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PubMed1987

Monoclonal antibodies inhibitory to rat hepatic cytochromes P-450: P-450 form specificities and use as probes for cytochrome P-450-dependent steroid hydroxylations.

Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1987

Human placenta estrogen synthetase (aromatase) purified by affinity chromatography.

Microsomal estrogen synthetase (cytochrome P-450ES), also known as aromatase, was purified from fresh human placenta microsomes by DEAE-Trisacryl and testosterone-agarose chromatography. Estrogen synthetase assays were done with androstenedione as substrate, NADPH as electron donor, and a partially purified P-450 reductase from human placenta as the electron carrier. The specific cytochrome P-450 content of the purified P-450 was 0.67 nmol mg-1 of protein, and the preparation contained no cytochrome P-420. The absorbance maximum was 448.5 nm. The specific estrogen synthetase activity of the purified P-450ES fraction was 35 nmol min-1 nmol-1 of cytochrome P-450 or 23.3 nmol min-1 mg-1 of protein. The latter value shows a 179-fold purification with a yield greater than 1% in the two-step procedure. Kinetic constants for the reaction were measured with androstenedione as the aromatizable substrate. The Km was 1.4 nM and the Vmax was 37 nmol min-1 nmol-1 of P-450. The purified enzyme aromatized androstenedione and testosterone at identical rates; androstenedione gave only estrone, and testosterone gave only estradiol-17 beta. Dehydroepiandrosterone was not detectably aromatized or otherwise metabolized. Neither 16 alpha-hydroxytestosterone nor 16 alpha-hydroxyandrostenedione was aromatized. No hydroxysteroid dehydrogenase or reductase was detected in direct assays. No free reaction intermediates were detected in aromatization assay incubation mixtures. The purity of the product and the simplicity of the preparation recommend it for use in further studies of the enzyme.

Aromatase↗
PubMed1985

[A special estrogen-binding protein in the rat liver: the effect of nuclear accumulation of estradiol-receptor complexes in vitro].

The accumulation of [3H]estradiol-receptor complexes by liver nuclei after preliminary incubation of the hormone with rat liver cytosol was studied. It was demonstrated that addition to female rat liver cytosol of a purified preparation of the unusual estrogen-binding protein (UEBP) from male rat liver causes a dose-dependent inhibition of subsequent accumulation of specifically bound [3H]estradiol in the nuclei. Addition to male rat liver cytosol of 1.5 microM 2 alpha-hydroxytestosterone, testosterone, 1-dehydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone, i. e. compounds possessing marked affinity for UEBP, resulted in a 2-5-fold increase of the subsequent nuclear accumulation of estrogen-receptor complexes. The use of UEBP-deficient female rat liver cytosol revealed that the afore-mentioned steroids are ineffective with respect to estrogen reception. It is concluded that UEBP of male rat liver is capable of modulating estrogen reception.

Animals↗
PubMed1982

Multiple forms of aromatase and response of breast cancer aromatase to antiplacental aromatase II antibodies.

Two distinct aromatase-active protein complexes are solubilized by use of deoxycholate and separated by diethylamino-ethyl-cellulose chromatography from lyophilized powder of 900 X g precipitate fraction of human term placenta. Aromatase activity to produce estriol, the major estrogen of human pregnancy, was designated to be aromatase I activity and measured by estriol formation from 16 alpha-hydroxytestosterone. Aromatases II activity was the designation for that which produces estrone plus estradiol and was measured by androstenedione aromatization. Aromatases II and I are eluted with 0.25 M and 0.5 M Tris buffer, respectively, from diethylaminoethyl-cellulose column in an Mr 2 million soluble complex. Each has a minimum active Mr 135,000 subunit, which is isolated by Bio-Gel filtration in the presence of detergents, and consists of a reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (Mr 83,000) and a cytochrome P-450 (Mr 52,000). Aromatase II was found to be the major aromatase, containing approximately five times more aromatase activity, reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity, cytochrome P-450, and protein than did aromatase I. Antibodies raised in rabbits against aromatase II and its reductase suppressed aromatase II activity of breast cancer tissues, as well as of adult male lung tissue, placental microsomes, and solubilized aromatase. The breast carcinoma specimens responded to the antibodies in different degrees, but there was no response to antibodies against rat liver cytochrome P-450. The results indicate similar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450.

Adult↗
PubMed1983

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification.

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.

Animals↗
PubMed1983

Regio- and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes.

High pressure liquid chromatographic systems capable of resolving at least 28 known and potential metabolites of 17 beta-hydroxy-4-androsten-3-one (testosterone) and 4-androstene-3,17-dione (androstenedione) were used to quantitatively assess the metabolism of the two steroids in monooxygenase systems reconstituted with five purified rat liver cytochrome P-450 isozymes. Cytochromes P-450a, -b, -c, -d, and -e catalyzed the oxidation of testosterone at overall rates of 21, 27, 2, 0.7, and 3 nmol/min/nmol of cytochrome P-450, respectively; while the corresponding rates for total androstenedione metabolism were 12, 62, 1.5, 0.3, and 5. Cytochrome P-450a catalyzed the oxidation of testosterone and androstenedione almost exclusively to their respective 7 alpha-hydroxy metabolites. Cytochrome P-450b catalyzed the oxidation of testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone in approximately equal molar ratios. However, this same hemoprotein exhibited a marked stereoselectivity in the metabolism of androstenedione since the molar ratio of 16 alpha- and 16 beta-hydroxyandrostenedione was greater than 1:10. Cytochrome P-450e catalyzed the oxidation of both steroids to the same products as cytochrome P-450b, but at approximately 10% of the rate. Cytochromes P-450c and P-450d catalyzed the oxidation of testosterone and androstenedione regio- and stereospecifically to their respective 6 beta-hydroxy metabolites. These results indicate that certain cytochrome P-450 isozymes show marked positional specificity in the metabolism of both testosterone and androstenedione, and that the rate as well as stereoselectivity of the oxidative reactions can be markedly dependent on subtle differences in the structure of the steroid substrate.

Androstenedione↗
PubMed1982

Metabolism of testosterone by GR mouse mammary tumors.

The metabolism of testosterone by GR mouse mammary tumors following serial transplantation was studied. Oophorectomized female recipients were maintained on estrone and progesterone (OEP) or without hormone maintenance (oophorectomized-only group) in order to assess whether the growth of the tumor was hormone dependent (HD) or hormone independent (HI). Tumors in the early generations of the OEP group were HD (generations 1 to 4), which became HI in the latter generations (G5 to G18). All tumors developed in the oophorectomized-only group (generations 1 to 18) were HI. All tumors investigated were capable of metabolizing testosterone to 4-androstenedione, 16 alpha-hydroxytestosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstanedione, and 5 alpha-androstanediol. Total 5 alpha-reduction in OEP group ranged between 50 and 60% of neutral metabolites in HD tumors and dropped to 13 to 28% in HI tumors (generations 5 to 18), similar to the activities (20 to 30%) of the HI tumors in the oophorectomized only group. Different patterns of estrogen synthesis were observed among these tumors. Although tumors showed the presence of appreciable amounts of estriol, estrone was synthesized only in 5 of the 9 HI tumors in the oophorectomized only group. The most striking contrast was that estradiol was synthesized by all HI tumors in the oophorectomized-only group and the OEP group but not in the HD tumors.

Androstanes↗
PubMed1995

Effect of model inducers on cytochrome P450 activities of human hepatocytes in primary culture.

The dealkylations of 7-ethoxy- and 7-pentoxyresorufin,p-nitrophenol hydroxylation, and regio- and stereoselective hydroxylation of testosterone were measured to study the stability and inducibility of cytochrome P450 activities in cultured human hepatocytes. The results showed that human hepatocytes in primary culture retain the ability to increase specific cytochrome P450 activities upon incubation with inducers. 3-Methylcholanthrene produced a strong increase (6- to 21-fold over control) in 7-ethoxyresorufin O-deethylase activity and a small enhancement (1.5- to 2.5-fold) of the p-nitrophenol hydroxylation rate. Incubation of cells with phenobarbital resulted in moderate increases in 7-pentoxyresorufin O-depentylation (1.5- to 2-fold) and in testosterone hydroxylation at 16 alpha (1.5- to 4.5-fold) and 16 beta (1.3- to 4-fold) positions. Ethanol specifically increased p-nitrophenol hydroxylase activity (1.5- to 3.5-fold) and reduced 15 beta- and 6 beta-hydroxylations of testosterone. Treatment of hepatocytes with dexamethasone produced an increase of almost all the activities studied, with 6 beta- (2- to 3-fold) and 16 beta-hydroxytestosterone (1.4- to 2.4-fold) formation showing the greatest enhancement. Clofibric acid exposure resulted in 1.5- to 3-fold increases in 7-pentoxyresorufin O-depentylase and in testosterone 6 beta- and 2 beta-hydroxylase activities. Isosafrol selectively increased 7-ethoxyresorufin O-deethylase activity (2- to 3-fold), and it moderately reduced the other activities studied.

Adult↗
PubMed1995

Induction of liver microsomal cytochrome P450 in cynomolgus monkeys.

The aim of this study was to determine whether treatment of cynomolgus monkeys (Macaca fasicularis) with phenobarbital, beta-naphthoflavone, or dexamethasone causes an induction of microsomal crytochrome P450 (CYP) enzymes that are structurally and functionally related to rat enzymes belonging to the CYP1A, CYP2B, and CYP3A gene families. Oral treatment of male and female monkeys with phenobarbital resulted in a marked induction of a protein recognized by antibody against rat CYP2B1, as determined by Western immunoblotting. This protein, presumably a CYP2B enzyme, was not detectable in untreated monkeys, and was modestly inducible by dexamethasone but not beta-naphthoflavone. Induction of this CYP2B enzyme by phenobarbital was associated with a relatively large increase (up to 5-fold) in the rate of testosterone 16 beta-hydroxylation. Antibody, against rat CYP2B1 markedly inhibited this reaction in liver microsomes from phenobarbital-treated monkeys, but not from control monkeys. Consequently, the antibody-inhibitable rate of testosterone 16 beta-hydroxylation increased 17-fold after treatment of monkeys with phenobarbital, which is comparable with the situation in rats. In contrast to the rat CYP2B enzymes, the monkey CYP2B enzyme had little or no capacity to convert testosterone to 16 alpha-hydroxytestosterone or androstenedione, and had negligible capacity to O-dealkylate 7-pentoxyresorufin and 7-benzyloxyresorufin. Oral treatment of male and female monkeys with beta-naphthoflavone resulted in a marked induction of a protein recognized by polyclonal and monoclonal antibodies against rat CYP1A1 or against both CYP1A1 and CYP1A2. This protein was apparently a mixture of CYP1A1 and CYP1A2, neither of which was readily detectable in liver microsomes from control monkeys or monkeys treated with phenobarbital or dexamethasone. Induction of monkey CYP1A1/2 was associated with a marked increase in the O-dealkylation of 7-methoxyresorufin (up to 65-fold), the O-dealkylation of 7-ethoxyresorufin (up to 30-fold), and the N3-demethylation of caffeine (up to 17-fold), but only a 2-fold increase in benzo[a]pyrene 3-hydroxylation. Polyclonal antibodies against CYP1A1 markedly inhibited the N3-demethylation of caffeine and the O-dealkylation of 7-methoxy- and 7-ethoxyresorufin by liver microsomes from beta-naphthoflavone-treated monkeys, and partially inhibited the 3-hydroxylation of benzo[a]pyrene, indicating that monkey CYP1A1 and/or CYP1A2, like the corresponding rat enzymes, can catalyze all four reactions. Treatment of monkeys with phenobarbital resulted in a 2- to 3-fold induction of a protein recognized by antibody against rat CYP3A1. This protein (CYP3A8 or an immunochemically related enzyme) was constitutively expressed in untreated monkeys of both sexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylation↗
PubMed1994

Effect of repeated injection of somatomedin C on drug metabolizing enzyme activities in rats.

The effect of somatomedin C on the hepatic drug-metabolizing enzyme system in rat liver after sc injection, a clinically applicable dosing route, was studied. Although liver weight was slightly increased after somatomedin C (1 mg/kg) was injected for 7 days, no significant changes were observed in other dosing group (0.1 and 10 mg/kg). There were no significant effects of somatomedin C on cytochrome P-450 and b5 contents, and NADPH-cytochrome c reductase, aminopyrine demethylase, aniline hydroxylase, and ethoxyresorufin deethylase. Additionally, somatomedin C treatment did not affect the activities of 2 alpha-, 2 beta-, 6 beta- and 16 alpha-hydroxytestosterone and androstenedione formation from testosterone. Changes relation to dosing period were also examined with 1 mg/kg of somatomedin C. In this case, hepatic levels of cytochrome P-450 and b5, NADPH-cytochrome c reductase and drug oxidations did not differ significantly among groups treated for 3, 7, or 14 days. These results suggest that somatomedin C has no effect on the hepatic mixed-function oxidase system.

Animals↗
PubMedDate not supplied

Evidence that famciclovir (BRL 42810) and its associated metabolites do not inhibit the 6 beta-hydroxylation of testosterone in human liver microsomes.

Famciclovir is the diacetyl 6-deoxy analog of the active antiviral compound penciclovir with potential use in the treatment of infections caused by the herpes family of viruses. The major pathway of metabolism of famciclovir is deacetylation to BRL 42359 followed by oxidation to penciclovir. It is possible that famciclovir may be coadministered with cyclosporin A to combat viral infections induced by immunosuppression in organ transplant and bone marrow transplant patients. As a result, information is required on possible interactions between the cytochrome P-450 3A substrate cyclosporin A and famciclovir and its metabolites in humans. In order to probe cytochrome P-450 3A activity, testosterone 6 beta-hydroxylation in two human liver microsomal preparations was measured. Nicardipine and ketoconazole, two drugs with known inhibitory interactions with cyclosporin A, were used as positive controls. Profiles of 6 beta-hydroxytestosterone production showed no inhibition effected by famciclovir, penciclovir, or BRL 42359 when marked inhibition was observed in incubations containing nicardipine, nifedipine, or ketoconazole. Further incubations of [14C]BRL 42359 with human liver cytosol and microsomes indicated that BRL 42359 is oxidized to penciclovir in cytosol but not in microsomes and that this reaction was not dependent on the presence of NADPH. Because P-450 resides mainly in the microsomal fraction and is dependent on the presence of cofactors for catalytic activity, it seems that this oxidation is not catalyzed by cytochrome P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Aminopurine↗
PubMedDate not supplied

Cyclosporine metabolism by rat liver microsomes. Evidence for involvement of enzyme(s) other than cytochromes P-450 3A.

Cyclosporine (CyA) metabolism was investigated in liver microsomes obtained from untreated male and female Sprague-Dawley rats, and rats pretreated with ethinyl estradiol (EE), dexamethasone (DX), and phenobarbital (PB). Total hepatic microsomal cytochrome P-450 content of DX- and PB-treated male and female rats was significantly higher than that of their respective control or EE-treated rats. However, CyA metabolism was significantly increased, by all drug pretreatments, both in male and female rats. EE increased (2-5 fold) the formation of AM9 (a hydroxylated metabolite) and AM1c (a cyclized-hydroxylated product) over the CyA concentration range tested (0.2-42 microM). DX and PB significantly increased (2- to 20-fold) all detected metabolites (AM1, another hydroxylated metabolite; AM9; AM4N, an N-demethylated product; and AM1c), especially at high substrate concentrations (above 1.25 microM). Immunoblot analyses revealed that the microsomal P-450 3A2 content was decreased in EE-treated male rats, but markedly induced in those treated with either DX or PB. P-450 3A1 was undetectable in untreated and EE-treated female rats, but greatly induced in DX-treated male and female rats. Examination of P-450 3A activity, using 6 beta-hydroxytestosterone formation as a probe, confirmed the immunoblot results. These studies suggest that enzyme(s), other than P-450s 3A1 and 3A2 also play a significant role in CyA metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Baculovirus-mediated expression and characterization of rat CYP2A3 and human CYP2a6: role in metabolic activation of nasal toxicants.

Cytochrome P450 2A3 (CYP2A3) was previously identified in rat lung by cDNA cloning and recently found to be expressed at a high level in the olfactory mucosa. In the current study, CYP2A3 was expressed in insect cells lacking endogenous cytochrome P450 (P450) activity, and the substrate specificity of the recombinant cytochrome was characterized and compared with that of CYP2A6, a human ortholog of rat CYP2A3, which has been detected in human olfactory mucosa as well as in liver. The CYP2A3 and CYP2A6 cDNAs were cloned into baculovirus, and recombinant viruses were used to produce active enzymes in Spodoptera frugiperta (SF9) cells. The metabolic activities of S. frugiperta cell microsomal fractions containing CYP2A3 or CYP2A6 were studied in a reconstituted system with purified rabbit NADPH-P450 reductase. CYP2A3 was found to be active toward testosterone, producing 15 alpha-hydroxytestosterone and several other metabolites, but it had only low activity toward coumarin. On the other hand, CYP2A6 was active toward coumarin but not toward testosterone. However, both enzymes were active in the metabolic activation of hexamethylphosphoramide, a nasal procarcinogen, and 2,6-dichlorobenzonitrile (DCBN), a herbicide known to cause tissue-specific toxicity in the olfactory mucosa of rodents at very low doses. In addition, both enzymes were active toward 4-nitrophenol, a preferred substrate for CYP2E1. Consistent with CYP2A3 being a major catalyst in microsomal metabolism of DCBN, the activities of both CYP2A3 and rat olfactory microsomes in DCBN metabolism were inhibited strongly by metyrapone and methoxsalen (ID50 < 1 microM, with DCBN at 30 microM), but only marginally by 4-methylpyrazole, an inhibitor of CYP2E1. In contrast, the activity of CYP2A6 was only weakly inhibited by metyrapone or methoxsalen (ID50 > 50 microM). Thus, rat CYP2A3 and human CYP2A6 have differences in substrate specificity as well as tissue distributor. These findings should be taken into account when assessing the risk of exposure to potential nasal toxicants in humans.

Animals↗
PubMed1998

Development of a non-high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes.

The major pathway of testosterone oxidation by human liver microsomes is the formation of 6beta-hydroxytestosterone, which is catalyzed by CYP3A4/5 and which accounts for 75-80% of all metabolites formed. In the present study, we describe a non-high pressure liquid chromatography assay (HPLC) of CYP3A4/5 activity based on the release of tritium (with formation of tritiated water) upon incubation of [1,2,6,7-3H]testosterone with human liver microsomes and NADPH. Unreacted testosterone and its metabolites were quantitatively extracted from the incubation mixture with activated charcoal under conditions that resulted in no extraction of tritiated water. The amount of tritiated water formed was quantified by liquid scintillation spectrometry and compared with the amount of hydroxylated testosterone metabolites formed, as determined by HPLC. Rates of tritium release from [1,2,6, 7-3H]testosterone paralleled rates of testosterone 6beta-hydroxylation as a function of incubation time, the amount of microsomal protein, and the concentration of substrate (which yielded identical apparent Km and Vmax values). The sample-to-sample variation in tritium release from [1,2,6,7-3H]testosterone with a panel of human liver microsomes was highly correlated with rates of testosterone 6beta-hydroxylation and terfenadine metabolism, two commonly used markers of CYP3A activity. Several recombinant human P450 enzymes were incubated with [1,2,6,7-3H]testosterone, and only cDNA-expressed CYP3A4 catalyzed a high rate of tritium release. The close agreement between the tritium-release assay and HPLC procedure for measuring testosterone oxidation indicates that tritium release from [1,2,6,7-3H]testosterone provides a simple and rapid alternative to the HPLC procedure for measuring CYP3A4/5 activity in human liver microsomes. However, the tritium-release assay may have limited value in measuring CYP3A activity in liver microsomes from other species due to the presence of other P450 enzymes that can catalyze tritium release from [1,2,6,7-3H]testosterone.

Aryl Hydrocarbon Hydroxylases↗
PubMed2000

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes.

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.

Atmospheric Pressure↗
PubMed2001

CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies.

The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1alpha,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 +/- 12.3 and 86.5 +/- 3.8 pmol 6beta-OH testosterone/min/mg cellular protein from 75 microM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6beta-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

Inhibition of androgen metabolism in stroma and epithelium of the human benign prostatic hyperplasia by progesterone, estrone, and estradiol.

It has been shown that progesterone, estrone, and estradiol are present in significant amounts in the human benign prostatic hyperplasia (BPH). Therefore it was of interest to determine the inhibitory effect of these steroids on the 5 alpha-reductase and 3 alpha (beta)-hydroxysteroiddehydrogenase (HSDH) activities, the enzymes responsible for the conversion of testosterone to 5 alpha-dihydrotestosterone (DHT), and DHT to 3 alpha (beta)-androstanediols, respectively. The enzyme inhibition was analyzed in vitro by measuring the 5 alpha-reductase in the presence of either progesterone, estrone, or estradiol, using testosterone as substrate. The DHT was used as substrate for HSDH. The metabolites were quantified by t.l.c. The main results were as follows: (1) Concerning 5 alpha-reductase in BPH, the mean inhibitor constants (KI; microM) of progesterone, estrone, and estradiol were 0.11, 15.5, and 5.1, respectively. (2) Analyzing epithelium and stroma of BPH separately, the inhibition of 5 alpha-reductase resulted in nearly identical KI's. (3) Concerning HSDH in BPH, the mean KI's of progesterone, estrone, and estradiol were 169, 63, and 192, respectively. (4) Analyzing epithelium and stroma of BPH separately, the inhibition of HSDH led to nearly identical KI's. (5) The kinetic parameters (KM, Vmax) of the 5 alpha-reduction of progesterone and testosterone were nearly identical. These results led to the suggestion that the endogenous concentrations of progesterone, estrone, and estradiol have no significant inhibitory effect on the 5 alpha-reductase and HSDH in vivo. Furthermore, the nearly identical inhibitor constants found for both enzymes in epithelium and stroma of BPH indicate that in both compartments the 5 alpha-reductase and HSDH are qualitatively identical.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Electron leakage from the adrenal cortex mitochondrial P450scc and P450c11 systems: NADPH and steroid dependence.

In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H2O2 in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased. However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11 alpha-hydroxy groups (11 alpha-OH-testosterone and 11 alpha-OH-cortisol) decreased leakage, and those with 11 beta-OH groups (11 beta-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H2O2 formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells.

Adrenal Cortex↗