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Drug Metabolism Reviews

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Sex-Related Differences in
Drug Metabolism
a

R. Kato
a

Research Laboratories Fujisawa Pharmaceutical

Co., Ltd., Osaka, 532, Japan
Published online: 08 Jun 2015.

To cite this article: R. Kato (1975) Sex-Related Differences in Drug Metabolism, Drug
Metabolism Reviews, 3:1, 1-32
To link to this article: http://dx.doi.org/10.3109/03602537408993737

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Sex-Related Differences in Drug Metabolism

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R. KATO
Research Laboratories
Fujisawa Pharmaceutical Co., Ltd.
Osaka 532, Japan

I. INTRODUCTION . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . .

11. STUDIES ON THE MECHANISM OF THE SEX

DIFFERENCE IN DRUG ACTION . . . . . . . . . . . . . . . . . . . . . . . . . .

111. SEX-RELATED DIFFERENCES IN MICROSOMAL

ENZYMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. SEX DIFFERENCES IN MICROSOMAL ELECTRON
TRANSPORT SYSTEM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. SEX DIFFERENCES IN SUBSTRATE BINDING WITH P-450. . . . .

6
8
10

VI. SEX DIFFERENCES IN ANIMALS OTHER THAN RATS. . . . . . . . 14

VII. EFFECT O F METHYLCHOLANTHRENE IN MALE AND

FEMALERATS .........................................

15

VIII. SEX DIFFERENCES IN PATHOLOGICAL CONDITIONS . . . . . . . 18

IX. SEX DIFFERENCES IN THE METABOLISM OF

STEROID HORMONES.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

.. . . . . . .. . .. .. . .. . . . .. .
XI. SUMMARY ............................................
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . .. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . .
X. MECHANISM OF SEX DIFFERENCE

26
28
29
29

1
Copyright fr 1974 by Marcel Dekker. !nc. All Rights Reserved. Neither this work nor any part may he
reproduced iir transmitted in any form or hy any means. electronic or mechanical. including photocopying.
microfilnung. and recording, or by any inlt~rniationstorage and retrieval system, without permission in
writing from the publisher.

KATO

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I. INTRODUCTION

It has been demonstrated by numerous investigators that the actions of a variety of drugs are more pronounced and persist longer
in female rats than in male rats, The first such observation was reported in 1932 by Nicholas and Barron [ l ] who found that amobarbital
anesthetized female rats in only half of the dose necessary for males.
Later, extensive studies by Holck e t al. [2] showed that the duration
of anesthesia caused by injecting barbiturates, such as amobarbital
hexobarbital and pentobarbital, is considerably longer in female than
in male rats. On the other hand, they observed no clear sex difference in the duration of anesthesia after the injection of certain other
barbiturates such a s barbital and phenobarbital [2]. In addition,
male and female rats responded similarly to the injection of chloral
hydrate.
Holck et al. [2] also reported that no distinct sex difference was
found for hexobarbital anesthesia in certain other species of animals
(dogs, cats, rabbits, guinea pigs, mice, and frogs). However, the
reasons for these differences were not quite conceivable at that time.
Another very interesting observation was that female rats showed
more pronounced responses to nicotine and strychnine than did
males [2, 81.
11. STUDIES ON THE MECHANISM OF THE SEX DIFFERENCE
IN DRUG ACTION
Holck e t al. [2] observed that: (1) the sex-related difference to
hexobarbital or pentobarbital anesthesia was characteristic of adult
rats and was not observed in immature rats of 3 to 4 weeks of age;
(2) adult female rats demonstrated a longer duration of anesthesia
induced by hexobarbital, amobarbital, o r phenobarbital than male
rats; and (3) castration of adult male rats lengthened the duration
of anesthesia induced by these barbiturates to that approximating
the duration observed in females. For barbital, a barbiturate which
does not demonstrate a sex-dependent response in intact rats, castration was without effect. In species which do not demonstrate a
sex-dependent response to the barbiturates, for example, male
rabbits, castration also was without influence. These results suggested that for the rat, the sex difference in the action of the barbiturates is dependent on the presence of the male sex hormone.
In support of this assumption, Holck et al. [21 demonstrated that the
administration of testosterone to intact and ovariectomized female
rats shortened the duration of anesthesia induced by hexobarbital
or pentobarbital. However, testosterone administration was in-

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SEX-RELATED DIFFERENCES IN DRUG METABOLISM

effective in modifying the response to these barbiturates in female

mica, a species which does not show a sex difference to these barbiturates.
Crevier e t al. [4] and Pellerin et al. [5] pioneered the study of
drug metabolism in relation to sex differences in responses to drugs.
They observed that pentobarbital is metabolized much faster by
liver slices from male rats than by those from female rats. They
also noted that castrating male rats abolished t h i s sex difference in
metabolism, and that the administration of testosterone increased
the metabolism of pentobarbital by liver slices in gonadectomized
rats of both sexes.
Further evidence of a sex-related difference for the liver metabolism of pentobarbital was reported by Brodie and his associates [6,
71. They observed that the plasma level of pentobarbital decreased
more rapidly in male than in female rats. Microsomes isolated from
the livers of the male rats showed considerably higher enzyme activity for hexobarbital oxidation than microsomes from females.
These investigators also demonstrated that the administration of
testosterone to female rats accelerated the disappearance of pentobarbital from the plasma and increased metabolism by the liver
microsomes 161. Conversely, the administration of estradiol to
male rats inhibited the rate of plasma disappearance of pentobarbital and decreased metabolism by liver microsomes. In mice o r
rabbits, where a sex-dependent anesthetic response to pentobarbital is not observed, the administration of testosterone o r estradiol induced no appreciable change, either in the duration of anesthesia o r in the rate of metabolism of pentobarbital. Brodie and his
associates therefore concluded that female rats a r e more susceptible to hexobarbital anesthesia because they a r e unable to metabolize hexobarbital as rapidly as males.
The sex-related difference demonstrated for the liver metabolism
of barbiturates has been observed for other drugs also. Axelrod [8]
found that liver microsomes from male rats N-demethylated some
narcotics, such a s morphine, methadone, and meperidine, more
rapidly than those from female rats.
Kato e t al. [9, 101 reported that the treatment of ovariectomized
rats with 4-chlortestosterone, a synthetic androgen, also increases
the oxidation of carisoprodol and strychnine by liver microsomes
and decreases the duration of paralysis of carisoprodol and the
toxicity of strychnine. In a related study, Booth and Gillette [ll]
demonstrated a clear correlation between the anabolic action and
the increase in drug-metabolizing activity of liver microsomes by
using various androgenic and anabolic hormones.
The sex difference in the toxicity of strychnine in rats was originally reported by Poe et al. [12)i n 1937. The difference was con-

KATO

sidered a t the time to be a sex-related differential in the sensitivity

of the central nervous system to strychnine, because the strychnine
toxicity occurred within 6 min after intraperitoneal injection. However, Kato e t al. [13] subsequently showed that the difference in
toxicity was due to a difference in the rate of metabolism of strychnine by liver microsomes. Male rats oxidized strychnine about 2.5
times faster than female rats; castration abolished the difference in
metabolism and toxicity of strychnine, a s shown in Fig. 1. Thus the
0
FEMALE RATS

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30

G
20
J

51

lo

c)

Y.
200

1 oa

3,
3

2
w

6
200

f
r

Iv)

100

normal

ca#raM

castrated

TcJfostemne

c&&d

&$Aronc

tastpTed
cstradid

FIG.1. Effects of castration and hormone treatments on the toxicity and metabolism
of strychnine in rats (Kato et al. [ lo]).a) LD,, of strychnine. b) Metabolism by liver
microsomes. c) Metabolism by liver slices. Male rats were castrated 25 days before
sacrifice and treated with 200 pg/kg (s.c.) of estradiol, or 2.5 mg/kg (s.c.) of testosterone
or 4-chlortestosterone for 15 days before sacrifice.

SEX-RELATED DIFFERENCES IN DRUG METABOLISM

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sex difference for strychnine toxicity appears to be related, not to

differences in the sensitivity of the central nervous system, but
more probably to the rate of strychnine metabolism by liver microsomes.
This interpretation has been supported by a number of experiments. The sex difference for strychnine toxicity was abolished o r
diminished when metabolism was minimized by intravenous administration of the drug o r by pretreatment of the animals with SKF 52"-A.
The data in Table 1 show that strychnine convulsion occurred 5 to 6
sec after intravenous administration of the drug, and that SKF 525-A
markedly inhibited strychnine metabolism by liver microsomes [lo].
TABLE 1
Strychnine Toxicity in Rats by Different Routes of
Administration (Kato et al. I131)
~~

Route of
administration

Sex

SKF 525-A
pretreatmenta

LD,,
(mg/kg)

(1) i.p.

1.62

(2) i.p.

2.82

(3) i.p.

1.33

(4) i.p.

1.38

(5) i.v.

0.57

(6) i.v.
(7) i.v.

M
F

+
+

(8) i.v.
~~

~~

~~

~~

Difference

X1.74
X1.04

0.57
0.58

x1.00

0.60

X1.04

~~

%KF 525-A (50 mg/kg) was given intraperitoneally 30 min before strychnine administration.

Similarly, it was demonstrated that the sex difference in picrotoxin toxicity is due to a higher rate of picrotoxin metabolism by
liver microsomes from male rats [14]. On the other hand, it is
interesting to note that guthion and octamethylpyrophosphoramide
(OMPA) a r e more toxic in male rats than in female rats. Guthion
and OMPA a r e converted to highly toxic choline esterase inhibitors
by liver microsomal drug-metabolizing enzymes. Since these metabolic transformations a r e more extensive in male rats, the toxicities
a r e also higher in male r a t s [lo, 15, 161. Accordingly, the administration of SKF 525-A markedly decreased the toxicity of OMPA in
both male and female rats [lo].

KATO

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111. SEX-RELATED DIFFERENCES IN MICROSOMAL ENZYMES

The activities of hepatic microsomal drug-metabolizing enzymes
a r e low in newborn and immature rats [17, 181, and clear sex differences a r e not observed until 30 days of age [lo]. The activities of
these enzymes a r e clearly increased in male rats over those of the
females a t 50 days of age [lo]. The appearance of these differences
in microsomal enzyme activities a r e coincident with the beginning of
sex maturation, and the difference persists in normal adult rats
until 600 days of age [191.
The drug- metabolizing enzymes a r e localized in microsomes and
oxidize a variety of drugs in the presence of oxygen and reduced
NADP [17]. Although the drug-metabolizing enzymes from r a t liver
microsomes metabolize a variety of drugs, Kato and Gillette [20\
demonstrated that the magnitude of the sex difference is dependent
on the substrates and metabolic pathways. For example, the N-demethylation of aminopyrine and the aliphatic hydroxylation of hexobarbital and pentobarbital are 3.5-fold higher in male rats than in
females, whereas there a r e no significant sex differences in the
aromatic hydroxylation of zoxazolamine or aniline. The N-demethylation of morphine is 2.5-fold higher in male rats and the N-demethylation of cocain, 0-demethylation of p-nitroanisole and N-demethylation of methylaniline a r e 85, 47, and 39%, respectively, higher
in male rats than in females.
On the other hand, Schenkman et al. [21] presented new evidence
that the Michaelis constants for hexobarbital hydroxylation and
aminopyrine N-demethylation by liver microsomal enzymes a r e
greater in female rats. However, they did not observe the sex difference in the Michaelis constant for aniline hydroxylation. Davies
e t al. [221 demonstrated a greater value of the Michaelis constant
for ethylmorphine N-demethylation by liver microsomes from
female rats. These results suggested that androgen may regulate
both the V,, (male > female) and K, (Michaelis constant) (female
> male) values for the oxidation of drugs. Indeed, Schenkman et al.
[21] observed that the K, value for aminopyrine N-demethylation by
liver microsomes from male rats which were castrated 2 weeks
earlier increased to a value intermediate between those found in
normal male and female rats. However, Davies et al. [221 did not
observe a significant change on the K, value for ethylmorphine Ndemethylation in castrated males although the V,, value had decreased to that of female rats.
Later, Kato and Onoda [23]demonstrated that the castration of
male rats decreased the V,, values and increased the K, values for
hexobarbital hydroxylation and aminopyrine N-demethylation, but

SEX-RELATED DIFFERENCES IN DRUG METABOLISM

did not produce any significant changes on the V,, and K, values for
aniline hydroxylation (Fig. 2). The administration of testosterone o r
methyltestosterone to the castrated rats increased the V,, value
and decreased the K, value for hexobarbital hydroxylation to the
level of intact male rats.

Hexobarbi tal hydroxylation

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C
E
.-

/x

C ont (F

1-0I

0~'CaSt

Cast +TS

-2.0 -1.5

-1.0

-0.5

0.5

1.0
1.5
11s ( r n M I-'

2.0

FIG.2. Effects of testosterone and estradiol on the reciprocal plots of hexobarbital

hydroxylation (Kato and Onoda [ 231). Male rats were castrated 23 days before sacrifice
and treated with testosterone propionate (5 mg/kg, s.c.) and estradiol benzoate
(500 pg/kg, s.c.) for 10 days before sacrifice. TS, Testosterone treatment; ED, estradiol
treatment; F, female rats; M, male rats.
Moreover, Kato and Onoda [23]showed that levels of estradiol
and diethylstilbestrol which were too low to antagonize the action of
the androgens on target organs such as prostate, seminal vesicle,
and muscle levator ani caused a clear inhibition of the action of
testosterone and methyltestosterone on the increase in the activity
of drug- metabolizing enzymes in castrated male rats. These results
indicate that estrogens directly antagonize the action of androgens,
and that their effects a r e not mediated through the depression of
androgen production by the testis [24]. Estrogens probably act
directly at the androgen receptor site. The treatment of mice, rabbits, guinea pigs, and hamsters with large amounts of androgen did
not cause significant increases in drug-metabolizing activities [25].

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KATO

These findings suggest that an androgen-dependent mechanism for

increasing liver microsomal drug-metabolizing activity may be
genetically lacking in mice, rabbits, guinea pigs, and hamsters.
Adopting the operon theory of Jacob and Monod [26], Kato and
Onoda [23] suggested that androgen may act as a depressor of an
active endogenous repressor which represses the action of a regulator gene responsible for the activities of microsomal drug metabolizing enzyme systems. If androgen plays a role as a depressor of
the repressor, it seems probable that estrogen may interfere with
the binding of androgen with the repressor. Since estradiol and diethylstilbestrol comparably counteract the stimulating action of
testosterone and methyltestosterone, the structural specificity of
the repressor seems to be relatively low.
The activity of liver microsoinal aniline hydroxylase of Sprague
Dawley male rats is only 10 to 20% higher than that of female rats;
t h i s difference is not statistically significant [20, 211. On the other
hand, Kato et al. [25] observed a rather small (30 to 35%), but significant, sex difference (male > female) in aniline hydroxylation by
liver microsomes from rats of the Wistar strain. Thus it seems
that the rat strain plays a minor quantitative role in sex differences
demonstrable by the hydroxylation of aniline.
IV. SEX DIFFERENCES IN
MICROSOMAL ELECTRON TRANSPORT SYSTEM
Kato e t al. [25] reported that the activity of the NADPH-linked
electron transport system of liver microsomes is slightly higher in
male W i s t a r rats than in females. The activities of NADPH oxidase
and NADPH-cytochrome c reductase are 37 to 38% higher in male
than in female rats. The cytochrome P-450 content of liver microsomes is 33% higher in male rats (Table 2).
Liver microsomes also can reduce nitro and azo compounds in
the presence of NADPH. The activities of nitro- and azo-reductases
a r e clearly higher in male rats than in females [25]. By contrast,
no sex difference was observed for the activities of NADH oxidase
and NADH-cytochrome c reductase, o r in the content of cytochrome
b, in liver microsomes from the Wistar rats [25]. Similarly, the
activity of NADPH-neotetrazolium reductase shows clear sex difference whereas none was observed in the activity of NADH-neotetrazolium reductase.
Since the magnitude of the sex difference in the activity of NADPHlinked electron transport system is similar to that of aniline hydroxylation, it is likely that the aniline hydroxylase sex difference may

SEX-RELATED DIFFERENCES IN DRUG METABOLISM

TABLE 2
Sex Difference in Drug-MetabolizingEnzyme Systems in Liver
Microsomes of Rats (Kato et al. [ 251; Gigon et al. [ 281).

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Enzymic activitya
Female

Male

Ratio

3.98

Hexobarbital hydroxylase

11.8 0.7

46.9 ? 3.5

Aniline hydroxylase

5.85 f 0.35

7.76 f 0.51

1.33

NADPH oxidase

102 f 5

140 f 8

1.38

1995 f 85

1.37

82 f 0.4

107 7

NADPH-cytochrornec reductase
NADPH-cytochrome P-450 reductase
NADH oxidase

1459 54
57.1 f 2.5

1.31

54.3 3.6

0.95

NADH-cytochrome c reductase

21.3 f 1.2

19.2 1.4

0.90

Cytochrome P-450b

72.8 4.1

96.6 f 3.9

1.33

aExpressed as lo-'' mole/mg microsomal protein/min.

b10-'0 mole/mg microsomal protein.

be due to the sex difference in the electron transport system. However, the far greater (3 to 4 times) sex differences in aminopyrine
N-demethylase and hexobarbital hydroxylase may be related to other
factors.
Gigon e t al. [27, 281 observed that the r a t e of reduction of cytochrome P-450 by NADPH in a system saturated with carbon monoxide was almost 30% faster by microsomes from male rats than
by those from female rats, and that the microsomal content of cytochrome P-450 was 27% higher in male rats than in females of the
Sprague-Dawley strain. Further, they observed that the addition of
ethylmorphine stimulated the reduction of cytochrome P-450 by
NADPH. The rate of cytochrome P-450 reduction by NADPH in the
presence of ethylmorphine was 1.69 times faster in microsomes
from male rats.
Ethylmorphine is not unique; drugs such as hexobarbital and
aminopyrine, which also produce the Type I spectral change by binding with cytochrome P-450 [29], also stimulate the reduction of cytochrome P-450 by NADPH. By contrast, drugs such as aniline, which
produce the Type 11 spectral change [29], decrease the r a t e of cytochrome P-450 reduction [27]. These observations by Gigon et al.
[28] suggested that the rate-limiting step of the microsomal drughydroxylating enzyme system is the reduction of cytochrome P-450
by an NADPH-linked electron transport system.

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10

KATO

Recently, the liver microsomal hydroxy lation enzyme system has

been solubilized and resolved by Lu et al. [30, 311 into three fractions containing cytochrome P-450, NADPH-cytochrome P-450
reductase, and lipid. A l l three fractions are essential for the maximal rate of hydroxylation of a variety of drugs. Moreover, Lu e t al.
[32] obtained interesting evidence that the reconstituted microsomal
hydroxylating enzyme system from rats treated with phenobarbital
exhibited high activity for benzamphetamine N-demethylation, but
very low activity for 3,4-benzpyrene hydroxylation. However, when
the cytochrome P-450 fraction from phenobarbital- treated rats was
replaced by the cytochrome P-448 fraction [33, 341 from rats treated
with 3-methylcholanthrene, the N-demethylation of benzamphetamine was markedly decreased while the hydroxylation of 3,4-benzpyrene was greatly increased. In addition, the administration of 3methylcholanthrene o r 3,4-benzpyrene into male r a t s reduced the
hydroxylation of hexobarbital and N-demethylation of benzamphetamine by liver microsomes [35, 361, although the rate of reduction of
cytochrome P-450 reductase was increased [36]. These results
scggest that the rate-limiting steps of the microsomal drug-hydroxylating enzyme system resides in the cytochrome P-450 fraction
rather than in the NADPH-cytochrome P-450 reductase.
V. SEX DIFFERENCES I N SUBSTRATE BINDING WITH P-450

Schenkman e t al. [29] and Imai and Sat0 [37] observed that a number of drugs which are substrates for hepatic microsomal hydroxylases react with cytochrome P-450 to give two characteristic types
of spectral change. These results suggest that the spectral changes
observed a r e indicative of substrate interaction with cytochrome
P-450, presumably representing the primary binding of substrate
for enzymatic hydroxylation [37.1.
The addition of hexobarbital to microsomes causes Type I spectral
change. As shown in Fig. 3, the magnitude of the spectral change
induced by the microsomes from male rats is about 3 times greater
than that from female rats, whereas the addition of aniline to microsomes causes Type I1 spectral change and only a slight sex difference is observed [21,23]. These results indicate that the cytochrome
P-450 of male rats has a greater ability to produce the Type I spect r a l changes than that of female rats, presumably due to a greater
capacity to bind with substrates [23].
Schenkman et al. [21] demonstrated that liver microsomes from
female rats have not only a greater Michaelis constant (K,) for
hexobarbital hydroxylase, but also have a greater spectral dissocia-

SEX-RELATED DIFFERENCES IN DRUG METABOLISM

11

tion constant (K,) for hexobarbital than do liver microsomes from

male rats. This contrasts with aniline hydroxylation, for which K,
and K, values were not significantly different for liver microsomes
from male and female rats. Castration of male rats decreases the
spectral change induced by hexobarbital, but not that induced by
aniline [21, 231. However, these findings indicate that for some
drugs the cytochrome P-450 of male rats has greater binding affinity.

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Hexobarbital

Aniline

Male

--- Female

FIG.3. Hexobarbital- or aniline-induced spectral changes of cytochrome P-450 in

liver microsomes from male and female rats (Kato and Onoda [ 231). Hexobarbital
(1.6 mM), aniline (2 mM), and microsomal suspension (1.2 mg proteidml).

Kato and Onoda [23] demonstrated that castration of male rats

decreases the AOD,,, and increases K, values for hexobarbital.
Treatment with testosterone o r methyltestosterone restores the
AOD,, and K, values to the level of intact male rats (Fig. 4 and
Table 3). In contrast, both the AOD,, and K, values for aniline
were not altered by castration and the subsequent treatment with
androgen. These investigators [23] also showed that the administration of estradiol and diethylstilbesterol together with testosterone
o r methyltestosterone to castrated rats blocked t h e restorative effect
of androgen on the diminished V,, and K, values for hexobarbital
hydroxylation resulting from castration.
The similarity between the alterations in V,, and K, values, and
and K, values for hexobarbital hydroxylation, indicates
of AOD

KATO

12

Hexobarbital- induced difference spectrum

.-C

200.

01

'Cast

z
E

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0
a
Y

+TS+E D

C ast+TS

-14

-12 -10

-8

-6

-4

-2

11s (rnM1-l
FIG. 4. Effect of testosterone and estradiol on reciprocal plots of the spectral change
induced by hexobarbital (Kato and Onoda [ 231). See the legend for Fig. 2.

that the sex difference in the hydroxylation of this drug may be

related to the magnitude of the spectral change produced in cytochrome P-450by hexobarbital [23].
Similar sex differences were observed in the N-demethylation of
aminopyrine and the magnitude of spectral change of cytochrome
P-450 induced by aminopyrine in rat liver microsomes. Moreover,
no sex difference was observed in either the hydroxylation of zoxazolamine o r the magnitude of spectral change induced by zoxazolamine [38].
Thus it is conceivable that the sex difference in the activity of
drug oxidation by liver microsomes may be related to the binding
affinity of cytrochrome P-450 and probably to the amount of drug
binding to cytochrome. However, many drugs a r e metabolized along
different metabolic pathways by microsomal drug-metabolizing
enzyme systems, and the magnitude of sex differences varies with
metabolic pathways [20,351. Therefore, it will be necessary to
employ many different kinds of compounds to investigate the correlation between the magnitude of sex differences in drug oxidation
with the spectral change of cytochrome P-450.
It might be speculated that the sex difference for the magnitude
of the spectral change may represent the sum of sex differences for

Km

2.85 (-36%)

0.28 ( +22%)

0.42 ( +83%)

0.46 (+loo%)

Castrated + TS

Castrated + TS + ED

Female control

4.97 (-32%)
4.11 (-43%)

1.42 (+19o%)

0.70 ( +43%)

1.28 (+161%)

1.59 (+224%)

Castrated + TS

Castrated + TS + ED

Female control

Ks

+32%)
0.187(+175%)

0.169 +149%)

0.090

0.163 +140%)

0.068

0.420 (+272%)

0.317 (+181%)

0.155 ( +37%)

0.324 (+187%)

0.113

(mM)
~

10.8(-44%)

11.4 (-41%)

17.9 ( -8%)

12.3 (-39%)

19.4

8.0 (-42%)

9.1 (-35%)

13.5 ( -3%)

8.4 (-40%)

13.9

ODmax
(AOD X l(Ylmr! protein/ml

aThe treatment of rats was the same as given in Fig. 2. The results are expressed as averages from four to Seven experiments. Pooled
livers from four to five rats were used for each experiment. The figures in parentheses indicate percentage differences from control
values.

7.02 ( -3%)

7.25
4.83 (-33%)

0.49

Castrated

B. Hexobarbital
Male control

3.12 (-2%)

0.40 ( +74%)

4.15 ( -6%)

4.42
2.83 (-39%)

0.23

Vmax
(mpmoles/mg proteidmin)

Male Control

(d)

Castrated

A. Aminopyrine

Group

Effects of Testosterone and Estrogen on the Michaelis Constant (Km), Maximum Velocity (Vmax),Spectral Dissociation
Constant (&), and Maximum Spectral Change (AODm,) for Aminopyrine and Hexobarbital (Kato and Onoda [ 23])a

TABLE 3

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the different metabolic pathways. The careful selection of suitable

drugs and the establishment of accurate methods for the quantitative
determination of each metabolic pathway will be prerequisites for
such an investigation. Such studies might be expected to provide
useful information for resolving problems about the multiplicity of
microsomal hemoproteins and drug-metabolizing enzymes [39, 40.1.

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VI. SEX DIFFERENCES IN ANIMALS OTHER THAN RATS

A sex difference in drug metabolism by liver microsomes seems
to be restricted to rats, A variety of drugs have been investigated
in mice, guinea pigs, hamsters, rabbits, dogs, and monkeys without
a clearcut demonstration of a sex difference of drug metabolism in
these species [7,10, 13, 14, 251. These negative results have been
confirmed repeatedly for many kinds of drugs and animal species
including man (Kato e t al., 1973, unpublished results). Therefore
one can predict with some certainty that if a drug shows a clear sex
difference in its effect in rats, but not in mice, the observed sex differences are related to the metabolism of the drug, Furthermore, it
is likely, for such a drug, that a sex difference does not occur in
other animal species or man. On the other hand, if a drug shows a
clear sex difference in its response in both rats and in mice, then
the difference may be related not only to the metabolism of the drug,
but also to differences in tissue sensitivity. Moreover, it might be
expected that the sex differences may occur in other species of animals and in man.
However, it is important to note that there a r e slight sex differences in the metabolism of some drugs in some strains of mice.
Although there seems to be no clear sex differences in the hydroxylation of hexobarbital, the oxidation of strychnine, or the N-demethylation of aminopyrine in NIH, Swiss, and dd strains of mice [7,13,
25, 411, Catz and Jaffe [42] reported a small sex difference in the
hydroxylation of pentobarbital of liver microsomes from mice of the
C,H and C,,B1 Strains. Noordhoek and Rumke [43] also found that
the hydroxylation of hexobarbital is slightly faster in female mice
than in male mice.
Consistent with the absence of a sex difference in drug-metabolizing enzyme activity in mice liver microsomes, Kato eta1 [25,41,44]
observed no clear sex difference in the cytochrome P-450 content o r
in the activity of NADPH oxidase, NADPH-cytochrome c reductase,
and NADPH-neotetrazolium reductase of liver microsomes prepared
from mice and rabbits.
Likewise, Castro and Gillette [45] reported no sex difference in

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SEX-RELATED DIFFERENCES IN DRUG METABOLISM

15

t h e K, and V,, values for ethylmorphine N-demethylation in guinea

pigs, rabbits, and monkeys. In addition, Kato et al. [41] concluded
that there is no sex difference in the binding capacity of cytochrome
P-450 with hexobarbital and aminopyrine in liver microsomes obtained from mice and rabbits. The administration of testosterone o r
methyltestosterone to mice, guinea pigs, and rabbits did not increase
the activity of drug-metabolizing enzymes in liver microsomes [lo].
On the other hand, Novick e t al. [46] reported that the repeated o r
single administration of high doses of some anabolic steroids to
female mice increased the metabolism of hexobarbital by liver
microsomes. However, they observed an increase in the hexobarbital metabolism not only in female mice, but also in male mice. The
repeated administration of high doses of testosterone to female
mice did not increase the metabolism of hexobarbital by their liver
microsomes. Moreover, they found that treatment with anabolic
steroids reduced zoxazolamine paralysis time. It is likely, therefore, that the observed increase in the metabolism of hexobarbital
by the anabolic steroids is due to a phenobarbitallike induction of
the activity of microsomal drug-metabolizing enzymes.

VII. EFFECT OF METHYLCHOLANTHRENE

IN MALE AND FEMALE RATS

In contrast to the effect of phenobarbital, the administration of

3-methylcholanthrene o r 3,4-benzpyrene increases only some
activities of the drug-metabolizing enzyme system of liver microsomes [47, 481. Kato and Takayanagi [35] demonstrated that the
androgen-dependent drug- metabolizing activity of liver microsomes
(such a s hexobarbital hydroxylation, pentobarbital oxidation, and
aminopyrine N-demethylation) is clearly decreased by treating male
rats with 3-methylcholanthrene, while the androgen-independent
activity of liver microsomes (such as aniline and zoxazolamine
hydroxylation) is markedly increased by the same treatment.
A s shown in Table 4, the effect of methylcholanthrene on enzyme
activity is related to the magnitude of the sex difference, The
metabolic activities displaying a relatively low sex difference a r e
increased by methylcholanthrene; the critical point of the magnitude of sex difference appears to be a male to female ratio of about
2.25. On the other hand, the treatment of female rats did not alter
significantly the androgen-dependent drug-metabolizing activities
but markedly increased those activities which have a low magnitude
of sex difference [35].
The administration of methyltestosterone to castrated female rats

KATO

16
TABLE 4

Effect of Methylcholanthrene on the Activity of DrugMetabolizing

Enzymes of Liver Microsomes in Male and Female Rats (Kato and Takayanaghi [ 351)

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Methylcholanthrene
treatment (%)
Sex difference
(male/female ratio)

Female

Male

Aminopyrine
N-demethylation

4.04

-5

-46*

Butynamine N-demethylation

5.96

-1 3

-51*

Diphenhydramine
N-demethy lation

2.38

-6

-26*

Merperidine N-demethylation

2.41

-14

-28*

N-Methylbarbital
N-demethylation

4.03

-6

-43*

Morphine N-demethylation

2.57

-7

-32*

N-Methylaniline
N-demethylation

1.51

+97*

+45*

N,N-Dimethylaniline
N-demethylation

2.07

+57*

+46*

N,N-Dimethylnaphylamine
N-demethylation

2.24

+8

+23*

Carisoprodol oxidation

2.53

-3

-34*

Pentobarbital oxidation

2.61

-8

-45*

Hexobarbital hydroxylation

2.96

-9

-30*

pNitroanizole
0-demethylation

1.66

+108*

+47*

Aniline hydroxylation

1.31

+75*

+53*

Zoxazolamine hydroxylation

1.15

+423*

+386*

*Significantly different ( p < 0.05) from the value for untreated rats.

(Fig. 5) markedly increased aminopyrine N-demethylation and

buty namine N-demethy lation, whereas N- methy laniline N- deme thy lation and zoxazolamine hydroxylation were only slightly or unaffected by the same treatment. Phenobarbital treatment additively
increased all the activities. Methylcholanthrene treatment antag-

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17

FIG.5. Effects of administration of phenobarbital or methylcholanthrene on the

N-demethylation of aminopyrine and butynamine, and on the hydroxylation of
zoxazolamine in castrated male rats (Kato and Takayanaghi [ 351). Female rats were
castrated 20 days before sacrifice and treated with methyltestosterone (10 mg/kg, s.c.)
every other day for 18 days or treated with phenobarbital (PB, 80 mg/kg, i.p.) or methyl.
cholanthrene (MC, 40 mg/kg, i.p.) 4 8 and 72 hr before sacrifice.
onized the effect of methyltestosterone on aminopyrine and butynamine N-demethylations, but additively increased the N-methylaniline N-demethylation and zoxazolamine hydroxylation [35].
These results would appear to indicate that methylcholanthrene
blocks the stimulatory action of androgen on the androgen-dependent
drug-metabolizing activity, and decreases these activities, whereas
some activities, which have a low sensitivity to the stimulatory action of androgen, a r e increased [35, 491.
Methylcholanthrene did not alter, significantly, the activity of
NADPH oxidase or NADPH-cytochrome c reductase, but did increase
slightly the microsomal content of P-450 of both male and female
r a t s [35, 50, 511.
Also, Kato e t al. [50, 511 and Shoeman et al. [52] demonstrated
that the administration of methylcholanthrene to male rats markedly
decreased the magnitude of the spectral change of cytochrome P-450
induced by hexobarbital and aminopyrine, but increased the magnitude of spectral change induced by aniline and zoxazolamine. These

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18

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results a r e in accord with previous observations that hexobarbital

hydroxylation and aminopyrine N-demethylation a r e decreased, and
aniline and zoxazolamine hydroxylations a r e increased in male rats
by methylcholanthrene treatment.
Furthermore, methylcholanthrene decreased not only the magnitude of the spectral change induced by hexobarbital and aminopyrine,
but also completely modified the pattern of the difference spectrum
[51, 521, thereby suggesting that methylcholanthrene produced some
alterations in the physicochemical nature of cytochrome P-450.
A shift of the peak in CO-induced difference spectrum of cytochrome P-450 from 450 to 448 mp anda shift of pH-dependent change
of the relative intensities of two peaks (430 and 455 mp) of ethylisocyanide-induced difference spectrum of cytochrome P-450 in liver
microsomes from methylcholanthrene-treated r a t s were reported by
Sladek and Mannering [33] and Alvares et al. [34]. The alteration of
the difference spectrum of the ethylisocyanide complex can be prevented by an inhibitor of protein synthesis, such a s ethionine or
actinomycin D [53]. The alteration of the difference spectrum of
cytochrome P-450 may be due therefore to the synthesis of a new
type of cytochrome P-450,i.e., cytochrome P-448,by treatment
with methylcholanthrene [33, 34, 52, 531. On the other hand,
Schenkman et al. [54]suggested that the alteration of the difference
spectrum of cytochrome P-450 may be due to some residues of
methylcholanthrene and its metabolites binding with cytochrome
P-450. Kato et al. [51] observed, however, that the change in the
difference spectrum induced by hexobarbital is not evident at 2 and
6 h r after administration of methylcholanthrene. Formation of cytochrome P-448 was not noted until 16 hr after methylcholanthrene
administration [ 551. Additional studies will be required to clarify
the mechanism of the alteration of difference spectrum of cytochrgme P-450 induced by various ligands [36,571.
The pattern of difference spectra of cytochrome P-450 induced by
aniline and zoxazolamine was not significantly modified by methylcholanthrene treatment in either male o r female rats [51]. It is of
interest, however, that the administration of methylcholanthrene to
male and female rats decreases the K, value for aniline-induced difference spectrum, whereas it increases K, value for aniline hydroxylation [51, 561.
VIII. SEX DIFFERENCES IN PATHOLOGICAL CONDITIONS

Decreases in the activities of drug-metabolizing enzymes in

liver microsomes isolated from adrenalectomized, alloxan, thyrox-

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SEX-RELATED DIFFERENCES IN DRUG METABOLISM

19

ine, or morphine-treated rats have been reported by several investigators [17, 571. These studies were carried out in male rats, using
hexobarbital, aminopyrine, morphine, and monomethyl-4-aminoantipyrine as the substrates.
Kato and Gillette [20,491 demonstrated that hexobarbital hydroxylation and aminopyrine N-demethylation a r e markedly decreased
in microsomes isolated from male rats under such pathological
conditions as starvation, alloxan diabetes, adrenalectomy, and treatment with thyroxine, morphine, o r formalin. However, similar results were not obtained with microsomes from female rats, and the
hydroxylations of aniline and zoxazolamine were not decreased in
either male o r female rats.
Castration of male rats decreases hexobarbital hydroxylation
and aminopyrine N-demethylation by liver microsomes. Treatment
of these rats with alloxan, morphine, o r thyroxine, however, does
not further decrease these drug-metabolizing activities [491, and
the effects of such treatment can be observed in castrated male rats
pretreated with methyltestosterone. These findings indicate that the
effects of alloxan, morphine, o r thyroxine treatment to decrease
hexobarbital hydroxylation and aminopyrine N-demethylation occurs
only in rats in which the activities of drug-metabolizing enzymes
a r e being stimulated by androgen, perhaps through a direct interference of the action of androgen [49]. Kato and Gillette [49] speculated that the action of androgen to increase the activities of drugmetabolizing enzymes is easily impaired under pathological conditions through some unknown mechanism(s).
It was of interest to investigate further this hypothesis, because
if the sex difference in the alteration of hexobarbital hydroxylation
and aminopyrine N-demethylation under pathological conditions is
due only to impairment of androgen action, then a sex difference
should not be observed in mice, guinea pigs, o r rabbits in which the
activities of drug-metabolizing enzymes a r e not stimulated by
androgen [7, lo]. Kato and Onoda [58]were able to confirm this
hypothesis by demonstrating that the administration of morphine
decreased aminopyrine N-demethylation and hexobarbital hydroxylation only in male rats, but not in male o r female mice, guinea pigs,
o r rabbits.
Kato et al. [59-611observed that hexobarbital hydroxylation and
aminopyrine N-demethylation a r e decreased in liver microsomes
from male and female rats fed a low protein diet o r bearing Walker
carcinosarcoma. The percentage decrease was greater for males.
Thyroidectomy decreased hexobarbital hydroxylation and aminopyrine N-demethylation in both sexes [62],and aniline and zoxazolamine hydroxylation were decreased in rats (male and female) which

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20

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were fed a low protein diet, were thyroidectomized, o r bearing

tumors. These results indicate that the effects of these pathological
conditions may be related to impairments of other processes for
the oxidation of drugs by liver microsomes a s well a s an impairment of androgen action.
A series of studies by Kato and his associates [38,63-65]demonstrated that the magnitude of the difference spectrum of cytochrome
P-450 induced by hexobarbital o r aminopyrine was markedly decreased in male r a t s but not in female rats in the pathological conditions noted above. Also, the magnitude of the difference spectrum
per content of cytochrome P-450 was decreased in the male rats
(Table 5). This suggests that the binding capacities of cytochrome
P-450 for hexobarbital and aminopyrine, which are normally stimulated by androgen, a r e decreased in microsomes from male rats,
due to the pathological conditions.
However, the magnitude of the difference spectrum of cytochrome
P-450 induced by aniline or zoxazolamine was not decreased in male
rats by the same pathological conditions (Table 5). A good correlation was observed between the magnitude of change of the difference
spectrum and the activity of drug-metabolizing enzymes induced by
the various treatment, thus suggesting that decreases in the activity
of drug-metabolizing enzymes a r e probably due to decreases in the
amounts of drug binding with cytochrome P-450 [66].
Kato et al. [38,64-66]found that treatment with alloxan, thyroxine, and morphine, starvation, and adrenalectomy not only decreases
the V,,, values for hexobarbital hydroxylation and aminopyrine Ndemethylation, but also affects the K, values for microsomes from
male rats. The K, values for hexobarbital- and aminopyrine-induced
difference spectra were also altered in the microsomes from these
male rats. In fact, both the K, and K, values were increased by the
pathological states toward the values obtained with microsomes
from normal female rats [38,64-66].
In contrast, the K, value for aniline hydroxylation and the K,
value for aniline-induced difference spectrum are not altered significantly in liver microsomes from male rats subjected to the
various pathological conditions. For female rats, however, the K,
and K, values for hexobarbital, aminopyrine, and aniline a r e unchanged. These results would indicate that the actions of androgen
to increase the binding capacity and affinity of cytochrome P-450
with the drug substrates a r e impaired by the pathological states.
On the other hand, Gram e t al. [67] showed that the K, values for
aminopyrine N-demethylation and ethylmorphine N-demethylation
were increased in microsomes from starved male rats, but the K,
value for hexobarbital hydroxylation was decreased, and that for
aniline hydroxylation was not affected.

17.0 f 1.1
9.7 k 0.5
17.4 f 0.5
9.0 f 0.4

Alloxan diabetes

*Significantlydifferent (p < 0.05)from control value.

~~

~~

16.7 f 0.5
8.5 f 0.4

M
F

Thyroxine

~~

17.4 f 0.5
9.0 f 0.4

M
F

Morphine

Starvation

17.0 f 1.1
9.7 f 0.5

Adrenalectomy

Control

Sex

Treatment

13.5 f 0.5 (-22%)*

9.2f 0.5 ( +2%)

10.5 f 0.8( +8%)*

12.0 f 0.9 (-29%)*

11.5 f 1.0 (-31%)*

9.3 f 0.5(+lo%)

f 0.7

14.3 f 0.6
13.2 f 0.5

14.9 f 0.8

14.2

14.0 f 0.5
13.4 f 0.5

14.3 f 0.6
13.2 f 0.5

13.0 f 0.7 (-25%)*

8.4 f 0.5 ( -6%)

Control

14.2 f 0.7
14.9 f 0.8

12.6 f 0.9(-26%)*
10.5 f 0.7 ( +8%)

Treated

Hexobarbital

Treated

14.7 f 0.8( +3%)

14.7f 1.0 (+11%)

16.4 f 1.2 ( +lo%)

15.3 f 1.1 ( +8%)

12.6 f 0.7 (-10%)

13.7 f 0.5 ( +2%)

14.2 f 0.6 ( 0%)

12.5 f 0.3 ( -5%)

14.2 ? 1.1 ( 0%)

15.2 f 1.0 ( +2%)

Aniline

Cytochrome P-450binding capacity (AOD X 103/mpmole P-450)

TABLE 5
Effect of Adrenalectomy,Morphine or Thyroxine Treatment, Alloxan Diabetes, and Starvation on the Binding Capacity of
Cvtochrome P450 for Hexobarbital and Aniline (Kato et al. 138.64-661)

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Kato e t al. [68-701, extending their studies to other species,

showed that hexobarbital hydroxylation and aminopyrine N-demethylation were not decreased in microsomes from male mice and
rabbits treated with alloxan, thyroxine, o r morphine, o r in fasted o r
adrenalectomized male mice and rabbits. Also, the magnitude of the
difference spectrum induced by hexobarbital or aminopyrine was not
decreased, nor was the binding capacity of cytochrome P-450.
Similar results were obtained with aniline.
It is probable that the sex differences observed with alloxan,
thyroxine, and morphine treatment, starvation, and adrenalectomy
are closely related to the sex differences present in normal animals.
The findings reported above support the hypothesis that the decreases in hexobarbital hydroxylation and aminopyrine N-demethylation, and also the difference spectra of cytochrome P-450 induced by
hexobarbital and aminopyrine, a r e due to an impairment of the action of androgen that normally increases the binding capacity of cytochrome P-450 with hexobarbital and aminopyrine [23, 661.
Wada e t al. 711, on the other hand, recently reported that aminopyrine N-demethylation in liver microsomes was slightly decreased
in female mice by adrenalectomy, although in male mice the activity
was not affected. However, these workers did not report the sex
difference for the activity of aminopyrine N-demethylase for intact
male and female mice of the strain used in their studies. Thus the
sex difference noted in this study, due to adrenalectomy, may be
related to that reported by Catz and Jaffe [42]for some strains of
intact mice.
Tumor-bearing rats of both sexes demonstrate decreases in hexobarbital hydroxylation and aminopyrine N-demethylation a s well a s a
decrease in the magnitudes of the difference spectra of cytochrome
P-450 induced by hexobarbital and aminopyrine. However, the percentage decreases in the difference spectra were greater for male
rats than for females [72]. These rats also demonstrate a decrease
in the magnitude of the difference spectra induced by aniline and
zoxazolamine.
A s the content of cytochrome P-450 in liver microsomes from
the tumor-bearing rats was decreased for both sexes, the decrease
in the difference spectrum of cytochrome P-450,induced by hexobarbital, was considered to be due mainly to the decrease in the
amount of cytochrome P-450. Also, the binding capacity of cytochrome P-450with hexobarbital o r aminopyrine was decreased, but
only in the tumor-bearing male rat. This was not the case with
aniline, however.
Thyroidectomy decreased hexobarbital hydroxylation, aminopyrine
N-demethylation, and aniline hydroxylation in liver microsomes from
both male and female rats. It has been reported that these effects

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SEX-RELATED DIFFERENCES IN DRUG METABOLISM

23

a r e due mainly to a decrease in the activity of any NADPH-linked

electron transport system of the microsomes [62, 731. In thyroidectomized male rats the binding capacity of cytochrome P-450
with hexobarbital and aminopyrine was slightly decreased but was
unaffected with aniline.
Sasame e t al. [741 observed that after the administration of carbon tetrachloride, the magnitude of decrease in ethylmorphine Ndemethylation by the liver microsomes was slightly higher in male
r a t s but the overall decrease in N-demethylation activity was due
mainly to a decrease in the amount of cytochrome P-450 in the
microsomes.
Recently, Stripp et al. [75] noted that the administration of spironolactone, a steroid presumably without direct hormonal effects on
the liver, increased drug-metabolizing activities in female rats. The
effect in male r a t s was variable, with both a small increase o r decrease being noted. Similarly, spironolactone increased the magnitude of the difference spectrum of cytochrome P-450 induced by
hexobarbital in female r a t s but not in males [76]. These findings
may be related to the observation by Kato and associates [35, 77,
781 of the effect of phenobarbital on the drug-metabolizing activity
of male and female rats. Phenobarbital increases the androgendependent drug-metabolizing activities more extensively in the
female than in the male rat. F o r example, phenobarbital increases
hexobarbital hydroxylation by liver microsomes by 4.2-fold in female rats, but only by 1.9-fold in male r a t s [35]. Morphine and
meperidine N-demethylations a r e increased by phenobarbital treatment in female r a t s only [35].
The oxidation of OMPA is increased in female r a t s and slightly
decreased in male r a t s by phenobarbital treatment [16]. Similar
treatment did not change N-demethylation of dimethylnitrosamine
activity in female rats but significantly decreased the activity in
male r a t s [78]. This would suggest that the additive effects of
androgen and phenobarbital may be due solely to an increased basal
activity induced by phenobarbital as illustrated in Fig, 6. With some
microsomal-metabolizing enzymes, such as OMPA oxidation and
dimethylnitroamine N-demethylation, the effect of androgen is more
easily impaired by nontoxic compounds such a s phenobarbital. These
enzyme activities seemed to be unstable a s indicated by the observation that the administration of methylcholanthrene clearly decreases
the activity in rats of either sex [16, 781.
IX. SEX DIFFERENCES IN THE METABOLISM
OF STEROID HORMONES
Liver microsomes contain a number of NADPH-dependent enzymes which catalyze the hydroxylation of a variety of steroids.

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FIG. 6. Schematic representation of the effect of phenobarbital on hexobarbital

hydroxylation, aminopyrine N-demethylation, OMPA oxidation, and aniline hydroxylation in male and female rats (Kato et al. [ 16, 35, 771).White bar, basal activity; hashed
bar, part of phenobarbital-induced activity; black bar, part of androgen-induced activity;
F, female rats; M, male rats. The metabolic activities are expressed as percentages of the
activities in control female rats.

For example, estradiol is hydroxylated to form 6g-, 60-, 160-, and

20-hydroxyestradiol [79]. Liver microsomal enzymes also convert
testosterone to 2a-, 6p-, 70-, and 160-hydroxytestosterone [80],
and progesterone to Sp- and 160-hydroxyprogesterone [81].
Kuntzman and Conney [82, 831 postulated that steroid hormones a r e
hydroxylated by liver microsomal enzymes that a r e similar or
identical to drug-metabolizing enzymes.
Sex differences in the hydroxylation of steroid hormones have
been reported by Leybold and Staudinger [84] and Kuntzman et al.
[82]. Jacobson and Kuntzman [85] reported that castration of male
rats decreases the hydroxylation activity of testosterone which is
restored by the administration of testosterone propionate.
Kato et al. [86] also reported that castration of male rats decreased the hydroxylation of testosterone and progesterone by liver
microsomes, and found that administration of testosterone o r
methyltestosterone restored the activities to the level of intact male
rats. However, administration of estradiol o r diethylstilbesterol
with testosterone o r methyltestosterone to castrated rats antago-

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25

nized the action of androgen. Lehmann and Breuer [87] observed

that castration markedly decreased the hydroxylation of estrone by
liver microsomes to the level of that of microsomes from intact
female rats.
The magnitudes of the difference spectra of cytochrome P-450,
induced by testosterone and progesterone, were higher in microsomes from male rats than from females, according to Kato et al.
[86, 881. Castration of male rats decreased these magnitudes, but
the administration of androgen restored them to the level of the
intact male rats [86].
Further studies by Kato and h i s associates [87-891 have shown
that the decrease in the hydroxylation of steroid hormones, and the
decrease in magnitude of the difference spectra of cytochrome
P-450 were observed only for liver microsomes isolated from
adrenalectomized, thyroxine-, o r morphine-treated male rats. This
was not the case in female rats a s observed in studies on hexobarbital metabolixm of female rats under similar conditions [88-901.
No sex differences in progesterone hydroxylation by liver microsomes of mice were observed by Kuntzman et al. [82]. Kato et al.
[go] also reported no sex difference in the activity of testosterone o r
progesterone hydroxylation in adrenalectomized o r intact mice,
lending support to the postulation of Kuntzman and Conney [82, 831
that steroid hormones a r e hydroxylated mainly by liver microsomal
enzymes which a r e identical to hexobarbital-hydroxylating enzyme.
For the hydroxylation of testosterone, the activities of 6p- and
16a-hydroxylation a r e higher in male than in female rats, whereas
the activity of 70-hydroxylation is similar for both sexes [85, 911.
Levin et al. [92] observed that 7a-hydroxylation of testosterone was
stimulated in liver microsomes of male rats treated with methylcholanthrene, whereas 16a-hydroxylation was markedly decreased.
These findings agree with previous observations made by Kato and
Takayanagi [35] on the hydroxylations of aniline and hexobarbital in
liver microsomes from methylcholanthrene-treated male rats.
Kato and Onoda (unpublished results, 1969) have observed that the
magnitude of spectral change of cytochrome P-450,induced by
testosterone or progesterone, was decreased in microsomes from
methylcholanthrene-treated male rats along with a decrease in the
total hydroxylation of these two substances.
Einarsson et al. [93] recently reported that 16a-hydroxylation
activity of 4-androstenedione by liver microsomes from male rats
was greater than in those from female rats, whereas a clear sex
difference was not obtained f o r the 6P-hydroxylation. They observed
that 6B-hydroxylation of progesterone by liver microsomes was
greater in male than in female rats. The reason for the different

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26

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results with the different substrates is not clear. They also observed that pseudohermaphroditic rats hydroxylated steroid hormones by liver microsomes in a manner identical to female rats.
The principal metabolic pathway for steroid hormones i s A4 reduction. The activities of microsomal 50-reductase of A4 steroid
hormones in female rats a r e greater than those in male rats [94 I,
but the activities in both sexes are affected differently by some treat
ments. The activity of 5a-reductase is increased in tumor-bearing
male rats, but unaltered in tumor-bearing female rats [95]. Thyroxine treatment increased 5a-reductase in female r a t s but decreased
it in male rats [96]. These findings should be taken into account
when determining and interpreting sex differences in the hydroxylation of steroid hormones.

X. MECHANISM OF SEX DIFFERENCE

A preponderance of evidence suggests that the sex difference in

the oxidation of drugs by liver microsomes is due mainly to the
higher binding capacity of cytochrome P-450 of male rats. In addition, a slight sex difference in the activity of the microsomalNADPHlinked electron transport system is a contributing factor.
The mechanism by which androgen increases the binding capacity
of cytochrome P-450 is unknown. The content of cytochrome P-450
in microsomes is increased 20 to 30% by androgen. Therefore, it is
conceivable that that cytochrome P-450, newly synthesized by androgen, has a very high binding capacity with hexobarbital and thus the
total binding capacity of the cytochrome P-450 is increased by 2.5fold [23]. It is possible that androgen stimulates the synthesis of
cytochrome P-450 of a normal binding capacity, but that it also
synthesizes factor(s) which may affect the binding capacity. It is
unlikely, though we cannot neglect the possibility, that androgen does
not increase binding capacity but instead increases the spectral
character of cytochrome P-450.
Recent studies present evidence that the alterations in the magnitude of drug-induced spectral change do not always parallel the
activity of drug oxidation, and therefore the meaning of the spectral
change should be studied more carefully [51, 52, 73, 76, 971. For
example, Chaplin and MaMering [98]showed that when Type I binding of microsomes was eliminated with phospholipase c treatment,
subsequent metabolism of ethylmorphine o r aminopyrine was not
completely eliminated; the residual activity retained had a higher

Km.

The activity of NADPH-cytochrome P-450 reductase is 20 to 30(Z

higher in male than in female rats, but the increase induced by the

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addition of hexobarbital or ethylmorphine is 3 to 5-fold greater in

males [28, 76, 1011. It might be possible, with the use of a purified
cytochrome P-450 and NADPH-cytochrome P-450 reductase system,
to resolve these problems [32, 99, 1001.
Lu et al. [30,321 have pointed out that a phospholipid fraction
(phosphatidycholine) from liver microsomes was important for the
oxidation of drugs by the liver microsomal drug-metabolizing enzyme system. It is of interest, therefore, that the biosynthesis of
phosphatidylcholine, via methylation of phosphatidylethanolamine, is
higher in female than in male rats. Also, the phosphatidylcholine
from female rats has a higher proportion of stearic and archidonic
acids [102].
Several other studies have implicated a function for phospholipids
in the binding of drug substrates to cytochrome P-450. Chaplin and
Mannering [98] eliminated the spectral change of Type I binding, but
a t the same time enhanced the spectral change of Type I1 binding,
with phospholipase c treatment of rat liver microsomes. They
postulated that the Type I binding site was closely associated with
membrane phospholipids that might be essential for a specific conformation of the binding sites.
Liebman and Estabrook [lo31 showed that extraction of microsoma1 phospholipid with isooctane caused the loss of Type I binding
and enhancement of Type I1 binding spectral change. Chaplin and
Mannering 1981 proposed that the conformational changes of the cytochrome P-450 that occurred in the presence of phosphatidylcholine
might facilitate the flow of electrons with a drug-hemoprotein complex in such a way as to enhance the reaction.
On the other hand, Kato et al. [951 observed that the administration of thyroxine to male rats increased the activity of 50-reductase
for steroid hormones in liver microsomes toward the level of female rats, whereas the activity in liver microsomes from female
rats was not affected. The activity of 5a-reductase in liver microsomes is regulated by androgen, and in contrast to the effect on the
hydroxylase, androgen depresses the activity of 50-reductase. The
effect of thyroxine may therefore be related to the impairment of
androgen action on microsomal enzymes as postulated in the case
of the effect of thyroxine on the steroid hydroxylases and drug
hydroxylases [62, 901.
The activity of 5a-reductase of steroid hormones in liver microsomes from male mice is identical to that in female mice, and Lle
effect of thyroxine on the activity of 5a-reductase is identical in
both sexes [95].
These results suggest that the effects of androgen to enhance the
hydroxylation of steroid hormones and to decrease the activity of
microsomal steroid 5a-reductase are carried out through a common mechanism.

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XI. SUMMARY

Hepatic microsomes isolated from male rats can hydroxylate

many drugs more rapidly than those from female rats. The spectral
changes of cytochrome P-450,induced by some drugs, is greater in
males than in females. These results indicate that the binding capacity of cytochrome P-450 for substrates is higher in male rats.
Castration of male rats decreases both the oxidation of some drugs
and the binding capacity of cytochrome P-450 for these compounds.
Androgen restores both deficiencies to the level of intact male rats,
whereas estrogen antagonizes these actions of androgen.
The hydroxylation of hexobarbital and aminopypine N-demethylation, as well as the binding capacity of cytochrome P-450 with hexobarbital and aminopyrine, a r e androgen-dependent. The hydroxylation of aniline and zoxazolamine, as well a s the binding capacity with
aniline and zoxazolamine, a r e androgen-independent. In castrated
male rats the K, values for the hexobarbital hydroxylation and
aminopyrine N-demethylation, and the K, values for hexobarbital
and aminopyrine-induced spectral changes, a r e increased, whereas
the same values for the aniline hydroxylation and for aniline-induced
spectral changes a r e not affected. Androgen produces these effects
in r a t s only; mice, guinea pigs, and rabbits a r e insensitive to both
actions of androgen.
The effect of methylcholanthrene treatment on the activities of
microsomal drug- metabolizing enzymes is apparently related to the
sex difference for the enzyme activities. The androgen-dependent
activities are decreased in male r a t s but not significantly affected
in female rats, whereas the androgen-independent activities are
increased in both sexes.
Treatment with thyroxine, morphine, and alloxan, adrenalectomy, and starvation decreases the metabolism of hexobarbital and
aminopyrine and the binding capacity of cytochrome P-450 for
drugs in male rats, but not in female rats. These pathological states
do not decrease the metabolism of aniline o r zoxazolamine o r the
binding capacity of cytochrome P-450 for these drugs in rats of
either sex. These findings indicate that the decrease in the binding
capacity of cytochrome P-450 with hexobarbital and aminopyrine
may be the factor responsible, in male rats, for the decrease in the
oxidation of drugs by liver microsomes in these patholigical conditions. These conditions may interfere with the action of androgen to
increase the binding capacity of cytochrome P-450.
Since the binding capacity of cytochrome P-450 for hexobarbital
and aminopyrine in mice and n b b i t s is insensitive to androgen, there
is no decrease in this activity, nor is a sex difference observed for

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the metabolism of hexobarbital and aminopyrine in these species in

pathological states.
Androgen has an effect on the hydroxylation of steroid hormones
by liver microsomes identical to its effect on the metabolism of
other drugs. The patterns of activity changes in pathological states
suggest that the activities of 5wreductase on steroid hormones and
that of hydroxylase on steroid hormones and drugs are regulated by
androgen, probably through a common mechanism, though the direction of the regulation is always opposite.
The mechanism by which androgen increases the binding capacity
of cytochrome P-450 is not well understood and deserves further
study.
Acknowledgment
The author greatly appreciates the extensive editorial effort put
forth by Prof. I. W. F. Davidson, Department of Physiology and
Pharmacology, Bowman Gray School of Medicine.
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