Oxidative metabolism of zolpidem by human liver cytochrome P450S

The aim of this study was to identify the form(s) of cytochrome P450 (CYP) responsible for the biotransformation of zolpidem to its alcohol derivatives which, after rapid conversion to carboxylic acids, represents the main way of metabolism in humans. In human liver microsomes, zolpidem was converted to alcohol derivatives. Production of these correlated with the level of CYP3A4 and with cyclosporin oxidation and erythromycin N-demethylation activities, but not with the level of CYP1A2 nor with ethoxyresorufin O-deethylation or S-mephenytoin 4'-hydroxylation activities. Liver microsomes from CYP2D6-deficient patients exhibited normal activity. Production of alcohol derivatives was significantly inhibited by anti-CYP3A antibodies and by ketoconazole. Antibodies directed against other CYP forms (including CYP1A1, CYP1A2, CYP2A6, CYP2B4, and CYP2C8), and CYP-specific substrates or inhibitors (including propranolol, coumarin, mephenytoin, sulfaphenazole, quinidine, aniline, and lauric acid) produced a moderate or no inhibitory effect. cDNA-expressed CYP3A4 and CYP1A2 generated significant amounts of one of the alcohol derivatives, whereas CYP2D6 generated both of them in similar amounts. In human hepatocytes in primary culture, zolpidem was extensively and almost exclusively converted to one of the carboxylic acid derivatives, the main species identified in vivo. Treatment of cells with inducers of CYP1A (beta-naphthoflavone) and CYP3A (rifampicin and phenobarbital) greatly increased the rate of production of this metabolite. We conclude that the formation of alcohol derivatives of zolpidem is rate-limiting and principally mediated by CYP3A4. Both CYP1A2 and CYP2D6 participate in alcohol formation; but, because of their low relative level of expression in the human liver, their contribution is minor.     

The traditional fermented milk products of the Sudan

Primary human hepatocytes contain a full complement of human drug-metabolizing enzymes and therefore represent a relevant experimental system for the evaluation of pharmacokinetic drug-drug interaction potential in human. In this study, the cytochrome P450 (CYP) induction potential of pantoprazole (PAN) was evaluated and compared to two other proton pump inhibitors (PPIs), omeprazole (OM) and lansoprazole (LAN). Primary human hepatocytes from three donors were studied. The hepatocytes were cultured for 3 days, followed by treatment for 3 days with the PPIs at 2, 5 and 10 microM. Two other known CYP inducers, 3-methylcholanthrene at 1 microM and rifampin at 50 microM, were also evaluated. Induction potentials of these chemicals for CYP1A and CYP3A were evaluated by isozyme activity and isozyme content. 7-Ethoxyresorufin-O-deethylase and testosterone 6beta-hydroxylase activities were used as endpoints for CYP1A and CYP3A, respectively. Isozyme protein contents of CYP1A and CYP3A were evaluated via Western blotting. The results showed that for CYP1A induction, the rank ordering in induction potential was consistently OM > LAN > PAN. CYP3A induction by the PPI's were observed in two of the three hepatocyte cultures, with no apparent differences in induction potency for the three compounds. Our results on CYP1A induction suggest that PAN has a lower drug-drug interaction potential than OM and LAN.     

Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture

The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.     

Major involvement of rabbit liver cytochrome P4501A in thiabendazole 5-hydroxylation

1. Thiabendazole is a widely used food preservative and anthelmintic drug for breeding animal species. In order to characterize precisely the cytochrome P450 isozyme(s) involved in its major route of metabolism, a rapid and sensitive spectrofluorimetric method was developed for the simultaneous determination of thiabendazole and its main hepatic metabolite 5-hydroxythiabendazole. 2. The kinetics of thiabendazole 5-hydroxylation were determined in microsomal preparations from control rabbits or animals previously treated with either beta-naphthoflavone, isosafrole, phenobarbital, rifampicin or clofibrate. These treatments led to specific induction of CYP1A1, 1A2, 2B4, 3A6 and 4A1 respectively. 3. By considering this panel of characterised microsomal preparations, only those obtained from BNF-treated rabbits exhibited an increase in thiabendazole 5-hydroxylase activity Ethoxyresorufin O-deethylation in these microsomes was solely inhibited by thiabendazole. These argue for a specific involvement of the CYP1A subfamily. 4. In the CYP1A subfamily, CYP1A2 appears to be responsible for basal 5-hydroxylation and further unidentified metabolism of thiabendazole in control livers. However, the major involvement of CYP1A1 is supported by the following characteristics of 5-hydroxylation of thiabendazole: (1) the correlation with CYP1A1 expression and (2) the inhibition by ellipticine and not by furafylline, inhibitors of CYP1A1 and CYP1A2 respectively. 5. All these data demonstrated that the rabbit cytochrome P4501A is predominantly involved in thiabendazole 5-hydroxylation which has been suspected to be critical in terms of safety of the parent drug.     

Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail (Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs)

Ergosterol biosynthesis inhibiting fungicides (EBIFs) have complex effects on the hepatic microsomal monooxygenase systems of vertebrate species, having been described as mixed inducers and inhibitors of cytochrome P450. In the current study, we examined the effects of two EBIFs in clinical use, clotrimazole and ketoconazole, and two agricultural EBIFs, propiconazole and vinclozolin, on hepatic monooxygenase activities and P450 apoprotein expression in the male Sprague-Dawley rat and the male bobwhite quail. EBIFs produced Type II binding spectra with hepatic microsomes from both species and were effective inhibitors of methoxyresorufin O-demethylase, an activity selective for P450 isozymes in gene family 1. However, the EBIFs varied widely in their effectiveness as inducers of P450 isozymes in gene families 1, 2, 3 and 4, both within the same species and between species. In the rat, clotrimazole was the most effective inducer, increasing expression of CYP 3A isozymes over 450-fold, CYP 2B1/2 30-fold and CYP 1A1/2 12-fold and suppressing expression of CYP 2C11 nearly 70%. By contrast, in the quail, clotrimazole was the least effective inducer. In quail, vinclozolin and propiconazole elevated total P450 content 10- and 7-fold, respectively. The induction response also appeared to be mixed, but in this case consisted of a 5-fold induction of P450s in gene family 1A, a 3-fold induction of P450s in gene family 3A and 4A, and induction of protein(s) from gene family 2, cross-reactive with antisera against rat CYP 2C11 and CYP 2A1. A protein that was cross-reactive with antibodies raised against rat CYP 2B1 was decreased with EBIF treatment. In conclusion, EBIFs have complex patterns of induction, suppression and inhibition of cytochrome P450 isozymes in both mammals and birds, which vary according to both the fungicide and the species.     

Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices

Human, rat, and dog phase I and phase II xenobiotic metabolism in precision-cut liver slices and freshly isolated hepatocytes was compared using a range of substrates. Carbamazepine (50 microM) and styrene (2 mM) were used as probes to study the maintenance of cytochrome P450 and epoxide hydrolase-mediated metabolism in male Sprague-Dawley rat, precision-cut liver slices and hepatocytes. Carbamazepine metabolism in both models resulted in the formation of the bioactive 10,11-epoxide (KM = 766 microM and Vmax = 2.5 pmol/min/mg protein in precision-cut slices). Epoxide formation was higher (2.4-fold) in hepatocytes than slices. Styrene was deactivated to styrene diol at a higher rate in hepatocytes (9.7-fold) than slices. The lower rate of metabolism in slices compared with hepatocytes confirms our previous observations using testosterone, 7-ethoxycoumarin, 1-chloro-2,4-dinitrobenzene and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone in the rat. Testosterone 6 beta-hydroxylation in human liver slices was similar to cultured hepatocytes, but lower than in freshly isolated hepatocytes. 7-Ethoxycoumarin O-deethylation was higher in freshly isolated human hepatocytes, as was the ratio of glucuronide to 7-hydroxycoumarin. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, and 1-chloro-2,4-dinitrobenzene conjugation were also lower in male beagle dog slices, compared with freshly isolated hepatocytes. Attempts at long-term preservation of dog liver slices using vitrification and storage for up to 9 days at -196 degrees C resulted in the retention of phase I and phase II metabolism, although conjugation was lower than in freshly prepared slices. Xenobiotic metabolism in short-term incubations is consistently lower in dog and rat precision-cut slices than in freshly isolated hepatocytes; whereas, in humans, this quantitative difference is partly hidden by the large interindividual variation.     

The effect of 1,2,3,4-tetrachlorodibenzo-p-dioxin on drug-metabolizing enzymes in the rat liver

The effects of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) on drug-metabolizing enzymes were studied in male and female rats. 1,2,3,4-TCDD (25, 50, 100 and 200 mumol/kg) was administered by i.p. injection once. Among the cytochrome P-450 (P450)-mediated monooxygenase activities tested, 7-ethoxyresorufin O-deethylase (EROD) activities in both male and female rats, which are associated with CYP1A1, were remarkably induced by all doses of 1,2,3,4-TCDD. The relative induction to each control activity were from 3.0- to 24.5-fold and from 2.2- to 16.5-fold, respectively. Also, 1,2,3,4-TCDD increased other CYP1A-mediated monooxygenase activities such as 7-ethoxycoumarin O-deethylase (ECOD) and 7-methoxyresorufin O-demethylase (MROD) in male and female rats dose-dependently (1.4- to 4.3-fold). Western immunoblotting showed that the levels of CYP1A1 and CYP1A2 proteins in liver microsomes were increased by 1,2,3,4-TCDD. Although the activities of other P450-mediated monooxygenases, namely 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND) and nitrosodimethylamine N-demethylase (NDAND) in both male and female rats were induced at high doses (> or = 50 mumol/kg) of 1,2,3,4-TCDD, the relative level was low compared with those of the CYP1A-mediated monooxygenase such as EROD, ECOD or MROD. In addition to P450-mediated monooxygenase, there was significant induction in the activities of the Phase II drug-metabolizing enzymes, UDP-glucuronyltransferase (UGT) activities towards 4-nitrophenol (4-NP) and 7-hydroxycoumarin (7-HC) and glutathione S-transferase (GST) towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and DT-diaphorase. These results indicate that 1,2,3,4-TCDD induces both Phase I (CYP1A-mediated monooxygenase) and Phase II drug-metabolizing enzymes (UGT, GST, DT-diaphorase) in the male and female rat liver, and that the alterations of drug-metabolizing enzyme are characteristic of PCDD toxicity.     

Isozyme selective alterations of the expression of cytochrome P450 during regeneration of male rat liver following partial hepatectomy

1. During liver regeneration in the male rat, the metabolic activities of imipramine were differentially affected depending on the specific metabolic pathways. Imipramine N-demethylation was markedly reduced whereas 2-hydroxylation showed only a moderate reduction following partial hepatectomy. 2. A slight decline was observed in the hepatic microsomal content of CYP2D apoprotein, whereas a substantial decrease occurred in CYP2C11 content during liver regeneration. Since imipramine 2-hydroxylation and N-demethylation are mediated by CYP2D and 2C11 respectively, metabolic pathway-specific alterations in the activities of imipramine metabolism are explained by the isozyme selective alteration in the levels of CYPs in regenerating liver. 3. No significant effect of regeneration was observed on expression of CYP2B1 and 2E1 apoproteins. CYP3A2 apoprotein, one of the male-specific CYP isoforms, was significantly suppressed in regenerating liver showing a similar pattern of alteration to the levels of CYP2C11. The alteration pattern of the CYP1A1 level was different to the above with a moderate decline at the first day post-operation and a marked rebound thereafter. 4. In the partially hepatectomized male rate, no significant increase in androstenedione 5-alpha reductase activity, an activity predominant in the female rat, was detected. It is concluded that the pattern of alterations of hepatic oxidative metabolism during liver regeneration was not related to the functional feminization of the liver.     

Characterization of the effects of musk ketone on mouse hepatic cytochrome P450 enzymes

Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/ kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later. While MX inhibited more than 90% of the PB-induced CYP2B activity in the microsomes, MK caused only a small (about 20%) reduction in PB-induced CYP2B enzyme activity. These results indicate that, like MX. MK is a PB-type inducer of mouse liver CYP2B isozymes, but unlike MX, MK does not effectively inhibit PB-induced CYP2B enzyme activity.     

High-level expression of rat farnesyl:protein transferase in Escherichia coli as a translationally coupled heterodimer

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immunoblot analyses showed that these antibodies were specific and sensitive, detecting both the human and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers of female Sprague-Dawley rats following either acute (single dose of 25 microg TCDD/kg) or chronic (125 ng TCDD/kg/day for 30 weeks) exposure to TCDD. CYP1B1 protein exhibited a dose-response to TCDD that was different from those of CYP1A1 and CYP1A2. CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher doses of TCDD than that required for induction of CYP1A1 or CYP1A2. Immunohistochemical analysis showed that in animals chronically exposed to TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centrilobular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochromes in livers of TCDD-treated rats and apparent differences in both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction.     

Cytochromes P450, P420 & mixed-function oxidases as biomarkers of polychlorinated biphenyl (PCB) exposure in harbour seals (Phoca vitulina)

Hepatic microsomal cytochrome P450, EROD and ECOD activity were investigated as biomarkers of PCB exposure in harbour seals (Phoca vitulina). Due to the difficulty of obtaining undegraded seal liver samples, standard spectrophotometric methodology was adapted to investigate P420 (degraded P450) as a PCB biomarker with partially degraded samples. Total PCB burdens in both blubber and liver had positive correlations with P450, P420 and MFO activity levels. The use of P420 biomarkers in this study supports the inclusion of samples from by-caught marine mammals for future biomonitoring studies. P450 isozymes CYP1A (P4501A) and CYP2B (P4502B) in conjunction with MFO activity were investigated as "specific" biomarkers of PCB exposure. They were found to reliably reflect levels of [MC] and [PB]-type PCB exposure in harbour seal liver.     

Comparative effects of omeprazole on xenobiotic metabolizing enzymes in the rat and human

Omeprazole induces CYP1A in the human liver and gut, which has led to concern about possible side effects. The purpose of this study was to compare the effects of omeprazole on phase 1 and phase 2 enzymes in the rat and human. Male rats were treated with intraperitoneal (40 or 80 mg/kg) or oral omeprazole (40 mg/kg) for 5 or 14 days, respectively. The activities and amounts of CYP1A, uridine diphosphate-glucuronosyltransferase, and glutathione transferase were determined in liver and gut. Enzyme activities were also determined in duodenal biopsy specimens from six healthy human volunteers before and after treatment with omeprazole (20 mg/day) for 10 days. Treatment with intraperitoneal omeprazole (40 mg/kg; 80 mg/kg) coinduced uridine diphosphate-glucuronosyltransferase (36%; 66%), glutathione transferase (22%; 50%), and CYP1A (26%; 50%) in rat liver. In rat small intestine, comparable levels of induction were observed for uridine diphosphate-glucuronosyltransferase and glutathione transferase; CYP1A was unaffected. Oral omeprazole had similar effects. Immunoblotting showed corresponding changes in the amounts of these enzymes. Omeprazole increased the activities of CYP1A (19% to 167%; p = 0.014) and uridine diphosphate-glucuronosyltransferase (11% to 68%; p = 0.04) in the duodenal biopsy specimens of all six human volunteers; glutathione transferase was unaffected. Thus, omeprazole coinduced multiple xenobiotic metabolizing enzymes in the rat and human. The pattern of induction differed in the rat and human, consistent with known differences in genetic regulatory elements in the two species.     

Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver

It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of rats with fluvastatin had no effect on hepatic levels of CYP3A1/2 mRNA or immunoreactive protein. However, treatment with 50 mg/kg/day fluvastatin induced CYP1A1 mRNA and protein. The effects of fluvastatin treatment on P450 expression seen in primary cultured rat hepatocytes thus largely recapitulated the effects seen in vivo. The differences in effects among the HMG-CoA reductase inhibitors suggest that simple inhibition of HMG-CoA reductase cannot explain all of the effects of these drugs on P450 expression.     

In vitro evaluation of the antigenotoxic activity of a strain of L. bulgaricus isolated from commercial yogurt

Sequential oxidations at the arylamine moiety of the procainamide molecule leading to the formation of N-hydroxyprocainamide and its nitroso derivative may be responsible for lupus erythematosus observed in patients treated with the drug. The objective of the present study was to characterize major cytochrome P450 isozyme(s) involved in the N-hydroxylation of procainamide. Firstly, incubations were performed with microsomes from either lymphoblastoid cells or yeast transfected with cDNA encoding for specific human cytochrome P450 isozymes. Experiments performed with these enzyme expression systems indicated that the highest formation rate of N-hydroxyprocainamide was observed in the presence of CYP2D6 enriched microsomes. Additional experiments demonstrated that the formation rate of N-hydroxyprocainamide by CYP2D6 enriched microsomes was decreased from 45 +/- 4% to 93 +/- 1% by quinidine at concentrations ranging from 30 nM to 100 microM (all p < 0.05 vs control) and by approximately 75% by antibodies directed against CYP2D6. Secondly, incubations were performed with microsomes prepared from 15 human liver samples. Using this approach, an excellent correlation was observed between the formation rate of N-hydroxyprocainamide and dextromethorphan O-demethylase activity (CYP2D6; r = 0.9305; p < 0.0001). In contrast, no correlation could be established between N-hydroxyprocainamide formation rate and caffeine N3-demethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methlhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), chlorzoxazone 6-hydroxylase (CYP2E1), dextromethorphan N-demethylase (CYP3A4), testosterone 6 beta-hydroxylase (CYP3A4/5) or lauric acid 12-hydroxylase (CYP4A11) activities. Furthermore, formation rate of N-hydroxyprocainamide was decreased in a concentration-dependent manner by quinidine (300 nM to 100 microM) and by antibodies directed against CYP2D6 but not by furafylline 20 microM (CYP1A2), ketoconazole 1 microM (CYP3A4), sulfaphenazole 10 microM (CYP2C9) or antibodies directed against CYP1A1/1A2, CYP2C, CYP2A6, CYP2E1 or CYP3A4/3A5. In conclusion, the results obtained in the present study demonstrate that CYP2D6 is the major human cytochrome P450 isozyme involved in the formation of the reactive metabolite of procainamide, namely N-hydroxyprocainamide.     

Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes

Although hyperketonemia and/or altered growth hormone secretion caused by diabetes have been implicated in enhanced CYP2E1, 2B, 3A and 4A expression, the effect of insulin on hepatic P450 expression, in the absence of associated metabolic/hormonal alterations, remains unknown. Primary cultured rat hepatocytes have been shown (Zangar et al., Drug Metab. Dispos., 23:681, 1995) to express stable and inducible CYP2E1 mRNA and protein levels, and provide an excellent system for mechanistic examination of the effect of insulin on CYP2E1, 2B, 3A and 4A expression. Maintaining primary rat hepatocytes in culture in the absence of insulin for 48, 72, or 96 h increased CYP2E1 mRNA levels 5-, 11-, and 4-fold, respectively, relative to cells maintained in the presence of the standard concentration of 1 microM insulin. In contrast, CYP2B mRNA levels increased only approximately 2-fold in the absence of insulin, when compared with the presence of 1 microM insulin. CYP2E1 and 2B protein levels were increased 6.7- and 3.8-fold, respectively, in cells cultured for 96 h in the absence of insulin as compared with those cultured in medium containing 1 microM insulin. Concentration-response studies revealed that decreasing the concentration of insulin below 10 nM (i.e. 1 nM, 0.1 nM, no insulin) increased CYP2E1 mRNA levels 4-, 7-, and 11-fold, respectively. In contrast, no such concentration-dependence was observed for CYP2B mRNA expression. As CYP3A and 4A expression is also elevated in diabetic rats, the effects of insulin on these P450s was also examined. CYP3A mRNA levels were unaltered and CYP4A mRNA levels were decreased marginally (approximately 50%) by the absence of insulin relative to levels in cells cultured in the presence of 1 microM insulin over 96 h in culture. The results of this study provide evidence that insulin itself, in the absence of other diabetes-induced metabolic or hormonal alterations, affects CYP2E1 and 2B, but not CYP3A or 4A, expression in primary cultured rat hepatocytes. Furthermore, CYP2E1 expression is differentially regulated by insulin relative to CYP2B, 3A or 4A. This study also demonstrates that decreasing the concentration of insulin in the culture medium provides a method by which CYP2E1 levels can be increased in primary cultured hepatocytes to facilitate mechanistic studies on the regulation of CYP2E1 expression.     

Impact of lipid and ACE inhibitor therapy on cardiovascular disease and metabolic abnormalities in the diabetic and hypertensive patient

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2alpha-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was < 10%. Furthermore, immunoblotting showed that 1,1-DCE (> or = 400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6beta-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40-70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid omega-hydroxylase (LAOH), and testosterone 7alpha-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60-66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (approximately 1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE.     

Induction of cytochromes P450 2B and 2E1 in rat liver by isomeric picoline N-oxides

Pyridine derivatives are widely used solvents and precursors for the synthesis of chemicals of industrial importance. Oxidized metabolites have been implicated in the observed toxicity of pyridines and are known to induce drug-metabolizing enzymes in rat liver. In this study the three isomeric picoline (methylpyridine) N-oxides, as major oxidized metabolites of 2-, 3- and 4-picoline, were evaluated as inducers of cytochrome P450 (CYP) enzymes in rat liver. After a single dose of 100 mg/kg 24 h before sacrifice the 3- and 4-isomers were effective inducers of microsomal substrate oxidations associated with the phenobarbital-inducible CYPs 2B; upregulation of CYP2B protein was confirmed by immunoblotting. In contrast, the 2-isomer did not increase CYP2B protein or activity in rat liver but CYP2E1 protein expression was upregulated by the isomers to 160-200% of control. The three chemicals increased aniline 4-hydroxylation activity in rat liver, which is consistent with induction of CYPs 2B or 2E1 and 4-nitrophenol 2-hydroxylation activity was increased in microsomal fractions from 3- and 4-picoline N-oxide-treated rats. The activities of several other CYPs were also determined and CYP1A-dependent 7-ethylresorufin O-deethylation was increased (to approximately 6- and 2-fold of control) by the 3- and 4-isomer, respectively, whereas the activity of CYP3A-mediated androstenedione 6beta-hydroxylation was decreased by the agents--most notably by the 2-isomer. During NADPH-supported oxidation of CCl4, lipid peroxidation was increased in microsomes from 3- and 4-picoline N-oxide-pretreated rats and was modulated in vitro by the CYP2B inhibitor orphenadrine, but not by the CYP2E1 inhibitor 4-methylpyrazole. These findings establish that particular isomers of picoline N-oxide rapidly upregulate CYP2B or, to a lesser extent, CYP2E1 and implicate CYP2B in the enhanced lipid peroxidation observed in microsomes from rats treated with 3- and 4-picoline N-oxides. Such induction process may contribute to the hepatotoxicity of pyridines by enhancing the capacity for microsomal lipid peroxidation.     

Changes in rat liver cytochrome P450 enzymes by atrazine and simazine treatment

1. We examined the effect of two chloro-s-triazines (atrazine and simazine) on hepatic microsomal cytochrome P450 enzymes in rat. Rats were treated intraperitoneally with atrazine or simazine daily for 3 days with 100, 200 and 400 mumol/kg. 2. Among the P450-dependent monooxygenase activities, testosterone 2 alpha-hydroxylase (T2AH) activity in rat, which is associated with CYP2C11, was significantly decreased at all doses of atrazine and simazine. The levels relative to control activities were 59-46 and 60-32% respectively. Similarly, oestradiol 2-hydroxylase (ED2H) activity was also significantly decreased by 28-51% by atrazine and simazine at all doses. However, no change in CYP2C11 protein level by either chloro-s-triazine was observed. K(m) for T2AH was significantly increased only by simazine (200 mumol/kg), whereas the Vmax and Cl(int) for T2AH were significantly decreased by atrazine and simazine at all doses. 3. 7-Ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were significantly increased by 1.4-1.6-, 1.7-3.2- and 1.5-2.2-fold respectively, by both chloro-s-triazines at 200 or 400 mumol/kg. Lauric acid omega-hydroxylase (LAOH) was also increased by 1.4-fold by simazine at 200 and 400 mumol/kg. Immunoblotting showed that only simazine induces CYP1A2 and CYP4A1/2 protein expression. 4. The activities of 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1'-hydroxylase (BF1'H), chlorzoxazone 6-hydroxylase (CZ6H), testosterone 6 beta-hydroxylase (T6BH) and testosterone 7 alpha-hydroxylase (T7AH) were not affected by either chloro-s-triazine. 5. These results suggest that the pattern of changes in P450 isoforms by chloro-s-triazines differs between atrazine and simazine, that these herbicides change the constitutive and/or male specific P450 isoform(s) in rat liver, and that these changes closely relate to the toxicity of chloro-s-triazines.     

Investigation on possible antioxidative properties of the NMDA-receptor antagonists ketamine, memantine, and amantadine in comparison to nicanartine in vitro

Possible antioxidative properties of three N-methyl-D-aspartate (NMDA)-receptor antagonists, the anesthetic ketamine and the antiparkinson drugs memantine and amantadine were investigated in vitro on the microsomal cytochrome P450 (P450) system of rat livers and on rat whole blood chemiluminescence in comparison to nicanartine, a substance with known antiatherosclerotic, hypolipemic and antioxidative capacity. For this purpose, the effects on NADPH- and iron-stimulated lipid peroxidation (LPO), hydrogen peroxide (H2O2) production, and NADPH- and iron-stimulated lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) were examined using rat liver microsomes. Additionally, the influence on LM amplified whole blood chemiluminescence after zymosan activation of polymorphonuclear leukocytes (WB-CL) was investigated. Furthermore, binding to P450 and effects on P450 mediated monooxygenase function, as measured by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (END), were assessed. Nicanartine concentration dependently reduced LPO and H2O2 production already at a concentration of 1 microM, whereas LC and LM amplified CL and WB-CL were not affected. EROD and END were concentration dependently diminished starting at 1 microM, and ECOD already at 0.1 microM. Ketamine decreased LPO, H2O2 production and LM and LC amplified CL, starting at 100 microM. WB-CL was significantly diminished already at 10 microM. EROD and ECOD were inhibited at 10 and 100 microM and END at 100 microM. With memantine a concentration dependent inhibition of LPO and WB-CL was seen at 100 and 1000 microM and a reduction of LC and LM amplified CL only at 1000 microM. H2O2 production was not affected. EROD and ECOD were significantly diminished by a concentration of 100 microM. No effect was observed on END. Amantadine significantly reduced LPO and WB-CL, but only at 1000 microM. H2O2 production and LC and LM amplified CL were not affected. EROD was significantly diminished at 100 microM, whereas no influence was seen on ECOD and END. Nicanartine displayed type II or reverse type I, ketamine, memantine and amantadine type I substrate binding to P450. The highest binding affinity to P450 was seen with nicanartine, followed by ketamine, memantine and then amantadine. These results demonstrate, that all four substances seem to act as radical scavengers and/or as inhibitors of the oxidative function of P450. All four substances seem to interfere with the monooxygenase function of P450. This may result in a possible influence on the biotransformation of endogenous as well as of foreign compounds. The effects of nicanartine were much more pronounced than those of ketamine, memantine, and amantadine.