Human cytochrome P-450IIE1 has been implicated in the oxidation of a number of substrates, including protoxins and -carcinogens. To date, no drugs have been identified that are exclusive substrates for the protein and are applicable for use as noninvasive probes of the in vivo function of the enzyme in humans. Chlorzoxazone was found to be oxidized only to 6-hydroxychlorzoxazone in human liver microsomes. Results of steady-state kinetics are consistent with the view that only a single enzyme catalyzes the reaction. The microsomal reaction was strongly inhibited by rabbit anti-P-450IIE1 and, in a competitive manner, by known P-450IIE1 substrates. Rates of chlorzoxazone 6-hydroxylation in different human liver microsomal preparations were well correlated with levels of immunochemically measured P-450IIE1 and rates of (CH3)2NNO oxidation. Chlorzoxazone 6-hydroxylation was also found to be catalyzed by purified human liver P-450IIE1. These results provide strong evidence that P-450IIE1 is the primary catalyst of chlorzoxazone 6-hydroxylation in human liver. Rates of chlorzoxazone 6-hydroxylation vary considerably among human liver samples, and chlorzoxazone 6-hydroxylation may have potential use as a noninvasive probe in estimating the in vivo expression of human P-450IIE1 and its significance as a risk factor in the toxicity and carcinogenicity of a number of solvents, nitrosamines, and drugs.

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Acetaminophen (APAP), a widely used analgesic and antipyretic agent, is bioactivated by cytochromes P450 to cause severe hepatotoxicity. APAP is oxidized by two pathways to form a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI), and a nontoxic catechol metabolite, 3-hydroxy-APAP (3-OH-APAP). We investigated the role of P450 2E1 and 2A6 in APAP oxidation by using baculovirus-expressed and highly purified forms of human P450 2E1 and 2A6. An electrochemical HPLC assay was developed to quantify both oxidative metabolites simultaneously. For the first time, it was demonstrated that human P450 2E1 selectively oxidized APAP to NAPQI (assayed as its glutathione conjugate, GS-APAP), whereas human P450 2A6 selectively oxidized APAP to 3-OH-APAP. At 1 mM APAP, the relative ratio for the formation of GS-APAP vs 3-OH-APAP with human P450 2E1 was approximately 6:1, whereas the ratio with human P450 2A6 was 1:3. Apparent Km and Vmax values for the formation of GS-APAP by human P450 2E1 were 1.3 mM and 6.9 nmol/min/nmol of P450, respectively, whereas they were 4.6 mM and 7.9 nmol/min/nmol of P450 for P450 2A6. Apparent Km and Vmax values for the formation of 3-OH-APAP by human P450 2E1 were 4.0 mM and 2.5 nmol/min/nmol of P450, respectively, whereas they were 2.2 mM and 14.2 nmol/min/nmol of P450, respectively, for P450 2A6. Thus, although at toxic doses of APAP P450 2E1 is the more efficient catalyst for the formation of the toxic metabolite NAPQI, P450 2A6 also can contribute significantly to NAPQI production.

Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P450 2C9 and P450 1A2, respectively. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P450 2E1 and appeared to be directed against mainly conformational epitopes. In addition, because cytochrome P450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P450 2E1 may be able to bypass the immunologic tolerance that normally exists against cytochrome P450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.

A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.

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Methapyrilene (MP), a 2-thiophene H 1 -receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([ 3 H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat Ͼ mouse Ϸ human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [ 3 H]MP intravenously. MP produced concentration-and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes. Fig. 1], a 2-thiophene H 1 -receptor antagonist, was not associated with hepatotoxicity in humans, but it causes unusual dose-dependent periportal damage in adult male rats (Graichen et al., 1985;Ratra et al., 1998a;Ratra et al., 2000). Typical hepatotoxic regimens (150 -300 mg/kg/day ϫ 3) produce a mild to moderate injury characterized by hepatocellular necrosis, bile duct proliferation, and inflammatory cell infiltration. MP is also toxic to isolated rat hepatocytes (McQueen and Williams, 1982;Ratra et al., 1998b), which are reported to be more susceptible than mouse hepatocytes (Kelly et al., 1992).MP has come to be regarded as a model investigatory compound among drug hepatotoxicants, and it is used fre- Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.135483.ABBREVIATIONS: MP, methapyrilene; P450, cytochrome P450; tGSH, total glutathione (GSH plus glutathione disulfide); MeOD, monodeuteromethanol; HPLC, high-performance liquid chromatography; [ 3 H]MP, methapyrilene tritiated at C-2 of the diaminoethane moie...