Oxidation Of Testosterone Research Articles

OMEPRAZOLE HYDROXYLATION BY CYP2C19 AND CYP3A4: PREDICTION TOWARDS HUMAN LIVER ACTIVITIES USING THE DATA OF RECOMBINANT P450 ENZYMES

Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Omeprazole 5-hydroxylation by liver microsomes of a human sample which contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, while a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive towards fluvoxamine and anti-CYP2C9 antibodies. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal P450 enzymes. When recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 μM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Different contributions of CYP2C19 and CYP3A4 In the oxidation of testosterone and progesterone by human liver microsomes are also reported.

High Rates of Substrate Hydroxylation by Human Cytochrome P450 3A4 in Reconstituted Membranous Vesicles: Influence of Membrane Charge

CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem. Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testosterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroyl-phosphatidylcholine micelles was unsuccessful. A higherVmaxfor nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence theKm.It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane.

Effects of erythromycin and roxithromycin on oxidation of testosterone and nifedipine catalyzed by CYP3A4 in human liver microsomes.

Roxithromycin and erythromycin were incubated with rat and human liver microsomal or reconstituted cytochrome P450 (P450 or CYP) monooxygenase systems in the presence of an NADPH-generating system, and the effects of these chemicals on testosterone 6 beta-hydroxylation and nifedipine oxidation activities were compared with those of typical CYP3A4 inhibitors including ketoconazole, troleandomycin, and gestodene. Roxithromycin and erythromycin were found to be relatively weak inhibitors of testosterone 6 beta-hydroxylation and nifedipine oxidation activities by rat and human liver microsomes or by reconstituted systems containing CYP3A4/5. Formation of an inhibitory P450-metabolite complex was determined spectrally by incubating troleandomycin with human liver microsomes; the extents of the complex formation were lesser in liver microsomes of humans than those of rats treated with dexamethasone. Erythromycin and roxithromycin were also activated slightly by rat liver microsomes to form P450.Fe(II)-metabolite complex, although these chemicals caused very little or undetectable levels, respectively, of spectral changes by human liver microsomes even when a human sample which contained relatively high levels of CYP3A4 was used. These results suggested that roxithromycin and erythromycin were relatively less potent to inhibit CYP3A4-catalytic activities in human liver microsomes, because of their low capabilities to form P450.Fe(II)-metabolite complex.

Sulfoxidation of mercapturic acids derived from tri- and tetrachloroethene by cytochromes P450 3A: a bioactivation reaction in addition to deacetylation and cysteine conjugate beta-lyase mediated cleavage.

In the present study we investigated the formation of sulfoxides from N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (N-Ac-TCVC), N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-Ac-1,2-DCVC), and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (N-Ac-2,2-DCVC), which are formed in the glutathione dependent bioactivation of tri- and tetrachloroethene. The first aim was to elucidate the enzymes involved in these oxidation reactions. N-Ac-TCVC, N-Ac-1,2-DCVC, and N-Ac-2,2-DCVC are oxidized to the corresponding sulfoxides mainly, if not exclusively, by cytochrome P450 enzymes in liver microsomes of untreated male rats, since no role for the flavin-containing monooxygenase (FMO) could be demonstrated by heat inactivation experiments and by the use of n-octylamine. The sulfoxidation rates were increased when using liver microsomes of phenobarbital and dexamethasone pretreated male rats as well as liver microsomes of dexamethasone pretreated female rats, while no sulfoxide formation was observed in liver microsomes of untreated female rats, suggesting an involvement of cytochrome P450 3A. Also, troleandomycin, a specific chemical inhibitor for cytochrome P450 3A, drastically reduced sulfoxidation rates. The observed rates of sulfoxidation also correlated well with the rates of oxidation of testosterone at the 6-beta-position, a specific marker for P450 3A activity. The second aim of this study was to compare the cytotoxicity of the sulfoxides with the cytotoxicity of the corresponding mercapturic acids in isolated rat renal epithelial cells. Both mercapturic acids and the corresponding sulfoxides were cytotoxic. Cytotoxicity of the mercapturic acids could be blocked by (aminooxy)acetic acid (AOAA), an inhibitor of cysteine conjugate beta-lyase, while the cytotoxicity of the sulfoxides was not influenced by this treatment. Moreover, the sulfoxides were significantly more cytotoxic than the corresponding mercapturic acids at equimolar doses. The results show that mercapturic acids derived from TRI and PER are oxidized to sulfoxides by microsomal monooxygenases from rat liver. The cytotoxicity of the produced sulfoxides could not be reduced by AOAA, consistent with a role of the sulfoxides as direct acting electrophiles (i.e., Michael acceptor substrates).

Roles of Cytochrome b5in the Oxidation of Testosterone and Nifedipine by Recombinant Cytochrome P450 3A4 and by Human Liver Microsomes

NADH-dependent testosterone 6β-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5(b5), NADH–b5reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH–b5reductase with NADPH–P450 reductase produced an eightfold increase in testosterone 6β-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6β-hydroxylation activities in the presence of NADPH–P450 reductase but resulted in complete loss of the activities in the absence of NADPH–P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH–P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5and NADH–b5reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH–P450 reductase. Anti-human b5immunoglobulin G (IgG) (purified using rabbit b5affinity chromatography) inhibited testosterone 6β-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH–P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2stimulated rates of reduction of b5by NADPH–P450 reductase, but not by NADH–b5reductase, in reconstituted systems. These results suggest that b5is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH–P450 reductase are accelerated by complexation with substrates and b5is supported in this study.

Effect of several metallothionein inducers on oxidative stress defense mechanisms in rats

One mechanism by which chemicals cause cellular injury is the formation of reactive oxygen species. In vitro studies have shown that metallothionein (MT), a small metal-binding, sulfhydryl-rich, readily inducible protein, can scavenge reactive oxygen species, especially hydroxyl radicals. Nevertheless, whether or not MT protects against oxidative stress in the intact animal is not known. Experimental induction of MT could help to clarify this question, however, it is unclear whether agents that induce MT also influence known antioxidant systems. Therefore, the present study was designed to determine whether the well-known MT inducers are specific for induction of MT or whether they might also influence other hepatic systems that protect against oxidative stress. Male rats were administered cadmium chloride (Cd; 30 pmol/kg s.c.), zinc chloride (Zn; 1000 pmol/kg s.c.), α-hederin (α-H, 30 pmol/kg s.c.) or lipopolysaccharide (LPS; 1 mg/kg s.c.) 24 h prior to measurement of antioxidant systems. Zn and α-H increased hepatic GSH concentration 20% and 55%, respectively. Cd significantly increased, whereas LPS reduced, the activities of selenium-dependent glutathione peroxidase and glutathione reductase. Glutathione S-transferases were not altered by any of the inducers. Cd also increased DT-diaphorase activity. Cd, Zn and α-H all decreased catalase activity 20–35%, while the activity of Superoxide dismutase was unaffected by the inducers. The amount of total cytochrome P450 enzymes and cytochrome b 5 were decreased by LPS, Cd and α-H, while Zn appeared to have no effect. The activities of P450 enzymes towards testosterone oxidation were also decreased by LPS, Cd and α-H. In conclusion, all four MT inducers examined affect systems known to protect cells against oxidative stress. Therefore, using these chemicals to determine the in vivo role of MT in protecting against oxidative stress poses difficulties.

Suppression of Liver Cytochrome P450 by α-Hederin: Relevance to Hepatoprotection

This study was designed to determine the protective effects of α-hederin on chemical-induced liver injury in CF-1 mice and to evaluate cytochrome P450 suppression by α-hederin as a means of protection. α-Hederin pretreatment (30 μmol/kg, sc × 3 days) protected mice from acetaminophen-, bromobenzene-, carbon tetrachloride-, furosemide-, and thioacetamide-induced liver injury, without affecting the hepatotoxicity of chloroform and dimethylnitrosamine. To examine the role of P450 in hepatoprotection by α-hederin, liver microsomes were prepared 24 hr following the last dose of α-hederin treatment (10 and 30 μmol/kg, sc × 3 days). Treatment of mice with α-hederin produced a dose-dependent suppression of liver cytochrome P450 (30-50%) and cytochrome b5 (20-30%) levels, as well as NADPH-cytochrome c reductase activity (15-25%). α-Hederin treatment also decreased the activities of P450 enzymes, such as 7-ethoxyresorufin O-dealkylation (65%), 7-pentoxyresorufin O-dealkylation (50%), coumarin 7-hydroxylation (40%), 7-ethoxycoumarin O-deethylation (45%), caffeine N3-demethylation (30-50%), chlorzoxazone 6-hydroxylation (35-55%), and the oxidation of testosterone to 2 α-, 6 α-, 15 α-, 15 β-, 16 α-, 16 β-, and 18/12 α-hydroxyltestosterone, androstenedione, and 6-dehydroxytestosterone (25-60%). Consistent with these observations, the levels of CYP1A, CYP2A, and CYP3A enzymes were also suppressed, as determined by immunoblotting with antibodies against rat P450 enzymes. These results demonstrate that treatment of mice with α-hederin decreases the levels and activities of several P450 enzymes. The suppression of P450 appears to be one of mechanisms by which α-hederin protects mice from the hepatotoxicity of some chemicals.

Expression of a Male-Specific Cytochrome P450 Isozyme (CYP2C11) in fa/fa Zucker Rats: Effect of Phenobarbital Treatment

The present study determined the effect of genetic obesity and phenobarbital (PB) treatment on the expression and regulation of the hepatic cytochrome P450 enzyme (CYP2CL1) in Fa/? and fa/fa Zucker rats. Hepatic CYP2C1 1 levels as determined by Western immunoblotting and associated enzymatic activity (testosterone oxidation at the 2α position) were significantly lower in untreated fa/fa Zucker rats compared with that observed in Fa/? Zucker rats. There was no significant difference in the constitutive CYP2C11 steady-state mRNA level hybridizable to the cDNA (P450 16α) or specific oligonucleotide probe (Northern and slot blot analyses) between fa/fa and Fa/? Zucker rats. The depressed constitutive CYP2C11 protein levels in fa/fa rats may be attributed to their low plasma testosterone and growth hormone levels; however, lack of differences in CYP2C11 steady-state mRNA suggest post-transcriptional regulatory mechanism(s). Treatment with PB further suppressed hepatic CYP2C11 protein levels and activities in both fa/fa and Fa/? Zucker rats in comparison with that seen in controls. The level of CYP2C11 steady-state mRNA was significantly higher after treatment with PB in Fa/? Zucker rats, while no change was observed in fa/fa animals. The mechanism by which PB treatment fails to increase CYP2C11 steady-state mRNA levels in the fa/fa Zucker rat is unknown; however, it may share a common molecular basis with the defect in nuclear transcription rate previously observed with CYP2B1/2B2.

Cytochrome P450 2B Enzyme (CYP2B) Induction Defect Following Phenobarbital Treatment in the fa/fa Zucker Rat: Molecular Characterization

The present study describes the mechanism of the dampened induction of the CYP2B1 and CYP2B2 genes following phenobarbital treatment in the phenotypically obese fa/fa Zucker rat. The fa/fa Zucker rat demonstrated a threefold lower level of CYP2B1/2B2 enzyme induction, as indicated by reduced testosterone oxidation at the 16β position and resorufin formation from pentoxy- and benzyloxyresorufin, protein concentration (Western blot analysis), and steady-state mRNA levels (Northern and slot blot analyses) following in vivo treatment with phenobarbital than the Fa/? littermate controls. A primary hepatocyte cell culture system was used to determine if the dampened induction of the CYP2B1/2B2 enzyme is dependent on hormonal influences. Phenobarbital-treated (0.75 mM) hepatocytes from fa/fa Zucker rats showed approximately a three-fold lower induction response based on measurements of CYP2B1/2B2 (R-17 cDNA probe) and CYP2B1 (oligo probe) mRNAs. In order to evaluate whether this dampened response was at the level of transcriptional activation or initiation, as opposed to altered message stability, we measured the rate of transcription of CYP2B1/2B2 genes in nuclei from cultured hepatocytes during run-off experiments. Compared to Fa/? controls, the fa/fa Zucker rat had a greater than threefold lower nuclear transcription rate of CYP2B1/2B2 mRNA. These results suggest that the defective induction of the CYP2B1 and CYP2B2 genes exists at the transcriptional level in the mutant obese fa/fa Zucker rat. These data provide strong evidence that at least two genes are involved. Multiple gene involvement would suggest that the defect is not due to a mutation of the CYP2B gene cis-acting sequence. Instead, the lack of binding of a trans-acting factor, the presence of a repressor, or a defect in transcriptional activation is more likely the molecular mechanism(s) for this enzyme induction defect.

The effects of sex hormones on the immune system in pregnancy.

The immune system is affected by sex steroids. Immunoglobulin synthesis, T-cell subsets, and a variety of cytokines are targets for sex steroids. Hormonal variations are found in patients with rheumatic diseases. These variations include the hydroxylation of estrone and the oxidation of testosterone, both of which are altered in rheumatic disease. These changes are magnified in the pregnant state. More estrogen metabolites of the C-16 variety can bind to cells (lymphocytes and erythrocytes) and his reaction is accelerated in pregnancy. The role of the catechol estrogens is discussed. These compounds may be important to some of the vasospastic symptoms seen during pregnancy in patients; moreover, oxidation of testosterone to weak androgens is found in women with systemic lupus erythematosus (SLE). Overall, interesting observations and conclusions will evolve from a better understanding of sex hormone metabolism and its effects on the immune system in pregnancy.

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)

Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1.

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16 alpha-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male greater than female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2 beta-, 6 beta-, and 15 beta-hydroxylation of testosterone.

The effect of budesonide and triamcinolone acetonide on hepatic microsomal testosterone metabolism in the rat

The hepatic microsomal metabolism of testosterone was studied in male rats after treatment with either budesonide or triamcinolone acetonide for 13 weeks. The in vitro metabolism was determined using a testosterone concentration of 35 nM which is comparable to the levels found in plasma. It was shown that the total microsomal testosterone metabolism was decreased in budesonide-treated rats and increased in rats treated with triamcinolone acetonide. The testosterone metabolites produced were measured and thus it was revealed that budesonide treatment brought about its effect through a 50% decrease in the activity of steroid 5α-reductase, but did not affect other reductive enzymes, or the oxidation of testosterone. Triamcinolone acetonide treatment decreased steroid 5α-reductase activity by 95% and also decreased the activities of steroid 3α- and 3β-reductases by more than 90%. In addition, treatment with triamcinolone acetonide caused a 50% increase in the oxidative metabolism of testosterone, which resulted in the observed increase in total testosterone metabolism. The presence of 0.1 μM budesonide in the microsomal incubations was without effect on testosterone metabolism. However, 0.1 μM triamcinolone acetonide inhibited testosterone oxidation by 65%, without affecting the reductive pathway of testosterone metabolism.

Cytochrome P450 IIIA1 (P450p) requires cytochrome b 5 and phospholipid with unsaturated fatty acids

In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b 5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2β-, 6β-, and 15β-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1) 2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b 5. A polyclonal antibody against purified cytochrome b 5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b 5 caused a ∼60% inhibition of testosterone 2β-, 6β-, and 5β-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by ∼,;70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b 5 and phospholipid containing unsaturated fatty acids.

The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers

Changes in the isoenzyme pattern of cytochrome P450 during culture were investigated in primary cultures of rat hepatocytes, measuring specific enzyme activities in microsomes prepared from cultured cells as well as in intact monolayers. Assays of 7-ethoxyresorufin O-deethylation (EROD), 7-pentoxyresorufin O-depentylation (PROD), aniline 4-hydroxylation (AH) and the specific regioselective hydroxylation of testosterone were used as representatives of the activities of seven isoenzymes of cytochrome P450. The isoenzyme profile expressed as catalytic activities was qualitatively and quantitatively similar in microsomes obtained from freshly isolated hepatocytes in comparison with microsomes obtained from whole livers of untreated rats. There was a relatively high activity in EROD, AH and the oxidation of testosterone at the 7α, 2α, 6β, 16α and 17 sites (androstenedione). During culture, these microsornal enzyme activities declined at a similar rate to ca. 50% of the activities of microsomes prepared from freshly isolated hepatocytes after 24 hr and to 15% after 96 hr. The overall decline of cytochrome P450-dependent activities during culture was not accompanied with gross changes in catalytic profile. Determining the same drug-metabolizing activities directly in intact hepatocyte monolayers revealed a much higher metabolic rate for all measured P450-dependent activities. The profile of the catalytic activities was essentially the same as measured in microsomes prepared from cultured hepatocytes. The relatively low activity towards the 7α site of testosterone measured in intact hepatocytes, however, remained constant during culture. Determination of enzyme activities directly in intact hepatocytes is a convenient way of studying changes in monooxygenase activities of different P450 isoenzymes in vitro.

Effects of cytochrome P-450 monooxygenase inducers on mouse hepatic microsomal metabolism of testosterone and alkoxyresorufins

The effects of treatment with phenobarbital, l,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), pregnenolone-16/ga-carbonitrile (PCN), 3-methylcholanthrene (3-MC) and isosafrole on the hepatic microsomal formation of nine monohydroxy metabolites of testosterone and the O-dealkylation of the ethyl and pentyl ethers of resourfin were evaluated in adult male C57BL/6J and DBA/ 2NCR mice. In both strains, phenobarbital, TCPOBOP and PCN induced testosterone 2β-, 6β-, 15β-and 16/gb-hydroxylases up to 5-fold, while phenobarbital and TCPOBOP increased the rate of dealkylation of pentoxyresorufin by approximately 30-fold. However, phenobarbital and TCPOBOP did not exhibit identical patterns of induction for the testosterone oxidation reactions. Hepatic microsomes from C57BL/6J mice treated with TCPOBOP displayed a depression in 6α-testosterone hydroxylase activity, which was also observed in PCN-treated animals, whereas phenobarbital-treated mice exhibited an elevation in this monooxygenase activity. A dose of TCPOBOP (0.5 μmol/kg) previously demonstrated to represent an ED 50 for mouse aminopyrine N-demethylase activity was also found to approximate the ED 50 for pentoxyresonifin O-dealkylase activity in the C57BL/6J mouse. Isosafrole or 3-MC treatment had little effect on testosterone metabolism or pentoxyresorufin O-dealkylase activity in either strain, while 3-MC induced ethoxyresorufin O-deethylase activity in C57BL/6J but not DBA/2NCR mice. This study confirms that TCPOBOP is a potent cytochrome P-450 inducer which most closely resembles phenobarbital in its mode of action. However, TCPOBOP and phenobarbital do not evoke identical modulations of cytochrome P-450-dependent monooxygenases in mice.

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 7α-, 16β-, 2α- and 6β-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 ∼ 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. The mechanism by which an increase in ionic strength or pH caused a decrease in testosterone oxidation by cytochromes P-450a, P-450b and P-450h may involve an impairment of the interaction between these enzymes and NADPH-cytochrome P-450 reductase, because the effects of ionic strength and pH were diminished when purified P-450a, P-450b and P-450h were incubated with saturating amounts of NADPH-cytochrome P-450 reductase. The mechanism by which an increase in ionic strength or pH caused an increase in testosterone (and digitoxin) oxidation by cytochrome P-450p is unknown, but may involve a stimulation of the interaction between NADPH-cytochrome P-450 reductase and cytochrome b 5.

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b 5, 200 μg/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 μg/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 m m potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6β-hydroxytestosterone (51%), 2β-hydroxytestosterone (18%), 15β-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1βhydroxytestosterone (3%), 16β-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16β-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited > 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.

Flumecinol, a novel inducer of testosterone 16a-hydroxylation in male rats

1. Flumecinol, a new inducer of the cytochrome P-450 monooxygenases, was studied in rats as a possible effector of liver microsomal testosterone oxidases. The drug enhanced the total content of liver cytochrome P-450 in immature and adult rats. 2. When total testosterone oxidation activity was compared in liver microsomes of treated and untreated rats, no differences in activities were observed in 60-day-old-rats, but a slight decrease was found in 35-day-old treated rats. 3. Several regio- and stereo-specific hydroxylases were modified by flumecinol administration; in 35-day-old rats only 16 alpha-hydroxylation was induced, whereas in 60-day-old rats a slight increase in 2 alpha-hydroxylation was also observed.

Comparison of the effects of acute and subacute treatment of phenobarbital in different strains of mice

A strain specificity has been demonstrated for the effect of subsequent administration of phenobarbital (PB), in which diethylnitrosamine (DENA)-initiated hepatocarcinogenesis was promoted in C3H mice, inhibited in B6C3F1 (C57BL × C3H) and not affected in C57BL mice. A correlation has been established between the ability of barbiturates and hydantoins to promote tumor formation and their ability to induce liver growth, hepatic DNA synthesis and mixed function oxidase activities. Therefore, we examined in these 3 strains of mice and in C3B6F1 (C3H × C57BL) mice the effect of PB administered in their drinking water for 4 days or 28 days. The liver weight to body weight ratio was increased by PB in all types of mice. Microsomal protein concentrations were increased in C57BL mice after 28 days of treatment, in C3H after both 4 days and 28 days and in B6C3F1 after 4 days of treatment. No effect upon microsomal protein content was observed in C3B6F1 mice. DNA content was increased in C3H mice, both in the 4-day and 28-day treatment groups, while the other strains showed either a decrease or no difference from control. DNA synthesis was elevated in all strains of mice after 4 days of treatment with PB, however, after 28 days of treatment there was either a much reduced increase (C57BL and C3B6F1) or no difference (C3H and B6C3F1) from controls. In all 4 types of mice after 4 and 28 days of treatment, PB increased the concentration of cytochrome P-450, the activity of aminopyrine-N-demethylase (AmDm) and 7-ethoxyresorufin-O-deethylase (ErDe) and the oxidation of testosterone (T). The oxidative metabolites of T were similar in the 4 types of mice.