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PubMed2000

Drug metabolism: in vitro biotransformation of anabolic steroids in canines.

Forensic drug testing of anabolic steroids in racing animals is required because of the potential for steroid abuse. Often when the metabolic products of an administered compound have not been identified, the analysis and verification of the administered compound is difficult. The objective of this study was to qualitatively identify the in vitro phase I biotransformation products of anabolic steroids that have a high potential for abuse in canines. The investigated steroids included testosterone, methyltestosterone, mibolerone and boldenone. Steroid biotransformation products were generated using beagle liver microsomes and analysed by high performance liquid chromatography (HPLC)/mass spectrometry (MS) with an electrospray ionization source. Characterization of steroid metabolites was based on HPLC retention, UV and mass spectra. The major testosterone metabolites were identified as androstenedione and 6beta- and 16alpha-hydroxytestosterone. 6beta-Hydroxymethyltestosterone was identified as a major metabolite in the methyltestosterone microsomal incubations. Several mibolerone metabolites were identified as monohydroxylated mibolerones as well as an oxidized mibolerone metabolite. Boldenone metabolites were identified as monohydroxylated boldenones, oxidized boldenone, and testosterone. This information should assist in the determination of anabolic steroid use in canines through the correlation of the urinary metabolites to the administered drug.

Anabolic Agents↗
PubMed2000

An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates.

Cytochrome P450(eryF) (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450(eryF) by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha-, 12-, and 16alpha-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450(eryF)-androstenedione complex. P450(eryF) is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450(eryF) mutants are also attractive templates for the engineering of novel P450 activities.

Amino Acid Substitution↗
PubMed2003

'Open access' generic method for continuous determination of major human CYP450 probe substrates/metabolites and its application in drug metabolism studies.

1. An 'open access' generic high-performance liquid chromatography method was developed for different combination sets each containing specific cytochrome P450 probe substrate and the corresponding metabolite. Method development, optimization and validation were carried out with the following combinations: phenacetin + paracetamol + internal standard (IS, celecoxib), bufuralol + hydroxybufuralol + IS, testosterone + 6beta-hydroxytestosterone + IS, chlorzoxazone + 6-hydroxychlorzoxazone + IS, coumarin + 7-hydroxycoumarin + IS, tolbutamide + hydroxytolbutamide + IS, and diazepam + desmethyldiazepam + IS. 2. The assay procedure involved a simple one-step liquid/liquid extraction followed by reverse phase chromatography (Inertsil ODS 3V column) employing a ternary gradient system and the eluate was monitored by a photodiode array/fluorescence detector. The standard curve for each compound, in the concentration range 0.1-10 microg ml(-1), in various sets was linear (r(2)>0.99) and absolute recoveries of all analytes were >90%. The lower limit of quantification was 0.1 microg ml(-1). The intraday precision and accuracy in the measurements of quality control were <15% relative standard deviation and <15% deviation from nominal values, respectively. 3. Each combination set was tested with individual chemical inhibitors (furafylline, quinidine, ketoconazole, disulfiram, diethyldithiocarbamate, sulphaphenazole and tranylcypromine) and all analytes were well resolved. Overall, the assay is simple, uses conventional instrumentation and provides a scope to analyse all cytochrome P450 combination sets continuously. The application of the method in the cytochrome P450 liability screen of novel compounds is also presented.

Calibration↗
PubMed2004

P-glycoprotein potentiates CYP3A4-mediated drug disappearance during Caco-2 intestinal secretory detoxification.

Human intestinal Caco-2 cell monolayers grown in the presence of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) were used to test the hypothesis that drugs which interact with the apical efflux pump P-glycoprotein (Pgp) may enhance CYP3A4-mediated disappearance of substrates. 6beta-hydroxytestosterone production, a marker of CYP3A4 activity, was approximately 3- and 7-fold greater in 1,25(OH)2D3-treated cells compared to untreated cells when incubated with 50 and 500 microM testosterone, respectively, and was unaffected by the addition of digoxin to reduce Pgp activity. In the presence of digoxin, secretory transport of vinblastine and erythromycin, substrates for both Pgp and cytochrome P450 3A4 (CYP3A4), was significantly reduced, whereas absorptive transport was unaffected. In contrast, no directional transport of testosterone, a substrate for CYP3A4 only, was observed, either in the presence or absence of digoxin. Over 2 h, disappearance of erythromycin and vinblastine from the incubation medium was significantly greater from the basolateral than from the apical compartments. In the presence of digoxin, disappearance of both compounds from the basolateral, but not from the apical compartments, was significantly reduced. In contrast, disappearance of testosterone was unaffected by the addition of digoxin, demonstrating that the effect of digoxin on erythromycin and vinblastine disappearance was via inhibition of Pgp function, rather than on CYP3A4 activity. Thus, evidence is provided for Pgp/CYP3A4 co-substrates, Pgp potentiates CYP3A4-mediated drug disappearance during intestinal secretory detoxification.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Neonatal exposure to xenobiotics alters adult hepatic protein kinase C alpha levels and testosterone metabolism: differential effects by diethylstilbestrol and phenobarbital.

Hepatic enzymes that metabolize endogenous and xenobiotic compounds have been shown to be altered in adult rats that had been exposed to xenobiotics as neonates. Protein kinase C (PKC) is important in intracellular signaling and has been implicated in the regulation of hepatic monooxygenases. Therefore, we examined the effects of neonatal exposure to diethylstilbestrol (DES) and phenobarbital (PB) on hepatic microsomal testosterone metabolism and on the alpha form of protein kinase C (PKC alpha) in adult rats. In adult males, neonatal exposure to DES altered adult testosterone metabolism such that 7 alpha-hydroxylation was increased by 58% but 2 alpha-, 16 alpha-, and 6 beta-hydroxylations and conversion to androstenedione were decreased 31-44%. In contrast, adult males neonatally exposed to PB showed increased (20-27%) testosterone 2 alpha- and 16 alpha-hydroxylations and androstenedione formation, but no effect was observed in the rate of 6 beta- or 7 alpha-hydroxylations. Western blot analyses indicated that cytosolic PKC alpha levels in male rats neonatally exposed to PB were decreased by approximately 63% relative to the vehicle control group but were not significantly altered in the DES males. The PKC alpha levels generally correlated (r = -.75) with 16 alpha-hydroxytestosterone formation in all samples. These results show that neonatal treatment with DES or PB differentially alters hepatic monooxygenase enzyme activities and PKC alpha levels in adult rats.

Animals↗
PubMed1988

Novel properties of human placental aromatase as cytochrome P-450: purification and characterization of a unique form of aromatase.

Aromatase has been purified to homogeneity from human placental microsomes based on detection of its catalytic activities in the eluates from columns of octylamino-Sepharose 4B, hydroxylapatite, Mono S, hydroxylapatite HCA, and Mono Q. The purified preparation shows only one band corresponding to the apparent subunit molecular weight of 51,000 daltons on sodium dodecyl sulfate-polyacrylamide gel. The aromatase in the presence of NADPH and NADPH-cytochrome P-450 reductase converts testosterone to 17 beta-estradiol with the high specific activity of 103 nmol/min/mg of protein. However, whether the preparation is reduced by sodium dithionate chemically or by NADPH and the reductase enzymatically, its reduced, CO-difference spectrum has no peak at about 450 nm and has only a small peak at about 420 nm, probably due to its inactivation in spite of the catalytically full activity in the same preparation. The absolute spectrum of the aromatase exhibits a Soret peak at 423 nm in the absence of testosterone and addition of testosterone to the aromatase sample makes its absorption peak shift gradually from 423 to 393 nm (high spin type peak), which is a usual characteristic in the spectrum of cytochrome P-450. The reconstituted aromatase system efficiently catalyzes aromatization of 4-androstenedione, 19-hydroxy-4-androstenedione as well as testosterone. 16 alpha-Hydroxy-4-androstenedione and 16 alpha-hydroxytestosterone are also aromatized less efficiently and 19-nortestosterone is aromatized least efficiently. The reconstituted aromatase could scarcely oxidize various xenobiotics examined, suggesting a strict and narrow substrate specificity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase↗
PubMed1988

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola.

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.

17-Hydroxysteroid Dehydrogenases↗
PubMed1990

Purification and characterization of two forms of 2,3,4,7,8-pentachlorodibenzofuran-inducible cytochrome P-450 in hamster liver.

Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0.107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[a]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed 7 alpha- and 2 alpha-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5-to a reconstituted system accelerated the formation of 7 alpha-hydroxytestosterone 5.3-fold with hamster P-450L and 2.2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Genetic findings and functional studies of human CYP3A5 single nucleotide polymorphisms in different ethnic groups.

OBJECTIVES: Genetic polymorphisms of cytochromes P450 (CYPs) are a principal reason for inter-individual variations in the metabolism of therapeutic drugs and environmental chemicals in humans. The present study identifies 34 single nucleotide polymorphisms (SNPs) of CYP3A5 including 27 previously unidentified SNPs by direct sequencing of the exons, intron-exon junctions and 5'-upstream region of CYP3A5 from 92 racially diverse individuals (24 Caucasians, 24 Africans, 24 Asians, and 20 individuals of unknown racial origin). RESULTS: Four new CYP3A5 SNPs produced coding changes: R28C, L82R, A337T, and F446S. CYP3A5 R28C occurred in African populations (allelic frequency of 4%). CYP3A5 A337T occurred in Asians (2% allelic frequency), CYP3A5 L82R (occurred in the racially unidentified group) and CYP3A5 F446S (identified in Caucasians with a 2% allelic frequency) were on an allele containing the splice change g.6986A>G known as CYP3A5*3. The newly identified allelic proteins were constructed by site-directed mutagenesis, expressed in Escherichia coli and purified. CYP3A5 L82R was expressed only as denatured CYP420, suggesting it may be unstable. CYP3A5*1 exhibited the highest maximal clearance for testosterone followed by CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S. CYP3A5*1 exhibited a higher V(max) for nifedipine oxidation than CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S. CYP3A5 A337T and CYP3A5 R28C exhibited a 42-64% lower V(max) for nifedipine oxidation than CYP3A5*1. CYP3A5 F446S exhibited a > 95% decrease in the intrinsic clearance for both 6beta-hydroxytestosterone and nifedipine oxidation. CONCLUSION: This study identifies four new potentially defective coding alleles. CYP3A5 F446S is predicted to be more catalytically defective than the splice change alone.

Alleles↗
PubMed2005

Anti-epileptic drug phenytoin enhances androgen metabolism and androgen receptor expression in murine hippocampus.

Epilepsy is very often related to strong impairment of neuronal networks, particularly in the hippocampus. Previous studies of brain tissue have demonstrated that long-term administration of the anti-epileptic drug (AED) phenytoin leads to enhanced metabolism of testosterone mediated by cytochrome P450 (CYP) isoforms. Thus, we speculate that AEDs affect androgen signalling in the hippocampus. In the present study, we investigated how the AED phenytoin influences the levels of testosterone, 17beta-oestradiol, and androgen receptor (AR) in the hippocampus of male C57Bl/6J mice. Phenytoin administration led to a 61.24% decreased hippocampal testosterone level as compared with controls, while serum levels were slightly enhanced. 17beta-Oestradiol serum level was elevated 2.6-fold. Concomitantly, the testosterone metabolizing CYP isoforms CYP3A11 and CYP19 (aromatase) have been found to be induced 2.4- and 4.2-fold, respectively. CYP3A-mediated depletion of testosterone-forming 2beta-, and 6beta-hydroxytestosterone was significantly enhanced. Additionally, AR expression was increased 2-fold (mRNA) and 1.8-fold (protein), predominantly in the CA1 region. AR was shown to concentrate in nuclei of CA1 pyramidal neurons. We conclude that phenytoin affects testosterone metabolism via induction of CYP isoforms. The increased metabolism of testosterone leading to augmented androgen metabolite formation most likely led to enhanced expression of CYP19 and AR in hippocampus. Phenytoin obviously modulates the androgen signalling in the hippocampus.

Androgens↗
PubMed1994

The effect of indobufen on the activities of selected rat liver phase I and phase II drug metabolizing enzymes, peroxisomal beta-oxidation and hepatic glutathione status.

Oral administration of indobufen to male rats for three days at daily doses of 5, 10 and 20 mg kg-1 resulted in no changes in liver total glutathione, cytosolic glutathione S-transferases or microsomal epoxide hydrolase. Reduced glutathione appeared slightly diminished to about 84% of control at the highest dose level. Microsomal cytochrome P450-dependent ethoxyresorufin O-de-ethylase and pentoxy-resorufin de-alkylase activities were decreased to 64% (not significantly) and 67% of control at the lowest dose level. 6 alpha- and 7 alpha-Hydroxytestosterone activities were decreased to 67 and 68% of control at the highest dose level. Cyanide-insensitive peroxisomal fatty acid beta-oxidation was increased to 223, 261 and 232% of control at doses of 5, 10, and 20 mg kg-1, respectively. The results obtained in this study are indicative of the action of indobufen as a weak peroxisome proliferator in male rat liver, and suggest a slight but toxicologically insignificant inhibitory action of this drug on microsomal cytochrome P450-dependent enzyme activities.

Animals↗
PubMed2006

Characterization of cytochrome P450 expression in murine embryonic stem cell-derived hepatic tissue system.

An in vitro system for liver organogenesis from murine embryonic stem (ES) cells has been recently established. This system is expected to be applied to the development of a new drug metabolism assay system that uses ES cells as a substitute for animal experiments. The objective of this study was to elucidate the drug metabolism profiles of the murine ES cell-derived hepatic tissue system compared with those of primary cultures of murine adult and fetal hepatocytes. The expression of the genes of the cytochrome P450 (P450) family, such as Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, Cyp3a11, and Cyp7a1, was observed in the murine ES cell-derived hepatic tissue system at 16 days and 18 days after plating (A16 and A18). To investigate the activities of these P450 family enzymes in the murine ES cell-derived hepatic tissue system at A16 and A18, testosterone metabolism in this system was analyzed. Testosterone was hydroxylated to 6beta-hydroxytestosterone (6beta-OHT), 16alpha-OHT, 2alpha-OHT, and 2beta-OHT in this system, and was not hydroxylated to 15alpha-OHT, 7alpha-OHT, and 16beta-OHT. This metabolism profile was similar to that of fetal hepatocytes and different from that of adult hepatocytes. Furthermore, pretreatment with phenobarbital resulted in a 2.5- and 2.6-fold increase in the production of 6beta-OHT and 16beta-OHT. Thus, evidence for drug metabolic activities in relation to P450s has been demonstrated in this system. These results in this system would be a stepping stone of the research on the development and differentiation to adult liver.

Animals↗
PubMed2006

Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.

CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.

Benzene Derivatives↗
PubMed2002

The effect of cyclophosphamide with and without dexamethasone on cytochrome P450 3A4 and 2B6 in human hepatocytes.

The purpose of this study was to characterize the concentration-response effects of cyclophosphamide (CPA) with and without dexamethasone (DEX; 10 microM) on the expression of CYP3A4 and CYP2B6 in cultured human hepatocytes at concentrations representative of standard- and high-dose CPA therapy (25 to 750 microM). CPA produced concentration-dependent increases in CYP3A4 and CYP2B6 activity and immunoreactive protein that peaked at 250 and 125 microM, respectively, and declined thereafter. The inductive effect of CPA alone and in combination with DEX was greater in magnitude for CYP2B6 compared with CYP3A4. To further examine the inductive effect of CPA on CYP3A4, CPA (250 microM) and DEX (10 microM) alone and in combination were examined in 10 hepatocyte preparations. The combination of CPA and DEX yielded higher rates of 6beta-hydroxytestosterone formation than either agent alone. However, the effect was less than additive in human hepatocyte cultures with relatively high baseline CYP3A4 activity and additive or synergistic in human hepatocyte cultures with relatively low baseline CYP3A4 activity. Induction index was highly correlated with CYP3A4 baseline activity for both CPA (r(2) = 0.75) and CPA plus DEX (r(2) = 0.85). To investigate the potential mechanism for CPA-induced increases in CYP3A4 activity, the ability of CPA alone and in combination with DEX to activate pregnane X receptor (PXR) was explored using transient transfection assays. CPA produced a dose-dependent increase in PXR activation that was maximal at the highest CPA concentration studied (500 microM). The addition of DEX to CPA resulted in a minor increase in PXR activation compared with CPA alone. These results indicate that CPA alone and in combination with DEX differentially induces the expression of CYP3A4 and 2B in a concentration-dependent manner, which may be mediated partially through activation of PXR. The impact of these effects on the efficacy and toxicity of CPA therapy warrants further investigation.

Aryl Hydrocarbon Hydroxylases↗
PubMed2003

In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes p450: role of cyp3a4 and cyp3a5.

Midazolam, triazolam (TRZ), testosterone, and nifedipine have all been widely used as probes for in vitro metabolism of CYP3A. We used these four substrates to assess the contributions of CYP3A4 and CYP3A5 to in vitro biotransformation in human liver microsomes (HLMs) and in recombinant enzymes. Recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) both produced 1-OH and 4-OH metabolites from midazolam and triazolam, 6 beta-hydroxytestosterone from testosterone, and oxidized nifedipine from nifedipine. Overall, the metabolic activity of CYP3A5 was less than that of CYP3A4. Ketoconazole potently inhibited midazolam, triazolam, testosterone, and nifedipine metabolite formation in HLMs and in rCYP3A4. The inhibitory potency of ketoconazole in rCYP3A5 was about 5- to 19-fold less than rCYP3A4 for all four substrates. In testosterone interaction studies, testosterone inhibited 1-OH-TRZ formation, but significantly activated 4-OH-TRZ formation in HLMs and rCYP3A4 but not in rCYP3A5. Oxidized nifedipine formation was inhibited by testosterone in rCYP3A4. However, in rCYP3A5, testosterone slightly activated oxidized nifedipine formation at lower concentrations, followed by inhibition. Thus, CYP3A4 and CYP3A5 both contribute to midazolam, triazolam, testosterone, and nifedipine biotransformation in HLMs, with CYP3A5 being metabolically less active than CYP3A4 in general. Because the inhibitory potency of ketoconazole in rCYP3A5 is substantially less than in rCYP3A4 and HLMs, CYP3A5 is probably less important than CYP3A4 in drug-drug interactions involving ketoconazole and CYP3A substrates.

Cytochrome P-450 CYP3A↗
PubMed2004

Silybin inactivates cytochromes P450 3A4 and 2C9 and inhibits major hepatic glucuronosyltransferases.

Silybin, a major constituent of the milk thistle, is used to treat several liver disorders. Silybin inactivated purified, recombinant cytochromes P450 (P450) 3A4 and 2C9 in a mechanism-based manner. The inactivations were time-, concentration-, and NADPH-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl-)coumarin O-debenzylation activity (P450 3A4) was characterized by a K(I) of 32 microM, a k(inact) of 0.06 min(-1), and a t(1/2) of 14 min. Testosterone metabolism to 6-beta-hydroxytestosterone (P450 3A4) was also inactivated with a K(I) of 166 microM, a k(inact) of 0.08 min(-1), and a t(1/2) of 9 min. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified human P450 2C9 was inactivated with a K(I) of 5 microM, a k(inact) of 0.14 min(-1), and a t(1/2) of 7 min. Parallel loss of heme was observed with both P450s. Activity of both P450 enzymes was not recovered after removal of silybin either by dialysis or by spin gel filtration. In addition, silybin inhibited the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin catalyzed by recombinant hepatic UDP-glucuronosyltransferases (UGTs) 1A1, 1A6, 1A9, 2B7, and 2B15, with IC(50) values of 1.4 microM, 28 microM, 20 microM, 92 microM, and 75 microM, respectively. Silybin was a potent inhibitor of UGT1A1 and was 14- and 20-fold more selective for UGT1A1 than for UGT1A9 and UGT1A6, respectively. Thus, careful administration of silybin with drugs primarily cleared by P450s 3A4 or 2C9 is advised, since drug-drug interactions cannot be excluded. The clinical significance of in vitro UGT1A1 inhibition is unknown.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Androgen metabolism in thymus of fetal and adult rats.

Cytochrome P450 (P450) monooxygenases play a role in target tissue metabolic activation of xenobiotics and/or endogenous compounds, such as vasoactive molecules or hormones. Indeed, tissue-specific metabolism of steroids is important in a variety of organs, including thymus, and may alter tissue-specific functions. Steroids have been shown to regulate thymus growth and function, but surprisingly little is known about expression of the responsible enzyme systems in thymus tissue, nor is the thymus-specific biotransformation of testosterone known. We therefore investigated gene and protein expression, total protein content, and enzyme activity of major P450 isoforms and other key steroid-metabolizing enzymes in thymus tissue of adult and fetal rats. We detected 6 beta-hydroxytestosterone (HT), 7 alpha-HT, 16 alpha-HT, 2 alpha-HT, and androstenedione to be major testosterone metabolites in the adult thymus. The high production of 7 alpha-HT and 16 alpha-HT correlated well with the gene and protein expression of CYP2A1/2 and CYP2B1/2 in thymus of adult animals. When compared with fetal thymic tissue, CYP2A1/2, 17beta-hydroxysteroid dehydrogenase isoform 1 (17 beta-HSDH1) and the androgen receptor were 8-, 3-, and 3-fold more highly expressed in adult rats, whereas 17 beta-HSDH2, 17 beta-HSDH3, and 5 alpha-reductase were reduced to 12%, 0%, and 32% of those in fetal thymus. In conclusion, we demonstrated that rat thymus expresses a variety of cytochrome P450 monooxygenases and other steroid-metabolizing enzymes, and it successfully metabolizes testosterone. Changes of the underlying steroid-metabolizing enzyme systems may aid in understanding the role of androgens in altering biological functions of the thymus.

Androgens↗
PubMed2003

Suppression of hepatic CYP3A1/2 and CYP2C11 by cyclosporine is not mediated by altering growth hormone levels.

Cyclosporine (CsA) suppresses drug metabolism by decreasing cytochrome P450 (P450) enzyme levels in rat liver. Growth hormone (GH) is known to pretranslationally regulate P450 expression. Thus, the suppression of P450 by CsA may involve GH as an intermediate. To address this question, we examined the effects of administering exogenous GH via twice daily subcutaneous injections and in conjunction with chronic subcutaneous CsA administration for 14 days on hepatic P450 expression. CsA alone decreased CYP3A1/2 and CYP2C11 significantly, in a manner similar to that previously found. When administered in the absence of CsA, GH also suppressed CYP3A1/2 and CYP2C11 protein levels as compared with GH vehicle. In the presence of CsA, GH did not cause further suppression of either CYP3A1/2 or CYP2C11 expression when compared with CsA treatment with GH vehicle. Testosterone in vitro catalytic assays confirmed that CsA and GH separately cause significant decreases in activity levels. Also, the concomitant administration of GH and CsA caused lowered production of 16alpha-, 2alpha-, 6beta-, and 2beta-hydroxytestosterone as compared with the administration of GH with CsA vehicle and as compared with the administration of GH vehicle with CsA. This study shows that GH is a dominating factor over CsA in determining hepatic P450 expression and activity. In addition, CsA does not seem to alter GH levels as a mediating event in suppressing P450 expression and activity. Since CsA given in combination with GH further suppressed P450 activity as compared with CsA given in combination with vehicle, this suggests that changes in hormonal status are likely to be one of the many factors that is responsible for the lack of a clear association between cyclosporine dosing and markers of toxicity.

Animals↗