Oxidative metabolism of seleno-L-methionine to L-methionine selenoxide by flavin-containing monooxygenases.

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-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. The formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean +/- S.D.; n = 4) of 0.91 +/- 0.29 mM and a Vmax value of 44 +/- 8.0 nmol MetSeO/mg protein/min. The 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, the 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 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest that FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.

Renal toxicity of perchloroethylene and S-(1,2,2-trichlorovinyl)glutathione in rats and mice: sex- and species-dependent differences.

Suspensions of renal cells from rats and renal mitochondria from rats and mice were used to assess the sex and species dependence of acute toxicity due to perchloroethylene (Perc) and its glutathione conjugate S-(1,2,2-trichlorovinyl)glutathione (TCVG). A marked sex dependence in the acute cytotoxicity of both Perc and TCVG was observed: Perc caused significant release of lactate dehydrogenase (LDH) in isolated kidney cells from male but not female rats, and TCVG caused much more LDH release from male than female rat kidney cells. Assessment of toxicity in suspensions of isolated mitochondria from kidneys of male and female rats revealed a generally similar pattern of sensitivity, with mitochondria from males exhibiting significantly more inhibition of State 3 respiration and decrease of respiratory control ratio than mitochondria from females. Respiratory function in mitochondria from male and female mice, however, was also significantly inhibited by Perc or TCVG but exhibited little sex dependence in the degree of inhibition. Comparison with results from similar studies using the congener trichloroethylene and its glutathione conjugate suggested that Perc and TCVG are more potent nephrotoxicants. Neither Perc nor TCVG produced any significant effects on cytotoxicity or mitochondrial function in isolated hepatocytes from rats or in isolated liver mitochondria from rats or mice, suggesting that the liver is not a major acute target for Perc or its glutathione conjugate. Thus, many of the species-, sex-, and tissue-dependent differences in toxicity of Perc and TCVG that are observed in vivo are also observed in these in vitro models.