Hormonal Regulation of Liver Cytochrome P450 Enzymes

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9 Hormonal Regulation of Liver Cytochrome P450 Enzymes

David J. Waxman and Thomas K.H. Chang

1. Introduction

Sex differences in hepatic drug metabolism have been known for more than 30 years, based on the early studies of Kato, Conney, Gillette, and others^~^. In rats and certain other species, the rate of drug metabolism is often several-fold higher in males as compared to females as revealed by in vivo pharmacokinetics and as demonstrated in vitro by assaying prototypic phase I c5^ochrome P450 drug substrates, such as ethylmorphine, benzo[a]pyrene, or hexobarbital using isolated liver microsome preparations (Figure 9.1). This sex-dependence of P450 metabolism is most striking in the rat, where sex differences in metabolic rates can be as high as 5-fold with some drug substrates. This finding was initially unexpected, because measurements of total liver P450 content indicated that the overall P450 levels in rat liver tissue are only —20% higher in males compared to females (Figure 9.1). Research carried out during the 1980s resolved this discrepancy with the finding that there are multiple liver-expressed P450 enzymes^' ^, each of which is encoded by a separate gene, and only some of which are expressed in a sex-dependent manner.

Based on the recently published human genome sequence, we now know that there are

57 ftinctional human cytochrome P450 genes, grouped into 17 distinct gene families^. Many of these P450 enzymes catalyze the biosynthesis of physiologically important endogenous substances, such as steroid hormones, whereas others are primarily involved in metabolism of environmental chemicals and other xenobiotics, notably drugs.

From the perspective of foreign compound metabolism, the most important cytochrome P450 ("CYP") genes are those in the CYPl, CYP2, and CYP3 families. These three families encompass

— 15-20 different cytochrome P450 enzymes, and collectively carry out essentially all of the phase I cytochrome P450 metabolic reactions in mam- malian liver^^. As individual liver P450 enzymes were purified and characterized, and subsequently, when their genes were cloned from multiple species, it became apparent that in certain species, such as the rat and mouse, a subset of these drug- metabolizing liver P450s is expressed in a sex- dependent fashion and subject to endocrine control^ ^ Human liver P450 enzyme levels and their associated drug metabolism activities may also be determined, in part, by age, sex, and hormone status^^~^^. Studies of the underlying mechanisms governing the endocrine regulation of rat liver P450 enzymes may thus be of general importance for our understanding of the hormonal

David J. Waxman • Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA. Thomas K.H. Chang • Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC, Canada.

Cytochrome P450: Structure, Mechanism, and Biochemistry, 3e, edited by Paul R. Ortiz de Montellano Kluwer Academic / Plenum Publishers, New York, 2005.

347

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12

^ o

.^ fl 8

N ^

0 E

^ H MALE EZZ2 FEMALE

i li 1. 1

M

1 1

L

M BP HB P450(10X)

Figure 9.1. Sex-differences in rat hepatic microsomal drug metabolism. Data shown are based on enzyme assays in rat liver microsomes using the three indicated xenobiotic substrates: ethylmorphine (EM)"*, benzo[a]

pyrene (BP)^, and hexobarbital (HB)^. Ethylmorphine A^-demethylase and benzo[(3]pyrene hydroxylase activities are expressed as nmol product formed per minute per mg microsomal protein, whereas hexobarbital hydroxylase activity is expressed as nmol product formed per 30 min per g liver. Also shown is hepatic microsomal total cytochrome P450 content, which is expressed as nmol per mg microsomal protein (values multiplied by 10)^. The data are shown as mean ± SD for 4 or 5 rats, except for ethylmorphine A^-demethylase and total P450 which are based on a pool of 6 livers.

regulation of liver-expressed genes in both rodent models and in man. Endocrine-regulated steroid hydroxylase P450s contribute in an important way to foreign compound metabolism in the liver, and studies of their hormonal control may shed light on the underlying basis for the influence of hormone status on a broad range of P450-catalyzed drug metabolism and carcinogen activation reactions.

This chapter reviews studies leading to the identification of the key endocrine regulatory factors and the underlying mechanisms through which these factors operate to control the expression of liver cytochrome P450 enzymes. Primary emphasis is given to studies on the hormonally regulated P450s expressed in rat liver, the best- studied model system. Also discussed are environmental and pathophysiological factors that can perturb hormonal status and the impact of these factors on the expression of sex-dependent cytochromes P450.

2. Steroid Hormones as

Substrates for Sex-Dependent Liver P450s

The precise physiological function of the endocrine-regulated rodent liver P450s is unclear;

however, steroid hormones are metabolized by liver P450 enzymes with a higher degree of regiospecificity and stereoselectivity than many foreign compound substrates^ ^ suggesting that these endogenous lipophiles serve as physiological P450 substrates. The physiological require- ments with respect to steroid hormone hydroxylation differ between the sexes, and not surprisingly, several steroid hydroxylase liver P450s are expressed in a sex-dependent manner^ ^' ^^.

Rat P450 enzymes CYP2C11 and CYP2C12 are prototypic examples of sex-specific liver P450 enzymes, and they have been a major focus of studies of the underlying endocrine factors, as well as the cellular and molecular regulatory mechanisms that govern sex-specific liver gene expression. CYP2C11 is the major male-specific androgen 16a- and 2a-hydroxylase in adult rat liver, and is induced at puberty in males but not females •^' ^^ under the influence of neonatal androgenic imprinting (programming)^ ^ By contrast, the steroid sulfate 15p-hydroxylase CYP2C12 is expressed in a female-specific manner in adult rats^'' ^^.

3. Developmental Regulation of Sex-Dependent Rat Liver P450S

Multiple rat liver cytochrome P450 enzymes are expressed in a sex-dependent manner, subject to complex developmental regulation and endocrine control (Table 9.1). CYP2C11, the major male-specific androgen 2a- and 16a- hydroxylase of adult liver, is not expressed in immature rats but is induced dramatically at puberty (beginning at 4-5 weeks of age) in male but not female rat liver^^' ^^. A similar developmental profile characterizes three other male- specific rat liver cytochromes P450, CYP2A2 (refs [27], [36]), CYP2C13 (refs [28], [37]), and CYP3A18 (refs [25], [38]). By contrast, another

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Table 9 . 1 .

CYP enzyme^

/. Male-specific 2A2

2C11 2C13 3A2 3A18 4A2

//. Female-specific 2C12

Hormonal Regulation of Sex-Dependent Rat Liver P450 Enzymes

///. Female-predominanf 2A1

2C7 3A9 5a-reductase

Testosterone hydroxylase activities^

15a 2a, 16a 6^, 15a 6P,2(3 6(3,2(3, 15P, (see footnote

15P^

7a 16a 6P

—

16a g)

Hormonal regulation^

Androgenic imprinting'^

+ + + + + + + + ND ND

- -

ND ND ND

Thyroid hormone^

+ / - + / - ND - - ND

-

4 - / -

- + + ND + +

''P450 gene designations are based on the systematic nomenclature of ref. [23]. Table is modified from ref [24].

^The major sites of testosterone hydroxylation catalyzed by the individual P450 proteins are shown. Testosterone metabolites specific to the P450's activity in rat liver microsomal incubations are underlined. Based on refs [8], [11], [21], [25], [26] and references therein.

cu_|_ _)_» in(jjcates a positive effect on adult enzyme expression, while " " indicates a suppressive effect. "—" indicates a lesser degree of suppression, while " + / - " indicates no major effect. ND—^not determined in a definitive manner.

'^For further details see refs [27]-[29].

^Based on refs [30]-[34].

^Purified CYP2C13 exhibits high testosterone hydroxylase activity in a reconstituted enzyme system, but this enzyme makes only marginal contributions to liver microsomal testosterone hydroxylation^^.

^CYP4A2 catalyzes fatty acid co-hydroxylation, but does not catalyze testosterone hydroxylation.

^15P-hydroxylation of steroid sulfates. CYP2C12 also catalyzes weak testosterone 15a- and la-hydroxylase activities.

'Liver expression of these enzymes is readily detectable in both male and female rats, but at a 3-10-fold higher level in females as compared to males.

adult male-specific liver P450, the steroid 6|3- hydroxylase CYP3A2, is expressed in prepubertal rat liver at similar levels in both sexes, but is selec- tively suppressed at puberty in females^ ^' ^^"^^ The steroid sulfate ISP-hydroxylase CYP2C12 is expressed at a moderate level in both male and female rats at 3 ^ weeks of age. Beginning at puberty, however, CYP2C12 levels are further increased in females while they are fully suppressed in males^^' '^^. Several other female-predominant liver enzymes have also been shown to increase at puberty in adult female rats. These include: CYP2C7 (refs [37], [42]), which catalyzes retinoic acid 4-hydroxylation'^^; CYP3A9 (ref [44]), which catalyzes steroid 6p-hydroxylation^^;

and steroid 5a-reductase, which is not a cytochrome P450 enzyme but plays an important role in steroid metaboHsm in adult female rats^^' ^^.

Finally, CYP2A1 is a female-predominant steroid 7a-hydroxylase that is expressed in both sexes shortly afler birth, but is suppressed at puberty to a greater extent in male than in female rat liver^^' ^^' ^^

(Table 9.1). Each of these sex-dependent P450 enzymes is primarily expressed in liver, although low-level extrahepatic expression may occur in some cases"^^^^. Studies of liver P450 expression during senescence have revealed a general loss of gender-dependent enzyme expression that reflects a decrease in male P450 levels and an increase in expression of female-predominant P450 enzymes

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in aging male rats^^' ^^. This appears to be related to the age-dependent reduction in the secretion of growth hormone (GH) releasing factor and the changes in plasma profile of GH^^, which is a major regulator in the sex-dependent expression of liver CYP enzymes (see Section 4.2).

4. Hormonal Control of Liver P450 Expression

4.1. Regulation by Gonadal Hormones

Initial studies on the endocrine regulation of the liver P450 enzymes demonstrated that the gonadal steroids play an important role in regulating enzyme expression, but only indirectly. Gonadal steroids do not act directly on the liver to confer the sex-dependent pattern of hepatic steroid and drug metabolism and P450 expression, but rather, act indirectly via the hypothalamus, which regulates the pituitary gland and its secretion of the polypeptide hormone, GH (see Section 4.2).

4.1.1. Testosterone

4.1.1.1. Distinct effects of neonatal androgen and adult androgen. Gonadal hor- mones play an essential role in determining the expression of the major sex-specific rat liver P450 forms at adulthood. In the case of testosterone, there are two distinct postnatal developmental periods of hormone production, neonatal and postpubertal, and each period makes a distinct con- tribution to the expression of the sex-dependent liver P450s at adulthood. Castration of male rats at birth eliminates both periods of androgen production, and this in turn abolishes the normal adult expression of each of the male-specific P450s:

CYP2A2 (ref [27]), CYP2C11 (refs [20], [21], [29], [37], [53]), CYP2C13 (ref [28]), and CYP3A2 (refs [21], [29], [53]). CYP2C13 mRNA levels^^

are also abolished in birth-castrated rats, indicating that enzyme expression is regulated at a pre- translational step. Treatment of birth-castrated male rats with testosterone during the neonatal period leads to a partial restoration of the expression of these male-specific P450 forms at adulthood^^' ^^' ^^. A brief period of neonatal androgen

exposure is thus sufficient to "imprint" or irre- versibly program the male rat to express these P450 enzymes later on in adult life. These effects of neonatal androgen on male-specific P450 enzymes are very similar to the androgenic imprinting effects observed in earlier studies of liver microsomal steroid hydroxylase activities'^' 54, 55^ several of which can be associated with specific liver P450 forms^^

However, neonatal testosterone given to birth- castrated male rats only partially restores CYP2C11 (refs [21], [53]) or CYP2C13 (ref [28]) to normal adult male levels, indicating that neonatal androgen alone is insufficient for full adult expression of these male-specific P450s. Con- sistent with this observation, the combination of neonatal androgen treatment with adult androgen exposure results in complete restoration of normal adult male expression of the male-specific P450s (ref [28]). Moreover, testosterone treatment of adult male rats that were castrated either neonatally or prepubertally, can substantially increase the expression of CYP2C11 (refs [29], [53], [56], [57]) and CYP2C13 (ref [28]). However, in contrast to the irreversible imprinting effects of neonatal androgen treatment, the effects of adult androgen exposure are likely to be reversible, as evidenced by the partial loss of CYP2C11 in male rats castrated at adulthood^^' ^' and by the reversal of this loss by the synthetic androgen methyltrienolone^^. Similarly, the continued presence of testosterone at adulthood is also required to maintain normal adult expression of CYP3A2, since castration at 90 days of age reduces hepatic CYP3A2 mRNA levels by >80%, but this can be restored by subsequent administration of testosterone to the adult rat^^. Thus, while neonatal testosterone imprints the rat for expression of the male-specific P450 enzymes beginning at puberty, a time when the demand for P450- dependent liver steroid metabolism is increased, the additional presence of androgen during the pubertal and postpubertal periods is required to maintain full enzyme expression during adult

life^O'^i.

4.1.1.2. Testosterone suppression of female enzymes. In contrast to the positive reg-

ulation by testosterone of the male-specific enzymes, testosterone suppresses expression of the female-specific CYP2C12 as well as the female-predominant enzymes CYP2A1 and

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steroid 5a-reductase. Hepatic CYP2C12 content is reduced in intact, adult female rats exposed chronically to testosterone^^ or to the synthetic androgen methyltrienolone^^. Similarly, treatment of neonatally or prepubertally ovariectomized rats with testosterone, either neonatally or pubertally, results in a major decrease in microsomal steroid 5a-reductase activity^^' ^^. Birth castration of male rats increases the adult levels of hepatic CYP2A1, but testosterone administration to these animals re-masculinizes (i.e., decreases) the levels of this P450 (ref [62]). Androgens thus exert a suppressive effect on liver CYP2A1 expression.

Studies of the effect of testosterone on the expression of the female-predominant CYP2C7 are inconclusive^^' ^^.

4.1.1.3. Mechanisms of testosterone regulation. The mechanisms by which neonatal testosterone imprints the expression of liver P450 enzymes during adulthood are only partially understood. Testosterone's primary effects on liver P450 profiles are mediated by the hypothalamic- pituitary axis^"^ and its control of the sex-dependent pattern of pituitary GH secretion^^' ^^.

Consistent with this conclusion, testosterone has only minor effects on liver enzyme profiles in hypophysectomized rats in most^^ but not all^^' ^^

instances. As discussed in Section 4.2, pituitary GH secretory patterns play a key role in regulating the expression of the sex-dependent P450 forms.

4.1.2. Estrogen

Whereas testosterone has a major positive regulatory influence on the male-specific P450 forms, estrogen plays a somewhat lesser role in the expression of the female-specific and the female-predominant liver P450 enzymes. Ovari- ectomy at birth reduces, but does not abolish, expression of hepatic CYP2C7, CYP2C12, and steroid 5a-reductase in adult female rats^^' ^^' ^^ and normal adult enzyme levels can be restored by estrogen replacement. Ovariectomy during adulthood^^ or neonatal administration of an estrogen receptor antagonist, tamoxifen^ \ reduces hepatic CYP3A9 expression in adult female rats. The reduced expression of CYP3A9 in ovariectomized rats can be restored by estrogen^^. By contrast, estradiol suppresses hepatic CYP2C11 in both intact and castrated male rats^^' ^^. However, the

absence of CYP2C11 in adult female rats is not due to a negative effect of estrogen. Thus, ovariectomy alone does not lead to CYP2C11 expression in female rats^^' ^^' ^^. In male rats, the suppression of CYP2C11 by estradiol may be irreversible, as demonstrated by the major loss of this P450 in adult male rats exposed to estradiol during the neonatal period or at puberty. However, this effect is not a consequence of a direct action of estradiol on the liver, since estradiol does not impact on hepatic CYP2C11 levels in hypophysectomized rats^^. Rather, the effects of estradiol on liver P450 expression involve the hypothalamo-pituitary

^^jg64,72^ ^j^^ j^Qg^ likely result from an estrogen- dependent increase in the interpeak baseline levels of plasma GH^"^' ^^. This effect of estradiol may be sufficient to alter the sex-specific effects of the GH secretory pattern since, as discussed in greater detail below, recognition of a "masculine" GH pulse by hepatocytes requires an obligatory recovery period during which there is no plasma GH and hence no stimulation of hepatocyte GH receptors (GHRs)^^. In addition, estrogen may antago- nize the induction of CYP2C11 by testosterone as suggested by the absence of androgen imprinting of this P450 in intact female rats treated with neonatal or pubertal testosterone^^' ^^. Indeed, the stimulatory effect of testosterone on pulsatile GH secretion can be blocked by the presence of intact ovaries in female rats^^. Interestingly, prepubertal treatment of intact (i.e., non-ovariectomized) female rats with tamoxifen enhances the induction of CYP2C11 and CYP3A2 protein expression by pubertal and postpubertal androgen^^. The precise neuroendocrine mechanisms responsible for the antagonistic effects of estrogen on androgen imprinting remain to be elucidated.

4.2. Regulation by Growth Hormone

4.2.1. Sex-Dependent GH Secretory Profiles

In many species, the pituitary gland secretes GH into plasma in a highly regulated temporal fashion that differs between males and females.

This sex-dependent secretion of GH is most striking in rodents^"*"^^, but key features are conserved in humans^^"^^. In the rat, GH is secreted by the pituitary gland in adult males in an intermittent, or

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pulsatile, maimer that is characterized by high (Figure 9.2(A)). By contrast, in the adult female peaks of hormone in plasma (150-200 ng/ml) rat, GH is secreted more frequently (multiple pitu- each 3.5-4 hr followed by a period of very low or itary secretory events per hour) and in a manner undetectable circulating GH ( < 1-2 ng/ml) such that the plasma GH pulses overlap and the

Females

9:00 11:00 13:00 15:00 17:00

I I I I" I I I I I I I I I' I I I' I I I I I I I I I I I I I I I I I I

9:00 11:00 13:00 15:00 17:00

Figure 9.2. Sex-dependence of plasma GH profiles in adult rats. Shown are plasma GH profiles measured during the course of an 8 hr day in unrestrained and unstressed male (panel A) and female rats (panel B). Data shown are fromref [73].

A GH Pulse Induction oiCYPICll: B Continuous GH Feminization Pulse Frequency Dependence

Male Hx + GH M Hx 6 P 2 P F

2 C 1 1 - mRNA

fir

1 2 3 4 5 6 7 8

Female Hx + GH F Hx 6 P 2 P M

2C11_

mRNA

Mrtl

M + M +

M F hGH rGH M

^ / ; '

W^M

4A2

I

1 2 3 4 5 6 7 8 9

2C11

W 3A2

2C12

Tubulin

1 2 3 4 5 6 7 8 9 10 11

Figure 9.3. Role of GH secretory profiles in the expression of sex-dependent rat hepatic GYP mRNAs. Shown are Northern blots probed with oligonucleotide probes specific to each of the indicated GYP RNAs. Panel A shows the male (M) specificity of GYP2G11, its absence in hypophysectomized rats (Hx) and its induced expression in either male or female (F) hypophysectomized rats given either 2 or 6 pulses (P) of GH/day for 7 days. Data based on ref [73]. Panel B shows the effects of continuous rat (r) or human (h) GH infusion in male rats (lanes 6-10) on the expression of CYPs 4A2, 2G11, and 3A2 (all male-specific; lanes 1, 2, 11 vs lanes 3-5), as well as the induction of GYP2G12. Tubulin RNA is shown as a loading control. Data based on ref [32].

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hormone is continually present in circulation at significant levels (~15^0ng/ml) at nearly all times^'' (Figure 9.2 (B)). Hypophysectomy and GH replacement experiments carried out in several laboratories have demonstrated that these sex- dependent plasma GH profiles are, in turn, responsible for establishing and for maintaining the sex-dependent patterns of liver P450 gene expression in rats^^' ^^' ^^' ^^' ^^ and mice^"^' ^^ (for earlier reviews, see refs [24], [86]). Clinical studies in humans also demonstrate a role for GH^^~^^

and its sex-dependent plasma secretory patterns^^

in regulating P450-dependent drug metabolism.

Studies in the rat model reveal three distinct responses of liver P450s to plasma GH profiles (Figure 9.3).

(1) Continuous plasma GH, a characteristic of adult female rats, stimulates expression of female specific enzymes, such as CYP2C12 and steroid 5a-reductase^^' ^^, and female-dominant liver enzymes, such as CYP2A1, CYP2C7, and CYP3A9 (refs [31], [62], [93], [94]). Hepatic levels of CYP2C12 and steroid 5a-reductase are undetectable in hypophysectomized female rats but can be restored to near-normal female level by continuous GH replacement^^' ^^~^^. This restoration can be achieved with as little as 12-25% of the physiological levels of GH^^. Higher levels of GH are required to induce expression of CYP2C12 and steroid 5a- reductase in hypophysectomized male rats^^.

(2) Intermittent plasma GH pulses, which are characteristic of adult male rats, induce

expression of the male-specific liver enzyme CYP2C11 (Figure 9.3(A)) and its associated testosterone 2a-hydroxylase activity^^' ^^' ^^' ^^. The stimulatory effects of intermittent GH stimulation on this "class I" male P450 enzyme can be distin- guished from the effects of GH pulses on a larger, second group ("class II") male-specific liver P450s (CYP2A2, CYP2C13, CYP3A2, CYP3A18, and CYP4A2). In contrast to the class ICYP2C11, class II male P450s are not obligatorily dependent on GH pulses, as judged by their high level of expression in the absence of GH, as shown in hypophysectomized rats of both sexes27'28,38,4i,97,98,100,101. Nevertheless, liver expression of the class II enzymes CYP2A2 and CYP3A2 is induced when intermittent GH pulses are given to adult male rats that are depleted of circulating GH by neonatal monosodium gluta- mate (MSG) treatment^^^.

(3) Continuous GH exposure exerts major negative regulatory effects on liver P450 enzyme expression, as revealed by the marked suppression of each of the class I and class II male-specific rat P450s following continuous GH treatment of intact male rats (Figure 9.3(B)). In some cases this effect can be achieved at fairly low GH levels, corresponding to only 3-12% of the physiological GH levels present in intact female rats^^. The high level expression of class II P450 mRNAs seen in the absence of GH pulses, that is, in hypophysectomized male rats, is also suppressed by continuous GH, indicating that continuous GH actively suppresses P450 gene expression, and does not simply abolish the stimulatory pulsatile plasma

Table 9.2. Response of Sex-Specific Rat CYPs to GH

GYP F

- _

Intact rats

M

+ + + +

M + G H _ ,

- _

Hypophysectomized rats

F M

-

+ + + + M

+ GH,„,

+ 4-

+ + M + GH,,„,

- + / -

MSG-treated rats

M

- _

M + GHi„,

+ + + 4- CYP2C1F

(Male class I) CYP2A2^

(Male class II) CYPC12^

(Female-specific) + +

F, female; M, male; GH^^j^^, continuous GH; GHj^^j, intermittent (pulsatile) GH.

"+ + " indicates a positive effect, "—" indicates a suppressive effect, or absence of expression, and "+/—" indicates no major effect.

''Data are based on refs [20], [73], [97], [98], [100], [101], [104]-[110].

^Data are based on refs [27], [97], [98], [100], [101], [104], [109]-[111].

'^Data are based on refs [95], [97], [98], [104], [107], [109]-[111].

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GH pattern. GH suppression is also a key determinant of the lower responsiveness of female rats to phenobarbital induction of CYP2B1 (refs [102], [103]), and probably also the lower responsiveness of the females to the induction of CYP4A enzymes by peroxisome proliferators such as clofibrate^^.

The response of the class II male P450 genes to hypophysectomy of female rats, which de- represses, that is, increases liver enzyme levels to near-normal intact male liver levels, demonstrates that the class II male liver P450s are subject to negative pituitary regulation in female rat liver, where their expression is strongly repressed by the near continuous pattern of plasma GH exposure. These patterns of hormonal regulation are summarized in Table 9.2, which presents the responses of repre- sentative sex-specific liver CYPs to continuous and intermittent GH treatment applied to intact, hypophysectomized and neonatal MSG-treated rats.

4.2.2. Transcriptional Effects of GH on CYP Genes

GH regulates liver P450 steady-state mRNA levels (e.g.. Figure 9.3) in parallel with P450 protein and P450 enzyme activity levels, all but ruling out major regulation by translational and post-translational mechanisms, such as GH regulation of P450 protein turnover. Induction of CYP2C12 mRNA by continuous GH requires ongoing protein synthesis^ *^, suggesting either an indirect induction mechanism or a requirement for one or more protein components that may have a short half-life. Analysis of liver nuclear RNA pools demonstrates that unprocessed, nuclear CYP2C11 and CYP2C12 RNAs respond to circulating GH profiles in a manner that is indistin- guishable from the corresponding mature, cytoplasmic mRNAs^^^. Consequently, transport of CYP2C11 and CYP2C12 mRNA to the cytoplasm, and cytoplasmic P450 mRNA stability are unlikely to be important GH-regulated control points for sex-specific P450 expression. More- over, nuclear run-on transcription analyses have established that GH regulates the sex-specific expression of the CYP2C11 and CYP2C12 genes at the level of transcript initiation^^^' ^^^.

Transcription is also the major step for regulation of the male class II CYP2A2 and CYP2C13

mRNAs^^^' ^^^, whose male-specific expression is primarily a consequence of the suppressive effects of continuous GH exposure in adult female rats^^.

Thus, transcription initiation is the key step at which the three distinct effects of GH outlined in Section 4.2.1 are operative: stimulation of 2C11 expression by pulsatile GH, suppression of both class I and class II male-specific P450s by continuous GH, and stimulation of CYP2C12 expression by continuous GH^^^.

Consistent with the finding that GH regulates sex-dependent liver CYPs by transcriptional mechanisms, the 5'-flanking DNA segments of the CYP2C11 (ref [115]) and CYP2C12 genes^^^

both contain specific DNA sequences that interact in a sex-dependent and GH-regulated manner with DNA-binding proteins (putative transcription factors) that are differentially expressed in male vs female rat liver nuclei^^^' ^^^. These DNA sequences are hypothesized to include GH response elements that contribute to the sex-specific transcription of the CYP2C11 and CYP2C12 genes. Two negative regulatory elements ("silencer elements") were also identified in the CYP2C11 promoter; however, their significance with respect to GH regulation and sex-specific P450 expression is as yet unclear^ ^^. Functional studies of the sex-specific CYP promoters and their interactions with liver-enriched and GH-responsive transcription factors are discussed below.

4.2.3. Cellular Mechanisms of GH Signaling

The cellular mechanisms whereby pituitary GH secretory profiles differentially regulate expression of the sex-dependent liver P450s are only partially understood. GH can act directly on the hepatocyte to regulate liver P450 expression, as demonstrated by the responsiveness of primary rat hepatocyte cultures to continuous GH-stimulated expression of CYP2C12 mRNA; however, these effects do not involve IGF-I, a mediator of several of GH's physiological effects on extrahepatic tissues'^^' ''^. Discrimination by the hepatocyte between male and female plasma GH profiles is likely to occur at the cell surface, where a higher level of GHRs (see below) is found in female as compared to male rats'^^. This sex difference in

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cell surface GHR abundance may, at least in part, be due to differential effects of intermittent vs continuous GH stimulation^^^ and could play a role in the activation of distinct intracellular signaling pathways by chronic (female) as compared to intermittent (male) GH stimulation.

4.2.3.1. Significance of GH pulse fi^equency. Studies have been carried out to deter-

mine which of the three descriptive features of a GH pulse—^namely, GH pulse duration, GH pulse height, and GH pulse frequency—is required for proper recognition of a GH pulse as "masculine."

Direct measurement of the actual plasma GH profiles achieved when GH is administered to hypophysectomized rats by twice daily s.c. GH injection (i.e., the intermittent GH replacement pro- tocol most commonly used to stimulate CYP2C11 expression) has revealed broad peaks of circulating GH, which last as long as 5-6 hr^^. These sustained GH "pulses" are effective in stimulating expression of the male-specific CYP2C11, provided that they are not administered in close succession. It is thus apparent that physiological GH pulse duration (<2 hr) is not required to elicit a male CYP response.

Studies carried out in GH-deficient rat models (either dwarf rats or rats depleted of adult circulating GH by neonatal MSG treatment) demonstrate that GH pulse height is also not a critical factor for stimulation of CYP2C11 expression^^^' ^^^. This finding can be understood in terms of the K^ of the GH-GHR complex, which at 10"^^ M (~2 ng/ml)^^^, is only 1% of the peak plasma hormone level in adult male rats. In contrast, GH pulse frequency is a critical determinant for GH stimulation of a male pattern of liver P450 expression, as shown in hypophysectomized rats given physiological replacement doses of GH for 7 days by intermittent intravenous injections at frequencies of 2,4, 6, or 7 times per day^^. Analysis of liver CYP2C11 levels in these rats revealed a normal male pattern of liver CYP2C11 gene expression in response to 6 GH pulses per day (which approximates the normal male plasma GH pulse frequency), as well as in response to GH pulses given at lower frequencies, 2 or 4 times per day (e.g., Figure 9.3). However, hypophysectomized rats are not masculinized by 7 daily GH pulses, indicating that the hepatocyte does not recognize the pulse as "masculine" if GH pulsation becomes too frequent. Hepatocytes thus require a minimum GH off time (—2.5 hr in the hypophysectomized rat model used in these

studies), which implies the need for an obligatory recovery period to effectively stimulate CYP2C11 expression. This condition is not met in the case of hepatocytes exposed to GH continuously (female hormone profile). This recovery period may serve to reset the cellular signaling apparatus, for example, by replenishing GHRs at the cell surface (see below).

4.2.3.2. RoleofGH receptor (GHR). The effects of GH on hepatocytes and other responsive cells are transduced by GHR, a 620 amino acid cell surface transmembrane protein^^^ belonging to the cytokine receptor superfamily^^"^. GHR is comprised of a 246 amino acid extracellular domain that binds GH, a single transmembrane segment, and a 350 amino acid intracellular domain that participates in the intracellular signaling events stimulated by GH^^^' ^^^. X-ray crystal- lographic and other studies establish that a single molecule of GH binds in a stepwise manner to two GHR molecules to yield receptor dimers:

+GHR

GH + GHR -> GH-GHR -> GH-(GHR)2 ^^^^ ^^7 (Figure 9.4).

The four helix bundle protein GH is proposed to initially bind to a single receptor molecule by contacts that involve amino acids comprising GH's Site 1, followed by binding of a second molecule of GHR, which interacts with Site 2 on the GH molecule to give a heterotrimeric GH-(GHR)2 complex. GH-induced receptor dimerization is reversible, and the equilibrium may be shifted in favor of monomer formation in the continued presence of excess GH:

CTH

GH-(GHR)2^2GH-GHR. Other, more recent studies suggest that GH may bind to, and thereby activate a preformed receptor dimer at the cell surface ^^^' ^^^. Independent of whether the receptor dimer is preformed, however, it is clear that the conformational changes that accompany formation of the active GH-(GHR)2 complex are neces- sary, and probably sufficient, for stimulation of GH-induced intracellular signaling events ^^^

(Figure 9.4). Although GHR dimerization is thus required for most GH responses, some GH responses might not require receptor dimerization^^ ^ and indeed, might be mediated by monomeric GH-GHR complexes. Conceivably, the distinct patterns of liver P450 gene expression induced by continuous plasma GH (female GH pattern; CYP2C12 expression) as compared to

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GHR

Physiological [GH]

i

Intermediate

i

High [GH]

Signaling via Protein Tyrosine Phosphorylation

Figure 9.4. Activation of GHR by GH-induced sequential dimerization mechanism. GHR is shown localized in the plasma membrane, and sites 1 and 2 of the GH molecule (see text) are as indicated. The active receptor dimer dominates at physiological GH concentrations. Model based on data presented in ref [128].

pulsatile GH (male GH pattern; CYP2C11 expression) might, in part, arise from distinct GH signaling pathways perhaps stimulated by monomeric (GH-GHR) as compared to dimeric (GH-(GHR)2) hormone-receptor complexes. GH mutants and analogs that bind GHR without effecting fiinctional receptor dimerization'^^~^^^

could be used to test this hypothesis.

In intact male rat liver, GHR internalizes to an intracellular compartment coincident with its stimulation by plasma GH pulses, and then reap- pears at the cell surface at the time of the next hormone pulse'^^' '^^. Other studies suggest that GHR undergoes endocytosis constitutively, that is, in a ligand-independent manner. GHR internalization involves coated vesicles and ultimately takes the receptor to lysosomes for degradation. GHR endo- C5^osis and degradation both require (a) an intact ubiquitin conjugation system, which targets a specific 10 amino acid-long cytoplasmic GHR tail sequence, and (b) 26S proteasome activity, as evidenced by the inhibitory effects of the proteasome

inhibitor IVIG132. Interestingly, although cellular ubiquitination activity is required for receptor endocytosis, GHR itself does not need to undergo ubiquitination, as shown using a mutant GHR devoid of its cytoplasmic lysine residue targets for ubiquitination'^''' '^^. Thus, the ubiquitin-proteasome system is a major regulator of intracellular GHR trafficking.

4.2.4. Role of STAT5b in Sex-Dependent CYP Expression

4.2.4.1. GH signaling pathways involving STAT transcription factors. How does GH impart sex-dependent transcriptional regulation to liver P450 genes? To answer this question, we may consider the following hypotheses: (a) that cell surface GHRs can discriminate between the two tem- porally distinct patterns of plasma GH stimulation, and (b) the receptors can then transduce this infor- mation to the nucleus, where both pulse and

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continuous GH-stimulated transcriptional events occur. Presumably, GH-activated GHR accom- plishes this by activating two distinct pathways of intracellular signaling, one in response to GH pulses and the other in response to continuous GH stimulation (Figure 9.5). Studies of GH-induced signal transduction pathways^^^^^^ have highlighted the importance of the GH-bound receptor dimer in activating JAK2, a GHR-associated tyrosine kinase that initiates several downstream pathways of intracellular protein tyrosine phosphorylation (Figure 9.4).

Other studies have shown that a distinct pattern of nuclear protein tyrosine phosphorylation is induced in rat liver when the effects of the male pulsatile plasma GH hormone profile are compared to those of the female pattern of continuous

GH stimulation^^^. This led to the discovery that an intracellular signaling protein and transcription factor, termed STATSb, is present in its nuclear, tyrosine phosphorylated form at a substantially higher level in male rat liver than in female rat liver^^^. STATs are latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation induced by a variety of cytokines and growth factors, and were first discovered as signal mediators that carry transcription signals into the nucleus in the interferon signaling pathway^"^^.

In hypophysectomized rat liver, where there is no endogenous GH signaling, there is little or no tyrosine phosphorylated STATSb protein in the nucleus; essentially all of the STAT5b protein is found in the C3^osolic fraction, where it resides in a

3.5 hr

I

Male-specific CYP Steroid OHases

(CYP2C11)

hr

I

Female-specific CYP Steroid OHases

(CYP2C12)

Figure 9.5. Distinct intracellular signaling pathways induced by GH are proposed to be activated by plasma GH pulses, leading to male-specific CYP expression (left), and by continuous GH stimulation, leading to female-specific CYP expression (right).

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latent, inactive (non-tyrosine phosphorylated) form. However, when a hypophysectomized rat is injected with a single pulse of GH, STATSb protein appears in the nucleus in its active, tyrosine phosphorylated state within 10-15 min^^^' ^"^^ This tyrosine phosphorylation reaction occurs on STATSb tyrosine residue 699, enabling two STATSb molecules to dimerize via mutual interactions between the phosphotyrosine residue on one STATSb molecule and the SH2 domain (a protein module that recognizes and binds specifically to phosphotyrosine residues) on a second STATSb molecule. The STATSb-STATSb dimer that is thus formed quickly enters the nucleus, where it binds with high affinity to DNA sites upstream of genes that are transcrip- tionally activated in response to the initial GH stimulus (Figure 9.6).

STATSb is not present in the nucleus at all times in male rat liver. Rather, STATSb is repeatedly activated in concert with the onset of each

male plasma GH pulse. It thus undergoes repeated cycles of translocation from the cytoplasm into the nucleus, and then back out to the cytoplasm^^^' ^'^^.

For example, if the liver is excised from a rat killed at the peak of a plasma GH pulse, then STATSb is tyrosine phosphorylated and localized to the nucleus, whereas if the liver is excised from a rat killed at a time point between successive plasma GH pulses, STATSb is inactive and cytoplasmic.

Moreover, in female rats there is generally a much lower level of active, nuclear STATSb protein (~S-10% that of the peak male liver level) ^'^^. This close temporal linkage between plasma GH pattern and the state of liver STATSb activation has been confirmed in intact male rats killed at times shown to be specifically associated with spontaneous peaks or troughs of the plasma GH rhythm^"^"^. The key difference between male and female rat liver is that STATSb is repeatedly, and efficiently, activated by plasma GH pulses in the case of the

GH

Figure 9.6. Role of complex formed by GH, GH receptor, and the tyrosine kinase JAK2 in activation of STATSb by tyrosine phosphorylation. JAK2 tyrosine phosphorylates itself and multiple tyrosines on the cytoplasmic tail of GHR. Several of these sites serve to recruit STATSb to the receptor-kinase complex. STATSb is then tyrosine phosphorylated, whereupon it dimerizes, translocates to the nucleus, and binds to DNA regulatory elements upstream of target genes.

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males, but is inefficiently activated by the more continuous GH profile of the females. The rela- tively weak STATSb activation pathway in female rat liver appears to reflect a partial desensitization of this signaling pathway in response to the chronic presence of GH in plasma (Figure 9.7)^'*^.

4.2.4.2. STATSb gene knockout mouse model. The proposal that STATSb is a critical factor in mediating GH-regulated liver P450 gene

expression is strongly supported by studies carried out in mice that are deficient in STAT5b (STATSb knockout mouse model) ^"^^ (see ref [147] for a review). Disruption of the STAT5b gene results in two striking phenotypes (Figure 9.8). These phenotypes are seen in STATSb-deficient males but not in corresponding females. First, there is a global loss of GH-regulated, male-specific liver gene expression, including male-specific P450

Activation of STATSb by Plasma GH Pulses

Proposed Role in Male-specific Liver Gene Transcriptionl

time

Plasma Membrane

Cytosol

Nucleus

r m :

Transcriptional Activation:

Male-specific LiverCVP Genes

Figure 9.7. Proposed cycle for STATSb activation and deactivation in response to sexually dimorphic plasma GH profiles. GH pulses, but not continuous GH, induce robust STAT5b tyrosine phosphorylation (pTyr). Nuclear STATSb is dephosphorylated by a tyrosine phosphatase and then returned to the cytoplasm, where it may undergo a subsequent round of activation/deactivation.

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Loss of STATSb

Direct

Impaired Pulsatile GH Signaling

in Liver

Indirect

Loss of Male-Characteristic Body Weight Gain Suppression of Male- Specific Gene Expression Stimulation of Female-Specific

Liver Gene Expression

II Impaired Feedback-Inhibition of Pituitary GH Release

Pertubation of Sexually Dimorphic Plasma GH Pattern

Figure 9.8. Impact of STAT5b loss in knockout (KO) mice. Shown in the box at right are the major effects of STAT5b deficiency. These effects all are a direct result of the loss of GH-induced liver STAT5b signaling (model I), rather than an indirect response to impaired feedback-inhibition of pituitary GH release (model 11), as demonstrated by the lack of responsiveness of hypophysectomized STATSb-deficient male mice to GH pulses^^^.

gene expression. Thus, in the absence of STATSb, the liver does not express the male-specific P450s.

Moreover, the expression of female-specific, GH- regulated liver P450s is increased to near-normal female levels in STAT5-deficient male mice.

Thus, the overall pattern of sexually dimorphic liver gene expression is critically dependent on the presence of STATSb. The elevated expression in STATSb-deficient males of female-specific P4S0s indicates that STATSb can serve as a negative regulator of female-specific liver P4S0s, in addition to its positive regulatory effects on male-specific P4S0 genes.

A second phenot3^e exhibited by STATSb- deficient mice is that the male pubertal growth spurt is absent^"^^. This growth deficiency does not emerge until the beginning of puberty, and is not seen in STATSb-deficient females. A male- enhanced pubertal growth spurt is characteristic of all mammals, including humans. In the case of rodents, the pubertal growth spurt is augmented by the strong growth stimulatory effects of the male pattern of pulsatile GH secretion, which explains why this growth response is enhanced in the males. The two major phenotypes that charac- terize STATSb knockout mice are also seen in STATSa/STATSb double knockout mice^"^^ but are not seen in mice where the disruption is limited to the STATS a gene^'*^' ^^^, whose protein-coding sequence is ~90% identical to that of STATSb^^^

Hypophysectomy and GH pulse replacement studies establish that both major phenotypes of STATSb-deficient mice are a direct response to the loss of STATSb-dependent GH signaling in the Hver, as opposed to indirect effects of the loss of STATSb on the overall pattern of GH secretion by the pituitary gland'^^ (Figure 9.8).

4.2.4.3. Interaction of GH-responsive CYP promoters with GH-activated STATSb.

The strong, repeated pulses of nuclear GH- activated STATSb that occur in adult male rats have been proposed to induce binding of STATSb directly to STATS response elements found in promoters of STATS target genes, which may include sex-dependent P4S0 genes, and thereby medi- ate GH pulse-stimulated gene transcription^ ^^.

Consistent with this hypothesis, STATS response elements matching the consensus sequence TTC-NNN-GAA have been found upstream of several male-specific rat liver P4S0 genes, including CYPs 2C11, 2A2, and 4A2 (ref. [1S2]). GH- stimulated CYP promoter-luciferase reporter activity has been demonstrated using the corresponding isolated STATS response elements, although the magnitude of the GH- and STATSb- dependent gene induction is small, generally only

~2-3-fold'^^' '^^. Moreover, although pulsatile STATSb signaling is first seen in young male rats at ~S weeks of age, when liver CYP2C11 expression is first detected, precocious activation of

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STAT5b, achieved in 3-week-old male rats given pulsatile GH injections, does not lead to preco- cious CYP2C11 gene induction^'^^. These and other findings suggest that STAT5b may in part act in an indirect manner, by regulating the tran- scriptional activity of liver-enriched transcription factors (HNFs, hepatocyte nuclear factors) that cooperate with STAT5b to control the expression of sexually-dimorphic liver P450 genes^^^' i^^-^^e 4.2.4.4. Interactions between STAT5b and liver transcription factors regulating sex- specific CYPs. Liver-enriched transcription factors are key determinants of the liver-specific expression of many hepatic P450s (ref. [157]), including sex-dependent P450s. Functional analysis of sex-specific liver CYP proximal promoters has led to the identification of the liver transcription factors HNFla and HNF3p as strong ^ra«5-activators of CYP2C11 (ref. [152]).

By contrast, HNFSp and HNF6 serve as strong

^ra«^-activators of CYP2C12 (refs [155], [158]), with synergistic interactions between these two liver transcription factors leading to an overall ~300-fold activation of CYP2C12 promoter activity^ ^^. Other studies indicate a role for C/EBPa^^^' ^^^ and HNF4^56 in the regulation of CYP2C12 trans- cription. Interestingly, two of the factors regulating CYP2C12 expression, HNF6 and HNF3P, require GH for maximal expression in liver, with HNF6 being expressed at 2-3-fold higher levels in female compared to male rat liver^^^. This sex difference in HNF6 levels is too small to account for the

>50-fold higher level of CYP2C12 in female compared to male liver. Similarly, the STAT5b-responsiveness of male CYP promoter-derived regulatory elements^^^' ^^^ is too weak to account for the high degree of male-specificity (> 100-fold) that characterizes male-specific CYP expression.

Accordingly, additional factors and GH regulatory mechanisms are likely to be required for the sex- specific pattern of gene expression seen in vivo.

One such mechanism may involve an as yet uncharacterized "regulator of sex-limitation"

("Rsl"), which has been defined genetically and appears to repress certain GH-regulated, sex- dependent liver CYP genes in female mouse liver in a manner that is independent of GH action^^^.

Another possible mechanism for GH-regulated sex-specific CYP expression involves interactions between STAT5b and HNFs. For example, studies

of the CYP2C12 promoter have shown that STAT5b can substantially block the induction of promoter activity by HNF3P and HNF6 (ref. [152]). This inhibitory action of male GH pulse-activated STAT5b on HNF3P- and HNF6- stimulated CYP2C12 transcription is consistent with the de-repression (i.e., upregulation) of certain female-specific, GH-regulated mouse P450 genes seen in male STAT5b-deficient mice^"^^' ^^^.

The synergistic interaction between HNF6 and HNF3P in stimulating CYP2C12 promoter activity illustrates how small differences in the levels of these factors in liver cells in vivo (cf. the 2-3- fold higher expression of HNF6 in females) might lead to much larger differences in CYP2C12 gene expression, particularly when coupled to the inhibitory action of GH pulse-activated STAT5b in males. Other studies point to additional com- plexities, based on the potential of STAT5b to upregulate CYP2C12 gene transcription via an upstream pair of STAT5 response elements^^^, and the potential role of an inhibitory, COOH-terminal truncated STAT5 form^^^

4.2.4.5. Downregulation of hepatic STAT5b signaling. Other issues relating to GH and the STAT5b signaling pathway that are of cur- rent research interest include how the cycle of STAT5b activation is turned off at the conclusion of each GH pulse, and how STAT5b is subsequently returned to the cytoplasm in an inactive form, where it apparently waits for ~2-2.5 hr until it can be re-activated by the next pulse of GH (Figure 9.7)^^^. Another important question is why robust activation of the STAT5b signaling pathway is not achieved in female liver. The answer to these questions may, in part, involve an intriguing family of inhibitory proteins, referred to as SOCS and CIS proteins, which turn off signals to various hormones and cytokines, including GH^^^' ^^^. In the case of GH signaling, SOCS proteins bind to the GHR-JAK2 tyrosine kinase complex, enabling them to inhibit GH signaling by a complex series of interrelated mechanisms ^^^. One of these inhibitory factors, CIS, may be induced to a higher level by the continuous (female) GH pattern than by the pulsatile (male) GH pattern. This inhibitor of STAT5b signaling has been implicated in the downregulation of GH-induced STAT5b signaling in liver cells exposed to the female GH pattem^^^

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4.3. Regulation by Thyroid Hormone

4.3.1. Cytochromes P450

Although GH is the major regulator of specific liver P450s, thyroid hormone also plays a critical role. The major thyroid hormones, T3 and T4, pos- itively regulate some^^' ^^ but not alP^ of the female-predominant liver P450 enzymes, while they negatively regulate several of the male- specific enzymes^^'^"^ (Table 9.1). These effects of thyroid hormone are operative at the level of mRNA expression, and are independent of the indirect effects that thyroid hormone has on liver P450 levels as a consequence of its effects on liver GHRs^^^ and its stimulation of GH gene transcription and GH secretion by the pituitary^^^.

Molecular studies of these effects of thyroid hormone have not been carried out.

animals^'^^. The induction of rat hepatic P450 reductase by thyroid hormone in rats occurs by transcriptional ^^^ and post-transcriptional mechanisms ^^^ and appears to involve enhanced protein stability in hyperthyroid rat liver* ^'^. P450 reductase levels are also modulated by thyroid hormone status in several extrahepatic tissues*^^ Conceivably, interindividual differences in P450 reductase levels may occur in response to physiological or pathophysiological differences in circulating th)Toid hormone levels and could be an important contributory factor to individual differences in P450 reductase/c54ochrome P450-catalyzed carcinogen metabolism and carcinogen activation reactions.

5. Alteration of Liver P450 Expression by Hormonal Perturbation

4.3.2. NADPH-Cytochrome P450 Reductase

Thyroid hormone is also required for full expression of NADPH-cytochrome P450 reductase, a flavoenzyme that catalyzes electron transfer to all microsomal P450s. P450 reductase is an obligatory, and oflen rate-limiting electron-transfer protein that participates in all microsomal P450- catalyzed drug oxidation and steroid hydroxylase reactions *^^' '^^. This thyroid hormone dependence of P450 reductase enzyme expression is evidenced by the major decrease (>80% reduction) in liver microsomal P450 reductase activity and P450 reductase mRNA levels that occurs following hypophysectomy'^^ or in response to methimazole- induced hypothyroidism^'''. It is further supported by the reversal of this activity loss when thyroxine (T4), but not GH or other pituitary-dependent factors, is given at a physiological replacement dose'^^' ^^K Restoration of liver P450 reductase activity in vivo by T4 replacement also effects a substantial increase in liver microsomal P450 steroid hydroxylase activities. A similar effect can be achieved when liver microsomes isolated from hypophysectomized rats are supplemented with exogenous, purified P450 reductase, which prefer- entially stimulates steroid hydroxylation catalyzed by microsomes prepared from thyroid-deficient

As discussed earlier in this chapter, many, although not all, liver P450s are under hormone regulatory controls. An individual's circulating hormonal profile can, however, be altered under certain situations, including drug therapy, exposure to chemicals found in the environment, and disease states such as diabetes and liver cirrhosis. The resultant changes in circulating hormone levels or alterations in hormone secretory dynamics could, therefore, influence the expression of specific liver P450s. The following sections describe some of the factors that are known to cause hormonal perturbation and discuss the impact of these changes on liver P450 expression and on P450-dependent drug and xenobiotic metabolism and toxicity.

5.1. Modulation by Drugs

The anticancer drugs cisplatin'^^' ''^^, cyclo- phosphamide''^^' '^^, and ifosfamide'^^ alter the profile of P450 enzyme expression in liver and perhaps other tissues, at least in part due to the hormonal perturbations that these cytotoxic agents induce. Treatment of adult male rats with a single dose of cisplatin depletes serum androgen, and this effect persists for up to 28 days after drug administration'^^. Serum androgen depletion by cisplatin is associated with a feminization of hepatic liver enzyme expression. Thus, cisplatin-treated male rats have elevated levels of the female-predominant