Reappraisal of human CYP isoforms involved in imipramine N-demethylation and 2-hydroxylation: a study using microsomes obtained from putative extensive and poor metabolizers of S-mephenytoin and eleven recombinant human CYPs

Cytochrome P450 (CYP) involved in the two major pathways of imipramine (IMI) was reappraised using human liver microsomes phenotyped for S-mephenytoin 4'-hydroxylation in vitro and 11 recombinant human CYP isoforms. Individual Eadie-Hoffstee plots for IMI N-demethylation and 2-hydroxylation showed a monophasic profile in microsomes obtained from three putative S-mephenytoin poor metabolizer (PM) livers, whereas the plots gave a biphasic relationship (except for one case in 2-hydroxylation) in those from the three extensive metabolizer (EM) livers. Effects of CYP-selective inhibitor/substrate probes on the two metabolic reactions were examined at the two IMI concentrations (2 and 400 microM) with microsomes obtained from the two PM and three EM livers. S-mephenytoin inhibited IMI N-demethylation by 50% at the low concentration in microsomes from the EM livers with no discernible effect on this pathway in those from the PM livers. Furafylline inhibited the N-demethylation by about 60% at the low and high substrate concentrations in microsomes from both the EM and PM livers. Quinidine abolished the 2-hydroxylation at the low and high concentrations in microsomes from both the EM and the PM livers. Among the recombinant human CYPs, CYP2C19, 2C18, 2D6, 1A2, 3A4 and 2B6 in rank order catalyzed the N-demethylation, whereas CYP2D6, 2C19, 1A2, 2C18 and 3A4 catalyzed the 2-hydroxylation. The Km values obtained from recombinant CYP2C19 and 1A2 approximated those of the high- and low-affinity components from human liver microsomes for IMI N-demethylation, respectively. For IMI 2-hydroxylation, the respective Km values obtained from recombinant CYP2D6 and 2C19 were close to those of the high- and low-affinity components from human liver microsomes. Our human liver microsomal study using the near-therapeutic IMI concentration (2 microM) suggests that 1) CYP2C19 and 1A2 are involved in the N-demethylation and the 2-hydroxylation is mediated exclusively by CYP2D6 and partially by CYP2C19 in the EM livers, and 2) CYP1A2 and 2D6 play a major role in IMI N-demethylation and 2-hydroxylation, respectively, in the PM livers. Our recombinant human CYP isoform study, in general, supports this conclusion.     

Regioselectivity and substrate concentration-dependency of involvement of the CYP2D subfamily in oxidative metabolism of amitriptyline and nortriptyline in rat liver microsomes

Kinetic analysis of the metabolism of amitriptyline and nortriptyline using liver microsomes from Wister rats showed that more than one enzyme was involved in each reaction except for monophasic amitriptyline N-demethylation. The Vmax values particularly in the high-affinity sites for E-10-hydroxylation of both drugs were larger than those for Z-10-hydroxylations. Their E- and E-10-hydroxylase activities in Dark-Agouti rats, which are deficient for CYP2D1, were significantly lower than those in Wistar rats at a lower substrate concentration (5 microM). The strain difference was reduced at a higher substrate concentration (500 microM). A similar but a smaller strain difference was also observed in nortriptyline N-demethylase activity, and a pronounced sex difference (male > female) was observed in N-demethylation of both drugs in Wistar and Dark-Agouti rats. The reactions with the strain difference were inhibited concentration-dependently by sparteine, a substrate of the CYP2D subfamily, and an antibody against a CYP2D isoenzyme. The profiles of these decreased metabolic activities corresponded to that of the lower metabolic activities in Dark-Agouti rats. These results indicated that a cytochrome P450 isozyme in the CYP2D subfamily was involved in E- and Z-10-hydroxylations of amitriptyline and nortriptyline in rat liver microsomes as a major isozyme in a low substrate concentration range. It seems likely that the CYP2D enzyme contributes to nortriptyline N-demethylation.     

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.     

The high-affinity binding of [3H]norharman ([3H]beta-carboline) to the ethanol-inducible cytochrome P450 2E1 in rat liver

High-affinity binding sites of [3H]norharman (synonymous: [3H]beta-carboline) were characterized in microsomal membranes from rat liver utilizing various beta-carboline (BC) derivatives and substances binding to enzymes of the cytochrome P450 (CYP) superfamily (EC 1.14.14.1). Saturation experiments demonstrated that [3H]norharman binds with high-affinity (dissociation constant 20.86 nM; maximum binding 21.40 pmol/mg protein). Displacement experiments with the beta-carboline derivatives 6-methyl-BC and 6-hydroxy-BC revealed a better adaptation to the two-site model, indicating that [3H]norharman binds to at least two sites, with an affinity of the high-affinity site in the low nM range. Substances binding with relative preference to isozymes of the CYP superfamily displaced [3H]norharman with a lesser potency than unlabeled norharman. Imidazole, pyrazole, and 4-methylpyrazole, known as inducers of the ethanol-inducible CYP2E1, displaced [3H]norharman with relative high potency. Furthermore, binding experiments with microsomes from human lymphoblast-expressed rat CYP2E1 revealed a high-affinity binding site [inhibition constant (Ki) 13.21 nM] comparable to that of microsomal membranes for norharman. It was displaceable by ethanol (Ki 14.25 microM), indicating that norharman and ethanol bind to the same binding site on CYP2E1. In vivo experiments with rats which had ingested ethanol for two weeks revealed that norharman blood plasma levels were significantly elevated at the end of this period, supporting the notion of an interaction of norharman and ethanol metabolism. Since it has been demonstrated in the Ames test that norharman's comutagenic action is connected with microsomal membranes (containing CYP isozymes), the present findings suggest that the observed increase in the levels of norharman in alcoholics leads to further CYP enzyme induction and thereby contributes to the increased risk of carcinomas in these patients.     

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.     

Phencyclidine metabolite irreversible binding in the rat: gonadal steroid regulation and CYP2C11

These studies were conducted to determine the effect of hormones on sex-related differences in phencyclidine (PCP) metabolite irreversible binding and to determine the cytochrome P450 isoform(s) involved in this process. Sprague-Dawley male rats were castrated or administered estradiol and Sprague-Dawley female rats were ovarectomized or ovarectomized and given testosterone. Liver microsomal metabolism studies demonstrated that PCP metabolite binding to proteins was significantly altered by testosterone and estrogen administration. Castration of male rats decreased metabolite binding to 57% of sham-operated male levels, and administration of testosterone to ovarectomized female rats increased metabolite binding to 41 % of normal male levels. No metabolite adducts could be detected in microsomes from male rats administered estradiol or from sham-operated females given vehicle. These hormone-induced changes in metabolite binding closely matched the hormone-induced changes in CYP2C11 function and expression in these same microsomes. PCP metabolite irreversible binding to microsomal proteins was highly correlated with CYP2C11 function (as assessed by the formation of 2alpha-OH-testosterone, r = 0.91) and with CYP2C11 expression (as assessed by Western blot analysis, r = 0.95). In addition, an anti-CYP2C11 monoclonal antibody almost completely inhibited PCP metabolite binding (down to 7% of control male values) in an antibody concentration-dependent manner. These data strongly implicate CYP2C11 as an isoform involved in PCP metabolism and the formation and/or binding of a reactive PCP metabolite to microsomal proteins.     

Identification of human cytochrome P450 isoforms involved in the 3-hydroxylation of quinine by human live microsomes and nine recombinant human cytochromes P450

Studies using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie-Hofstee plots for the formation of 3-hydroxyquinine exhibited apparently monophasic behavior for all of the 10 different microsomal samples studies. There was interindividual variability in the kinetic parameters, as follows: 1.8-, 3.2- and 3.5-fold for K(m) Vmax and Vmax/K(m), respectively. The mean +/- S.D. values for K(m), Vmax and Vmax/K(m) were 106.1 +/- 19.3 microM, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 microliters/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine and the metabolic activities for substrates of the respective CYP isoforms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testosterone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4'-hydroxylase activities was also observed. However, no significant correlation existed between the 3-hydroxylation of quinine and the oxidative activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and troleandomycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinine by human liver microsomes with respective mean IC50 values of 0.026 microM and 28.9 microM. Anti-CYP3A antibodies strongly inhibited quinine 3-hydroxylation, whereas weak inhibition was observed in the presence of S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to the 3-hydroxylation of quinine, compared with the minor activity of CYP2C19 and little discernible or no effect of other CYP isoforms. Collectively, these data suggest that the 3-hydroxylation of quinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance in this major pathway of quinine in humans.     

Ring hydroxylation of [o-3H]methoxychlor as a probe for liver microsomal CYP2B activity: potential for in vivo CYP2B assay

An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.     

A combination of a common splice site mutation and a frameshift mutation in the COL7A1 gene: absence of functional collagen VII in keratinocytes and skin

Musk xylene (2,4,6-trinitro-1-t-butylxylene; MX) is a synthetic nitromusk perfume ingredient that induces and inhibits mouse cytochrome P4502B (CYP2B) enzymes in vivo. The purpose of the present work was to determine whether amine metabolites of MX contributed to the enzyme inhibition and, if so, to define the nature and kinetics of this inhibition. When dosed orally to phenobarbital (PB)-treated mice, MX (200 mg/kg) inhibited > 90% of the PB-induced O-dealkylation of 7-pentoxyresorufin (PROD), and [14C]MX equivalents bound covalently to microsomal proteins. However, when this experiment was repeated in mice pretreated with antibiotics to eliminate the gastrointestinal flora, no decrease in PB-induced PROD activity and no covalent binding to microsomal proteins were observed. Thus, the ability of antibiotic treatment to eliminate the enzyme inhibition and covalent binding implicated amine metabolites of MX formed by nitroreduction in anaerobic intestinal flora as obligatory for these effects. Two monoamine metabolites of MX were synthesized to study enzyme inhibition directly. These metabolites were 2-amino-4,6-dinitro-1-t-butyl-xylene and 4-amino-2,6-dinitro-1-t-butylxylene, referred to as o-NH2-MX and p-NH2-MX, respectively, reflecting the position of the amine substitution relative to the t-butyl function. In the in vitro studies with PB-induced mouse liver microsomes, both amines inhibited PROD activity when preincubated in the absence of NADPH. However, only p-NH2-MX caused a time- and NADPH-dependent loss of PROD activity, and the inactivation rate was a pseudo-first-order process that displayed saturation kinetics. These results indicate that p-NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the Ki was calculated to be 10.5 microM and the Kinact was 1.2 min-1. As final confirmation of the inhibitory effects of p-NH2-MX on mouse CYP2B enzymes, the amine (0.67 mmol/kg) was dosed orally to PB-induced mice. At 2 hr after dosing, p-NH2-MX inhibited essentially all of the PB-induced PROD activity, whereas an equimolar dosage of parent MX had no effect at this early time. Thus, although MX is an inducer of mouse CYP2B enzymes, an amine metabolite of MX is a mechanism-based inactivator of mouse CYP2B10. Furthermore, it is likely that the amine is responsible for the lack of functional CYP2B enzyme activity associated with induction of this enzyme by MX.     

C-21- and 6-hydroxylation of progesterone by rabbit liver subcellular fractions

In vitro C-21-hydroxylation of [3H]progesterone (P) has been demonstrated for the first time with rabbit liver microsomes and mitochondria. Deoxycorticosterone (DOC) was rigorously characterized as a metabolite of both mitochondrial and microsomal metabolism, whereas 6alpha-hydroxy DOC and 6alpha-hydroxy P were only identified as microsomal metabolites. 6beta-Hydroxy metabolites were also detected but were of less quantitative significance. Formation of 6alpha-hydroxy P and 6alpha-hydroxy DOC increased steadily between 5 and 120 min of incubation with the microsomal fraction, whereas DOC increased up to 30 min of incubation and then declined. Maximal yield of DOC was 25.9 and 22.5 pmol/mg protein with the mitochondrial and microsomal fractions, respectively.     

Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin

Dextromethorphan and dextrorphan elicited a stereotyped behavioral syndrome in rats indistinguishable from that produced by PCP and other non-competitive NMDA antagonists. The rank order of potency for the induction of stereotyped behavior in male Sprague-Dawley rats was: MK-801>PCP>(+/-)cyclazocine>dextrorphan>(+/-)ketamine>dextromethorphan. These behavioral potencies were significantly correlated (0.91; P<0.05) with their respective affinities for high affinity [3H]dextrorphan-labelled NMDA receptors in rat forebrain membranes. To address the propensity of dextromethorphan to induce stereotyped behavior, dextrorotatory-opioid induced stereotypies were investigated in female Dark Agouti and female Sprague-Dawley rats. The female Dark Agouti lacks CYP2D1, the cytochrome P450 enzyme which catalyses the oxidative O-demethylation of dextromethorphan to dextrorphan. No differences were observed in either potency or time to peak effect for dextromethorphan to induce stereotyped behavior in the rat strains, suggesting that the affinity of dextromethorphan for NMDA receptors adequately accounts for its ability to induce stereotyped behavior. Female Dark Agouti rats were, however, more sensitive to the effects of dextrorphan, which may reflect differences in the ability of this strain to metabolize dextrorphan. We find no evidence to suggest that dextromethorphan produces a behavioral syndrome in rats that is distinct from that induced by dextrorphan. The commonality between the pharmacologic profiles of these compounds suggests that the abuse potential of dextromethorphan containing antitussive preparations is related to the non-competitive NMDA antagonist activity of dextromethorphan and its metabolites.     

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.     

Protein-reactive metabolites of carbamazepine in mouse liver microsomes

The character of reactive metabolites formed from carbamazepine (CBZ) was sought in incubations of [14C]CBZ in hepatic microsomes prepared from adult female mice of a strain (SWV/Fnn) susceptible to CBZ-induced teratogenicity. The formation of radio-labeled protein adducts was used as an index of reactive metabolite exposure. A dependence on cytochrome P450 was shown by a requirement for NADPH and inhibition by carbon monoxide, 1-aminobenzotriazole, piperonyl butoxide, and stiripentol. The addition of ascorbic acid, caffeic acid, N-acetylcysteine, and glutathione decreased the rate of binding of the radiolabel from [14C]CBZ to microsomal protein by more than 50%. The addition of glutathione transferases diminished protein adduct formation beyond that seen with glutathione alone. Evidence for the formation of an arene oxide was sought through the use of inhibitors of epoxide hydrolases, including cyclohexene oxide, chalcone oxides (with the addition of cytosol as appropriate), and by the addition of recombinant human soluble and microsomal epoxide hydrolases and recombinant rat microsomal epoxide hydrolase. The microsomal epoxide hydrolases decreased the velocity of 14C-labeled protein adduct formation by approximately 23%, whereas inhibitors had no effect, most likely because of the low native activity of microsomal epoxide hydrolase in mice. Both DT-diaphorase and catechol-O-methyltransferase diminished 14C-labeled protein adduct formation by 54% and 45%, respectively. The data suggest that the major reactive metabolites formed from CBZ by adult female SWV/Fnn liver microsomes are quinones and arene oxides.     

Effect of a supernatant protein on microsomal squalene epoxidase and 2,3-oxidosqualene-lanosterol cyclase

Squalene epoxidation catalyzed by rat liver microsomes requires oxygen NADPH, and the 105,000 x g supernatant (S105). The supernatant can be replaced by a partially purified S105 protein (SPF) and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). When washed microsomes are preincubated anaerobically with [14C]squalene and S105 without NADPH, followed by centrifugation and washing to remove the unbound squalene and S105, epoxidation in the presence of O2 and NADPH occurs subsequently at the same rate as in direct assays containing all required components from the start. Partially purified SPF (65-fold) shows the same effect. Washed microsomes preincubated anaerobically with squalene alone, or with bovine serum albumin instead of S105, also take up large amounts of squalene, but the squalene so incorporated is only poorly converted to epoxide. The epoxidation of endogenous squalene formed in liver homogenates from [14C]mevalonate is also stimulated by S105. The incorporation of squalene into microsomes is temperature dependent. 2,3-Oxidosqualene-lanosterol cyclase (cyclase) also requires S105 for optimal activity. It is suggested that the S105 protein acts internally within the microsomal membrane system facilitating the access of substrate to specific enzyme sites.     

(+)-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.     

Strain differences in adrenal microsomal steroid metabolism in guinea pigs

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17alpha-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17alpha-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17alpha-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.     

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.     

Involvement of CYP2D1 in the metabolism of carteolol by male rat liver microsomes

1. The metabolism of carteolol, a beta-adrenoceptor blocking drug, was investigated in male Sprague-Dawley rat liver microsomes. 2. The formation of 8-hydroxycarteolol was the principal metabolic pathway of carteolol in vitro and followed Michaelis-Menten kinetics with a K(m) = 11.0 +/- 5.4 microM and a Vmax = 1.58 +/- 0.64 nmol/min/nmol P450 respectively (mean +/- SD, n = 5). Eadie-Hofstee plot analysis of carteolol 8-hydroxylase activity confirmed single-enzyme Michaelis-Menten kinetics. 3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies. Quinine (Ki = 0.06 microM) and quinidine (Ki = 2.0 microM), selective inhibitors of CYP2D1, competitively inhibited 8-hydroxycarteolol formation. Furthermore, only anti-human CYP2D6 antibody inhibited this reaction. 4. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by CYP2D1. The K(m) of carteolol for CYP2D1 in male rat liver microsomes was much greater than those of propranolol or bunitrolol, indicating that carteolol has a lower affinity for CYP2D1 compared with these other beta-adrenoceptor blocking drugs.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.