Evidence for the involvement of a distinct form of cytochrome P450 3A in the oxidation of digitoxin by rat liver microsomes.

The preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1. J. Biochem. Toxicol., 7 (43-52).) described the regulation of two rat liver microsomal proteins (50- and 51-kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51-kDa 3A protein were usually paralleled by changes in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation. The present study demonstrates that age- and sex-dependent changes in the 50-kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50-kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this conclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone-16 alpha-carbonitrile (PCN), which induces both the 50-kDa and the 51-kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16- to 18-fold) in the 6 beta-hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN-induced rats with chloramphenicol caused a approximately 70% decrease in liver microsomal testosterone 6 beta-hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51-kDa protein) was also inhibited by chloramphenicol in a time- and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized troleandomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for the involvement of a distinct form of cytochrome p450 3a in the oxidation of digitoxin by rat liver microsomes

The preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1. J. Biochem. Toxicol, 7 (43–52).) described the regulation of two rat liver microsomal proteins (50- and 51-kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51-kDa 3A protein were usually paralleled by changes in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation. The present study demonstrates that age- and sex-dependent changes in the 50-kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50-kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2β-, 6β- and 15β-hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this coclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone-16α-carbonitrile (PCN), which induces both the 50-kDa and the 51-kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16- to 18-fold) in the 6β-hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN-induced rats with chloramphenicol caused a ～70% decrease in liver microsomal testosterone 6β-hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51-kDa protein) was also inhibited by chloramphenicol in a time- and reduced nicotinamite adenine dinucleotide phosphate (NADPH)-dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized trole-andomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes. We propose that the 2SbT-, 6β-, and 15β-hydroxylation of testosterone by rat liver microsomes is primarily catalyzed by the 51-kDa 3A proteins (either 3A1 or 3A2 depending on the source of microsomes), whereas digitoxin oxidation is primarily catalyzed by the 50-kDa protein.     

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.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes

The influence of age, sex, and hormonal status on the expression of eight rat hepatic cytochrome P-450 (P-450) isoenzymes was evaluated by both catalytic and immunochemical methods. The male specificity of P-450 2c(male)/UT-A, the major microsomal steroid 16 alpha-hydroxylase of uninduced rat liver [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], was shown to reflect its greater than or equal to 30-fold induction at puberty in male but not in female rats. The female specificity of P-450 2d(female)/UT-I was shown to reflect its developmental induction in females. P-450 PB-2a/PCN-E was shown to mediate greater than or equal to 85% of microsomal steroid 6 beta-hydroxylase activity; the male specificity of this P-450 largely reflects its developmental suppression in female rats. Neonatal gonadectomy and hormonal replacement experiments established that neonatal androgen "imprints" or programs the male rat for developmental induction of P-450 2c(male)/UT-A, for maintenance of P-450 PB-2a/PCN-E, and for suppression of P-450 2d(female)/UT-I, all of which occur in male rats at puberty. By contrast, the expressed levels of P-450 isoenzymes PB-1/PB-C, 3/UT-F, PB-4/PB-B, ISF-G, and beta NF-B were mostly unaffected by the rats' age, sex, and hormonal status. Studies on the sex specificity of P-450 induction established that the response of these latter five isoenzymes to the P-450 inducers phenobarbital, beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile, and isosafrole is qualitatively and quantitatively equivalent in females as in males.     

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.     

Effects of pregnenolone-16 alpha-carbonitrile on drug metabolizing enzymes in hypophysectomized female rats

Treatment of intact and hypophysectomized female rats with pregnenolone-16 alpha-carbonitrile (PCN) resulted in a significant increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, the total cytochrome P-450 concentration, as measured by CO difference spectra, was increased to a greater extent in hypophysectomized rats than in intact rats. Total cytochrome P-450 was found to be 0.82 +/- 0.16 vs 2.43 +/- 0.31 nmoles/mg protein for control and PCN-treated hypophysectomized rats, respectively, and 0.68 +/- 0.23 vs 1.28 +/- 0.05 nmoles/mg protein for control and PCN-treated intact rats respectively. The concentration of metyrapone complex in microsomes from intact control and PCN-treated rats was found to be 0.4 +/- 0.11 vs 1.88 +/- 0.23 M respectively. Treatment of hypophysectomized rats with PCN resulted in an approximate 10-fold increase in the concentration of the metyrapone complex (0.42 +/- 0.15 M for control and 4.46 +/- 0.44 M for PCN-treated). Microsomal NADPH and NADPH cytochrome c reductase activities were also altered by PCN-treatment. Aminopyrine demethylase activity was stimulated approximately three-fold by PCN treatment in both intact and hypophysectomized rats. Benzphetamine demethylase activity was not significantly affected by PCN treatment. The results of these studies suggest that the absence of the pituitary gland can markedly influence PCN induction of cytochrome P-450 in the liver in female rats. PCN also differentially affects microsomal mixed-function oxidase activities associated with drug and xenobiotic metabolism.     

Rat liver microsomal progesterone metabolism: evidence for differential troleandomycin and pregnenolone 16 alpha-carbonitrile inductive effects in the cytochrome P-450 III family

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.     

Lack of inducibility of brain monooxygenase activities including parathion desulfuration

The ability of phenobarbital and beta-naphthoflavone to induce parathion desulfuration, aminopyrine N-demethylation, and NADPH-cytochrome-c reductase activity in the brain and liver of male and female rats was investigated. Activities of all three enzymes were found in similar levels in both the mitochondrial and microsomal fractions of brain. There were no sex differences in brain activities. Liver activities were from 10- to 30-fold higher than brain activities when computed on a tissue-wet-weight-equivalent basis. Although exposure to both inducers increased all three enzyme activities and cytochrome P-450 in liver, neither inducer increased the enzyme activities in mitochondrial or microsomal brain fractions of either sex. Thus, these brain monooxygenase activities appear to be refractory to induction by two classical types of cytochrome P-450 inducers. This lack of inducibility could serve to protect the animal against environmentally enhanced increases in the activation of xenobiotics to neurotoxic metabolites, such as parathion desulfuration to paraoxon.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone

The effect of pituitary factor on the constitutive and inducible levels of hepatic phenobarbital (PB)-inducible major cytochrome P-450, P-450b and P-450e, in male and female rat livers was studied by immunoblot analyses. Although only trace amounts (approximately 4 pmol/mg protein) of P-450b and P-450e were detected in untreated adult rats, hypophysectomy increased the contents of P-450b and P-450e 58- and 14-fold, respectively, in male rats and 118- and 30-fold, respectively, in female rats. The increases were also observed in treatment with dexamethasone, which suppressed the pituitary function. Treatment with PB increased more effectively the hepatic contents of P-450b and P-450e, but their contents were still 4-fold higher in the male than the female. Treatment of hypophysectomized female rats with PB increased the contents of P-450b and P-450e 4-fold higher than the contents in PB-treated nonhypophysectomized female rats. Consequently, the sex-related difference in their contents was reduced less than 1.4-fold in the hypophysectomized rats treated with PB. Similar results were also obtained from the quantitation of microsomal O-pentylresorufin O-depentylation and testosterone 16 beta-hydroxylation. Either intermittent injection or continuous infusion of human growth hormone, but not of ovine prolactin, into hypophysectomized male and female rats decreased the contents of both cytochromes. These results indicate that growth hormone acts as a repressive factor for the constitutive and inducible levels of P-450b and P-450e in a manner different from the regulation of P-450-male and P-450-female.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Induction of cytochrome P-450 forms by prolonged administration of nifedipine

In experiments on male Wistar rats it has been found that nifedipine applied in a dose of 10 mg/kg body weight i.p. daily for 20 days did not significantly increase the total amount of cytochrome P-450 but markedly increased the 7 alpha-, 16 beta- and 6 beta-hydroxylation of androstenedione in liver microsomes, suggesting the induction of cytochromes P-450a, P-450b and P-450p respectively. The induction of cytochrome P-450b was also confirmed immunochemically with polyclonal antibodies against cytochrome P-450b/e.     

Induction of the hepatic microsomal and nuclear cytochrome P-450 system by hexachlorobenzene, pentachlorophenol and trichlorophenol.

The application of hexachlorobenzene (HCB), pentachlorophenol (PCP) and 2,4,5-trichlorophenol (TCP) to female rats led to an induction of both the microsomal and the nuclear cytochrome P-450 system in the liver. The increase of th mixed-function hydroxylase activities examined (7-ethoxycoumarin deethylase, 7-ethoxyresorufin deethylase, NADPH-dependent cytochrome c reductase, aminopyrine demethylase, benzpyrene hydroxylase) did not correlate strictly with the cytochrome P-450 content. Depending on the inducers and the substrates used, the content and the activity of the cytochrome P-450 were essentially smaller in the nuclei than in the microsomes. It was striking that in the nuclei those activities (benzpyrene hydroxylase, 7-ethoxyresorufin deethylase, 7-ethoxycoumarin deethylase) were preferably induced which can be attributed to the methyl-cholanthrene-induced form of the cytochrome P-450 (cytochrome P-448). These results suggest, also in the light of findings of other authors, the induction of different species of cytochrome P-450 in the nuclei and microsomes.     

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.     

Structural and regulatory analysis of the male-specific rat liver cytochrome P-450 g: repression by continuous growth hormone administration

Complementary DNA clones encoding the male-specific rat liver cytochrome P-450 g have been isolated by cross-hybridization with sequences from the female-specific rat liver cytochrome P-450 15 beta. Tissue distribution analysis indicates the liver as the organ with major expression of this cytochrome P-450 gene. Minimal P-450 g expression was also detected in prostate, kidney, heart, and brain. A developmental analysis reveals liver expression in the 8-week-old male and to a lesser extent in the 4-week-old male, but no detectable expression is seen in females of these ages or in 1- and 2-week-old rats from both sexes. Hypophysectomy of female rats dramatically increases hepatic expression of P-450 g, whereas continuous GH administration represses hepatic expression in male or female hypophysectomized rats. In similarity to P-450 15 beta and P-450 16 alpha, therefore, the cytochrome P-450 g gene in liver is GH regulated.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.