Single Cytochrome P450 Research Articles

Identification, characterization, and ontogeny of a second cytochrome P450 3A gene from the fresh water teleost medaka (Oryzias latipes).

Multiple copies of cytochrome P450 gene family 3 have been identified from numerous mammalian species. Often these genes exhibit differential catalytic activities and gene regulation. To date however, little information is available regarding multiple forms of this gene family in teleost fishes. In this study, a second isozyme of cytochrome P450 3A has been cloned from the teleost fish Oryzias latipes and designated CYP3A40. Screening of a cDNA library to medaka liver resulted in the identification of a full length cDNA clone containing a 2316 base pair (bp) insert with an open reading frame encoding a single peptide of 502 amino acids. Comparisons of the deduced amino acid sequence to other known cytochrome P450 sequences indicate that this gene product is most similar to the CYP3A gene family and shares a 90% identity to CYP3A38 previously identified from medaka liver. Consistent with Northern blot and Western blot analysis, Southern blots of medaka genomic DNA demonstrated the presence of two CYP3A genes. Gene expression studies demonstrated that CYP3A38 and CYP3A40 are differentially regulated according to embryonic development. Northern blot analysis, using a probe to a conserved region of both CYP3A genes, demonstrated the presence of a single CYP3A transcript for early and late embryonic stages and two CYP3A transcripts in larvae and adult liver. Similarly, Western blots show a single faint immunoreactive cytochrome P450 3A protein in microsomes from early and late embryos and two abundant protein bands in microsomes from larval and adult liver. To further examine the transcriptional differences in CYP3A expression, RT-PCR analysis was performed on embryonic stages 11-35, 1- and 14-day-old larvae, and adult liver using primer sets specific for CYP3A38 and CYP3A40. These results demonstrate that CYP3A40 is expressed early in embryonic development and continues throughout adult stages. CYP3A38, however, is tightly regulated during embryonic development and is only expressed post-hatch.

Identification of human cytochrome P450 isoforms involved in the metabolism of S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid

1. To identify the cytochrome P450 (CYP) isoenzymes responsible for the major metabolic pathways of S-2-[4-(3-methyl-2-thienyl)phenyl] propionic acid (S-MTPPA) in man, the metabolism of S-MTPPA was examined using human liver microsomes and microsomes containing cDNA-expressed CYP isozymes (CYP1A2, 2A6, 2B6, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2E1 and 3A4). 2. S-MTPPA was mainly oxidized to the 5-hydroxylated metabolite of the thiophene ring (MA6) with human liver microsomes in the presence of NADPH. The formation of MA6 was inhibited by SKF 525-A, suggesting that CYP plays role in the formation of MA6. 3. Eadie-Hofstee plots for the 5-hydroxylation of S-MTPPA in the range 5-100 μM were linear for all samples studied, suggesting that the formation of MA6 by human liver microsomes follows simple Michaelis-Menten kinetics. Apparent Vmax = 1.42±0.64 nmol/min/mg protein; Km = 12±5 μM. 4. Among the CYP inhibitors examined (α-naphthoflavone, sulphaphenazole, omeprazole, quinidine and troleandomycin), sulphaphenazole (a CYP2C9 inhibitor) showed the most potent inhibitory effect on the 5-hydroxylation of S-MTPPA by human liver microsomes. 5. When incubated with microsomes containing cDNA-expressed CYP isozymes, S-MTPPA was substantially oxidized to MA6 only by CYP2C9. 6. These results suggest that formation of the major metabolite of S-MTPPA, MA6, in human liver microsomes is catalysed predominantly by a single CYP isoenzyme, namely CYP2C9.

Macrolide biosynthesis: A single cytochrome P450, PicK, is responsible for the hydroxylations that generate methymycin, neomethymycin, and picromycin in Streptomyces venezuelae

The final step in the biosynthesis of methymycin, neomethymycin, and picromycin is an hydroxylation, shown to be carried out by the cytochrome P-450 monooxygenase, PicK. Direct comparison of the relative k cat K m values for the two substrates, YC-17 and narbomycin, showed a threefold rate preference of picK for narbomycin.

Use of cDNA-Expressed Human Cytochrome P450 Enzymes to Study Potential Drug-Drug Interactions

Complementary DNA (cDNA)-expressed human cytochrome P450 enzymes provide a reproducible, consistent source of single enzymes for many types of studies. The use of single enzymes systems, relative to multienzyme systems, has distinct advantages and disadvantages depending on the specific application. cDNA-expressed materials have advantages in the analysis of cytochrome P450 form-selective metabolism of a drug or drug candidate. This analysis can be accomplished by direct incubation of the drug with microsomes prepared from cells expressing a single cytochrome P450 form coupled with analysis of either metabolite formation or loss of parent compound. This approach allows the unambiguous assignment of specific biotransformations to specific enzymes. However, extending these data to the balance of enzymes present in human liver microsomes can be problematic. New approaches for relating rates of metabolism for cDNA-expressed enzymes to human liver microsomes metabolism are being developed (Crespi, 1995). In addition, cDNA-expressed enzymes can be used to study the cytochrome P450 form-selective inhibition by drugs or drug candidates. This analysis is accomplished through the study of the inhibition of the metabolism of a model substrate by the drug or drug candidate. Through these analyses, apparent Ki values can be obtained and compared to Ki values for known, clinically significant inhibitors of the same enzyme. For this application, cDNA-expressed, single enzyme systems have distinct advantages because of greater flexibility in the choice of model substrates and the lack of competing pathways of metabolism. Specific data for the use of cDNA-expressed CYP2C9, CYP2D6, and CYP3A4 are presented.

The Use of Macroporous Microcarriers for the Large-Scale Growth of V79 Cells Genetically Designed to Express Single Human Cytochrome P450 Isoenzymes and for the Characterization of the Expressed Cytochrome P450

In this study, macroporous microcarriers were used for the large-scale growth of parental V79 cells and V79 cells genetically engineered to express a single human cytochrome P4501A1 isoenzyme (V79h1A1). Starting from 2 × 105cells/ml, approximately 1 × 107cells/ml could easily be harvested after 6 days in the case of the parental V79 cells, or after 11 days in the case of the V79h1A1 cells, resulting in a total of 3.6 × 1010cells. For the first time, the presence of cytochrome P450 (CYP) in the expressed V79 cells could be demonstrated by CO difference spectra with a Soret maximum around 450 nm. CYP levels in microsomes derived from the V79h1A1 cells of 14 pmol/mg protein were achieved. Importantly, no CYP was detected in microsomal fractions of the parental V79 cells. Cytochrome b5 levels could also be measured by difference spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growth of V79h1A1 cells and parental V79 cells, i.e., 16.7 ± 7.9 vs 14.5 ± 7.6 pmol/mg protein. The presence of human cytochrome P4501A1 (CYPh1A1) in microsomal fractions derived from the large-scale growth of V79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testosterone-6β-hydroxylation activities. EROD, ECOD, and testosterone-6β-hydroxylation activities of the V79h1A1 microsomes were 40 pmol resorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6β-hydroxytestosterone/min/pmol CYPh1A1, respectively, indicating the presence of a highly active human CYP1A1 enzyme system. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporous microcarriers is suitable for large-scale growth of V79 cells expressing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes containing human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes containing human CYP isoenzymes may substantially facilitate the production of sufficient quantities of human metabolites to allow isolation and identification in an early stage of development of pharmacologically interesting drugs.

Idiosyncratic drug reactions. Metabolic bioactivation as a pathogenic mechanism.

The metabolism of drugs to chemically reactive metabolites may play a pivotal role in the pathogenesis of idiosyncratic drug toxicity. A large number of in vitro studies and a limited number of in vivo studies have demonstrated that many drugs are not toxic per se, but produce toxicity after undergoing enzyme-mediated bioactivation to chemically reactive species. Such reactive species may inflict a toxic insult on the cell either directly or indirectly by acting as a hapten and initiating an immune-mediated reaction. The enzymes responsible for bioactivation have been widely studied, both quantitatively and qualitatively, the most important being the enzymes of the cytochrome P450 (CYP) mixed function oxidase system. CYP enzymes are the most predominant drug metabolising enzymes in the liver and are also present in most other tissues of the body. The diversity of this enzyme system means that a wide range of xenobiotic substrates can be bioactivated by either a single CYP isoform or multiple isoforms of this enzyme superfamily. Other enzymes do, however, play an important role in drug bioactivation. In white blood cells, for example, myeloperoxidase has been shown to bioactivate a wide range of drugs. In other tissues low in CYP activity, prostaglandin H synthase may also be responsible for bioactivation; e.g. in the kidney paracetamol (acetaminophen) toxicity is though to result from activation via this enzyme. The phase II or conjugation enzymes may also be important in the ultimate bioactivation of drug molecules. Whilst activation by these enzymes is, to date, apparently confined to chemicals, most drugs are also substrates for these enzymes and bioactivation by them must remain a possibility.

Selective Inhibition of a Cytochrome P450 Enzyme in Wheat That Oxidizes Both the Natural Substrate Lauric Acid and the Synthetic Herbicide Diclofop

Earlier studies from our laboratory strongly suggested that a single or similar cytochrome P450 isoform(s) isolated from microsomes of wheat catalyze the oxidation of the medium-chain fatty acid, lauric acid, and the wheat selective herbicide diclofop. Lauric acid is mainly hydroxylated at the subterminal position C11 (ω-1) and for that reason P450-dependent reactions were initially designated lauric acid (ω-1)-hydroxylase ((ω-1)-LAH). This report presents data on thein vitroandin vivoirreversible inhibition of both lauric acid and diclofop oxidation by mechanism-based inhibitors targeting lauric acid hydroxylation. Incubation of microsomes from etiolated wheat seedlings with 10-dodecynoic acid (10-DDYA) produces a dramatic inhibition of lauric acid hydroxylation. The inhibition is both time- and concentration-dependent in a process typical for mechanism-based inhibitors. A half-life of 3 min and an apparent inhibition constant of 14 μMwere determined from pseudo-first order kinetic studies of (ω-1)-LAH inhibition. Similar results were obtained by incubating microsomes with a terminal acetylene, 11-dodecynoic acid (11-DDYA). Based on results ofin vitroexperiments we have developed a series of new mechanism-based inhibitors to inhibit diclofop metabolism in developing wheat seedlings. To protect the inhibitors from degradation by α- and β-oxidation systems, water-soluble sodium salts of undec-10-yne-1-sulfonic acid (10-UDYS), undec-9-yne-1-sulfonic acid (9-UDYS), and undecan-1-sulfonic acid (SULAUR) were synthesized. Following treatment of wheat coleoptiles for 6 hr with these modified inhibitors, diclofop and chlortoluron metabolism was measured. Both compounds selectively inhibited diclofop metabolism without affecting chlortoluron metabolism. The importance of triple bonds in the inhibition process is clearly demonstrated by the fact that SULAUR had very small inhibitory effects. A 100 μMconcentration of both inhibitors resulted in about 50% inhibition of diclofop metabolism. Increasing the concentration of inhibitors to 2.5 mMincreased inhibition of diclofop oxidation to about 90%. Our results clearly show that one or similar forms of cytochrome P450 that are naturally involved in lauric acid oxidation are responsible for diclofop hydroxylation.

Congenital hypoaldosteronism: the Visser-Cost syndrome revisited.

In 1964, H. K. A. Visser and W. S. Cost were the first to suggest a defect of the terminal aldosterone (Aldo) biosynthesis in patients with hypoaldosteronism. In the last years, the molecular basis of the terminal Aldo biosynthesis has been elucidated. Aldo biosynthesis requires 11beta-hydroxylation of 11-deoxycorticosterone to form corticosterone, hydroxylation at position C-18 to form 18-hydroxycorticosterone (18-OHB), and finally oxidation at position C-18. One single cytochrome P450 enzyme (P450aldo) catalyzes all three reactions in the zona glomerulosa. The coding gene is termed CYP11B2. Two inborn errors of terminal Aldo biosynthesis characterized by overproduction of corticosterone and deficient synthesis of Aldo have been described. Corticosterone methyl oxidase deficiency type I (CMO I) is distinguished by decreased production of 18-OHB while CMO II is characterized by overproduction of 18-OHB and an elevated ratio of 18-OHB to Aldo. Both disorders are inherited by an autosomal recessive trait and cause salt-wasting and failure to thrive in early infancy. Our present series includes 14 CMO deficient infants diagnosed by multisteroid analysis (RIA after extraction and automated high performance gel chromatography) which provides precise biochemical criteria for the differentiation of the two CMO variants. So far, three different mutations within the CYP11B2 gene in patients with P450aldo deficiency have been described. Introduction of these mutations into a CYP11B2 cDNA expression vector construct and subsequent expression in COS cells revealed loss of 11beta-hydroxylase, 18-hydroxylase, and 18-dehydrogenase activity of P450aldo. Further molecular studies on more P450aldo-deficient patients might clarify in the future the still existing discrepancies in CYP11B2 (P450aldo) structure-function relationship.

Multisteroid analysis in children with terminal aldosterone biosynthesis defects.

Aldosterone (Aldo), the most potent mineralocorticoid, is synthesized in the adrenal zona glomerulosa, requiring 11 beta-hydroxylation of 11-deoxycorticosterone (DOC) to form corticosterone (B), hydroxylation at position C18 to form 18-hydroxycorticosterone (18-OHB), and finally oxidation at position C18. There is a single cytochrome P450 enzyme (P450aldo) catalyzing all three reactions in the zona glomerulosa. The gene encoding for this enzyme is termed CYP11B2. There are two inborn errors of terminal aldosterone biosynthesis characterized by overproduction of B and deficient synthesis of Aldo. Corticosterone methyl oxidase deficiency type I (CMO-I) is characterized by decreased production of 18-OHB, whereas CMO-II is characterized by overproduction of 18-OHB and an elevated plasma ratio of 18-OHB to Aldo. Both disorders have an autosomal recessive inheritance and are rare causes of salt-wasting and failure to thrive in early infancy. In the last 10 yr, we diagnosed 16 infants with CMO deficiencies by simultaneous multisteroid analysis in a small plasma sample (RIA after extraction and automated high performance gel chromatography). All patients presented with severe failure to thrive in the first 3 months of life, associated with severe hyponatremia, hyperkalemia, and increased PRA. Basal Aldo levels were decreased (range, 0.055-0.11 nmol/L), whereas B was elevated (range, 19-154 nmol/L). Plasma 18-OHB, ranging from 0.063-0.44 nmol/L, was decreased or in the lower normal range in seven patients, whereas the other seven patients had elevated 18-OHB levels (range, 12.1-57.7 nmol/L). 18-OH-DOC (range, 0.81-7.8 nmol/L) and DOC (range, 0.7-9.53 nmol/L) levels were elevated in all patients. In seven patients, we found an elevated ratio of 18-OHB/Aldo (range, 286-900) and a low ratio of B/18-OHB (range, 1.1-5.8), whereas seven other patients had a low 18-OHB/Aldo ratio (range, 1.1-6.95) and a high B/18-OHB ratio (range, 41-1360). These findings confirmed the diagnosis of CMO-I in seven patients (low 18-OHB, 18-OHB/Aldo ratio < 10, and B/18-OHB ratio > 40) and the diagnosis of CMO-II in seven other patients (high 18-OHB, 18-OHB/Aldo ratio > 100, and B/18-OHB ratio < 10), whereas 18-OHB could not be determined in two patients. The B/18-OHB ratio appears particularly useful in CMO cases with undetectably low Aldo plasma levels and uncalculable 18-OHB/Aldo ratios. In conclusion, the simultaneous multisteroid determination method allows the precise differentiation of CMO-I and CMO-II in a small plasma sample during early infancy.

A single cytochrome P-450 system is involved in degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine by Rhodococcus sp. strain NI86/21

During atrazine degradation by Rhodococcus sp. strain N186/21, N-dealkylated metabolites and an hydroxyisopropyl derivative are produced. The cytochrome P-450 system that is involved in degradation of thiocarbamate herbicides by strain N186/21 (I. Nagy, G. Schoofs, F. Compernolle, P. Proost, J. Vanderleyden, and R. De Mot, J. Bacteriol. 177:676-687, 1995) is also required for atrazine degradation. Atrazine-degrading activity was conferred on the atrazine-negative strains, mutant FAJ2027 of Rhodococcus sp. strain N186/21 and Rhodococcus erythropolis SQ1, upon transformation with the genes encoding the cytochrome P-450 system.

Biotransformation of the Naturally Occurring Lignan (-)-Arctigenin in Mammalian Cell Lines Genetically Engineered for Expression of Single Cytochrome P450 Isoforms

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450 1A1, P450 1A2, and P450 2B1, and human P450 1A2 were used to analyse the metabolism of (-)-arctigenin. Concentrations of metabolites formed in the medium were determined by TLC, HPLC, GC, and GC-MS. Only in the cell line expressing rat P450 2B1 was (-)-arctigenin metabolized to a significant extent. The lignan was converted by O-demethylation to (-)-3'-O-demethylarctigenin.

Biotransformation of methylxanthines in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Allocation of metabolic pathways to isoforms and inhibitory effects of quinolones

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2 were used to allocate metabolic pathways of methylxanthines to specific isoforms and to test the suitability of such cell lines for investigations on drug interactions occurring at the cytochrome expressed. The cell lines were exposed to caffeine and/or theophylline and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity. Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine metabolism in vivo and in human liver microsomes, reduced formation rates of all metabolites of caffeine in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1.

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg −1 (±SD, n = 4), reconstituted aromatase activity of 111 ± 19 nmol·min −1·mmg −1 and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min −1·mg −1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K m of 1.58 μM and V max of 8.9 nmol·min −1·mg −1. A significant shift of the optimum pH and V max, but not the K m , for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.

Species differences in 5α-androstane-3β,17β-diol hydroxylation by rat, monkey, and human prostate microsomes

The 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5α-androstane-3β,17β-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6α-hydroxylation of 5α-androstane-3β,17β-diol varied independently of both the 7α- and 7β-hydroxylation, which varied in unison. The 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with β-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7α- and the 7β-hydroxylation of 5α-androstane-3β,17β-diol without increasing the 6α-hydroxylation. The 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by human and monkey prostate microsomes, but not the 6α-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by human prostate microsomes, but not the 6α-hydroxylation, was markedly inhibited (>85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6α-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5α-androstane-3β,17β-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia. This suggests that an impairment of androgen inactivation is not the mechanism for the proposed accumulation of 5α-dihydrotestosterone in the hyperplastic prostate. The rate of 5α-androstane-3β,17β-diol hydroxylation by human prostate microsomes ranged from 1 to 10 pmol/mg protein/min, which is considerably less than the rate catalyzed by rat prostate microsomes (~600 pmol/mg protein/min). This marked species difference in the rate of inactivation of androgens by prostate microsomes could potentially explain why humans, in contrast to rats, are prone to develop benign prostatic hyperplasia.

Hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes: Effects of antibodies and chemical inhibitors of cytochrome P450 enzymes

The purpose of the present study was to test the hypothesis that rat prostate microsomes contain a single cytochrome P450 enzyme responsible for the conversion of 5α-androstane-3β,17β-diol to a series of trihydroxylated products. The three major metabolites formed by in vitro incubation of 5α-[ 3H]androstane-3β,17β-diol with rat prostate microsomes were apparently 5α-androstane-3β,6α,17β-triol, 5α-androstane-3β,7α,17β-triol, and 5α-androstane-3β,7β,17β-triol, which were resolved and quantified by reverse-phase HPLC with a flow through radioactivity detector. The ratio of the three metabolites remained constant as a function of incubation time, microsomal protein concentration, ionic strength, and substrate concentration. The ratio of the three metabolites was dependent on pH, apparently because the hydroxylation of 5α-androstane-3β,17β-diol shifted from the 6α-to the 7α-position with increasing pH (6.8–8.0). The V max values were 380, 160, and 60 pmol/mg microsomal protein/min for the rate of 6α-, 7α-, and 7β-hydroxylation, respectively. Similar K m values (0.5–0.7 μ m) were measured for enzymatic formation of all three metabolites, which suggests that formation of all three metabolites was catalyzed by a single, high-affinity enzyme. Testosterone, 5α-dihydrotestosterone, and 5α-androstane-3α,17β-diol did not appreciably inhibit the hydroxylation of 5α-androstane-3β,17β-diol, suggesting that this enzyme exhibits a high degree of substrate specificity. Formation of all three metabolites was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase (85%) and by a 9:1 mixture of carbon monoxide and oxygen (60%). Several chemical inhibitors of cytochrome P450 enzymes, especially the antimycotic drug clotrimazole, also inhibited the formation of all three metabolites. Polyclonal antibodies that recognize liver cytochrome P450 1A, 2A, 2B, 2C, and 3A enzymes did not inhibit 5α-androstane-3β,17β-diol hydroxylase activity. Overall, these results are consistent with the hypothesis that the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes is catalyzed by a single, high-affinity P450 enzyme. This cytochrome P450 enzyme appears to be structurally distinct from those in the 1A, 2A, 2B, 2C, and 3A gene families.

Hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes: Potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5α-reductase inhibitors

5α-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5α-reductase. The inactivation of 5α-dihydrotestosterone involves reversible reduction to 5α-androstane-3β,17β-diol by 3β-hydroxysteroid oxidoreductase followed by 6α-, 7α-, or 7β-hydroxylation. 5α-Androstane-3β,17β-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity ( K m ~ 0.5 μM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5α-androstane-3β,17β-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5α-reductase (4-azasteroid analogs; 10 μ m) or inhibitors of 3β-hydroxy-steroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 μ m) had no appreciable effect on the 6α-, 7α-, or 7β-hydroxylation of 5α-androstane-3β,17β-diol (10 μ m) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1–10 μ m) all markedly inhibited 5α-androstane-3β,17β-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1–10 μ m) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole > clotrimazole > ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a K i of 0.03 μ m. The imidazole-type antimycotic drugs inhibited all three pathways of 5α-androstane-3β,17β-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5α-androstane-3β,17β-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.

Regioselectivity of a plant lauric acid omega hydroxylase. Omega hydroxylation of cis and trans unsaturated lauric acid analogs and epoxygenation of the terminal olefin by plant cytochrome P-450

The study of the stereochemistry and regioselectivity of plant fatty acid hydroxylases is hampered by the difficulty to purify plant cytochrome P-450 enzymes. To provide an alternative, we have now defined an experimental plant system which expresses only one hydroxylase activity towards lauric acid: microsomes from clofibrate-induced Vicia sativa seedlings hydroxylate this fatty acid exclusively at the methyl terminus. To explore the catalytic capabilities of this laurate oxidase, a series of 1- 14C-radiolabeled unsaturated lauric acid analogs (7-, 8-, 9-, 10- and 11-dodecenoic acids) were synthesized. Microsomes from clofibrate induced Vicia sativa seedlings catalyzed the omega-oxidation of the lauric acid analogs in the presence of O 2 and NADPH. The cis and trans forms of the four in-chain unsaturated analogs of lauric acid were 12-hydroxylated with similar efficiency. The terminal olefin was readily converted to the epoxide with only marginal autocatalytic inactivation of the enzyme. The formation of each metabolite was inhibited to the same extend when microsomes were incubated in presence of carbon monoxide or a suicide-substrate for omega LAH, suggesting that a single cytochrome P-450 isoenzyme from Vicia sativa microsomes is able to omega hydroxylate lauric acid and in-chain unsaturated analogs, and to epoxygenate 11-dodecenoic acid.

Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms

Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. Theophylline was metabolized mainly via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1. These results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

Testosterone-mediated regulation of mouse renal cytochrome P-450 isoenzymes

We have studied the extent to which mouse renal cytochrome P-450 isoenzymes are sexually differentiated, and the factor(s) regulating this dimorphism. Intriguingly, sex differences were not seen in the expression of a single cytochrome P-450 enzyme, but were observed in the expression of all P-450 isoenzymes detectable, encoded by six gene families or sub-families. This effect was mediated by testosterone, which had the capacity to both induce and repress P-450 gene expression, and which was independent of growth hormone. The changes in protein content were mirrored in all but one case by changes in the levels of mRNA, indicating that these genes contain hormone-responsive elements. These findings are consistent with numerous reports of sex differences in the susceptibility of the mouse kidney to the toxic and carcinogenic effects of drugs and environmental chemicals, many of which are metabolized to cytotoxic products by the cytochrome P-450-dependent mono-oxygenases. These data imply that circulating androgen levels will be an important factor in susceptibility of the kidney to toxic or carcinogenic compounds which require metabolic activation.

Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450 BM-1 from Bacillus megaterium

The gene encoding barbiturate-inducible cytochrome P-450 BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH 2-terminal sequence of P-450 BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an M r of 47 439. The sequence is most, but less than 27%, similar to that of P-450 CAM from Pseudomonas putida, so that P-450 BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450 BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH- P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987–10995).