Induction of testosterone 16β-hydroxylase in rat liver microsomes by phenobarbital pretreatment

Oxidation products of testosterone in control rat liver microsomes were 16α-, 2α-, 6β-, 7α-hydroxytestosterone and 4-androstene-3,17-dione, but no 2β-hydroxytestosterone was detected. Increased testosterone 16β-hydroxylase activity and 4-androstene-3,17-dione production were found upon incubation of testosterone with phenobarbital-pretreated rat liver microsomes.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

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.     

Induction of liver cytochrome P-450 (CYP) 3A in male and female rats by a steroidal androgen receptor antagonist,Zanoterone

The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6β-, 7α-, and 16α-hydroxytestosterone (6β-, 7α-, and 16α-OHT) and the de-alkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes form rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and β-naphthoflavone (β-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6β-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than that produced by PB or β-NF (⊆threefold increases). Z (high dose), PB, and β-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2–2.9-fold in females. β-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6β position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6β-OHT suggests that Z induced CYP 3A activity. Theses findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily. © 1997 John Wiley & Sons, Inc.     

The facile bioconversion of testosterone by alginate-immobilised filamentous fungi

The potential for biotransformation of the substrate 17β-hydroxyandrost-4-en-3-one (testosterone) by six filamentous fungi, namely, Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and Whetzelinia sclerotiorum ATCC 18687, was investigated. In this study both free cells and macerated mycelia immobilised in calcium alginate were utilised and the results (products, % yields, % transformation) were compared. In general the encapsulated cells of the microorganisms effectively generated products similar to those found using free cells. However, with immobilised macerated mycelia, isolation of the transformation products was expedited by the simple work up procedure, and their purification was facilitated by the absence of fungal secondary metabolites. Twenty seven analogues of testosterone were generated, wherein the androstane skeleton was functionalised at C-1β, -2β, -6β, -7α, -11α, -14, -15α, -15β and -16β by the moulds. Redox chemistry was also observed. Seven of the analogues, 6β,11α,17β-trihydroxyandrost-4-en-3-one, 6β,14α,17β-trihydroxyandrost-4-en-3-one, 2,6β-dihydroxyandrosta-1,4-diene-3,17-dione, 2β,16β-dihydroxyandrost-4-ene-3,17-dione, 2β,6β-dihydroxyandrost-4-ene-3,17-dione, 2β,15β,17β-trihydroxyandrost-4-en-3-one and 2β,3α,17β-trihydroxyandrost-4-ene, were novel compounds. Five others, namely, 7α,17β-dihydroxyandrost-4-en-3-one, 6β,14α-dihydroxyandrost-4-ene-3,17-dione, 15α,17β-dihydroxyandrost-4-en-3-one, 16β,17α-dihydroxyandrost-4-en-3-one and 2β,16β,17β-trihydroxyandrost-4-en-3-one, were fully characterised for the first time.     

Differential modulation of hepatic cytochrome P-450 enzymes in rat and syrian hamster by 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl

The effects of a single injection (40 mg/kg) of 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7α-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6β, 16α, and 16β-hydroxylases, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15α, and 16α-hydroxylases, and androstenedione production, whereas ErND, testosterone 6β, and 7α-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6β2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6β2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.     

Determination of testosterone and 6β-hydroxytestosterone by gas chromatography–selected ion monitoring–mass spectrometry for the characterization of cytochrome p450 3A activity

A method for the determination of testosterone and its metabolite, 6β-hydroxytestosterone, in liver microsomal incubates employing gas chromatography with selected ion monitoring mass spectrometric detection (GC–SIM–MS) has been developed. The method is more rapid than previously reported methods. Testosterone and its metabolites are extracted from the incubation mixture in a single step with methylene chloride. The method does not require derivatization and testosterone and its metabolites are separated on a HP-5MS fused-silica capillary column in less than 15 min. The retention times of testosterone (m/z 288), methyltestosterone (m/z 302), and 6β-hydroxytestosterone (m/z 304) are approximately 12.7, 12.8, and 13.4 min, respectively. There are no interferences from other known CYP450 metabolites of testosterone. In addition, the selectivity and specificity of the mass spectrometer helps eliminate possible interferences from drugs and new chemical entities evaluated using this methodology. Calibration curves for testosterone and 6β-hydroxytestosterone are linear from 0.25 to 100 μM. Extraction recoveries are better than 92% for both analytes and the internal standard, methyltestosterone. Over the course of five separate runs, within-day and inter-day precision (expressed as relative standard deviation) was less than 5% for all concentrations of testosterone and 6β-hydroxytestosterone. Accuracies ranged from 95.8 to 105.8% for testosterone and 94.6 to 104.2% for 6β-hydroxytestosterone. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog liver microsomes.     

Steroid metabolism by human breast and rat mammary carcinomata

The metabolism of dehydroepiandrosterone (DHA) and testosterone by both human breast carcinomata and dimethylbenzanthracene (DMBA)-induced rat mammary carcinomata has been investigated.The rat and human carcinomata converted DHA to testosterone and both DHA and testosterone to 5α-dihydrotestosterone, 5α-androstanediol and 16α-hydroxytestosterone. Tentative evidence is also presented to indicate that some rat adenocarcinomata can convert androgen precursors into estradiol-17β.Although quantitative differences between incubations occurred, the spectrum of steroid transformations was similar in both human and rat tumours. The DMBA-induced rat tumour may therefore prove to be a valuable experimental model for human carcinoma tissue with regard to further steroidogenic studies.     

Glutathione Oxidase from Bovine Skim Milk Membranes: Its Copurification with γ-Glutamyltransferase

Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2α-, 2β-, 6β-, 7β-, 11β-, 12β-, 15β-, 16α-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2α-, 2β-, 6β-, 11β-, 15β-, 16α-, and 17-hydroxylation are reactions common to both human and bacterial P450s. Most of the testosterone hydroxylation catalyzed by bacterial P450s is on the β face.     

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4

Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy- and 19-oxo derivatives and the resulting Δ(1,10)-, Δ(5,10)-, and Δ(9,10)-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ～10-fold higher than reported for 3α-reduction by 3α-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5β-, and 9β-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the Δ(1,10)-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the Δ(4,5) bond is remarkable in shifting the course of hydroxylation from the 6β-, 2β-, 1β-, and 15β-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(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-16α-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 > 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 2β, 6β-, and 15β-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 2β-, 6β-, and 15β-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 2β-, 6β-, and 15β-hydroxylation of testosterone.     

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.     

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V_max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K_m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b₅ increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels (r²=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.     

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6β,11α-dihydroxyprogesterone or 17β-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5β-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17β-alcohols; activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) has significant impact on the amount of the formed ring-D δ-lactone.     

Identification by capillary gas chromatography-mass spectrometry of eleven non-ecdysteroid steroids in the haemolymph of larvae of Sarcophaga bullata

$\text{O-}\text{Pentafluorobenzyloxime}$ (OPFB)-heptafluorobutyrylester (HFB) derivatives and $\text{OPFB}\text{-O-}\text{methyloxime}$ (MO)-trimethylsilylether (TMS) derivatives of non-ecdysteroid steroids were prepared from haemolymph extracts of last instar larvae of the fleshfly Sarcophaga bullata. Using a negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS) technique the following steroids could be identified: progesterone, testosterone, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, androst-5-ene-3β,17β-diol, androstenedione, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, 17α-hydroxyprogesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxyprogesterone. Although the technique is very sensitive, estrogens could not be detected. These results suggest an active metabolism of progesterone and testosterone.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.     

Kinetic study of HCG induced decrease of microsomal 7α-hydroxylase activity in rat testes

Microsomes from rat testes were incubated with varying concentrations of ¹⁴C labelled testosterone and androstenedione. The production of 7α(-hydroxytestosterone and 7α-hydroxyandrostenedione was followed; K_m and V_m values were calculated from Lineweaver-Burk curves.A sustained treatment of rats with HCG resulted in a considerable decrease of the maximal 7α-hydroxylation rate (V_m) whereas the K_m value was not changed. V_m of microsomes from normal rats, when incubated with microsomes from HCG-treated animals, was also decreased substantially. It is concluded that HCG-induced depression of 7α-hydroxylation capacity of testicular microsomes is at least in part due to non-competitive inhibition of the enzyme.     

Neonatal phenobarbital imprinting of rat hepatic microsomal testosterone hydroxylations

The effects of neonatally administered phenobarbital (PB) on adult rat hepatic microsomal metabolism of testosterone were examined in 60-, 90-, and 120-day-old animals. Phenobarbital-induced imprinting was evident at all ages; however, female rats appeared to be more susceptible to the neonatal effects of phenobarbital than did male rats. In 60-day-old female rats, increased testosterone 2α-hydroxylase activity was observed in microsomes from noninduced rats, whereas decreased testosterone oxidation at all positions except 2α and 15β was observed in microsomes from Aroclor 1254-induced rats. The decreased oxidation of testosterone at specific sites in response to Aroclor 1254 induction was quite dramatic, decreasing the activities to near or below control levels. By contrast, phenobarbital-treated 60-day-old males exhibited approximately a twofold increase in Aroclor 1254-induced 16α and 2α-hydroxylase activities. The pattern of changes in testosterone metabolism observed in phenobarbital-treated animals was different at both 90 and 120 days from that at 60 days. Only minor alterations in the oxidation of testosterone were observed in 90-day-old animals of either sex. In 120-day-old animals the greatest effects of neonatal phenobarbital exposure were on Aroclor 1254–induced 16β-hydroxylase activities. In induced female rats 16β-hydroxylase activity was again decreased to noninduced levels, while in induced male rats a fourfold increase in this activity was observed. These results demonstrate that neonatal exposure to phenobarbital can alter both constitutive and Aroclor 1254–induced testosterone metabolism in adult rats and that the effects of neonatal phenobarbital exposure are age and sex differentiated.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes: Gas chromatographic–mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17α-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17α-methyltestosterone, produced 6β-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography–mass spectrometry (GC–MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6β-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6β-hydroxyl metabolites with testosterone and 17α-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6β-hydroxylation.