Age-dependent expression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Characterization of a cDNA for rat P-450g, a highly polymorphic, male-specific cytochrome in the P-450IIC subfamily

Cytochrome P-450g (IIC13) is a highly polymorphic, male-specific rat liver isozyme which is a member of the P-450IIC subfamily. A cDNA, c5126 (1737 bp), for P-450g was isolated from a lambda gt11 library synthesized from (+g) male rat liver mRNA. Sequence analysis of the clone, c5126, revealed an open reading frame of 1473 nucleotides, which encodes for a 490 amino acid polypeptide possessing the 30 NH2-terminal residues reported for cytochrome P-450 (M-3) (P-450g) [Matsumoto et al. (1986) J. Biochem. 100, 1359-1371]. A high degree of sequence similarity (greater than 70%) exists between c5126 and the published sequences of cDNAs for members of the IIC subfamily, while its sequence similarity to other subfamilies (IA, IIB, and IIIA) was much lower (less than 55%). RNA blot analysis utilizing an oligonucleotide probe specific for P-450g revealed that P-450g mRNA was expressed in livers of male but not female Sprague-Dawley (CD) and ACI rats, indicating that the sex difference was regulated pretranslationally. Furthermore, expression of P-450g mRNA was age dependent in livers of male ACI rats (a homozygous, phenotypically high P-450g strain). However, the mRNA for P-450g was expressed equally in livers of outbred male CD rats representing either the high (+g) or the low (-g) phenotype and of inbred ACI rats (+g) representing the high phenotype, indicating that the defect in (-g) rats does not reflect differences in expression of P-450g mRNA.     

Inactivation of rat liver microsomal steroid hydroxylations by 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine: evidence for selectivity among steroid-inducible cytochrome P450IIIA forms

Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6- trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) via destruction of the heme prosthetic group. This is an important component of these compounds' porphyrinogenic mechanism. In an attempt to map the P-450 isozyme selectivities of DDC analogues, we have examined the effects of these compounds on the regioselective and stereoselective hydroxylation of androstenedione (AD) and progesterone (PG) in rat liver microsomal systems. In microsomes from phenobarbital-treated male rats, DDC analogues did not cause time-dependent inactivation of AD 7 alpha-hydroxylase, AD 16 beta-hydroxylase, and PG 21-hydroxylase, selective markers for P450IIA 1/2, IIB1, and IIC6, respectively. In contrast, DDC analogues were effective inactivators of PG 2 alpha-hydroxylase and steroid 6 beta-hydroxylases, selective markers for P450IIC11 and IIIA forms, respectively. We conclude that differences in porphyrinogenicity observed with various DDC analogues are not likely to be due to the selective destruction of different P-450 isozymes by different analogues, but rather to properties of the DDC analogues themselves. 4-Ethyl DDC was found to be capable of discriminating between P450IIIA subfamily forms. In microsomes from untreated male rats, which express P450IIIA2 but not IIIA1, 4-ethyl DDC inactivated both AD and PG 6 beta-hydroxylases. However, in microsomes from dexamethasone-treated female rats, which express P450IIIA1 but not IIIA2, no inactivation of the steroid 6 beta-hydroxylases was observed. Thus, 4-ethyl DDC appears to be a potentially valuable tool for differentiating between P450IIIA forms.     

Pituitary regulation of the male-specific steroid 6 beta-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretranslational leve;

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.     

Hepatic P450 expression in hypothyroid rats: differential responsiveness of male-specific P450 forms 2a (IIIA2), 2c (IIC11), and RLM2 (IIA2) to thyroid hormone

Studies carried out in hypophysectomized adult rats have demonstrated that both thyroid hormone and GH can suppress hepatic expression of the steroid 6 beta-hydroxylase P450 2a (IIIA2). The present study further characterizes the influence of thyroid hormone on the expression of P450 2a and two other male-specific hepatic P450s, a steroid 2 alpha/16 alpha-hydroxylase, designated P450 2c (IIC11), and a steroid 15 alpha-hydroxylase, designated P450 RLM2 (IIA2). These studies were carried out in rats rendered hypothyroid by treatment with methimazole, which allows for the nonsurgical depletion of circulating T4, and in hypophysectomized rats. Hypothyroidism led to an increase in hepatic P450 2a (IIIA2) protein and mRNA in both male and female rats that was fully reversed by T4 replacement. In contrast, hypothyroidism decreased by 70-80% the expression of P450 2c (IIC11) activity and mRNA, but did not significantly alter the expression of P450 RLM2 (IIA2). The decrease in P450 2c (IIC11) was not reversed by T4 replacement, suggesting that it is a consequence of the loss of plasma GH pulses that occurs secondary to hypothyroidism. In agreement with these findings, T4 given to hypophysectomized rats partially suppressed the expression of P450 2a (IIIA2) mRNA, but not P450 2c (IIC11) or P450 RLM2 (IIA2) mRNA. A more complete suppression of P450 2a (IIIA2) mRNA as well as P450 2c (IIC11) mRNA was achieved when the hypophysectomized rats were treated with T3 at a supraphysiological, receptor-saturating dose. Although GH administered to intact male rats by continuous infusion fully suppressed all three male-specific P450 proteins and their mRNAs, the same treatment given to hypothyroid rats was only partially suppressive in the case of P450 2a (IIIA2) and P450 RLM2 (IIA2), unless combined with T4. In the case of P450 2c (IIC11), substantial suppression of the residual P450 present in hypothyroid rats was achieved by treatment with GH alone, despite persistent thyroid hormone deficiency. These studies demonstrate that while thyroid hormone is a negative regulator of P450 2a (IIIA2) expression and is required for the full suppression of that P450 and P450 RLM2 (IIA2) by the continuous plasma GH profiles associated with adult female rats, the suppression of P450 2c (IIC11) by continuous plasma GH is largely independent of the presence of thyroid hormone.     

Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes

We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E.G. Schuetz et al., 1990 J. Biol. Chem. 265, 1188-1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or "phenobarbital-like" inducers to the culture medium inhibited induction of P450b/e mRNA (46-90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with delta-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1(IIIA1) and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1) mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1) mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1) and P450PCN2(IIIA2) are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis.     

Differences in the cytochrome P-450 isoenzymes involved in the 2-hydroxylation of oestradiol and 17 alpha-ethinyloestradiol. Relative activities of rat and human liver enzymes.

The metabolism of oestradiol and 17 alpha-ethinyloestradiol to their 2-hydroxy derivatives is an important determinant in their biological effects. In this work, we have investigated which rat or human cytochrome P-450 isoenzymes are involved in catalysing these reactions. Oestradiol 2-hydroxylation was catalysed by a wide variety of rat cytochrome P-450s from gene families P450IA, P450IIB, P450IIC and P450IIIA. Interestingly, 17 alpha-ethinyloestradiol, which only differs structurally from oestradiol at a position distant from the site of oxidation, was metabolized predominantly by members of the P450IIC gene subfamily. In order to establish which enzymes are responsible for the oxidation of these substrates in man, antibodies to rat liver cytochrome P-450 isoenzymes were used to inhibit these reactions in a panel of human liver microsomal fractions. Also, possible correlations between the proteins recognized by the antibodies and the 2-hydroxylation rate were determined. These experiments provide evidence that 2-hydroxylation of 17 alpha-ethinyloestradiol in man is catalysed by cytochromes from the P450IIC, P450IIE and P450IIIA gene families. In contrast, the major proteins involved in oestradiol metabolism are from the P450IA gene family, although members of the P450IIC and P450IIE gene families may also play a role. These data demonstrate that the differences in the capacity of rat P-450s to metabolize these substrates are also present in the comparable enzymes involved in man, and that a variety of factors will determine the rate of disposition of these compounds in man.     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a second gene product related to rabbit cytochrome P-450 1

A cDNA, p1-88, was cloned from a library constructed using rabbit liver mRNA. Sequence analysis indicates that p1-88 is highly similar (congruent to 95%) to the cDNA, p1-8, that encodes rabbit liver cytochrome P-450 1 and that had been isolated from the same library. The predicted amino acid sequence of the protein encoded by p1-88, P-450 IIC4, differs at 25 of 487 amino acids from that encoded by p1-8. P-450 IIC4 was synthesized in vitro using rabbit reticulocyte lysate primed with RNA transcribed from the coding sequence of p1-88 using a bacteriophage T7 RNA polymerase/promoter system. P-450 IIC4 reacts with two monoclonal antibodies that recognize P-450 1 and exhibits the same relative electrophoretic mobility as P-450 1. In contrast, the reactivity of a third monoclonal antibody recognizing P-450 1, 1F11, toward P-450 IIC4 synthesized in vitro is greatly diminished. The latter antibody extensively inhibits hepatic progesterone 21-hydroxylase activity and recognizes phenotypic differences among rabbits in the microsomal concentration of P-450 1. This difference in the immunoreactivity of P-450 IIC4 and P-450 1 with the 1F11 antibody suggests that P-450 IIC4 does not contribute significantly to hepatic progesterone 21-hydroxylase activity. S1 nuclease mapping demonstrates that the expression of mRNAs corresponding to p1-88 are expressed to equivalent extents in rabbits exhibiting high and low expression of mRNAs corresponding to p1-8. Thus, P-450 1 differs from the protein encoded by p1-88, in its regulation, immunoreactivity, and by inference its catalytic properties although the amino acid sequences of P-450 1 and P-450 IIC4 are highly similar (congruent to 95%).     

Regulation of the expression of the sex-specific isoforms of cytochrome P-450 in rat liver

The hepatic metabolism of steroid hormones and of xenobiotics frequently depends on the expression of the sex-specific isoforms of cytochrome P-450 and on differences in sex hormones. Following biochemical, immunological and molecular biological investigations, it was shown that in adult rat liver there exist at least four male-specific and one female-specific isoforms of cytochrome P-450. The designation of these sex-specific genes is IIC11, IIIA2, IIC13 and IIA2 in males, and IIC12 in females. The irreversible programming of the expression of these isoforms of cytochrome P-450 in adulthood occurs during the perinatal period of life, and is named enzyme imprinting. One of the main factors that regulates the expression of the sex-specific isoforms of cytochrome P-450 is the level of androgens in the blood. Castration of adult rats decreased the level of the male isoforms of cytochrome P-450 and the activity of the monooxygenase enzyme system that remained higher than in intact females. The mechanism of enzyme imprinting can be explained as follows: neonatal androgens program the secretion of hypothalamic hormones, somatostatin and growth-hormone-releasing factor. These factors determine the type of growth hormone secretion in adult rats, and this controls the type of sex-specific isoforms of cytochrome P-450 expressed in adulthood. Metabolic regulation similar to that outlined above was shown to occur for several metabolism-dependent chemical carcinogens. Such a pathway may explain the different sensitivity displayed by male and female rats to treatment with these carcinogenic agents. One possible way of modulating the expression of some isoforms of cytochrome P-450 in adult rats is by treating neonates with specific xenobiotics that change the constitutive expression of neonatal androgens. It appears that this enzyme imprinting plays an important role in determining the individual sensitivity to the carcinogenic effects of chemicals.     

Age-dependent exression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2α-, 2β-, 15α-, 16α-, and 16β-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7α-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible form) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6β-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Mouse liver cytochrome P-450 (P-450IIIAM1): its cDNA cloning and inducibility by dexamethasone.

A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone.     

Inhibition of cholesterol 7 alpha-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC.

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification.     

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.