The potential roles of human hepatic and renal flavin-containing monooxygenases (FMOs) in the metabolism of the cysteine S-conjugates S-allyl cysteine (SAC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) were investigated. Incubations of human cDNA-expressed FMO1, FMO3, FMO4, and FMO5 with SAC resulted in detection of SAC sulfoxide, with FMO3 exhibiting approximately 3-, 4-, and 10-fold higher activity than FMO1, FMO4, and FMO5, respectively. DCVC sulfoxide formation was only detected with FMO3 and was 59-fold lower than SAC sulfoxide formation. Incubations of human liver microsomes with SAC or DCVC resulted in detection of the corresponding sulfoxides and provided evidence for the involvement of FMO3. Incubations of SAC or DCVC with human kidney microsomes, however, led only to the detection of SAC sulfoxide. Immunoblots with monospecific antibodies to FMO1, FMO3, and FMO5 in kidney microsomes from 26 humans showed that the average expression levels for FMO1, FMO3, and FMO5 were 5.8 Ϯ 2.3, 0.5 Ϯ 0.4, and 2.4 Ϯ 1.4 pmol/mg (means Ϯ S.D.), respectively. Interestingly, African-American kidney samples (n ϭ 8) exhibited significantly higher FMO1 levels than Caucasian samples (n ϭ 17), whereas no difference in expression level between males and females was observed with any of the examined FMO isoforms. Collectively, the results provide evidence for the expression of three FMO isoforms in the human kidney and show that the contribution of renal FMOs in cysteine Sconjugate metabolism is likely to vary depending upon the cysteine S-conjugate and the relative expression levels of the active FMOs.Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze oxidation of sulfur-, selenium-, and nitrogen-containing compounds (Ziegler, 1993). Five active isoforms have been identified and are present in most mammalian tissues. However, species-, sex-, tissue-, and agedependent differences exist in expression levels of these isoforms (Hines et al., 1994;Dolphin et al., 1996;

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S-(1,2-Dichlorovinyl)-L-cysteine (DCVC)is the penultimate nephrotoxic metabolite of the environmental contaminant trichloroethylene. Although metabolism of DCVC by the cysteine conjugate ␤-lyase is the most studied bioactivation pathway, DCVC may also be metabolized by the flavin-containing monooxygenase (FMO) to yield DCVC sulfoxide (DCVCS). Renal cellular injury induced by DCVCS was investigated in primary cultures of human proximal tubular (hPT) cells by assessment of time-and concentration-dependent effects on cellular morphology, acute cellular necrosis, apoptosis, mitochondrial function, and cellular glutathione (GSH) status. Confluent hPT cells incubated with as little as 10 M DCVCS for 24 h exhibited morphological changes, although at least 100 M DCVCS was required to produce marked changes. Acute cellular necrosis did not occur until 48 h with at least 200 M DCVCS, indicating that this is a high-dose, late response. The extent of necrosis was similar to that with DCVC. In contrast, apoptosis occurred as early as 1 h with as little as 10 M DCVCS and the extent of apoptosis was much less than that with DCVC. Mitochondrial function was maintained with DCVCS concentrations up to 100 M, consistent with hPT cells only being competent to undergo apoptosis at early time points and relatively low concentrations. Marked depletion (Ͼ50%) of cellular GSH content was only observed with 500 M DCVCS. These results, combined with previous studies showing protection from DCVC-induced necrosis and apoptosis by the FMO inhibitor methimazole, suggest that formation of DCVCS plays a significant role in trichloroethylene-induced renal cellular injury in hPT cells.

The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-L-methionine (SeMet) to L-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenylderivatives of SeMet and MetSeO were synthesized and characterized by 1 H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. Formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet K m value (mean ±S.D.; n=4) of 0.91 ± 0.29 mM and a V max value of 44 ± 8.0 nmol MetSeO/ mg protein/min. Inclusion of 1-benzylimidazole, superoxide dismutase or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (K m = 0.11 mM, V max = 280 nmol/mg protein/min) and rat liver FMO1 (K m = 7.8 mM, V max = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.

The metabolic fate of 3-butene-1,2-diol (BDD), a secondary metabolite of the industrial carcinogen, 1,3-butadiene, is unclear. The current study characterizes BDD oxidation to hydroxymethylvinyl ketone (HMVK), a reactive Michael acceptor. Because of its instability in aqueous medium, HMVK was trapped by conjugation with GSH, a reaction that occurred readily at physiological conditions (pH 7.4, 37 degrees C) to yield 1-hydroxy-2-keto-4-(S-glutathionyl)butane. The results show that BDD was oxidized to HMVK by mouse, rat, and human liver microsomes and by cDNA-expressed human cytochrome P450s. Eadie-Hofstee plots demonstrated biphasic kinetics of BDD oxidation with mouse and rat liver microsomes and one of three individual human liver microsomes; BDD oxidation by the other two human liver microsomal samples was best described by monophasic kinetics. Of the human P450 enzymes examined, only P450 2E1 exhibited activity at 1 mM BDD. P450 3A4 was capable of catalyzing the reaction at a high BDD (10 mM) concentration; P450 1A1, 1A2, 1B1, 2D6-Met, and 2D6-Val produced only trace amounts of HMVK-GSH whereas P450 2A6, 2C8, 2C9, and 4A11 had no detectable activity. Detection of HMVK or the HMVK-GSH conjugate was dependent on reaction time, protein, and BDD concentrations, and the presence of NADPH. Collectively, the results provide clear evidence for BDD bioactivation to yield HMVK. Because mouse, rat, and human liver microsomes exhibited K(m) values of 50-80 microM, the results also suggest that HMVK could be formed after rodent or human exposure to BDD or its parent compound, BD.