Pituitary regulation of sex-specific forms of cytochrome P-450 in liver microsomes of rats

Effects of hypophysectomy and treatment with testosterone or estradiol on the sex-specific forms of cytochrome P-450, P-450-male and P-450-female, were examined. The amounts of P-450-male as well as drug oxidation activities were decreased by hypophysectomy of male rats. In female rats, drug oxidation activities were increased by hypophysectomy, which was associated with the disappearance of P-450-female and the appearance of P-450-male. Treatment of hypophysectomized female rats with testosterone or estrodiol effected minor changes in the amounts of P-450-male.     

Diazepam metabolism by multiple forms of cytochrome P-450 in rat liver microsomes

The synthesis of pharmacologically active diazepam metabolites (oxazepam, 4-hydroxydiazepam, N-demethyldiazepam) in liver microsomes of intact and phenobarbital-, 3-methylcholanthrene- and dexamethasone-induced male and female Wistar rats as well as in a reconstituted system with isolated forms of cytochrome P-450 (P-450a, P-450b, P-450c, P-450d and P-450k according to the Ryan nomenclature) was studied. Marked sex-dependent differences in the rates of diazepam metabolism in liver microsomes of intact and induced animals were revealed. The changes in the spectrum of diazepam metabolites in liver microsomes of induced rats (as compared to control animals) were revealed. In a reconstituted system only phenobarbital-induced cytochromes P-450b and P-450k were found to be active participants of diazepam N-demethylation; none of the isoenzymes tested were shown to be involved in diazepam hydroxylation.     

Induction of hepatic cytochrome P-450 mediated alkoxyresorufin O-dealkylase activities in different species by prototype P-450 inducers

The induction of cytochrome P-450-mediated alkoxyresorufin O-dealkylase activities by various xenobiotics was examined in liver from a variety of animal species in order to gain insights into the substrate specificities of the induced P-450s. We found that forms of cytochrome P-450 capable of mediating the O-dealkylation of the short-chain phenoxazone ethers methoxy-, ethoxy- and propoxyresorufin were highly induced by 3-methylcholanthrene-type inducers and by Aroclor-1254 in all species tested, although there were species differences in the relative turnover rates for the various substrates. For example, in hamster liver the turnover rates for the short-chain resorufin ethers decreased in the following order: methoxy greater than ethoxy much greater than propoxy, while in the rat liver almost the exact opposite order was observed: ethoxy = propoxy much greater than methoxy. In contrast, the degree of induction by phenobarbital-type inducers of isozymes catalyzing the O-dealkylation of pentoxy- or benzyloxyresorufin was highly species-dependent. Thus, F344/NCr rats, B6C3F1 mice and NZB rabbits showed the greatest (greater than 20-fold) induction of these activities, either by phenobarbital or Aroclor-1254, while Mongolian gerbils showed intermediate levels of induction and Syrian golden hamsters exhibited very low induction. In the Japanese quail, phenobarbital- or DDT-treatment resulted in minimal induction of pentoxy- or benzyloxyresorufin O-dealkylase activity, although significant induction of the latter activity occurred following treatment with 5,6-benzoflavone or with Aroclor-1254. Since substrate specificities of most enzymes can be rationalized based upon differences in the steric requirements at the enzyme active site, we employed molecular modeling techniques to calculate the molecular dimensions of the alkoxyresorufins. Surprisingly, the minimal energy conformations in vacuo of each of the resorufin ethers examined are essentially planar. However, alternative configurations, especially for the pentoxy- and benxyloxy-ethers, having greater three-dimensional bulk are also energetically possible.     

Inactivation of rat hepatic cytochrome P-450 by spironolactone

Administration of antimineralocorticoid spironolactone (SPL) to rats results in modest destruction of hepatic cytochrome P-450 with parallel loss of heme. This process is accentuated by pretreatment with dexamethasone (DEX), an inducer of cytochrome P-450p and is associated with marked functional loss of cytochrome P-450p-dependent hydroxylases. Cytochrome P-450 destruction may be replicated in vitro when microsomes from DEX-pretreated rats are incubated with SPL and NADPH and is impaired when these rats are given triacetyloleandomycin, an inhibitor of cytochrome P-450p. In vitro SPL-mediated cytochrome P-450 destruction is accompanied by a loss of heme, which appears to be converted to reactive intermediates which covalently bind to microsomes or are converted to polar metabolites.     

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.     

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Testosterone metabolism by microsomal cytochrome P-450 in liver of rats treated with some inducers

1. The stereoselective hydroxylation of testosterone by microsomal cytochrome P-450 and the changes in level of components participated in the microsomal electron transport system were observed in the microsomes induced unique P-450 isozymes. 2. Flavone- and hesperetin-inducible P-450 catalyzed the hydroxylation of testosterone more effectively than other chemicals-inducible ones. 3. The P-450 in all the microsomal preparations tested most rapidly oxidized testosterone to 6 beta-monohydroxy form. 4. Particularly, MC- and BNF-inducible P-450 showed high stereoselectivity on C6-position of testosterone, and PB-, flavone- and hesperetin-inducible one showed that on C2-position of this compound, respectively. 5. This specificity of two flavonoid-inducible P-450 for the formation of 2 alpha- and 2 beta-epimer of monohydroxytestosterone was opposite to each other. 6. The content of P-450 and the activity of NADPH-cytochrome P-450 reductase were high in PB-, MC- and BNF-microsomes, whereas NADH-cytochrome b5 reductase activity was high in two flavonoid-microsomes and the content of cytochrome b5 was not changed except the PB-treated rats. 7. It is suggested that the increasing activities of testosterone hydroxylases in flavonoid-microsomes seems to be closely related to NADH-cytochrome b5 reductase.     

Inhibition of cytochrome P-450-linked monooxygenase systems by naphthoquinones

Several naphthoquinones, except 2-hydroxy-1,4-naphthoquinone, were found to inhibit microsomal cytochrome P-450-linked monooxygenase activities in rabbit liver and human placenta. In particular, 5-hydroxy-1,4-naphthoquinone inhibited placental estrogen biosynthesis more effectively than it did hepatic drug oxidation reactions. There was little contribution by superoxide radicals to these enzyme inhibitions by naphthoquinones. Spectrophotometric studies revealed that naphthoquinones bind to the cytochrome P-450 component of the monooxygenase complex in both microsomal systems, suggesting that the inhibition is caused by direct interaction of these compounds with the heme.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography

14 microsomal cytochromes P-450 were purified from the liver of untreated and phenobarbital- or 3-methylcholanthrene-treated male rats. Following solubilization of microsomes with sodium cholate, poly(ethylene glycol) fractionation and aminohexyl-Sepharose 4B chromatography, cytochromes P-450 were purified by high-performance liquid chromatography (HPLC), using a preparative DEAE-anion-exchange column. The pass-through fraction was further purified by HPLC using a cation-exchange column. Other fractions eluted on preparative DEAE-HPLC were further applied onto an HPLC using a DEAE-column. Five kinds (P-450UT-2-6), four kinds (P-450PB-1,2,4 and 5) and five kinds (P-450MC-1-5) of cytochromes P-450 were purified from untreated rats or rats treated with phenobarbital or 3-methylcholanthrene, respectively. HPLC profiles of tryptic peptides of cytochromes P-450UT-2 and P-450MC-2 were identical and the other profiles obtained from seven purified cytochromes P-450 were distinct from each other. Amino-terminal sequences of eight forms of cytochrome P-450 (UT-2, UT-5, PB-1, PB-2, PB-4, PB-5, MC-1 and MC-5) were distinct except for cytochromes P-450PB-4 and P-450PB-5.     

Microheterogeneity of a male-specific rat hepatic cytochrome P-450: existence of three allozymic forms

Preparations of hepatic cytochrome P-450 h [D. E. Ryan, et al. (1984) J. Biol. Chem. 259, 1239] and cytochrome P-450 2c [D. J. Waxman (1984) J. Biol. Chem. 259, 15481] from outbred Sprague-Dawley rats were analyzed using two-dimensional electrophoresis and in situ peptide mapping. Both preparations consisted of the same isozyme which was previously characterized as a developmentally regulated, male-specific cytochrome P-450 active in the 16 alpha-hydroxylation of steroids. Each preparation evidenced microheterogeneity which was shown, in part, to result from the existence of two genetically determined variant forms of cytochrome P-450 h/2c. Analyses of hepatic microsomes from several inbred strains of rat revealed that each was characterized by a single variant form of this isozyme, with some strains expressing a variant that was not present in Sprague-Dawley rats. Genetic crosses indicated that these electrophoretic variants represent allozymic forms of cytochrome P-450 h/2c which are codominantly expressed at a single autosomal locus. Additional microheterogeneity of each allozymic form of cytochrome P-450 h/2c was shown to result from a specific in vitro modification that may involve limited proteolysis near its C terminus by a microsome-bound protease.     

Gamma-glutamyltransferase induction by dexamethasone in cytochrome P-450-depleted rat liver

The effects of the cytochrome P-450 depletion by cobaltic protoporphyrin IX on the postnatal glucocorticoid-inducibility of the membrane-bound enzyme gamma-glutamyltransferase have been assessed in the rat liver. Dexamethasone-induced gamma-glutamyltransferase activity in 14-, 28- and 77-day-old rats was high, weak and absent, respectively, and inversely correlated with the physiological cytochrome P-450 activity. In the liver acinus, the enzyme was reexpressed by the zone 1 and zone 2 hepatocytes in suckling rats, substantially only by the zone 1-hepatocytes in just weaned rats. Following cytochrome P-450 depletion, gamma-glutamyltransferase induction by dexamethasone was more rapid, more intense and more extended in the liver, acinus, occurring also in the zone 3 hepatocytes in suckling rats, in the zone 2 and a few zone 3 hepatocytes in just weaned rats. Further, the enzyme induction occurred also in adult rats in the zone 1 and in some zone 2 cells. This shows that cytochrome P-450 modulates the extent of hepatic gamma-glutamyltransferase induction by dexamethasone in postnatal rat-hepatocytes. The phenomenon may be consequent on hormone biotransformation changes caused by the cytochrome P-450 depletion.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

Multi-functional property of rat liver mitochondrial cytochrome P-450

To solve the problem of whether a common enzyme catalyzes both 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1 alpha-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol competitively inhibited 25-hydroxylation of 1 alpha-hydroxy-vitamin D3 and vice versa. From these results it was concluded that 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 1 alpha-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.     

Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p

We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of liver microsomal cytochrome P-450 (P-450) examined to date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the major form of P-450 induced in liver microsomes of rats treated with pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies to P-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN, the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and the ethylmorphine and erythromycin demethylase activities. Antibodies to P-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p.     

Identification of cross-linked cytochrome P-450 in rat liver microsomes by enzyme-immunoassay

The topography of microsomal proteins was studied by 2-dimensional gelelectrophoresis. The second dimension was run in the presence of 2-mercaptoethanol, thus allowing detection of proteins previously cross-linked by disulfide bonds as off-diagonal spots. With hepatic microsomes from phenobarbital pretreated rats, several off-diagonal spots were seen. The most intense spot, with a molecular weight of 52,000, was derived from a dimer of this protein. It was identified as cytochrome P-450 (P-450) by a double antibody enzyme-immunoassay. The dimer is probably formed by oxidation of sulfhydryl groups of P-450 molecules during the preparation of microsomes. P-450 can also be cross-linked to form 105,000, 167,000, and 240,000 dal oligomers by treating microsomes with dithiobis(succinimidyl propionate) at 0°C. Cross-linking of P-450 to other proteins was also observed with one-dimensional gel-electrophoresis. The results suggest that the cross-linked proteins are close neighbors of P-450 in the membrane.     

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-2 hydroxylation of prostaglandins [K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489]. The current study examined the regioselectivity of hydroxylation of PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH-P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b5 requirement.