Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis.

The molecular mechanism(s) underlying induction of the hepatic microsomal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone) is controversial. Proposed mechanisms include increased rates of enzyme synthesis due to elevated 2E1 mRNA levels, enhanced translation of pre-existing mRNA, or stabilization of 2E1 protein. To further assess which, if any, of these events predominates during the initial stages of 2E1 protein induction, we investigated the effects of acetone treatment on 2E1 content in cultured rabbit hepatocytes, an in vitro system that allows for precise control of the cellular mileau. Hepatocytes harvested from female rabbits and plated on plastic dishes with serum-supplemented medium were 90-100% viable for at least 48 hr in culture. Analysis of immunoreactive 2E1 content and aniline hydroxylase activity in microsomes isolated from hepatocytes cultured for up to 24 hr revealed that 2E1 expression was equal to that of microsomes from unplated cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Moreover, microsomes isolated from cells exposed to 17 mM acetone for 24 hr exhibited a 53 and 62% increase in aniline hydroxylase activity and 2E1 content, respectively, compared to untreated cells. To explain these increases, the rate of 2E1 protein synthesis was determined in untreated cells or in cells treated with 17 mM acetone by first exposing hepatocytes to medium supplemented with 35S-labeled methionine and cysteine ([35S]Met/Cys) and subsequently assessing radiolabel incorporation into 2E1 protein. While no difference was found between untreated and acetone-treated cells in the incorporation of [35S]Met/Cys into trichloracetic acid-precipitable microsomal proteins, immunoaffinity purification of 2E1 revealed that incorporation of 35S-labeled amino acids specifically into 2E1 was elevated by acetone to 200% of control values. Treatment of hepatocytes with the transcriptional inhibitor, alpha-amanitin, markedly inhibited this acetone-mediated increase in [35S]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA revealed that acetone increased 2E1 mRNA to 130 and 160% of control levels at 6 and 24 hr, respectively, and that these increases were prevented by pretreatment with alpha-amanitin. Our results indicate that acetone increases 2E1 protein levels in cultured rabbit hepatocytes by stimulating its rate of de novo synthesis. Since this increase in 2E1 synthesis stems, at least in part, from the acetone-mediated enhancement of hepatocyte 2E1 mRNA content and is inhibitable by alpha-amanitin, transcriptional activation of the rabbit CYP2E1 gene is apparently involved in the induction of 2E1 protein by acetone.

Risk assessment: toxicity from chemical exposure resulting from enhanced expression of CYP2E1.

Humans are continuously exposed to a wide variety of xenobiotics either voluntarily or from environmental exposure. Many xenobiotics including pesticides, nitrosamines, polycyclic aromatic hydrocarbons and halogenated hydrocarbons, require bioactivation by P450 enzymes to elicit toxicity. CYP2E1 is considered to be toxicologically important in humans because of its capacity to produce intermediates that promote cytotoxicity and/or carcinogenicity from a number of xenobiotics. Importantly, CYP2E1 is present constitutively and its content can be modulated by a variety of factors including xenobiotics such as alcohol. Because hepatic concentrations of CYP2E1 can vary considerably from one individual to another, the extent of formation of toxic products also varies. Indeed, as hepatic concentrations increase so does the risk of toxicity from chemicals activated by this P450 enzyme. Many chemicals modulate CYP2E1 expression and exposure to one compound may alter the toxicological impact of another. Considering that CYP2E1 content is related to toxicity from chemicals, identifying subjects with elevated levels may lead to minimizing exposure in high risk individuals.

Extensive alteration of genomic DNA and rise in nuclear Ca2+ in vivo early after hepatotoxic acetaminophen overdose in mice.

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis. In this in vivo study, 600 mg/kg acetaminophen caused total nuclear Ca2+ and % fragmented DNA to rise in parallel from 2-6 hr, followed by large later increases mirroring frank liver injury. Agarose gel electrophoresis revealed substantial loss of large genomic DNA from 2 hours onward, with accumulation of DNA fragments in a ladder-like pattern resembling apoptosis. Extensive late cleavage of DNA probably resulted from cell death, whereas degradative loss of large genomic DNA at 2 hours arose at an early enough point to contribute to acetaminophen-induced liver necrosis in mice.

Recent advances in P450 research.

P450 enzymes comprise a superfamily of heme-containing proteins that catalyze oxidative metabolism of structurally diverse chemicals. Over the past few years, there has been significant progress in P450 research on many fronts and the information gained is currently being applied to both drug development and clinical practice. Recently, a major accomplishment occurred when the structure of a mammalian P450 was determined by crystallography. Results from these studies will have a major impact on understanding structure-activity relationships of P450 enzymes and promote prediction of drug interactions. In addition, new technologies have facilitated the identification of several new P450 alleles. This information will profoundly affect our understanding of the causes attributed to interindividual variations in drug responses and link these differences to efficacy or toxicity of many therapeutic agents. Finally, the recent accomplishments towards constructing P450 null animals have afforded determination of the role of these enzymes in toxicity. Moreover, advances have been made towards the construction of humanized transgenic animals and plants. Overall, the outcome of recent developments in the P450 arena will be safer and more efficient drug therapies.

Variations among untreated rabbits in benzo(a)pyrene metabolism and its modulation by 7,8-benzoflavone.

The metabolism of benzo(a)pyrene by rabbit liver microsomes can be stimulated or inhibited by 7,8-benzo(a)flavone (ANF) depending on the distribution of specific P-450 enzymes present within the microsomes. Treatment of rabbits with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or rifampicin leads to an increase of hepatic microsomal metabolism of benzo(a)pyrene. ANF stimulates the rate of benzo(a)pyrene metabolism catalyzed by microsomes isolated from rabbits treated with rifampicin by 3-fold. In contrast, ANF moderately inhibits the activity of microsomes from TCDD-treated rabbits. Variations in the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits apparently reflect differences in the expression of P-450 1, a constitutive form of P-450. Thus, the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits, which varies from 0.40 to 1.5 nmol/min/mg of protein, is directly correlated with the microsomal concentration of P-450 1. The metabolism of benzo(a)pyrene by microsomes containing high concentrations of P-450 1 is inhibited by a monoclonal antibody specific for this cytochrome to approximately the rate exhibited by microsomes with a low concentration of P-450 1. The benzo(a)pyrene activity stimulated by ANF in microsomes with a low concentration of P-450 1 is not inhibited by the monoclonal antibody. The activity of P-450 1 is inhibited by ANF at concentrations that stimulate other constitutive forms of P-450. Thus, ANF produces offsetting effects on benzo(a)pyrene metabolism in microsomes from untreated animals by stimulating the activity of at least one cytochrome and inhibiting P-450 1-mediated activity.

Identification of lanosterol 14 alpha-methyl demethylase in human tissues.

Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite. The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation. Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied. Kinetic parameters were determined and shown to differ between two separate liver samples. Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite. The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene. The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.

Bioactivation of halogenated hydrocarbons by cytochrome P4502E1.

Numerous halogenated hydrocarbons of the alkane, alkene, and alkyne classes are metabolized by P450 enzymes to products that elicit cytotoxic and/or carcinogenic effects. Such halogenated hydrocarbons include anesthetics (e.g., halothane and enflurane) and industrial solvents (e.g., carbon tetrachloride, chloroform, and vinylidine chloride). Formation of reaction intermediates from these compounds occurs via P450-promoted dehalogenation, reduction, or reductive oxygenation, with certain hydrocarbons undergoing all three reaction types. Of the multiple forms of P450 present in liver microsomes, P4502E1 has been identified as the primary catalyst of hydrocarbon bioactivation in animals and, most likely, in humans as well. As hepatic concentrations of this P450 enzyme are highly inducible by ethanol and similar agents, prior exposure to 2E1-inducing compounds can play a pivotal role in halogenated hydrocarbon toxicity. Considering that metabolism governs the cytotoxicity and carcinogenicity of halogenated hydrocarbons, an understanding of the mechanism(s) underlying 2E1 induction in man becomes all the more important.

Modulation of rabbit and human hepatic cytochrome P-450-catalyzed steroid hydroxylations by alpha-naphthoflavone.

Rifampicin induces cytochrome P-450 3c, progesterone 16 alpha- and 6 beta-hydroxylation, 17 beta-estradiol 2-hydroxylation, benzo[a] pyrene hydroxylation, and erythromycin N-demethylation in rabbit liver microsomes. Kinetic analysis of the 6 beta-hydroxylation of progesterone as catalyzed by liver microsomes prepared from rifampicin-treated B/J rabbits exhibits a curvilinear double-reciprocal plot, suggestive of substrate activation. Further experimentation demonstrated that alpha-naphthoflavone could augment the catalytic efficiency [Vmax/Km] observed for the 16 alpha- and 6 beta-hydroxylation of progesterone and the 2-hydroxylation of 17 beta-estradiol, whereas erythromycin N-demethylase activity was partially inhibited. Allosteric activation of these steroid hydroxylases by alpha-naphthoflavone is also found for human liver microsomes, indicating that the activation of these enzymes is conserved in man and rabbit.

Human hepatic microsomal metabolism of delta 1-tetrahydrocannabinol.

Hepatic microsomal metabolism of delta 1-tetrahydrocannabinol (THC) has been extensively studied in many rodent species, but there have been few reports describing such metabolism in humans. Because several THC metabolites are known to be pharmacologically active, identifying the P-450 subfamilies responsible for their formation is of clinical importance. We have found that, in addition to catalyzing the formation of significant amounts of 7-hydroxy-THC, hepatic microsomes from nine human livers also formed 6 beta-hydroxy-THC at approximately the same rate. In addition, 1 alpha,2 alpha-epoxyhexahydrocannabinol (EHHC) was formed at approximately one-third the rate of 7-hydroxy- and 6 beta-hydroxy-THC, and small amounts of 6 alpha-hydroxy- and 6-keto-THC were also found. Immunoinhibition studies with antibodies raised against human hepatic P-450 2C9, or a mouse hepatic P-450 isozyme belonging to the P-450 3A subfamily, revealed that P-450 2C9 catalyzed the formation of 7-hydroxy-THC, whereas P-450 3A catalyzed the formation of 6 beta-hydroxy-THC, EHHC, and the relatively minor metabolites. In contrast, antibodies raised against human P-450 2C8 had no affect on human microsomal THC hydroxylation. Excellent correlations were found between hepatic microsomal P-450 2C9 and 3A content and 7-hydroxy- and 6 beta-hydroxy-THC formation, respectively. In addition, purified P-450 2C9 catalyzed the formation of 7-hydroxy-THC at a 7-fold higher rate than that observed with microsomes. Microsomal 7-hydroxy-THC formation varied less than 5-fold between the livers, suggesting that this activity is normally expressed and probably not subject to environmental influences.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of lymphocytes for assessing ethanol-mediated alterations in the expression of hepatic cytochrome P4502E1.

The ethanol-inducible cytochrome P4502E1 (2E1) is involved in the bioactivation of numerous hepatotoxins and hepatocarcinogens. Because high levels of expression may enhance the degree and severity of hepatotoxicity from exposure to chemicals metabolized by this enzyme, a relatively noninvasive method to phenotypically distinguish those individuals exhibiting elevated concentrations of 2E1 may be useful. With this in mind, we examined whether ethanol exposure could alter 2E1 in rabbit white blood cells and liver in a similar manner. Microsomes prepared from freshly isolated, rather than cultured cells, were used to immunochemically detect 2E1. The enzyme was found in lymphocytes and neutrophils. Lymphocytes, which comprise the majority of the white cell population in rabbits, were monitored for changes in 2E1 protein levels after ethanol exposure and compared with alterations of the hepatic enzyme. Results presented herein demonstrate that the degree of enhancement in 2E1 expression of lymphocytes and liver was dependent on the length and dose of alcohol exposure. Indeed, correlations were observed between blood alcohol concentrations and 2E1 content in lymphocytes (r = 0.65, p < 0.01) and liver (r = 0.60, p < 0.01). The greatest increase in 2E1 (6- to 10-fold) occurred in both liver and lymphocytes at a dose of 15% ethanol for 12 days of treatment. This induction was evident regardless of whether blood was taken from treated and compared with untreated rabbits or if white cells were obtained from the same animal before and after ethanol exposure. The latter findings demonstrate that changes in lymphocyte 2E1 were caused by ethanol exposure and not to variability in enzyme expression among rabbits. Interestingly, at the 10% dose, elevation of 2E1 was noted as early as 3 days, declined at 6 days, and at 12 and 24 days returned to slightly higher levels than those seen at the 3-day exposure period. This pattern of 2E1 elevation was observed in both the liver and lymphocytes. In fact, at all exposure periods and at the two doses of alcohol examined, a correlation (r = 0.70, p < 0.01) was observed between lymphocyte and liver 2E1 content. Collectively, these studies show that induction of 2E1 in lymphocytes and liver occurs in a parallel fashion. Furthermore, results suggest that blood 2E1 may be used in humans as a phenotypic marker for xenobiotic-promoted alterations in the expression of the liver enzyme. These findings should have a significant impact on in vivo monitoring of this P450 enzyme.

Expression, induction, and catalytic activity of the ethanol-inducible cytochrome P450 (CYP2E1) in human fetal liver and hepatocytes.

The mechanisms responsible for ethanol-mediated teratogenesis have not been resolved. However, possible etiologies include the local formation of the teratogen acetaldehyde or oxygen radicals by fetal ethanol-oxidizing enzymes. As alcohol dehydrogenases are expressed at very low concentrations in human embryonic tissues, the ethanol-inducible P450 enzyme, CYP2E1, could be the sole catalyst of fetal ethanol oxidation. With this in mind, we examined the expression of this P450 in liver samples from fetuses ranging in gestational age from 16 to 24 weeks. Immunoblot analysis of fetal liver microsomes revealed the presence of a protein immunoreactive with CYP2E1 antibodies that exhibited a slightly lower molecular weight than that found in adult liver samples. Embryonic CYP2E1 expression was further confirmed by the reverse transcriptase reaction with RNA from a 19-week gestational fetal liver used as template. Catalytic capabilities of human fetal microsomes were assessed by measurement of the rate of ethanol oxidation to acetaldehyde, which were 12-27% of those exhibited by adult liver microsomes. Immunoinhibition studies with CYP2E1 antibodies revealed that the corresponding antigen was the major catalyst of this reaction in both fetal and adult tissues. We then assessed whether embryonic CYP2E1 was, like the adult enzyme, inducible by xenobiotics. Treatment of primary fetal hepatocyte cultures with either ethanol or clofibrate demonstrated a 2-fold increase in CYP2E1 levels compared with untreated cells. Collectively, our results indicate that CYP2E1 is present in human fetal liver, that the enzyme is functionally similar to CYP2E1 from adults, and that fetal hepatocyte CYP2E1 is inducible in culture by xenobiotics, including ethanol. Because fetal CYP2E1 mediates ethanol metabolism, the enzyme may play a pivotal role in the local production of acetaldehyde and free radicals, both of which have potential deleterious effects on the developing fetus.

Regio- and stereoselective epoxidation of arachidonic acid by human cytochromes P450 2C8 and 2C9.

In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with [1-14C]-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68% of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69% of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorbenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J.T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R), 15(S)-EET with 86.2% and 62.5% selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1% selective for 11(R), 12(S)-EET; P450 2C9 is 69.4% selective for the 11(S), 12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.

Cloning and expression of complementary DNAs for multiple members of the human cytochrome P450IIC subfamily.

The present study characterizes the profile of cDNAs from the human P450IIC subfamily in a library from one individual, and it describes three new members of this subfamily (IIC17, IIC18, and IIC19) isolated from two human cDNA libraries. cDNA libraries were constructed from two human livers which differed phenotypically in the hepatic content of P450 HLx (IIC8). The library from the phenotypically low HLx individual was screened by using a cDNA for rat liver P450IIC13 and an oligonucleotide probe for human IIC8. One clone, 245c, was isolated which clearly represents a new member of the human P450IIC subfamily (IIC17). This clone lacked the first 358 nucleotides at the N-terminus but was only 91% homologous in its nucleic acid sequence to IIC9 and 79% homologous to IIC8. Near-full-length clones for IIC9 were also isolated from this library, but no clones for IIC8 were found. Northern blots indicated that the mRNA for IIC8 was low or absent in this individual. A second cDNA library (from a liver phenotypically high in HLx) was then screened. Eighty-three essentially full-length (greater than 1.8 kb) clones belonging to the IIC subfamily were isolated from this library. These include full-length clones for two additional new members of the IIC subfamily. Clones 29c and 6b appear to be allelic variants (IIC18), differing by one nucleotide (one amino acid change) in the coding region. Clone 11a represents a full-length clone for a third new P450 (IIC19).(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol-mediated transplacental induction of CYP2E1 in fetal rat liver.

We examined the potential for the widely consumed xenobiotic ethanol to transplacentally induce fetal rat CYP2E1. Throughout gestation, rat dams were fed a liquid diet containing 5% ethanol or two separate control diets. At 2 days before term, the dams were killed, and maternal and embryonic tissues were collected. Immunoblot analysis of microsomes from fetal liver, placenta and maternal brain revealed a band that comigrated with adult liver CYP2E1. The identity of the immunoreactive protein in placenta, brain and fetal liver was substantiated as CYP2E1 through restriction enzyme digestion of a reverse transcription-polymerase chain reaction product. Quantification of immunoblots containing microsomes from maternal and fetal liver of ethanol-treated dams displayed a 1.4- and 2.4-fold increase in CYP2E1, respectively, compared with microsomes from pair-fed controls. Chlorzoxazone and low substrate concentrations of N-nitrosodimethylamine were used as metabolic probes for CYP2E1. The rate of chlorzoxazone metabolism by maternal hepatic microsomes from dams fed the 5% ethanol diet was 2.6-fold greater than that of controls. Conversely, a negligible increase was observed in the rate of metabolism by hepatic microsomes from ethanol-exposed fetuses compared with pair-fed animals. When N-nitrosodimethylamine demethylation was examined, these same fetal samples exhibited greater rates of activity (1.5-fold) compared with microsomes from control animals. However, this increase was not as great as expected considering the 2.4-fold increase in CYP2E1 protein. Collectively, fetuses exposed to a 5% ethanol diet throughout gestation exhibited transplacental induction of an hepatic CYP2E1 that may possess different catalytic properties from the analogous adult enzyme.

Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51.

The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6. Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18. The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation. Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards. Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected.

Diazepam metabolism by cDNA-expressed human 2C P450s: identification of P4502C18 and P4502C19 as low K(M) diazepam N-demethylases.

The present study provides a detailed kinetic analysis of diazepam metabolism by all four known members of the human P4502C subfamily expressed from their cDNAs in Escherichia coli. Both P4502C18 and P4502C19 were found to be low K(M) diazepam N-demethylases with apparent K(M) values of 24 +/- 4 microM and 21 +/- 3 microM, respectively. These values closely resemble the low K(M) component of diazepam N-demethylase activity exhibited by human liver microsomes. In addition, P4502C19 also catalyzed diazepam 3-hydroxylation with a K(M) value of 21 +/- 9 microM. Although P4502C8 was essentially inactive in catalyzing diazepam metabolism, P4502C9 catalyzed the N-demethylation with a relatively high K(M) of 80 +/- 15 microM and an overall 3- to 6-fold lower catalytic efficiency, compared with P4502C18 and P4502C19, respectively. At a substrate concentration of 10 microM, diazepam N-demethylation in a panel of human liver microsomes was inhibited 42 +/- 12% (mean +/- SD, N = 6) by a polyclonal anti-CYP2C antibody. In the same experiment, 3-hydroxylation remained unaffected (<10% inhibition). 1 microM of the CYP3A inhibitor ketoconazole inhibited 37 +/- 19% of the N-demethylation and 86 +/- 5% of 3-hydroxylation. Estimates of relative contributions to diazepam N-demethylation of P4502C9 (8 +/- 4%), P4502C18 (<2%), and P4502C19 (33 +/- 14%) and to diazepam 3-hydroxylation of P4502C19 (9 +/- 3%) based on the kinetic parameters of the recombinant enzymes and on specific contents of the individual 2C P450s determined in immunoblots are consistent with the inhibition data. In conclusion, these data confirm that both P4502C19 and P4503A are major contributors to human liver microsomal diazepam N-demethylation at low substrate concentrations, whereas P4503A is the major enzyme responsible for 3-hydroxylation.

CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes.

Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.

Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/- 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/- 5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid.