Comparison of CYP3A activities in a subclone of Caco-2 cells (TC7) and human intestine

PURPOSE: To compare the activity of the CYP3A enzyme expressed by TC7, a cell culture model of the intestinal epithelial cell, to the activity of human intestinal CYP3A4, using terfenadine as a substrate. METHODS: The metabolism of terfenadine was investigated in intact cells and microsomal preparations from TC7, human intestine, and liver. The effect of two CYP3A inhibitors, ketoconazole and troleandomycin (TAO), on the metabolism of terfenadine was also examined. RESULTS: Only hydroxy-terfenadine was detected in TC7 microsomal incubations. In contrast, azacyclonol and hydroxy-terfenadine were detected in human intestinal and hepatic microsomal incubations. The Km values for hydroxy-terfenadine formation in TC7 cells, intestine and liver microsomes were 1.91, 2.5, and 1.8, microM respectively. The corresponding Vmax values were 2.11, 61.0, and 370 pmol/min/mg protein. Km values for azacyclonol in intestinal and hepatic samples were 1.44 and 0.82 microM and the corresponding Vmax values were 14 and 60 pmol/min/mg protein. The formation of hydroxy-terfenadine was inhibited by ketoconazole and TAO in human intestine and TC7 cell microsomes. The Km and Vmax values for terfenadine metabolism in intact TC7 cells were similar to those from TC7 cell microsomes. CONCLUSIONS: Our results indicate that TC7 cells are a potentially useful alternative model for studies of CYP3A mediated drug metabolism. The CYP3A expressed by TC7 cells is not CYP3A4, but probably CYP3A5, making this cell line suitable for studies of colonic drug transport and metabolism.     

Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism

Cytochrome P450 3A (CYP3A) metabolizes a diverse array of clinically important drugs. For some of these (e.g., cyclosporine, verapamil, midazolam), CYP3A in the intestinal mucosa contributes to their extensive and variable first-pass extraction. To further characterize this phenomenon, we measured CYP3A content and catalytic activity toward the probe substrate midazolam in mucosa isolated from duodenal, jejunal and ileal sections of 20 human donor intestines. For comparison, the same measurements were performed for 20 human donor livers, eight of which were obtained from the same donors as eight of the intestines. Excellent correlations existed between homogenate and microsomal CYP3A content for the three intestinal regions. Median microsomal CYP3A content was greatest in the duodenum and lowest in the ileum (31 vs. 17 pmol/mg of protein). With respect to midazolam 1'-hydroxylation kinetics, the median Km for each intestinal region was similar to the median hepatic Km, approximately 4 microM. In contrast, the median Vmax decreased from liver to duodenum to jejunum to ileum (850 vs. 644 vs. 426 vs. 68 pmol/min/mg). Intrinsic clearance (Vmax/Km) followed a similar trend for the intestinal regions; median duodenal intrinsic clearance was comparable to hepatic intrinsic clearance (157 and 200 microl/min/mg, respectively). Vmax correlated with CYP3A content for all tissues except the ileum. Duodenal and jejunal Vmax and CYP3A content varied by >30-fold among donors. Microsomes prepared from every other 1-foot section of six intestines were also analyzed for CYP3A as well as for two coenzymes. In general, CYP3A activity, CYP3A content and CYP reductase activity rose slightly from duodenum to middle jejunum and then declined to distal jejunum and ileum. Cytochrome b5 content and cytochrome b5 reductase activity varied little throughout the intestinal tract. Regional intrinsic midazolam 1'-hydroxylation clearance was greatest for the jejunum, followed by the duodenum and ileum (144, 50 and 19 ml/min, respectively). Collectively, these results demonstrate that the upper small intestine serves as the major site for intestinal CYP3A-mediated first-pass metabolism and provides a rationale for interindividual differences in oral bioavailability for some CYP3A substrates.     

Zidovudine azido-reductase in human liver microsomes: activation by ethacrynic acid, dipyridamole, and indomethacin and inhibition by human immunodeficiency virus protease inhibitors

AZT (zidovudine, 3'-azido-3'-deoxythymidine), although metabolized primarily to AZT-glucuronide, is also metabolized to 3'-amino-3'-deoxythmidine (AMT) by reduction of the azide to an amine. The formation of the myelotoxic metabolite AMT has not been well characterized, but inhibition of AMT formation would be of therapeutic benefit. The aim of this study was to identify compounds that inhibit AMT formation. Using human liver microsomes under anaerobic conditions and [2-14C]AZT, K(m) values of AZT azido-reductase, estimated by radio-thin-layer chromatography, were 2.2 to 3.5 mM (n = 3). Oxygen completely inhibited this NADPH-dependent reduction. Thirteen of the 28 compounds tested inhibited the formation of AMT. In addition to the CYP3A4 inhibitors ketoconazole, fluconazole, indinavir, ritonavir, and saquinavir, metyrapone strongly inhibited AMT formation. An unexpected finding was the more-than-twofold increase in AMT formation in the presence of ethacrynic acid, dipyridamole, or indomethacin. Such activation of toxic metabolite formation would impair drug therapy.     

Enzymatic reduction of chromium(VI) by human hepatic microsomes

The reduction of chromium(VI) by human hepatic microsomes was investigated. The reduction rates were proportional to the amount of microsomes added and reduction was mediated by an NADPH-dependent enzymatic system which exhibited a Km for chromate of 1.04 +/- 0.18 microM and a Vmax of 5.03 +/- 0.49 nmol/min/mg protein. Relative to incubation under 0% O2, 21% O2 inhibited microsomal Cr(VI) reduction in three individuals by 53, 36 and 37%. Cr(VI) reduction was not inhibited by metyrapone, carbon monoxide, aminopyrine, piperonyl butoxide or chloroform, suggesting that cytochrome P450s did not play a major role. Thallium trichloride (0.13 and 0.26 mM), a known flavoprotein inhibitor, caused a complete inhibition of both Cr(VI) reduction and NADPH:cytochrome P450 (c) reductase activity. A partial inhibition of Cr(VI) reduction was seen in the presence of n-octylamine, which may suggest a possible role for flavin-containing monooxygenase (FMO). Overall, human microsomal Cr(VI) reduction is very different from the P450-mediated microsomal reduction observed in rodents. Specifically, the human system is much less oxygen-sensitive, has a much greater affinity for chromate and is apparently mediated by flavoproteins.     

Metabolism of epinastine, a histamine H1 receptor antagonist, in human liver microsomes in comparison with that of terfenadine

Epinastine is a non-sedative second-generation antiallergic drug, like terfenadine. In the present study, the metabolism of epinastine in human liver microsomes was investigated and compared with that of terfenadine. Terfenadine was extensively metabolized to terfenadine acid with a Km value of 1.78 microM, a Vmax value of 173.8 pmol/min/mg and a metabolic clearance (Vmax/Km) of 103.9. Epinastine, in contrast, was poorly metabolized by microsomes from the same source with a high Km value of 232 microM. Metabolic clearance of epinastine was only 0.832, which was lower by three orders of magnitude than that of terfenadine. Studies with microsomes expressing recombinant cytochrome P450 (CYP) species revealed that the CYP isoforms responsible for epinastine metabolism are CYP3A4, 2D6 and (to a minor extent) 2B6. Epinastine and terfenadine had no effect on CYP1A2 (theophylline 1-demethylation), 2C8/9 (tolbutamide hydroxylation) or 2E1 (chlorzoxazone 6-hydroxylation) activity, but weakly inhibited CYP2D6 (debrisoquine 4-hydroxylation) activity. CYP3A4 (testosterone 6 beta-hydroxylation) activity was strongly inhibited by terfenadine with a Ki value of 25 microM, whereas epinastine had no effect at up to 100 microM. Thus, epinastine is very poorly metabolized compared to terfenadine in human liver microsomes and does not inhibit CYP3A4 activity in vitro, unlike terfenadine.     

Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.     

(+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502) as a novel substrate for cytochrome P450 2A6 in human liver microsomes

(+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) was oxidized by human liver microsomes to produce the S-oxide as a sole metabolite. Indirect evidence suggested that the S-oxidation was catalyzed by cytochrome P450 (CYP). Eadie-Hofstee plots showed biphasic pattern, suggesting that at least two enzymes were involved in the S-oxidation in human liver microsomes. Kinetic parameters of the S-oxidase with high-affinity showed Km and Vmax values of 20.9 +/- 4.4 microM and 0.111 +/- 0.051 nmol/min/mg microsomal protein, respectively. The S-oxidase activity was inhibited by coumarin and anti-CYP2A antibody. Among the contents of forms of CYP 20 samples of human liver microsomes, the content of CYP2A6 correlated with S-oxidase activity measured with 50 microM SM-12502 (r = .808, P < .0005). A close correlation (r = .908, P < .0001) was observed between activities of SM-12502 S-oxidase and coumarin 7-hydroxylase. Microsomes from genetically engineered human B-lymphoblastoid cells expressing CYP2A6 metabolized SM-12502 to the S-oxide efficiently. The results indicate that CYP2A6 isozyme is a major form of CYP responsible for the S-oxidation of SM-12502 in human liver microsomes. Thus, SM-12502 will be a useful tool in further research to analyze a human genetic polymorphism of CYP2A6.     

Enzymology of the reduction of the novel fused pyrazine mono-n-oxide bioreductive drug, RB90740 roles for P450 reductase and cytochrome b5 reductase

RB90740 is the lead compound in a series of fused pyrazine mono-N-oxide bioreductive drugs. Theses agents have potential application in cancer therapy, since they are more toxic to hypoxic than to aerobic cells as a consequence of their bioactivation by cellular reductase enzymes within the hypoxic regions of a tumour. In this study, mouse liver microsomes have been used to characterise the enzymology of the reductive activation of RB90740. Under hypoxic conditions, the reduction of RB90740 to its stable 2-electron reduced product RB92815 was supported by both NADH and NADPH, the former supporting a rate approximately 80% of the latter. Combining the two cofactors had no additive effect. Neither carbon monoxide nor metyrapone inhibited reduction of RB90740, indicating that P450 isozymes were not involved in the reduction of this compound. 2' AMP, and inhibitor of P450 reductase, did not inhibit formation of RB92815, whereas DPIC, another inhibitor but with a different mode of action, inhibited both the NADH, and NADPH-dependent reduction of RB90740. Similarly, two selective inhibitors of NADH: cytochrome b5 reductase, pHMB and PTU, completely inhibited both NADH and NADPH-dependent reduction of RB90740. Our findings implicate P450 reductase, cytochrome b5 reductase, and cytochrome b5 in the activation of the compound. However, there is no clear relationship between the intracellular levels of P450 reductase and cytochrome b5 reductase and the hypoxic toxicity of RB90740, which implies that other factors, in addition to drug activation, play a major role in controlling the toxicity of this particular bioreductive drug.     

Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation

AIMS: Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. METHODS: Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. RESULTS: Km and Vmax values determined in human liver microsomes were lower for the demethylation (61 +/- 21 microM, 159 +/- 42 pmol min(-1) mg protein(-1) mean +/- s.d.; n = 4), than for the N-oxidation of CLZ (308 +/- 1.5 microM, 456 +/- 167 pmol min(-1) mg protein(-1); n = 3). Formation of DCLZ was inhibited by fluvoxamine (53 +/- 28% at 10 microM), triacetyloleandomycin (33 +/- 15% at 10 microM), and ketoconazole (51 +/- 28% at 2 microM) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34 +/- 16% at 10 microM) and ketoconazole (51 +/- 13% at 2 microM), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. Km and Vmax values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. CONCLUSIONS: It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

Characterization of the cytochrome P450 isoenzymes involved in the in vitro N-dealkylation of haloperidol

AIMS: The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). METHODS: In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). RESULTS: There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. Km and Vmax values ranged respectively from 50 to 78 microM and from 180 to 412 pmol mg-1 min-1 CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (Ki=0.1 microM), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC50 > 100 microM). CPHP formation was, moreover, enhanced by apha-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC50 values between 2.7 and 8.5 microM, but not (IC50 > 100 microM) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). CONCLUSIONS: These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.     

Recognition of the blood group H type 2 trisaccharide epitope by 28 monoclonal antibodies and three lectins

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductaseI (NQOI or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQOI because of a lack of correlation between NQOI (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH-cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).     

Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occurring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the p450 proteins and the corresponding C. maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (Ks= 26 micron) and n-octadecane (Ks = 27 microM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks = 127 microM) and myristic acid (Ks = 134 microM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined for n-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax = 27 microM x min-1, Km = 54 microM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax = 30 microM x min-1, Km = 61 microM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids.     

Formation in vitro of an inhibitory cytochrome P450 x Fe2+-metabolite complex with roxithromycin and its decladinosyl, O-dealkyl and N-demethyl metabolites in rat liver microsomes

1. Roxithromycin and its major metabolites found in rat and human urine, namely the decladinosyl derivative (M1), O-dealkyl derivative (M2) and N-demethyl derivative (M3), were incubated with rat liver microsomes and formation of an inhibitory cytochrome P450 (CYP)-metabolite complex and of formaldehyde (measurement of N-demethylation) were determined in vitro. Troleandomycin and erythromycin were also used for comparison. 2. Dexamethasone very significantly induced the microsomal N-demethylations of these macrolide antibiotics. The order of magnitude for the Vmax/Km ratio of N-demethylations by liver microsomes from dexamethasone-treated rats was troleandomycin > erythromycin = M2 > roxithromycin > M3, M1. 3. Formation of an inhibitory P450 x Fe2+-metabolite complex was detected on incubation of these macrolide antibiotics with rat liver microsomes in the presence of an NADPH-generating system and the order of maximum complex formation was troleandomycin > erythromycin > M2 > roxithromycin > M3 > M1. 4. Troleandomycin, erythromycin and M2 inhibited CYP3A-dependent testosterone 6beta-hydroxylation catalysed by liver microsomes from the dexamethasone-treated rat by 54, 33 and 23%, respectively, but roxithromycin, M3 and M1 were very weak by comparison. In the untreated rat, only testosterone 6beta-hydroxylation, but not testosterone 16alpha- and 2alpha-hydroxylation and androstenedione formation, activities were inhibited, indicating that inhibitory actions of these antibiotics are specific for CYP3A enzymes in liver microsomes. 5. These results support the view that formation of an inhibitory P450-metabolite complex is prerequisite for the inhibition of CYP3A-dependent substrate oxidations by rat liver microsomes and that M2 (and M3, to a lesser extent) may be the active metabolite that can form an inhibitory P450-metabolite complex by CYP3A enzyme(s).     

Effects of triphenyltin and other organotins on hepatic monooxygenase system in fish

The interaction of the organotin fungicide triphenyltin chloride (TPT) with fish microsomal monooxygenase systems has been studied in vitro and in vivo in the marine fish scup (Stenotomus chrysops). In vitro incubation of fish liver microsomes with TPT resulted in the conversion of about 40% of the native total spectral P450 to P420. In addition, a strong concentration-related inhibition of ethoxyresorufin O-deethylase (EROD) activity was observed, with a complete loss at 1.0 mM TPT. Pentoxyresorufin-O-dealkylase (PROD) activity was inhibited only at the highest concentration tested. This suggests either some specificity for the EROD catalyst CYP1A1, or a loss of reductant NADPH cytochrome c reductase as the cause. Further in vitro incubations showed that NADPH, but not NADH, cytochrome c reductase was strongly inhibited at 100 microM TPT and higher. To further investigate this effect, fish were injected with single doses of 5, 25 and 50 microM TPT (1.9, 9.6 and 19.3 mg kg-1 TPT), and 24 and 48 h later, hepatic microsomes were analyzed for total P450 content, EROD activity, NAD(P)H cytochrome c reductase, and the content of three CYP forms. EROD activity tended to be decreased in TPT-treated scup, with the response being stronger after 48 than 24 h. No significant conversion of spectrally determined P450 to cytochrome P420 was found, and cytochrome b5 was not affected. However, both NAD(P)H cytochrome c reductases were significantly inhibited at all concentrations. Immunoblot analysis showed reduction of CYP1A1 content at all doses, being significant at 25 mM after 48 h, but no decrease in CYP3A-like protein, the dominant catalyst of testosterone 6 beta-hydroxylation, nor CYP2B-like protein, the major contributor to indicates significant effects of TPT at high concentrations on fish hepatic CYP1A1 protein, EROD activity and the reductases. TPT seems to act more specifically on CYP1A1 than on other CYP forms. These findings combined with those of our previous studies (Brüschweiler BJ, Würgler FE, Fent K. Environ Toxicol Chem 1996;15:827-735; Fent K, Bucheli TD. Aquat Toxicol 1994;28:107-126; Fent K, Stegeman JJ. Aquat Toxicol 1991;20:159-168; Fent K, Stegeman JJ. Aquat Toxicol 1993;24:219-240) indicate a general degenerative effect of organotins on the fish microsomal monooxygenase system, although some differences are seen between the organotins, and between species. We conclude that these effects of organotins have consequences for use of CYP1A as a biomarker and endocrine disruption.     

Characterization of NAD(P)H-dependent ubiquinone reductase activities in rat liver microsomes

Exogenous ubiquinone-10 was efficiently reduced by rat liver microsomes in the presence of NADH and NADPH under anaerobic conditions. Ubiquinone-10 reduced under anaerobic conditions was rapidly re-oxidized by the re-aeration. The reduction and re-oxidation were not observed when the reactions were carried out with the boiled microsomes or without microsomes, suggesting that the reactions were enzymatically catalyzed by the electron transport system(s) from NAD(P)H to O2 through the ubiquinone. The Km and Vmax of the reductase activity for NADH were 0.4 mM and 1.7 nmol/min per mg of protein, and those for NADPH were 19 microM and 2.1 nmol/min per mg of protein, respectively. The NADH-dependent oxidoreduction system was different from the NADPH-dependent system because of the following observations; (1) rotenone inhibited only the NADH-dependent ubiquinone-10 reductase, (2) dicoumarol inhibited the NADPH-dependent ubiquinone-10 reduction more potently than the NADH-dependent reduction and (3) the activity oxidizing the reduced ubiquinone-10 in the presence of NADH was less than that in the presence of NADPH. Endogenous ubiquinone-9 was also reduced and re-oxidized in essentially the same manner as exogenous ubiquinone-10. Thus, ubiquinone-10 oxidoreductase in rat liver microsomes acts on endogenous ubiquinone-9.     

Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: application of the relative activity factor approach

The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activity factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4'-hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, respectively. The kinetics of these reactions were studied in human liver microsomes, in microsomes from human lymphoblastoid cells, and in insect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of Vmax (reaction velocity at saturating substrate concentrations) in human liver microsomes of the isoform-specific index reaction divided by the Vmax of the reaction catalyzed by the cDNA expressed isoform. RAFs were also determined for SUPERMIX, a commercially available mixture of cDNA expressed human drug metabolizing CYPs formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes. Lymphoblast RAF2C9 in human liver microsomes ranged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF2C9 in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg protein (mean value: 49), while a value of 201 pmol CYP/mg protein was obtained for SUPERMIX. Both lymphoblast and insect cell RAF2C19 in human liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean values: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system used had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or of CYP2C19 as a S-mephenytoin hydroxylase. However insect cell expressed CYP2C19 (which includes oxidoreductase) had substantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphoblast-determined RAF2C9 to RAF2C19 in human livers was 3.0 (range 1.6-17.9; n = 10), while this ratio for SUPERMIX was 8.6. The ratio of mean insect cell-determined RAF2C9 to RAF2C19 in human livers was 1.7 (range 0.04-16.2; n = 10), while this ratio for SUPERMIX was 7.0. Neither ratio is in agreement with the 20:1 ratio of immunoquantified levels of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels higher than those in human liver microsomes.     

Platelet-activating factor levels in term and preterm human milk

Tolterodine, a new muscarinic receptor antagonist, is metabolized via two pathways: oxidation of the 5-methyl group and dealkylation of the nitrogen. In an attempt to identify the specific cytochrome P450 enzymes involved in the metabolic pathway, tolterodine was incubated with microsomes from 10 different human liver samples where various cytochrome P450 activities had been rank ordered. Strong correlation was found between the formation of the 5-hydroxymethyl metabolite of tolterodine (5-HM) and CYP2D6 activity (r2, 0.87), as well as between the formation of N-dealkylated tolterodine and CYP3A activity (r2, 0.97). When tolterodine was incubated with human liver microsomes in the presence of compounds known to interact with different P450 isoforms, quinidine was found to be the strongest inhibitor of the formation of 5-HM. Ketoconazole and troleandomycin were found to be the strongest inhibitors of the formation of N-dealkylated tolterodine. A weak inhibitory effect on the formation of N-dealkylated tolterodine was found with sulfaphenazole, whereas tranylcypromine did not inhibit the formation of this metabolite. Microsomes from cells overexpressing CYP2D6 formed 5-HM, whereas N-dealkylated tolterodine was formed by microsomes expressing CYP2C9, -2C19, and -3A4. The Km for formation of N-dealkylated tolterodine by CYP3A4 was similar to that obtained in human liver microsomes and higher for CYP2C9 and -2C19. We conclude from these studies that the formation of 5-HM is catalyzed by CYP2D6 and that the formation of N-dealkylated tolterodine is predominantly catalyzed by CYP3A isoenzymes in human liver microsomes.     

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV)

In this study we have investigated the occurrence of cytochrome P450 isoforms and of related cytochrome P450 reductase in human hepatic stellate cells (hHSC), a type of cell having relevant roles in physiopathological conditions of the liver. By performing immunoblotting of hHSC microsomes and immunofluorescence analysis associated to confocal laser microscopy we detected only P450 enzymes belonging to the cytochrome P450 3A subfamily (CYP3A) as well as cytochrome P450 reductase. The presence of CYP3A was further indicated by detection of testosterone 6beta-hydroxylase activity in hHSC microsomes. Other important human P450 forms were either undetectable (CYP1A2, CYP2E1, CYP2C8/9/19 and CYP4A) or bearly detectable (CYP1A1) in hHSC. This is the first study showing existence of active cytochrome P450 isoforms in human HSC.     

Female exposure to high G: chronic adaptations of cardiovascular functions

The substrate structure-activity relationships described for the major human drug-metabolizing cytochrome P450 (P450 or CYP) enzymes suggest that the H1 receptor antagonist terfenadine could interact with CYP2D6 either as a substrate or as an inhibitor, in addition to its known ability to act as a substrate for CYP3A4. Based on this substrate structure-activity relationship, computer modeling studies were undertaken to explore the likely interactions of terfenadine with CYP2D6. An overlay of terfenadine and dextromethorphan, a known substrate of CYP2D6, showed that it was possible to superimpose the site of hydroxylation (t-butyl group) and the nitrogen atom of terfenadine with similar regions in dextromethorphan. These observations were substantiated by the ease of docking of terfenadine into a protein model of CYP2D6. Experimentally, terfenadine inhibited CYP2D6 activity in human liver microsomes with an IC50 of 14-27 microM, depending on the CYP2D6 substrate used. The inhibition of CYP2D6 was further defined by determining the Ki for terfenadine against bufuralol 1'-hydroxylase activity in four human livers. Terfenadine inhibited bufuralol 1'-hydroxylase activity with a Ki of approximately 3.6 microM. The formation of the hydroxylated metabolite (hydroxyterfenadine) in microsomes prepared from human liver and specific P450 cDNA-transfected B lymphoblastoid cells indicated that only CYP2D6 and CYP3A4 were involved in this transformation. As expected, the rate of formation was greatest with CYP3A4 (Vmax = 1257 pmol/min/nmol of P450), with CYP2D6 forming the metabolite at a 6-fold lower rate (Vmax = 206 pmol/min/nmol of P450). The two enzymes had similar KM values (9 and 13 microM, respectively). These data indicate that, as predicted from modeling studies, terfenadine has the structural features necessary for interaction with CYP2D6.     

Model calculation of the crystal-field magnetostriction and its temperature dependence in the itinerant uniaxial ferromagnet Y2Fe17

A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the monooxygenase reaction with benzphetamine as substrate. Cytochrome b5 also can increase the monooxygenase coupling efficiency. At low ionic strength cytochrome b5 did not much influence the reduction of P450, but the rate constant of the cytochrome b5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b5 on the cytochrome P450-mediated monooxygenation reaction is due to an increase in the efficiency of the electron transfer reaction: With cytochrome b5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b5 complex in a single interaction, obviating the need for a second interaction with the reductase.