 0.2% Emulgen 911. Peak heights at 450 nm
were measured in relation to the steady baseline at 480 to 500 nm.

90
Radiometric assay for determination of testosterone hydroxylase
activities. Testosterone hydroxylase activities at the 6a, 63, 15a,
153 and 16a positions viere determined by a radioassay procedure
described previously (59). Briefly, 0.4-0.5 mg of microsomal protein
viere incubated at 37° C in a shaking water bath with NADPH (1.0 ymol),
MgClo (10.0 ynol), potassium phosphate (final concentration, 0.1 M, pH
7.4) and [-^C]-enriched testosterone (0.131 nmol, 0.5 yCi in 10 yl
ethanol) all in a final volume of 1.00 ml. Reactions were initiated by
the addition of NADPH and terminated 10 min later by the addition of 5
ml of ice-cold methylene chloride. Each sample was then transferred to
a 10 ml Teflon-lined screw-capped test tube, shaken vigorously for 30
min and centrifuged to separate phases. The organic phase was removed
and evaporated to dryness at room temperature under nitrogen. Residues
were resuspended in 50 yl ethyl acetate and the radiolabelled
metabolites of testosterone were separated by TLC on plastic-backed
silica gel plates developed three times in ether/benzene/methanol,
55:44:0.5 (v/v).
Calculation and statistical evaluation of results. Testosterone
hydroxylase activities were expressed as apparent molecular activities
or nmoles of product formed per min per nmole of cytochrome P-450
assuming that 1 nmole [^C] hydroxytestosterone was derived from 1
nmole of [^C] testosterone. This was done to minimize the impact of
variation in cytochrome P-450 content (81). We do not know whether the
relatively small day-to-day variation in P-450 content/ng microsomal
protein is biological or methodologic. Expression of results in terms
of specific activity or product formed per mg microsomal protein

91
increased intrastrain variation a small amount but did not change basic
conclusions. The fluctuation in cytochrome P-450 content is not due to
induction by environmental exposure to polycyclic aromatic hydrocarbons
since the AKR/J strain is non-responsive (64). Statistical analysis of
the data was performed according to Student's t-test; P < 0.05 was
taken as significant. All values represent the mean of five
determinations and are expressed with standard errors.
The number of genes involved in determining quantitative
differences in testosterone hydroxylase activity between the RALB/cJ
and AKR/J mouse strains was estimated by the method of Falconer (108),
where N, the number of independent loci involved, is given by the
following relationship:
_ 1/8 (mean parent 1 - mean parent 2)^
(F2 variance - FI variance)

92
Results
Site-specific hydroxylation of testosterone by hepatic microsomes
from AKfi/J and BALB/cJ mice. Testosterone 6a-, 6b-, 7a-, 15a-, 15s-
and 16a-hydroxylase activities of hepatic microsomes prepared from
untreated male and female AKR/J and BALB/cJ mice are summarized in
Table 11. The various hydroxylase activities of preparations from
AKR/J males differ from those of the BALB/cJ males by less than 30%.
In contrast, only the rate of 7a hydroxylation of testosterone was
similar in microsomes from female mice of the two strains. The rate of
15a hydroxylation was 2-fold higher in microsomes prepared from female
AKR/J mice than in those from female BALB/cJ mice. The opposite held
for 16a hydroxylation, which was 2-fold higher in hepatic microsomes
from female BALB/cJ mice. Microsomal hydroxylase activities at the 6a,
6b and 153 positions of testosterone were 50-62% higher in female AKR/J
than in female BALB/cJ mice.
Comparisons of males and females within each strain revealed a 2-
and 3-fold predominance in the 16a-hydroxylase activity in males of the
3AL3/cJ and AKR/J strains, respectively. In the AKR/J strain female
predominance was observed in hydroxylase activities at the 153, 6a, 63
and especially 15a (3-fold) positions of testosterone; no sexual
dimorphism was exhibited in the BALB/cJ strain with regard to any of
these site-specific hydroxyl ations. Microsomal cytochrome P-450
content did not differ significantly between strains or sexes (in
nmoles P-450 per mg microsomal protein: AKR/J male, 0.71; BALB/cJ
male, 0.75; AKR/J female, 0.63; BALB/cJ female, 0.72). We now consider

TABLE 11
STRAIN AND SEX DIFFERENCES IN TESTOSTERONE HYDROXYLATIONS
Position of
Hydroxyl at ion
Male3
Femal ea
BALB/cJ
AKR/J
BALB/cJ
AKR/J
(nmole product per
10 min per nmole
P-450)
15a
2.42
1.74
2.57
5.21
16a .
6.47
5.10
3.30
1.63
7a
1.60
1.43
1.68
1.41
15B
1.54
1.45
1.60
2.59
6a
3.87
3.56
3.39
5.07
60
25.90
33.30
29.90
43.40
aThe values depicted represents the mean of 5 determinations.
In each case, standard deviation was 10-15% of the mean.

94
in sequence the endocrine and genetic factors controlling expression
of 16a-hydroxylase and 15a-hydroxylase activities.
Effects of gonadectomy and testosterone propionate on
16a-hydroxylase activity. Microsomal protein content and cytochrome
P-450 concentrations were not significantly altered in either male or
female mice following gonadectomy. Postpubertal orchiectomy
significantly decreased 16a-hydroxylase activity in microsomes from
AKR/J males to values near those of female preparations; ovariectomy
had no influence on AKR/J female microsomal 16a-hydroxylase activity
(Figure 14). These results suggest that the higher 16a-hydroxylase
activity of microsomes from males is androgen-dependent. In the
experiment depicted in Figure 15, 16a-hydroxylase activity in
sham-operated/vehicle control microsomes from males was found to be the
same in both AKR/J and BALB/cJ mice. Following orchiectomy BALR/cJ
activity decreased only slightly so that 16a-hydroxylase activity in
castrated BALB/cJ male microsomes was about 2-fold higher than that
found in castrated AKR/J male preparations. Administration of
testosterone propionate to castrated animals restored testosterone
ISa-hydroxylase activity to control values in AKR/J mice while values
somewhat higher than control were obtained in 3ALR/cJ.
The effect of treatment with testosterone propionate on
16a-hydroxylase activity in intact female AKR/J and BALB/cJ mice is
depicted in Figure 16. Although 16a-hydroxylase activity increased
significantly in both strains, the percent increase was less in the
BALB/cJ strain; this decreased the two-fold difference in activities
typical of control AKR/J and BALB/cJ female preparations.

nmoles PRODUCT/nmole P-450
95
c? $ 9 §
TREATMENT GROUP
Figure 14. The effect of postpubertal gonadectony on testosterone
16a-hydroxylase activity of liver microsones from male and
female AKR/J mice. Gonadectomies (*$,§) were performed at
age 56 days, 21 days before study; control animals were
sham-operated. Values represent the mean of 5
determinations with standard error.

nmoles PRODUCT/nmole P-450
96
(5 $ Sf+TP
AKR/J
C? $ tSf+TP
BALB/cJ
TREATMENT GROUP
Figure 15. Comparison of testosterone 16a-hydroxylase activities in
liver microsomes prepared from sham-operated, ochiectomized
(*$), and testosterone propionate-treated (TP)-castrated
males from the AKR/J and RALR/cJ strains. Operations were
performed at age 56 days, 21 days before study. All
animals received either vehicle or TP (100 pg) sc daily for
7 days before study. Values represent the mean of 5
determinations with standard error.

nmoles PRODUCT/nmole P-450
97
9 9+tp
AKR/J
9 9+Tp
BALB/cJ
TREATMENT GROUP
Figure 16. The effect of treatment with testosterone propionate on
testosterone 16ct-hydroxylase activities in liver microsomes
from female AKR/J and BALB/cJ mice. Animals were treated
with vehicle or testosterone propionate (TP, 100 yg) sc
daily for 7 days before study. Values represent means of 5
determinations with standard error.

98
Inheritance of 16g-hydroxylase activity in BALB/cJ and AKR/J
female mice» Basal 16a-hydroxylase activities, namely those in either
untreated female or castrated male BALB/cJ mice, were two-fold greater
than the corresponding values in AKR/J mice. Therefore female AKR/J
and BALB/cJ mice were selected for studies to analyze the genetic basis
of this strain difference. The inheritance pattern of hepatic
microsomal testosterone 16a-hydroxylase activity in untreated BALB/cJ,
AKR/J, CAKFj/J, CAKF2, and backcross female mice is shown in Figure 17.
There was no overlap in hydroxylase activity between the two parental
strains, and the first generation CAKF^/J hybrid mice exhibited
activities that were intermediate with respect to the parental strains.
The distribution of activities in progeny among parent, Fj backcross,
and populations, and the statistical analysis of these results in
Table 18, suggest segregation at a single locus with two alleles
showing additive inheritance. Classification of individuals in the
F2 and backcross generations into discrete phenotypic classes was not
possible because the difference in parental activities was only 8-fold,
and there was overlap between the intermediate hybrid type and both the
lav and high parental types. From the data in Table 18 the number of
genes (N) involved in the C x AK cross was estimated to be 0.79; this
also indicates that only a single gene accounts for the difference in
testosterone 16a-hydroxylase activities in females of each strain. The
underlying principle in the statistical analysis of quantitative traits
is the observation that the greater the number of genetic loci
contributing to a quantitative trait, the less chance there is of

99
TESTOSTERONE ISa-HYDROXYLASE ACTIVITY
(nmoles PRODUCT/min mg PROTEIN)
Figure 17. Inheritance of hepatic microsomal testosterone
16a-hydroxylase activity in female AKR/J and BALB/cJ
mice, and Fj, F2 and backcross offspring. The abscissa
represents 0.02 unit groupings (1 unit equals 1 nmole
product formed per min per nmole cytochrome P-450) of
16a-hydroxylase activity. The ordinate depicts the
number of animals with activity within each grouping.

TABLE 12
STATISTICAL ANALYSIS OF INHERITANCE OF TESTOSTERONE
16a-HYDROXYLASE ACTIVITY IN LIVER MICROSOMES AKR/J
AMD BALB/CJ MICE
Activitya
SDb
Vari anee
Coefficient of
Variation
BALB/cJ
0.339
0.050
0.0025
0.147
AKR/J
0.161
0.021
0.0004
0.130
CAKFi/J
0.274
0.036
0.0013
0.131
cakf2
0.223
0.079
0.0063
0.354
aMean testosterone 16a-hydroxylase activity (nmoles product
formed per min per nmole cytochrome P-450).
^Standard deviation.

101
observing in the population the extreme phenotype of one of the
parental classes. This results in a decrease in the F2 variance
which is then inversely proportional to the number of genes involved in
the expression of the trait.
Inheritance of 15a-hydrox,ylase activity in BALB/cJ and AKR/J
female mice. Figure 18 shows the segregation of testosterone
15a-hydroxylase activity in hepatic microsomes prepared from untreated
BALB/cJ, AKR/J, CAKF^/J, CAKF2 and backcross female mice. A pattern
similar to that seen for 16a-hydroxyl ase activity was observed except
in this case the AKR/J has higher basal values than the BALB/cJ.
15a-Hydroxylase activity in AKR/J was about 2-fold higher than BALB/cJ
parental activity while the mean activity in CAKFi/J hybrid mice was
intermediate. This suggests the absence of dominance. Considerable
overlap between the F]_ hybrids and the AKR/J and BALB/cJ parental
strains made it difficult to sort the backcross and F2 animals into
distinct phenotypic classes. Nonetheless the observed distribution of
phenotypes among parental, Fj, F2 and backcross generations again
suggests segregation at a single locus with additive inheritance. The
number of genes involved in the inheritance of 15a-hydroxyl ase activity
in the C x AK cross was estimated from the data in Table 13 to be 0.43.
The low N value is probably the result of the unusually high variance
observed in the F2 generation in this experiment. The observation
that seven F2 progeny have 15a-hydroxyl ase activities more than two
standard deviations higher than the mean activity of the parental AKR/J
strain suggests that gene interaction or minor modifying genes have
influenced variance in the F2 animals.

102
0.2 0.4 0.6 08 1.0
TESTOSTERONE 15a-HYDROXYLASE ACTIVITY
(nmoles PRODUCT/min mg PROTEIN)
Figure 18. Inheritance of hepatic microsomal testosterone
15a-hydroxylase activity in female AKR/J and BALB/cJ
mice, and F^, and backcross offspring. The abscissa
represents 0.05 unit groupings (1 unit equals 1 nmole
product formed per min per nmole cytochrome P-450) of
15a-hydroxylase activity. The ordinate depicts the
number of animals with activity within each grouping.

TABLE 13
STATISTICAL ANALYSIS OF INHERITANCE OF TESTOSTERONE
15a~HYDR0XYLASE ACTIVITY IN LIVER MICROSOMES AKR/J
AND BALB/cJ MICE
Activitya
SDb
Variance
Coefficient of
Variation
BALB/cJ
0.264
0.061
0.0037
0.168
AKR/J
0.565
0.059
0.0035
0.104
CAKFi/J
0.434
0.058
0.0033
0.134
cakf2
0.539
0.172
0.0297
0.319
aMean testosterone 15ct-hydroxyIase activity (nmoles product
formed per min per nmole cytochrome P-450).
^Standard deviation.

104
Effects of gonadectomy and testosterone propionate on
15ct-hydroxylase activity. The rates of 6a, 63, 7a, 15a and 15b
hydroxylation of testosterone were not affected significantly by
ovariectomy in either AKR/J or BALB/cJ female mice (data not shown).
These activities, therefore, are neither stimulated beyond male values
in the AKR/J strain nor suppressed to male values in the BALB/cJ strain
by circulating levels of ovarian estrogens. However, in males
postpubertal orchiectomy increased several of these activities; the
operation nearly doubled 15a-hydroxylase in AKR/J males, but had no
effect on activity in BALB/cJ (Figure 19). These results suggest a
suppressive effect of androgen on 15a-hydroxylase activity in AKR/J
mice. Since gonadectomy of males caused 15a-hydroxyl ase activity to
approach, but not reach, female values, other factors must also
contribute.
Treatment with testosterone propionate is also more effective in
AKR/J than BALB/cJ females in decreasing 15a-hydroxylase activity.
Figure 20 depicts the effect of testosterone propionate on 15a
hydroxylation in microsomes from intact AKR/J and BALB/cJ female mice.
The 15a-hydroxylase activity in AKR/J microsomes was suppressed to
control male values. Experiments with ovariectomized AKR/J females
yielded similar results. In the BALB/cJ strain a small decrease in
activity v/as observed. These results indicate that 15a-hydroxylase
activity in AKR/J, in comparison with BALB/cJ, is more sensitive to
suppression by testosterone.
Association of the loci controlling 15a- and 16a-hydroxylase
activities. Although the overlap in the CAKF^/J x AKR/J backcross

nmoles PRODUCT/nmoie P-450
105
0.4
<3 $
AKR/J
3 3
BALB/cJ
TREATMENT GROUP
Figure 19. Effect of castration on testosterone 15a~hydroxylase
activities in liver microsornes from male AKR/J and BALB/cJ
mice. Orchiectomies (§) and sham-operations were performed
at 55 days, 21 days before studies. Values represent the
means of 5 determinations with standard errors.

nmoles PRODUCT/nmole P-450
106
0.6-1
9 9+Tp 9 9+tp
AKR/J BALB/cJ
TREATMENT GROUP
Figure 20. The effect of treatment with testosterone propionate on
testosterone 15a-hydroxylase activity in liver microsomes
from female AKR/0 and BALB/cJ mice. Animals were treated
with vehicle or testosterone propionate (TP, 100 yg) sc
daily for 7 days before study. Values represent means of 5
determinations with standard errors.

107
animals made precise phenotypic classification difficult, no females in
this group exhibited the combination of high 16a-hydroxylase and low
15a-hydroxylase activities characteristic of the BALB/cJ strain.
Therefore females were screened for recombinants with respect to these
two activities, i.e. females having the combination of high AKR/J type
15a-hydroxylase activity and intermediate 16a-hydroxylase activity, or
the combination of intermediate 15a-hydroxylase activity and lev/ AKR/J
type 16a-hydroxylase activity. From a total population of 36 backcross
females, approximately 55% exhibited the AKR/J type combination of
activities while only 30% of the mice were recombinants. Hon-linkage
between the two loci would have yielded 25% and 50% of animals in these
groups, respectively. The nonrandom assortment of these two activities
might indicate an association for the two controlling loci although the
small quantitative differences and the indication of modifying genes or
gene interaction preclude any definitive analysis.
With the same reservations, analysis of the CAKF2 population did
provide further support for an association between the two loci.
Figure 21 depicts 15a- versus 16a-hydroxylase activities for each of
the 42 female F2 progeny. A correlation coefficient of 0.46 was
obtained. Although this was statistically significant at the P < 0.01
level, recombinants, e.g. animals 1 and 2 (high 16a- and high
15a-hydroxylase activities) and animal 3 (low 16a- and low
15a-hydroxylase activities) emphasize the independence of these two
catalytic activities.

TESTOSTERONE 16a-HYDROXYLASE ACTIVITY
ins
TESTOSTERONE I5a-HYDROXYLASE ACTIVITY
gure 21, Comparison of hepatic microsomal testosterone 15a- and
16a-hydroxylase activities in individual animals in CAKF2.
Animals 1, 2 and 3 are labelled for reference to text.
Activities are expressed in terms of nmoles product formed
per min per nmole cytochrome P-450.

109
Discussion
Our results indicate that an intriguing set of interactions
between genetic and sexual factors combine to regulate the expression
of testosterone hydroxylase activities in mice. Sex-dependent
differences in steroid and drug metabolism by the hepatic monoxygenase
system have been well characterized in the rat (36,37,69). Higher
rates of metabolism in males are thought to be due to the presence of
testicular androgen (70,109,110). Reports of such sexual dimorphisms
in mice are sparse, and typically report only small sex differences
(ca. 30-69%; 41,111,112). In contradistinction to the rat, a female
predominance is often observed in mice; results are often inconsistent,
and the difference between activities in the sexes is strain-dependent
(40,71,72). In CPB-SE mice, female predominance in hexobarbital
metabolism was attributed to suppression of male activity by testicular
androgens (40,113).
The experiments described here have focused particularly on the
factors that regulate expression of 16a- and 15a-hydroxylase
activities. Testosterone 16a-hydroxylase seems to be regulated by
independent hormonal and genetic factors. In both the AKR/J and
RALB/cJ strains 16a-hydroxylase activity is reversibly induced by
androgens. Furthermore, single genetic locus seems to be responsible
for the strain difference in the basal level of 16a-hydroxylase in
female mice. We suspect that this conclusion would apply also to the
strain difference between castrated males. It is interesting that
treatment with testosterone propionate masks the strain difference in
16a-hydroxylase activity between either female or castrated male AKR/J

no
and RALR/cJ mice. We have earlier reported that differences among
eight isogenic mouse strains in 16a-hydroxylase activity were
restricted to females (81). Perhaps potential differences between
males of the various strains were masked by the action of endogenous
androgens.
The female predominance observed in rates of 6a, 6b, 15a and 15b
hydroxylation of testosterone in the AKR/J strain but not in the
BALR/cJ strain is typical of the strain-dependent sex differences in
hepatic cytochrome P-450-mediated drug metabolism that have been
reported in mice. These results suggest that the isozymes involved in
these steroid hydroxyl ations may be similar, with regard to regulation
or identity, to those involved in the oxidation of certain xenobiotics.
Our results concerning 15a hydroxylation of testosterone have
demonstrated that the strain-specific sexual dimorphism of this
activity depends on an interaction between genetic and endocrine
factors. Studies on the inheritance of 15a-hydroxylase activity in
AKR/J and BALB/cJ female mice suggest the involvement of only one gene
in the control of the level of 15a-hydroxylase in these two strains of
mice. The mechanism underlying this genetic difference in the
regulation of 15a-hydroxylase activity remains unknown, but the AKR/J
strain may be more sensitive to regulation of 15a-hydroxylase activity
by sex steroids. Treatment of females with testosterone propionate
suppresses AKR/J 15a-hydroxylase activity but has little or no action
on RALR/cJ; likewise, castration of males results in an increase in
15a-hydroxylase activity only in the AKR/J strain. Our results
indicate that 16a-hydroxylase activity is also more responsive to

Ill
androgens in AKR/J mice than BALB/cJ animals, although in this case
androgen induces rather than suppresses activity. Differences in
responsiveness to androgens between strains of mice has already been
observed with regard to enhancement of another P-450-mediated reaction,
ethylmorphine N-demethylase activity (41,114). Several factors can
influence responsiveness to androgens, especially neonatal imprinting.
Many hepatic enzyme activities in the rat have been shown to be
irreversibly imprinted or programed during the neonatal period by
testicular androgen(s) (36,109,115). Frequently, imprinting is
expressed in the adult as altered androgen responsiveness (116-118).
This usually results in increased levels of enzymatic activity in
males. However, negative neonatal imprinting of 153-hydroxylase
activity toward 5a-androstane-3a,173-diol 3,17-disulfate has been
reported in the rat (75,119); perhaps there is analogy between this and
the decreased male 15a-hydroxylase activity reported above. The
relatively small strain-dependent sex differences that have been
reported for cytochrome P-450-mediated drug oxidations in mice have
hampered studies into the mechanism(s) by which these sex differences
occur. The results presented here suggest that comparison of hepatic
steroid hydroxylations in isogenic mouse strains may provide an
advantageous system for study.
The degree of polymorphism we have observed in site-specific
hydroxylations of testosterone by hepatic microsomes from inbred mice,
although appreciable, may be an underestimate due to our experimental
approach. Firstly, our results indicate the polymorphism in
16a-hydroxylation is likely masked in males by an overriding

enhancement of activity caused in ail strains by testicular androgens.
It seems quite possible that similar physiological or environmental
factors are also impacting on other activities. Secondly, each
hydroxylase activity probably represents the catalytic contribution of
more than one cytochrome P-450. Each of these isozymes may be
polymorphic but measurement of the conglomerate effects of all forms at
a particular site of hydroxyl ation might mask changes in one of the
enzymes.
Finally, preliminary genetic analysis of the difference in 16a-
and 15a-hydroxylase activity between the AKR/J and BALB/cJ strains is
consistent with the existence of an association between the two genetic
loci controlling the levels of 15a- and basal 16a-hydroxylase
activities, respectively. The nature of this association is unknown,
although linkage of these two genes on the same chromosome is a
possible explanation. Studies of recombinant inbred mouse strains
derived from inbreeding the generation of the BALB/cJ x AKR/J
cross should resolve this question.

CHAPTER FIVE
ENDOCRINE FACTORS IN THE REGULATION OF HEPATIC MICROSOMAL TESTOSTERONE
15a- AND 16a-HYDR0XYLASE ACTIVITIES IN AKR/J and BALB/cJ MICE
Introduction
Much of the recent progress in establishing heterogeneity in the
cytochromes P-450 has focused on the isolation and purification of
inducible P-450 isozymes from the liver microsomes of rat (43,50) and
rabbit (51,52). The extent of this multiplicity, as well as the
physical, chemical, and biological basis for constitutive P-450
heterogeneity in untreated animals, remains largely unknown. Only
recently have some of these efforts been applied to the constitutive
forms of cytochrome P-450 in untreated rats (46) and rabbits (45,53).
Some evidence suggests that the endogenous forms may be more
site-specific and regioselective than inducible forms in the oxidation
of endogenous substrates. It is likely that some constitutive forms of
P-450 may also differ in their physiological function and regulation
from the inducible isozymes. The forms of cytochrome P-450 in
untreated animals, while important in the biotransformation of
xenobiotics in the constitutive state, may also be involved in
biosynthetic and catabolic processes essential to maintaining internal
homeostasis. Enzymes serving such roles are often stringently
controlled.
Sex-dependent differences in steroid and drug metabolism by the
hepatic microsomal monoxygenase system have been well characterized in
113

114
the rat (35,37,69,70) and some progress has been made in the
distinction and resolution of male and female forms of cytochrome P-450
(120,121). In the rat, several hepatic enzyme activities have been
shown to be irreversibly imprinted during the neonatal period by
testicular androgen(s) (36,109,115). Reports of sex differences in
mice have been rare and inconsistent, perhaps reflecting the relatively
small differences and strain-dependency associated with this phenomenon
iri mice (40,71,72). Recent studies have suggested that the higher
activity in females, which is characteristic of many monoxygenase
activities in mice, may be due to a suppression of activity in males by
testicular androgens (40,113). The relatively small sex differences
that have been reported for cytochrome P-450-dependent drug oxidations
in mice have hampered studies into the mechanism(s) by which these
strain-dependent sex differences occur.
He are studying the constitutive forms of cytochrome P-450 by
examining the site-specific monohydroxylations of testosterone in
isogenic strains of mice. We found previously (81) the rate of
16a-hydroxylat ion of testosterone to be 2 to 3-fold higher in males of
all eight isogenic strains of mice. In contrast, the rate of
15a-hydroxyl ation was found to be 2 to 3-fold higher in female mice of
certain strains only. Other less prominent strain-dependent sex
differences were also observed for other sites of hydroxylation. These
results suggested that independent P-450 isozymes were involved in
these hydroxylations and that they could be regulated independently.
Later results emphasized important interactions between a very few
genes and endocrine factors in determination of these strain and sex

115
differences. For example, treatment of castrated males or females with
testosterone propionate increased 16a-hydroxylase activity in all cases
but the magnitude of the response did vary with strain (73).
Curiously, adult castration of either AKR/J male or female mice did
not eliminate the sexual dimorphism in testosterone 15a-hydroxylase
activity, although treatment of either intact or ovariectomized female
AKR/J mice with testosterone propionate completely suppressed the high
15a-hydroxylase activity to values typical of intact males (73). These
results suggested that regulation of 15a-hydroxylase in mice was like
that described by Gustafsson and Ingelman-Sundberg (74,75,76) for the
15-hydroxylase of female rats that acts on 5a-androstane-3a, 170-diol
3,17-disulfate. An interesting observation of relevance to our results
was that the negative neonatal imprinting of the 150-hydroxylase system
in male rats could be partly overcome by the administration of
pharmacological amounts of estradiol benzoate to postpubertally
castrated male rats (75).
In this chapter, we extend our investigation of the endocrine
regulation of testosterone 15a- and 16a-hydroxylase activity in hepatic
microsomes of mice. Advantage is taken of the strain differences in
sexual dimorphism, but interpretations are more or less complicated by
the probability that more than one form of P-450 contributes to
hydroxylation at each site. Experiments concentrate on the timing of
castration, the duration of castration, and treatment with sex steroids
in the AKR/J and BALB/cJ mice. The results, of course, promise to
contribute to our appreciation of the heterogeneity of constitutive

116
/
P-4501s, as well as to probe the complex mechanisms involved in the
expression of sexual dimorphism in the liver.

117
Materials and Methods
Materials. Testosterone [4-^C] (56.1 Ci/mol) v/as purchased from
New England Nuclear, Waltham, Ma. The radiochemical purity was greater
than 98% as established by TLC on silica gel F 254Plates with the
solvent system ether/benzene/methanol, 55:44:0.5 (v/v). Testosterone
was obtained from Sigma Chemical Co., St. Louis, Mo., and was
recrystallized twice from acetone/hexane. Reference steroid standards
were obtained through sources previously described (59).
Precoated (0.2 mm) silica gel 60 F254 TLC plates (E. Merck,
Darmstadt, Germany) were purchased from Scientific Products, Brunswick,
N.J. SB-5 X-ray film for autoradiography was obtained from the E. Kodak
Co., Rochester, N.Y., and Liquiscint liquid scintillation mixture was
obtained from National Diagnostics, Somerville, N.J. Emulgen 911 was
purchased from the Kao-Atlas Co., Tokyo, Japan.
NADPH, testosterone propionate and estradiol benzoate were
purchased from Sigma Chemicals, St. Louis, Mo. All other materials and
reagents viere purchased from the Fisher Scientific Co., Pittsburgh,
Pa., and were of the highest quality commercially available.
Animals. Male and female AKR/J and BALB/cJ mice (6-8 weeks old,
20-25 g) were purchased from Jackson Laboratories, Bar Harbor, Maine.
Mice of the same sex were housed in groups of six or less per cage on
corncob Sani-cel bedding in an animal room with a controlled
environment. Smoking and the use of insecticides were prohibited, and
a 12-hour 1ight/12-hour dark cycle was maintained. Animals were fed
Purina Rodent Laboratory Chow and tap water ad 1ibiturn. Mice were kept
under these conditions for at least three weeks before use.

118
In one series of experiments male and female AKR/J mice were
castrated or sham-operated at 54 or 56 days of age under methoxyflurane
anesthesia. All mice were 77 days of age at the time of study.
Adequacy of surgery was assessed by inspection at autopsy.
In a second series of experiments intact male and female AKR/J and
BALB/cJ mice received subcutaneous injections of 100 ug of testosterone
propionate and/or 100 ug of estradiol benzoate in 0.1 ml of sesame seed
oil daily for seven days before study at age 77 days. Control animals
received vehicle only. Mice were killed 24 hours after the last
injection.
In a third series of experiments male AKR/J mice were castrated at
1, 4, 10, 21 or 54 days of age. Up to 10 days of age, animals were
castrated under hypothermia. For this series of experiments ten AKR/J
time-pregnant female mice were obtained from Jackson Laboratories, Bar
Harbor, Me., between the 13th and 15th day of pregnancy. Pregnancy had
been ascertained by the observation of a vaginal plug the day after
mating and gross observation or palpation after the tenth day of
gestation. Females and their litters viere housed in separate cages on
corn cob Sani-cel bedding, were maintained on a 12 hr light/12 hr dark
photoperiod and were allowed both food (Purina Rodent Chow) and water
ad 1ibitum. The pups were weaned at 21 days of age and then housed 4-6
per cage according to sex. Each experimental age group consisted of
five males selected randomly from among the ten litters. All castrated
male animals, randomly selected female control mice and day 54
s'nam-operated male mice were studied at 77 days of age.

119
The last type of experiment involved of two groups of male AKR/J
mice, one castrated at 54 and the other at 106 days of age, and female
AKR/J mice ovariectomized at day 54. All mice in this series of
experiments were subsequently killed at 130 days of age along with day
54 sham-operated controls.
No discernible differences in hydroxylase activity were observed
between sham-operated, untreated or sesame seed oil treated control
mice of either sex or strain.
Preparation of microsomes. Mice were killed by decapitation
between 8 and 10 am, livers were excised immediately, and gall bladders
were carefully removed. The livers were then rinsed and homogenized
with 5 ml/gram liver of ice-cold 150 mM potassium chloride - 250 mM
potassium phosphate buffer, pH 7.4, using a motor-driven glass-Teflon
homogenizer. Microsomes were prepared by differential centrifugation
(27). Microsomal pellets from 1 gram of liver were resuspended in 2 ml
of 100 mM phosphate buffer, pH 7.4, and were used immediately for
assay. Microsomal suspensions contained approximately 8-10 mg
microsomal protein per ml as determined by the method of Lowry et al.
(28). Cytochrome P-450 content was determined by the method of Omura
and Sato (29) with a Beckman 5260 Spectrophotometer after microsomal
suspensions were diluted ten-fold in 100 mM phosphate buffer, pH 7.4,
containing 20% glycerol and 0.2% Emu!gen 911. Peak heights at 450 nm
were measured in relation to the steady baseline occurring between 480
and 500 nm.
Radiometric assay for determination of testosterone hydroxylase
activities. The amount of testosterone hydroxylase activity at the 6a,

120
63, 15a, 153 and 16a positions was determined by the radioassay
procedure described previously (59). Briefly, 0.4-0.5 mg of microsomal
protein were incubated at 37° C in a shaking water bath with NADPH (1.0
nmol), MgCl2 (10.0 pmol), potassium phosphate buffer (final
concentration, 0.1 M, pH 7.4) and [^C] enriched testosterone (0.131
pmol containing 0.5 pCi in 10 pi ethanol) in a final volume of 1.00
ml. Reactions were initiated by the addition of NADPH and terminated
10 min later by the addition of 5 ml of ice-cold C^C^* Each sample
was then transferred to a 10 ml Teflon-lined screw-capped test tube,
shaken vigorously for 30 min and centrifuged for 15 min to separate
phases. The organic phase was removed and evaporated to dryness at
room temperature under nitrogen.
Sample residues were resuspended in 50 pi ethyl acetate and the
radio!abelled metabolites of testosterone were separated by TLC on
plastic-backed silica gel plates developed three times in
ether/benzene/methanol, 55:44:0.5 (v/v).
Calculation and statistical evaluation of results. The amount of
the various hydroxytestosterone metabolites was expressed as the
percentage of counts in a zone relative to the total counts applied to
the TLC plate. Testosterone hydroxylase activities are expressed as
nmoles of product formed per min per nmole of cytochrome P-450 assuming
that 1 nmole [^C] hydroxytestosterone is derived from 1 nmole of [^C]
testosterone substrate. Results were expressed in this manner to
minimize the impact of day-to-day variation in cytochrome P-450
content. Expression of results in terms of microsomal protein usually
increased intrastrain variation, but did not change any conclusions.

121
The fluctuation in cytochrome P-450 content is not due to induction by
environmental exposure to polycyclic aromatic hydrocarbons as the AKR/J
strain is a non-responsive mouse strain (64), and direct studies in
this laboratory with known inducers and responsive mice support this
conclusion. Statistical analysis of the results was performed with
Student's t-test or Duncan's multiple range test; P < 0.05 was taken as
significant, and all values are expressed as means with standard
errors.

122
Results
The effect of castration on sexual dimorphism of testosterone
hydroxyl ation in AKR/J mice. We have described previously (59,81)
sexual dimorphism in site-specific hydroxylation in hepatic microsomes
from AKR/J mice: 16a hydroxylation predominates in males whereas 6a,
63, 15g and especially 15a hydroxylation predominates in females.
There was no sexual dimorphism in 7a hydroxylation or in microsomal
protein content per gram of liver or in cytochrome P-450 content per mg
microsomal protein. Microsomal protein content and cytochrome P-450
concentration were not significantly affected in either male or female
mice following long or short term gonadectomy. As we had found in an
earlier experiment that female predominance in 6a, 63, 15a and 153
hydroxylation was unaffected by postpubertal ovariectomy, but we have
observed a trend toward a small (19%) decrease in 15a-hydroxylase
activity three weeks after castration; all other hydroxylase activities
were unchanged (Table 14).
Changes in testosterone hydroxylase activities were more
substantial following castration of adult male AKR/J mice (Table 15).
Three weeks after postpubertal orchiectomy there was a 58% decrease in
16a-hydroxylase activity. In contrast, 6a- and 15a-hydroxylase
activities increased 168% and 188%, respectively, in castrated males;
the rates of 7a and 153 hydroxylation of testosterone were unaffected
by castration. Treatment of postpubertally-castrated male mice with
testosterone propionate reversed the selective effects of castration on
the rates of 16a, 6a and 15a hydroxylation (Table 15). A comparison of
the results depicted in Tables 14 and 15 reveals that postpubertal

123
TABLE 14
THE EFFECT OF OVARIECTOMY ON SITE-SPECIFIC HYDROXYLATION OF
TESTOSTERONE BY HEPATIC MICROSOMES FROM AKR/J MICE
Hydroxytestosterone
Metabolite
Rate of Hydroxylation
(nmoles/10 min/nmole P-450)
Sham-operateda Ovariectomized3
N = 7
15a
5.00
+
0.44
4.07
+
0.21
16a
1.56
+
0.12
1.54
+
0.16
7a
1.45
+
0.08
1.38
+
0.03
153
2.57
+
0.20
2.11
±
0.09
6a
5.02
+
0.18
4.88
±
0.14
63
39.24
+
2.21
37.91
±
1.74
aAnimals were sham-operated or ovariectomized at age 56
days, 3 weeks before study at age 77 days. Values
represent the mean ± SE. N indicates the number of
animals in each group. No differences between sham-operated
and ovariectomized groups were statistically significant.

TABLE 15
THE EFFECT OF ORCHIECTOMY ANO TESTOSTERONE PROPIONATE ON SITE-SPECIFIC HYDROXYLATION OF
TESTOSTERONE BY HEPATIC MICROSOMES FROM AKR/J MICE
Hydroxytestosterone
Metabolite
Rate of Hydroxyl ationa
(nmoles/10 min/nmole P-450)
Sham-operated
N = 5
LO
X
O II
2:
Cx + Oi1
N = 7
Cx + TP
N = 3
15a
1.55 ± 0.07*
2.92 ± 0.26A
2.94 ± 0.17A
1.87 ± 0.06*
16a
5.89 ± 0.55*
2.46 ± 0.13A
2.90 ± 0.11A
6.18 ± 0.35*
7a
1.63 ± 0.08*A
&
O
•
o
+1
CO
•
rH
1.70 ± 0.06*
2.38 ± 0.07
150
1.46 ± 0.06
2.13 ± 0.16*
1.98 ± 0.10*
1.90 ± 0.08*
6a
3.11 ± 0.16*
5.22 ± 0.12"
5.77 ± 0.09"
4.04 ± 0.13A
60
30.88 ± 1.60
39.53 ± 2.58A
37.71 ± 1.84A
38.51 ± 2.46A
Operations were performed at age 56 days, 3 weeks before study at age 77 days. Where
indicated, castrated animals (Cx) were treated with testosterone propionate (TP), 100
ug sc daily for 7 days before study; controls received oil vehicle. Values represent
means ± SE. N signifies the number of animals in each group. ®, a and a signify
treatment groups for each hydroxylase activity that are indistinguishable by Duncan's
multiple range test with alpha equal to 0.05.

125
castration of AKR/J males only partially feminizes 15a- and
16a-hydroxylase activities. In contrast, 6a-hydroxylase activity is
completely feminized in these animals.
An experiment was conducted to determine if neonatal imprinting
was important in mediating the residual sex differences observed in
15a- and ISa-hydroxylase activities after postpubertal gonadectomy
(Table 16). Although orchiectomy on days 1, 4 and 10 failed to
completely feminize the 15a-hydroxylase activity in the adult (P <
0.01), activity was significantly greater in these mice compared to
those castrated postpubertally (P < 0.01). An interesting finding v/as
that castration on day 21 was as effective as neonatal castration in
increasing the rate of 15a hydroxylation. Unlike 15a-hydroxylase
activity, the high 16a-hydroxylase activity in male AKR/J mice was
completely feminized by castration on day 1, 4, 10 or 21 of age.
The quantitative differences between the feminizing effects of
prepubertal (day 1, 4, 10, 21) and postpubertal castration (day 54) on
the rates of 15a and 16a hydroxylation of testosterone could indicate
that the length of castration may be a more important factor than the
age at which male mice are castrated. As seen in Table 17, eleven
weeks after postpubertal gonadectomy on day 54, the rates of 68 and 7a
hydroxylation were essentially unchanged in both male and female mice.
The increase in male 6a-hydroxylase activity to the female level was
found previously to occur within three weeks of castration (Table 15).
Although three weeks of postpubertal castration failed to completely
feminize the rate of 16a hydroxylation in adult male castrates,
11-weeks of castration did result in lower values that were

126
TABLE 16
THE EFFECT OF AGE AT ORCHIECTOMY OH SITE-SPECIFIC HYDROXYLATION OF
TESTOSTERONE BY HEPATIC MICROSOMES FROM ADULT AKR/J MICE
Age (days) at
(N)
Rate of Hydroxyl ation3
(nmoles/10 min/nmole P-450)
Orchiectomy3
15a Hydroxyl at ion
16a Hydroxylation
i
J.
(4)
4.80
± 0.32*
2.03
± 0.27®
4
(4)
5.06
± 0.42*a
1.76
± 0.19®
10
(5)
4.53
± 0.25®A
2.04
± 0.17®
21
(4)
4.49
± 0.49®A
1.68
± 0.10®
54
(3)
3.53
± 0.17A
2.82
± 0.42a
Control male
(5)
1.72
± 0.15
6.83
± 0.43A
Control female
(10)
5.92
± 0.28"
1.68
± 0.09°
aAnimals were castrated at the indicated age; all were studied at
age 77 days. (N) indicates the number of animals per group. Values
represent means ± SE. •, a , a signify treatment groups that are
indistinguishable by Duncan's multiple range test with alpha equals
0.05.

TABLE 17
THE EFFECT OF LONG-TERM CASTRATION ON TESTOSTERONE HYDROXYLATION BY HEPATIC
MICROSOMES FROM AKR/J
MICE
Rate of Hydroxyl ationa
Hydroxytestosterone
(nmoles/10 min/nmole P-450)
Metabolite
Male
Female
Sham-operated
Castrated
Sham-operated
Castrated
(N = 5)
(N = 5)
(N = 6)
(N
= 6)
15a
1.40 ± 0.08
2.96 ± 0.14
5.61 ± 0.33
3.54
± 0.20
16a
4.32 ± 0.15
1.95 ± 0.14
1.66 ± 0.09
1.71
± 0.07
7a
1.37 ± 0.04
1.16 ± 0.08
1.45 ± 0.06
1.31
± 0.06
153
1.81 ± 0.15
2.14 ± 0.09
2.43 ± 0.09
1.29
± 0.08
6a
4.40 ± 0.21
5.59 ± 0.24
5.10 ± 0.14
5.43
± 0.10
63
36.16 ± 2.21
36.32 ± 0.99
40.77 ± 3.66
40.87
± 1.60
aAll animals were sham-operated or castrated at age 54 days, eleven weeks before study
at age 130 days. N indicates the number of animals studied. Values represent means ±
SE.

128
indistinguishable from those in females. In contrast, prepubertal
castration was still more effective than long-term postpubertal
castration in increasing 15a-hydroxylase activity in males toward the
female value. The 2-fold increase in 15a-hydroxylase activity in males
after 11-weeks of castration was the same as that seen within the first
3-weeks of postpubertal orchiectomy. What surprised us was a highly
significant decrease in 15a-hydroxylase activity in females 11-weeks
after ovariectomy, an effect that nearly eliminated the difference
between male and female castrates. Except for 15p-hydroxylase, other
activities in the female remained unchanged and emphasize the
specificity of this effect.
Effects of testosterone propionate and estradiol administration on
testosterone 15g- and 16a-hydroxylase activities in sexually-intact
mice. Treatment of adult male AKR/J mice with 100 yg of estradiol
benzoate or estradiol benzoate combined with testosterone propionate
(100 yg) daily for 7 days resulted in a 2-fold increase in
15a-hydroxylase activity (Figure 22). As noted above, postpubertal
castration of males also elicited a 2-fold increase in 15a-hydroxylase
activity. Testosterone propionate (100 yg) was administered with
estradiol to determine if the increase in activity by estradiol
reflected only an indirect action of estrogen by hypothalamo-pituitary
feedback on the production of testicular androgens. This dose of
testosterone did maintain the low male level of 15a-hydroxylase
activity in castrates and had no effect on activity in intact males.
Administration of estradiol benzoate to intact female AKR/J mice
had no effect on the level of 15a-hydroxyl ase activity. Conversely,
treatment with testosterone propionate completely suppressed the high

nmoles PRODUCT/nmole P-450
129
0.4-,
C? C?+EB Cf+TP Cf+EB+TP
TREATMENT GROUP
Figure 22. The effect of treatment of intact AKR/J males with
estradiol benzoate, testosterone propionate and the
combined hormones on hepatic microsomal testosterone
15a-hydroxyl ase activity. Animals received 100 yg of
either estradiol benzoate (EB), testosterone propionate
(TP) or both sc daily for 7 days before study at age 77
days. Controls received injections of oil vehicle.
Values represent the mean of 4 determinations with SE.

130
15a-hydroxylasa activity typical of females to values like those of
control males (Figure 23). When the same doses of estradiol and
testosterone v/ere administered together, 15a-hydroxylase activity in
females was only partially suppressed. The observation that virtually
the same level of 15a-hydroxylase activity was attained in
postpubertally castrated males, intact males treated with estradiol
benzoate, or intact male and female mice given the combination of
estradiol and testosterone is interesting and may indicate an
anti-androgenic action of estrogen on the androgen mediated suppression
of 15a-hydroyxlase activity.
We have previously reported that 16a-testosterone hydroyxlase
activity in male mice was reversibly induced by androgen and that the
activity can be induced in female mice treated with testosterone
propionate. As seen in Figures 24 and 25, the anti-androgenic action
of pharmacological doses of estrogen was also apparent with this
androgen-dependent activity. Administration of estradiol benzoate
alone or in conjunction with testosterone propionate to intact male
mice caused a significant decrease in the rate of 16a hydroxylation
(Figure 24). Likewise, the induction of 16a-hydroxylase activity in
females by testosterone propionate could be prevented by the
co-administration of estradiol benzoate (Figure 25).
The specific effect of ovariectomy on 15a-hydroxylase activity in
AKR/J mice suggested a female hormonal influence on the level of this
activity in hepatic microsomes. Nonetheless, induction of
15a-hydroxylase activity in intact males with pharmacological doses of
estradiol benzoate was not sufficient to achieve values typical of

nmoles PRODUCT/nmoie P-450
131
9 9+EB 9+TP 9+EB+TP
TREATMENT GROUP
Figure 23. The effect of treatment of intact AKR/J females with
estradiol benzoate, testosterone propionate and the
combined hormones on hepatic microsomal testosterone
15a-hydroxyl ase activity. Animals received 100 pg of
either estradiol benzoate (EB), testosterone propionate
(TP) or both sc daily for 7 days before study at age 77
days. Controls received injections of oil vehicle.
Values represent the mean of 4 determinations with SE.

nmoies PRODUCT/nmole P-450
132
i
0.6 -j
0.5-
0.4-
0.3-
0.2-
0.1 -
0 â– 
C? Cf+EB Cf+TP CJ+EB+TP
TREATMENT GROUP
Figure 24. The effect of treatment of intact AKR/J males with
estradiol benzoate, testosterone propionate and the
combined hormones on hepatic microsomal testosterone
16a-hydroxylase activity. Animals received 100 yg of
either estradiol benzoate (EB), testosterone propionate
(TP) or both sc daily for 7 days before study at age 77
days. Controls received injections of oil vehicle.
Values represent the mean of 4 determinations with SE.

nmoles PRODUCT/nmole P-450
133
9 Q+EB Q+TP 9+EB+TP
TREATMENT GROUP
Figure 25. The effect of treatment of intact AKR/J females with
estradiol benzoate, testosterone propionate and the
combined hormones on hepatic microsomal testosterone
loct-hydroxylase activity. Animals received 100 yg of
either estradiol benzoate (EB), testosterone propionate
(TP) or both sc daily for 7 days before study at age 77
days. Controls received injections of oil vehicle.
Values represent the mean of 4 determinations with SE.
U Stfv.

nmoies PRODUCT/nmole P-450
134
0.7
0.6
0.5
0.4
0.3
0.2
0. I
0
TREATMENT GROUP
Figure 26. The effect of castration of male AKR/J on response to
treatment with estradiol benzoate with regard to
testosterone 15a-hydroxylase activity of hepatic
microsomes. Males were sham-operated or castrated at age
56 days. At age 70 days both groups were treated with
estradiol benzoate (EB), 100 ug sc daily for 7 days until
study on day 77. Concurrent male and female controls were
included. Each value represents the mean of 4
determinations with SE. All groups were significantly
different (P < 0.02) except intact females and castrated
males treated with EB.

135
females, perhaps because of residual suppressive action of androgen.
However, the same dose of estradiol benzoate did fully "feminize"
15a-hydroxyl ase activity when administered to postpubertal ly castrated
males (Figure 26).
Hormonal effects on 15a-hydroxy1ase activity in BALB/cJ mice. We
have previously reported the lack of a sex difference in the
15a-hydroxylase activity of BALB/cJ mice (81). We have also
demonstrated the apparent lack of an effect of androgen in this strain
in that 15a-hydroxylase activity did not increase after postpubertal
orchiectomy. As seen in Figure 27, administration of estradiol
benzoate (100 yg) to castrated BALB/cJ mice daily for 7 days did induce
15a-hydroxylase activity in males. The results in Figure 27 confirm
our earlier finding that castration itself did not cause an increase in
15a-hydroxylase activity in BALB/cJ males. Lower doses of estradiol
benzoate were less active in the induction.
Since BALB/cJ animals seemed to be unresponsive to the suppressive
effect of androgen on 15a-hydroxylase activity, it was of interest to
see whether intact males or females were as responsive as male
castrates to estradiol benzoate. The results of this experiment are
depicted in Table 18, which summarizes the results of estrogen
treatment on the rates of 6a, 63, 7a, 15a, 153 and 16a hydroxylation of
testosterone per nanomole of cytochrome P-450. Cytochrome P-450
concentrations were not affected by the administration of estradiol
benzoate, 100 yg daily for 7 days, to either castrated or intact
BALB/cJ mice. 15a-Hydroxylase activity was induced more than 2-fold in
both sexes by estradiol benzoate treatment (perhaps more in the male);

SPECIFIC ACTIVITY
(nmoles PRODUCT/mln-mg PROTEIN)
136
d d $+TP d+EB d+EB
(10/i.g) (100/i.g)
TREATMENT GROUP
Figure 27. The effect of castration or treatment with hormones on
hepatic microsomal testosterone 15a-hydroxyl ase activity in
BALB/cJ males. Where indicated, animals were castrated
(*$) at age 56 days, 3 weeks before study at age 77 days.
Castrated animals were treated with oil vehicle,
testosterone propionate (TP) or estradiol benzoate (EB) at
the indicated doses sc daily for 7 days before study.
Values represent means of 4 determinations with SE.

TABLE 18
EFFECT OF TREATMENT WITH ESTRADIOL BENZOATE ON SITE-SPECIFIC HYDROXYLATION OF
TESTOSTERONE BY HEPATIC MICROSOMES FROM INTACT BALB/CJ MICE
Rate of Hydroxylationa
Hydroxytestosterone (nmoles/10 min/nmole P-450)
Metabolite
Males
Females
Oil
EB
Oil
15a
2.42
(0.24)
6.32
(0.70)
2.28
(0.11)
5.19
(0.27)
16a
4.00
(0.23)
3.26
(0.48)
2.94
(0.16)
2.38
(0.30)
7a
1.29
(0.13)
1.93
(0.28)
1.57
(0.09)
2.24
(0.26)
150
1.98
(0.32)
1.90
(0.37)
1.80
(0.14)
1.15
(0.14)
6a
4.60
(0.33)
3.91
(0.28)
4.59
(0.54)
3.92
(0.42)
6e
33.60
(3.64)
27.08
(3.65)
30.45
(0.41)
19.69
(1.38)
aIntact males and females were treated with oil vehicle or estradiol benzoate (EB),
100 ug sc daily, for 7 days before study at age 77 days. There were 3 animals in
each treatment group except EB females, where N = 4. Values are means with SE in
parentheses. * indicates P < 0.05 relative to vehicle controls.

138
7a-hydroxylase activity also increased a small amount in males and
females as well. Tne rates of hydroxylation at the other positions of
testosterone either remained constant or decreased.

139
Discussion
Sex-dependent differences in steroid and drug metabolism by the
hepatic microsomal monoxygenase system have been well characterized in
the rat (36,37,69,70,120); the higher rates of metabolism in males have
been attributed to testicular androgen. We are not aware of any
previously published reports of ovarian regulation of cytochrome P-450
dependent steroid or drug metabolism in the liver. While
pharmacological amounts of estrogen have been shown to inhibit hepatic
drug and steroid metabolism (122-125), no significant effect of
ovariectomy has been demonstrated in studies on the regulation of
either 4-androstene-3,17-dione (36,75) or lidocaine and imipramine
metabolism (126).
Reports of such sexual dimorphisms in mice have been rare and have
usually described only small differences, i.e. 30-60% (41,111,112).
Until recently, all sex differences in mice were shown to be
strain-dependent and generally in the opposite direction to that found
in rats (40,71,72). For instance, in some strains of mice the higher
female rate of hexobarbital metabolism was found to be a result of the
suppression of male activity by testicular androgens (40,113).
Therefore, as in the rat, androgens may mediate many of the sex
differences seen in certain strains of mice, although suppression
rather than induction predominates. The relatively small
strain-specific sex differences that have been reported for cytochrome
P-450 dependent drug oxidations in mice have hampered studies to
elucidate the genetic and endocrine mechanisms by which these sex
differences occur.

140
In the present study, the rate of 7a-hydroxylation of testosterone
was unaffected by either orchiectomy or ovariectomy, and no sex
difference was observed for this hydroxylase activity in AKR/J or
BALB/cJ mice. We did find (81) a slight female predominance in
7a-hydroxylase activity in other strains of mice; this situation may be
analogous to that in the rat where no sex difference is observed in the
rate of 7a hydroxylation of cholesterol in Sprague-Dawley rats (36) but
a slight female predominance was observed in King X-Holtzman (127).
The female predominance we observed in the rates of 6a, 65, 15a and 153
hydroxylation of testosterone in the AKR/J mouse strain (Tables 14 and
15) but not in the BALB/cJ strain (Table 18) typifies the
strain-dependent sex differences that have been reported by others in
murine hepatic cytochrome P-450 dependent drug metabolism. In
contrast, the higher male 16a-hydroxylase activity of both strains
resembles the sex differences usually reported in rats.
Hydroxylase activity at any position on the testosterone molecule
is likely to represent the cumulative action of more than a single form
of cytochrome P-450 due to the overlapping substrate specificity of
these isozymes. Any independence between the various hydroxylase
activities which suggests "functional multiplicity" probably reflects
the independent regulation of a specific isozymic form of cytochrome
P-450 in the circumstance that this form represents the predominant
catalyst for the activity at any single site of hydroxylation. At
least some of the female predominance in the AKR/J strain in 6a, 73 and
153-hydroxylase activities is probably due to a form of cytochrome

141
P-450 that mainly catalyzes the 15a-hydroxylation of testosterone. But
not all of the female predominance in these activities can be
attributed to a single isozyme. For example, postpubertal castration
of male AKR/J mice resulted in significant increases in 6a- and
15a-hydroxylase activities, while 60- and 150-hydroxylase activities
were influenced very little.
Although 16a-hydroxylase activity is reversibly induced by
testicular androgens, several weeks were required after castration
before a complete "feminization" of activity was observed (Table 16).
In other studies, we have found that testoterone-induced levels of
16a-hydroxyl ase activity in intact BALB/cJ female mice persist for more
than two weeks after treatment was stopped (unpublished observation).
Possible explanations for such results include the transcription of a
specific messenger with a long half-life or the persistence or slow
elimination of an active intermediate that participates in the
induction. In summary, complete (6a) and partial (15a) androgen
suppression and complete androgen stimulation (16a) of testosterone
hydroxylase activity can be demonstrated in AKR/J mice.
Regulation of the sexually-dimorphic 15a-hydroxylase activity in
AKR/J is interesting, albeit complex. There is no sex difference in
15a-hydroxylase in the BALB/cJ. In AKR/J, male and female mice exhibit
comparable 15a-hydroxylase activities only after the prepubertal
castration of males and long-term castration of females. Evidence
exists for both an androgen suppression and estrogen induction of
15a-hydroxylase in AKR/J, with direct or physiological interaction
between pharmacological doses of the sex steroids as well.

142
Interestingly, Duffel crt al_. (128) have described both male and female
regulation of dimethylani1ine N-oxidase in mouse liver; this activity
is catalyzed by the flavin-containing monoxygenase system. Briefly,
the female predominance of N-oxidase activity was due to a combination
of both repression by testicular androgens in males and a small but
significant ovarian stimulation in females (128). Although
testosterone was capable of suppressing N-oxidase activity,
progesterone or estrogen alone could not account for the higher
activity of intact female mice, and unidentified ovarian factors were
implicated. The regulation of hepatic dimethylaniline N-oxidase
activity seems to parallel that of testosterone 15a-hydroxyl ase
activity in AKR/J mice. The report of ovarian regulation of hepatic
cytochrome P-450 is unique, and a long period after ovariectomy was
required before this effect became apparent. Ovarian steroid hormones
may not directly mediate these effects; this possibility is further
substantiated by our finding that only pharmacological amounts of
estradiol benzoate were capable of the specific induction of
15a-hydroxylase activity. It is possible that in the absence of any
feedback control on the secretion of pituitary hormones by ovarian
steroid hormones, the secretory patterns of these and other pituitary
factors might change and in turn influence hepatic steroid metabolism.
The use of pharmacological amounts of estradiol benzoate could also
elicit actions that are not strictly estrogenic. The antiandrogenic
effects of high doses of estrogen on either the suppression of
15a-activity or the stimulation of 16a-activity by testosterone
suggests a common binding site for these two hormones. Two classes of

143
estrogen receptors have been characterized in rat liver cytosol, a high
affinity-low capacity receptor system specific for estrogens (129,130)
and a low affinity-high capacity receptor system found predominantly in
male liver cytosol with affinity for both androgens and estrogens
(131,132). Our observation that day-21 castration of males (but not
day-54 orchiectomy) was about as effective as neonatal castration in
eliciting increased adult levels of 15a-hydroxylase activity is
important in this regard since "imprinting" of the high capacity
receptor occurs after the first week, at least in rats (133). It has
been suggested that in vivo high concentrations of estradiol are
required for receptor binding because of the rapid rate of hepatic
estrogen metabolism, but some metabolites of estradiol also seem
capable of high affinity receptor binding (134,135). It is important
that in BALB/cJ mice, which exhibit no androgen suppression of
15a-hydroxyl ase activity, this activity can be specifically induced in
males and females by estradiol. Consequently, the lack of sex
difference in 15a-hydroxylase activity in the BALB/cJ strain is not due
to a genetic inability to produce a "specific" 15a-hydroxylase since
the activity can be evoked specifically by treatment with estradiol
benzoate. It seems most likely to us that the difference between AKR/J
and BALB/cJ with regard to regulation of testosterone 15a-hydroxylase
activity relates to a receptor capable of participating in both
estrogenic induction and androgenic suppression of activity.

GENERAL CONCLUSIONS
This thesis provides evidence for the existence of several
catalytic forms of constitutive cytochrome P-450 involved in the
site-specific monohydroxylations of testosterone by mouse hepatic
microsomes.
15a-HydroxyIase activity is not only distinguishable from the
other hydroxylases by several aspects of its regulation, but also
appears to be different in male and female microsomes. Male steroid
16a-hydroxylase is stimulated in vitro by high potassium phosphate
concentrations, is reversibly inducible by testicular androgen, and
demonstrates activity towards testosterone but not progesterone as
noted by the distinct lack of any male predominance in activity with
the latter substrate. Conversely, female steroid 16a-hydroxylase does
not increase after puberty and is not affected by high concentrations
of potassium phosphate j_n vitro. Presumably male microsomes contain
both types of 16a-hydroxylase since the more rapid postnatal develop¬
ment of this activity relative to postnatal increases in cytochrome
P-450 and the development of the other hydroxylase activities is seen
in microsomes from both males and females. The basal or female level
of activity is likely regulated by a single genetic locus.
7a-Hydroxylase activity is unique in that it remains rather
impervious to most of the effects described throughout these studies.
Indeed 7a-hydroxylase activity, when expressed per nanomole of
144

145
cytochrome P-450, even remains constant during neonatal development.
7a-Hydroxylase is also distinguishable from the other hydroxylases by
its great sensitivity to inhibition by potassium phosphate in vitro in
preparations from males or females.
The 63 and 153 hydroxyl at ions of testosterone may represent the
activity of a less specific hydroxylase which predominates in
microsomes from untreated male and female mice. Features
characteristic for both hydroxyl at ions include 1) high Km's for
testosterone (20 to 40 times greater than the apparent Km values for
tne other hydroxylases; 2) a relative insensitivity to changes in
potassium phosphate concentration; 3) slightly higher activity in
female microsomes; and 4) both hydroxylations undergo coordinate
neonatal development which parallels postnatal increases in cytochrome
P-450 content. 63-Hydroxylase activity accounts for more than half of
all of the testosterone metabolites formed, and it is intriguing to
speculate that both beta-hydroxylations are catalyzed by a low affinity
isozyme present in relatively high concentrations in microsomes from
uncreated mice. This form may also catalyze the biotransformation of a
large variety of xenobiotics by virtue of its broad substrate
specificity.
Finally, steroid 15a-hydroxyl ase may also be different in
microsomes from males and females. Clearly the ten day delay in the
development of this activity in microsomes from males and females
suggests the existence of a separate isozyme that catalyzes the 15a
hydroxylation of testosterone. The postpubertal increase in activity
in female AKR/J mice may signify the emergence of a distinct

146
sex-dependent form of cytochrome P-450 which can be characterized by
even higher rates of catalysis when progesterone is used as a substrate
and which is stimulated in vitro when low potassium phosphate
concentrations are used. This is in contrast to the situation in
microsomes from males where no increase in activity is observed in the
rate of 15a-hydroxylation postpubertally, when progesterone is
substrate, or when low potassium phosphate concentrations are used.
15a-Hydroxylase remains most unique in that it is inducible by
estradiol and suppressed by androgen. Physiologically, sexual
dimorphism in this activity is strain-specific with differences
seemingly reflecting a single genetic locus.
In all probability, hydroxylase activity at each position
represents the cumulative action of a number of isozymes which vary in
their relative abilities to hydroxylate testosterone at any given
position. Any independence between the various hydroxylase activities
that suggests catalytic multiplicity is likely due to the independent
regulation of a relatively specific isozyme of cytochrome P-450 such
that the activity of this form comes to represent a major component of
the site-specific hydroxylase activity. Although the heterogeneity and
regulation of the hepatic microsomal cytochromes P-450 represent
complex problems involving both genetic and endocrine influences, the
studies described in this thesis suggest an even greater amount of
diversity is to be found in studies of the isolated forms of cytochrome
P-450.

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BIOGRAPHICAL SKETCH
April 30, 1951, was hardly an auspicious day, my birth coming as
no big surprise to my mother and causing very little fanfare in
Gainesville, Florida, where my father was finishing his college
education. The next eighteen years of my life were rather forgettable,
except if my promotion to the third grade at Hibiscus Elementary in
Miami, Florida, appears significant. Graduation from Eau Gal lie High
School, Eau Gallie, Florida (1969), was only a momentous event in that
it marked the end of one of the most bizarre and confusing periods in
my life.
My own life began in 1970 when I enlisted into the United States
Navy. After months of being investigated, I was given top secret
security clearance and trained in the art of electronic espionage at
Pensacola, Florida. While stationed in the South Pacific to keep track
of Soviet submarines, scuba diving became a hobby and shell collecting
a full-time passion. In 1974, I met my future wife, Lynne Twiest while
stationed in Hawaii, and later that year I was discharged from the Navy
to start school. A major driving force in my early college studies was
the fear of being forced to re-enlist. In 1976, I obtained my marriage
certificate and two years later a B.S. in chemistry at the University
of West Florida. Lynne played no small part in either instance.
I returned to Gainesville in 1978 and entered graduate school
without knowing what pharmacology and magic bullets were. To my
long-lasting good fortune, Allen Neims had also recently come into the
155

156
Department of Pharmacology as its new chairman. He remains one of the
most influential factors in my life and under his tutelage my research
quickly fell into place.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Allen H. Neims', Chairman
Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
A
—-y
Margaret 0. James
Assistaitt-'Professor of Medicinal
Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Kenneth C. Leibman
Professor of Pharmacology
and Therapeutics

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
SJuahj>(/(
Kathleen T. Shi verick
Associate Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
úr—
David N. Silverman
Professor of Pharmacology
and Therapeutics
This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
August 1983






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