Oxidation Of Testosterone Research Articles

THE IMPACT OF 2-AMINOETHANESULFONIC ACID, GUARANINE METHYL THEOBROMINE 1, 3, 7-TRIMETHYLXANTHINE THEINE AND/OR ISOBUTYLPHENYL PROPIONIC ACID ON MASCULINE FECUNDITY, AND MORPHOPATHOLOGICAL DISORDERS IN RATS

Objectives: This study aims to evaluate the impact of caffeine (CAF), taurine (TUR), and ibuprofen (IBF) consumption on the masculine fecundity in rats and fetuses development.
 Methods: The 1st group was kept as a normal control received distilled water. The 2nd, 3rd, and 4th groups received orally 250 mg/kg, 40 mg/kg, and 270 mg/kg of TUR, CAF, and IBF, respectively. In addition, the 5th, 6th, 7th, and 8th groups received TUR with CAF, TUR with IBF, CAF with IBF, and TUR, CAF, IBF combination, for 45 days. Blood samples were taken to determine testosterone and follicle-stimulating hormone levels and oxidation levels. Moreover, in testes, malondialdehyde and nitrite/nitrate were estimated. Besides, the abnormality of sperms, sperm counts, and percentage of sperm motilities was characterized. These masculines were allowed to mate with untreated masculine rats to determine the rate of pregnancy and study any malformation in their fetuses.
 Results: The study revealed that each of CAF and/or IBF decreased the weights of fetuses, as well as sperm counts and motilities significantly. Besides, percentages of head and tail abnormalities were increased in CAF and/or IBF. The histopathological examination revealed the presence of marked degenerative changes in testes in CAF and/or IBF-treated groups.
 Conclusion: CAF and IBF have a teratogenic effect on spermatozoa and masculine fecundity, whereas TUR almost improved undesirable impacts induced by IBF or/and CAF.

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h.Graphical

Analogs and Antagonists of Male Sex Hormones

The steroid testosterone is the major circulating sex hormone in males and is the prototype for the androgens, the anabolic agents, and androgen antagonists. Endogenous androgens are biosynthesized from cholesterol; the majority of the circulating androgens are produced in the testes under the stimulation of luteinizing hormone (LH). The reduction of testosterone to dihydrotestosterone is necessary for androgenic actions of testosterone in many androgen target tissues such as the prostate; the oxidation of testosterone by the enzyme aromatase produces estradiol. The androgenic actions of testosterone and dihydrotestosterone are due to their binding to the androgen receptor, followed by nuclear localization, dimerization of the receptor complex, and binding to specific DNA sequences. This binding of the homodimer to the androgen response element leads to gene expression, stimulation, or repression of new mRNA synthesis, and subsequent protein biosynthesis. The synthetic androgens and anabolics were prepared to impart oral activity to the androgen molecule, to separate the androgenic effects of testosterone from its anabolic effects, and to improve on its biological activities. Novel nonsteroidal androgens, termed selective androgen receptor modulators, were developed to impart agonist activity in selective tissues. Drug preparations are used for the treatment of various androgen-deficient diseases and for the therapy of diseases characterized by muscle wasting and protein catabolism. Androgen antagonists include antiandrogens, which block interactions of androgens with the androgen receptor, and inhibitors of androgen biosynthesis and metabolism. Such compounds have therapeutic potential in the treatment of acne, virilization in women, hyperplasia and neoplasia of the prostate, and baldness. Keywords: androgens; testosterone; dihydrotestosterone; anabolics; antiandrogens; selective androgen receptor modulators; 5α-reductase inhibitors

Oxidation of testosterone by permanganate and its implication in sports drug testing

Adulteration of urine with permanganate can lead to depletion of testosterone and formation of new reaction products (4α,5α- and 4β,5β-dihydroxytestosterone).

Electrochemical detection in vitro and electron transfer mechanism of testosterone using a modified electrode with a cobalt oxide film

The voltammetric oxidation of testosterone (4-androsten-17β-ol-3-one) has been studied at an edge plane glassy carbon electrode modified with cobalt oxide. One sensitive and well-defined peak is obtained with a peak potential of 0.50V vs. SCE attributed to the reduction reaction CoIV/CoIII, which is responsible for oxidation of the steroid. A calibration curve was obtained for testosterone, with the measurements performed in triplicate, in the range from 0.33 to 2.00μМ, yielding a correlation coefficient >0.99 and detection limit of 0.16μM, with relative standard deviations (R.S.D) from 1.33×10−5 to 4.00×10−4% with 95% significance (p<0.05). The modified electrode showed a stable and reproducible response toward the detection of this steroid. Thus, the results showed that the electrode modified with cobalt oxide acts as a promising alternative for simple, cheap and rapid analysis of testosterone.

Cyclin D1 regulates hepatic estrogen and androgen metabolism

Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.

Concordant up‐regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment

Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug-hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2alpha-, 2beta-, 6beta-, 16alpha- and 16beta-hydroxytestosterones, while 6alpha- and 15alpha-hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chloramphenicol, ketoconazole and nifedipine as well as antibodies against CYP3A- and 2B-isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippocampus, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC-12 cells. Inhibition of phenytoin-induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so-called drug-hormone crosstalk.

The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes

The preparation of hepatocytes using the two-stage perfusion technique usually involves the use of collagenase (CII) alone or in combination with dispase (C/D) or trypsin inhibitor (CA/TI) as digestion enzymes. The effect of CII, C/D and CA/TI on cell viability, yield, cytochrome P450 mediated oxidation of testosterone, glucuronidation and sulfation of 7-hydroxycoumarin, glutathione content, glutathione-S-transferase activity and glutathione-conjugation capacity of hepatocytes has been assessed. Cytochrome P450 mediated oxidation of testosterone was significantly ( p < 0.05) decreased with CII isolated hepatocytes (81.7 ± 3.3 nmol/10 6 cells, mean ± S.E.M., n = 3), compared with those isolated using CA/TI (96.6 ± 1.9 nmol/10 6 cells) or C/D (95.1 ± 2.1 nmol/10 6 cells). In contrast, glutathione conjugation of the non-specific substrate 1-chloro-2,4-dinitrobenzene was significantly ( p < 0.05) increased with CII isolated hepatocytes (56.9 ± 5.9 nmol/10 6 cells, mean ± S.E.M., n = 3), compared with those isolated using CA/TI (36.0 ± 3.7 nmol/10 6 cells) or C/D (31.6 ± 3.7 nmol/10 6 cells). These findings have significant implications for the interpretation of metabolism data derived from hepatocytes in suspension, particularly in terms of glutathione conjugation of potentially toxic reactive intermediates of xenobiotic metabolism. Indeed, data presented show that the presence of trypsin inhibitor in the preparation of isolated rat hepatocytes significantly affects the formation of glutathione conjugates of reactive intermediate products of troglitazone metabolism.

The CYP3A4*18 Genotype in the Cytochrome P450 3A4 Gene, a Rapid Metabolizer of Sex Steroids, Is Associated With Low Bone Mineral Density

Osteoporosis is influenced by genetic factors. The interindividual variability in the activity of CYP3A, the metabolic enzyme of sex hormones, may result from genetic polymorphisms. In a study of 2,178 women of ages 40-79 years, the presence of the CYP3A4*18 variant was found to be significantly associated with low bone mass. In vitro functional analyses indicate that CYP3A4*18 is a gain-of-function mutation in sex steroid metabolism, resulting in rapid oxidation of estrogens and testosterone; in vivo pharmacokinetics using midazolam (MDZ) verify the altered activity of the CYP3A4*18, showing lower metabolic turnover in the mutant than in the wild type. Molecular modeling reveals the structural changes in the substrate recognition sites of CYP3A4*18 that can cause changes in enzymatic activity and that potentially account for the difference between the catalytic activities of estrogen and MDZ, depending on the genotype. The results indicate that a genetic variation in the CYP3A4 gene--as a gain-of-function mutation in the metabolism of certain CYP3A substrates, including sex steroids--may predispose individuals to osteoporosis.

Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis

An electrophoretically mediated microanalysis method for the determination of CYP3A4 activity using testosterone and nifedipine as substrates was developed. Initially, the enzymatic reaction was performed off-line and the samples were subsequently injected into the capillary by pressure. The CYP3A4 activity was determined by quantitation of the reactant cofactor, NADPH. To further optimize, speed-up and miniaturize the enzyme assay, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product cofactor, NADP, employing the plug-plug mode of electrophoretically mediated microanalysis. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 260 nm. This setup resulted in a fully automated assay, which can be carried out in less than 35 min. Using the Lineweaver-Burk equation, the Michaelis constants (K(m)) for the oxidation of testosterone and nifedipine by CYP3A4 were calculated to be 58.6+/-8.3 and 19.1+/-2.4 microM, respectively, which are consistent with off-line assay and previously reported values.

Defective Activity of Recombinant Cytochromes P450 3A4.2 and 3A4.16 in Oxidation of Midazolam, Nifedipine, and Testosterone

Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4(*)2 (S222P), (*)7 (G56D), (*)16 (T185S), and (*)18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower V(max) and increased K(m) relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower V(max) values for MDZ and NIF (60 and 84%, respectively) and a higher K(m) (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low V(max) values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio V(max)(/K)(m). In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.

Experimental approaches to evaluate activities of cytochromes P450 3A

Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b5. The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro.

Inhibitory Effects of Saturated and Polyunsaturated Fatty Acids on the Cytochrome P450 3A Activity in Rat Liver Microsomes

This study was conducted to investigate the inhibitory effects of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on cytochrome P450 3A (CYP3A). The inhibition of 6beta-hydroxy testosterone formation from testosterone in rat liver microsomes was used as an index of CYP3A activity. In the present study, among the three types of fatty acids, the rank order of inhibitory effects of fatty acids was SFAs<MUFAs<PUFAs. Among the four PUFAs examined (linoleic acid, gamma-linolenic acid, retinoic acid, and docosahexaenoic acid; DHA), the highest inhibitory effect of CYP3A-catalyzed testosterone metabolism was observed with DHA, which inhibited testosterone metabolism by 94%. The percentage inhibition (I) of fatty acids on testosterone 6beta-hydroxylation was predicted from the number of double bonds and the carbon chain length of the fatty acids. The correlation between the predicted and observed I values yielded the r(2) values of 0.759 and 0.907 using the linear and non-linear equations, respectively. Moreover, the goodness-of-fit assessed using the mean absolute error (MAE) was 17.6 with the linear model and 11.3 with the non-linear model. These observations suggested that fatty acids showed the variable inhibitory effects on CYP3A-mediated testosterone oxidation and that the number of double bonds, as well as the carbon chain lengths of fatty acids, may be important in the inhibition of CYP3A.

Functional genome analysis indicates loss of 17beta-hydroxysteroid dehydrogenase type 2 enzyme in the zebrafish

Among the family of 17beta-hydroxysteroid dehydrogenases, the type 2 (17beta-HSD 2) is the main enzyme responsible for inactivation of estrogens and androgens, catalyzing the oxidation of the C17 hydroxyl group. 17beta-HSD 2 has been studied only in mammals, its occurrence and function in other vertebrates hardly known. We investigated the presence of homologs in non-mammalian species and found sequences of 17beta-HSD 2 and its closest homolog 11beta-HSD 2 in zebrafish ( Danio rerio), Takifugu rubripes, Tetraodon nigroviridis, Xenopus tropicalis and chicken databases. Furthermore, we cloned zebrafish 17beta-HSD 2 from ovarian tissue and found high expression also in the testis of adult fish and throughout embryogenesis. The enzyme, though, is inactive likely due to a non-sense N-terminal region including a dysfunctional cofactor binding motif. Replacement of the affected part by the corresponding human 17beta-HSD 2 sequence fully restored enzymatic activity. Comparison of all retrieved 17beta-HSD 2 sequences indicates that this functional loss may have occurred only in zebrafish, where steroid inactivation at position C17 seems to pursue without the protein studied. The closely related 11beta-HSD 2 is unlikely to substitute for 17beta-HSD 2 since in our hands it did not catalyze the respective oxidation of testosterone or estradiol.

Effects of steatosis on drug-metabolizing capability of primary human hepatocytes

The suitability of liver grafts discarded for transplantation because of macrosteatosis for preparing human hepatocyte cultures for in vitro drug metabolism studies has been examined. Lower cell viability and yield of isolation procedure were obtained from fatty livers (>40% steatosis) with respect to normal tissue. Significant reductions in 7-ethoxycoumarin O-deethylation (ECOD) and testosterone oxidations were found in hepatocytes prepared from steatotic livers. The potential impact of lipid accumulation on P450 enzymes was studied in vitro by incubation of cultured hepatocytes with long chain free fatty acids (FFA). Treatment of cells with 0.25–3 mM FFA induced dose-dependent accumulation of lipids in the cytosol. Decreased ECOD and testosterone oxidation were found after 14 h of exposure to 1 mM or 2 mM FFA (about 60–70% and 30–60% of control, respectively). The effects of fat-overloading on individual P450s were analyzed both at activity and mRNA level. CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities were reduced after hepatocyte incubation with 1 mM (to 45–65% of control) or 2 mM (to 20–50%) FFA for 14 h. Reductions in P450 transcripts were also found in hepatocytes treated with 1 mM FFA. Our findings showed a general down-regulation of P450s involved in drug metabolism in fat-overloaded hepatocytes. The results suggest that, despite their reduced P450 function, human hepatocytes obtained from donors with steatosis are metabolically competent and could be used for drug metabolism studies.

Activities of Rat Cytochrome P450 3A and 2C Isoforms are Increased In Vivo by Magnesium Sulfate as Evidenced by Enhanced Oxidation of Bupivacaine and Testosterone in Liver Microsomes

We previously reported that magnesium sulfate (MgSO(4)) increases the threshold dose of bupivacaine in inducing seizure in rats. Cytochrome P450 (P450) isoforms involved in the biotransformation of bupivacaine to three oxidative metabolites and the effects of MgSO(4) in vivo on the P450 activities in rats were investigated. Of six cDNA-expressed rat P450 isoforms tested, CYP3A2 and CYP2C11 had high rates for N-debutlylation and 3'-hydroxylation of bupivacaine, respectively. The liver microsomes prepared from male rats pretreated with intravenous administration of MgSO(4) (a bolus dose of 25 mg/kg, followed by infusion of 2.0 mg/kg/min for 6 h) showed increased V(max) values for N-debutylation and 3'-hydroxylaiton of bupivacaine compared to the liver microsomes from control rats. Administration of MgSO(4) also increased the activities of testosterone 6beta- and 16alpha-hydroxylation. Although the level of expression of CYP3A and CYP2C isoforms in the liver microsomes were unchanged, NADPH-P450 reductase and cytochrome b(5) were found to be induced by intravenous administration of MgSO(4). These results suggest that CYP3A and CYP2C isoforms are activated by MgSO(4) in vivo as a consequence of enhanced microsomal electron transfer due to induction of NADPH-P450 reductase and cytochrome b(5), leading to the increased metabolism and clearance of bupivacaine.

Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.

Testosterone 1 beta-hydroxylation by human cytochrome P450 3A4.

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6 beta-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6 beta- and 2 beta-hydroxy products, was identified as 1 beta-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of approximately 6 microg of this product. Mass spectrometry established the formula as C(19)H(29)O(3) (MH(+) 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the beta-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1 alpha-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.

Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole

This unit describes methods for measuring CYP3A4 activity using testosterone as a specific substrate, and for measuring CYP3A4 inhibition using ketoconazole as a selective inhibitor of testosterone oxidation. CYP3A4 is one of the most important and most abundant drug-metabolizing CYP isoforms in human liver microsomes (∼40% of total CYP), and it has the broadest substrate specificity. It is important to determine whether CYP3A4 is involved in its metabolism.

CYP3A5 Contributes Significantly to CYP3A-mediated Drug Oxidations in Liver Microsomes from Japanese Subjects

The purpose of this study was to evaluate a contribution of polymorphic cytochrome P450 (CYP) 3A5 to the oxidation of diltiazem, midazolam and testosterone by liver microsomes from Japanese subjects. Twenty-seven liver samples were classified into three groups according to the CYP3A5 genotypes; CYP3A5(*)1/(*)1 (n=3), (*)1/(*)3 (n=12) and (*)3/(*)3 (n=12). The results of genotyping and immunochemical quantitation of CYP3A5 protein showed a good accordance between the CYP3A5 genotype and CYP3A5 content but not CYP3A4 content in liver microsomes. The expression levels of hepatic CYP3A5 protein ranged from 20 to 60% of the sum of CYP3A4 and CYP3A5 contents in subjects with at least one wild type allele ((*)1). The CYP3A5 contents correlated well with liver microsomal activities of diltiazem N-demethylation, midazolam 1'- and 4-hydroxylations and testosterone 6beta-hydroxylation among subjects carrying at least one (*)1 allele. In addition, the correlation coefficients of CYP3A5 contents with the rates of diltiazem N-demethylation, midazolam 1'-hydroxylation and testosterone 6beta- hydroxylation were higher than those of CYP3A4, although the value of CYP3A5 with the midazolam 4-hydroxylation rate was similar to that of CYP3A4. Kinetic analyses revealed a biphasic diltiazem N-demethylation in liver microsomes from subjects carrying the (*)1 allele. The apparent V(max)/K(m) values for recombinant CYP3A5 indicated the greater contributions to diltiazem N-demethylation and midazolam 1'-hydroxylation as compared with CYP3A4. These results suggest that polymorphic CYP3A5 contributes markedly to the drug oxidations, particularly diltiazem N-demethylation, midazolam 1'- hydroxylation and testosterone 6beta-hydroxylation by liver microsomes from Japanese subjects.