Flavin-containing monooxygenases: Isoform-dependent differences in kinetics, localization, and regulation in mammalian renal and hepatic tissues

We investigated the isoform distinct oxidative metabolism, localization, and expression of flavin-containing monooxygenases (FMOs). First, FMO3 was purified from rat kidney and the physical, immunological, and enzymatic properties of the isolated protein were characterized. Renal FMO3 exhibited kinetics similar to hepatic FMO3 with methionine, seleno-L-methionine, S-allyl-L-cysteine, and methimazole suggesting that renal FMO3 may play an important role in the metabolism of sulfur- and selenium-containing xenobiotics. Second, polyclonal antibodies specific to rat FMO1 and FMO4 were developed and characterized through immunoblotting and immunoprecipitation. Development of the isoform specific antibodies led to the immunochemical localization of FMO1, FMO3, and FMO4 in rat kidney and liver. These data provided a compelling visual demonstration of the isoform-specific localization patterns of FMOs in areas that are optimal for encountering and metabolizing xenobiotics. The immunoreactivity of the anti-rat FMO4 antibody in mouse and human tissues were also investigated and provided the first evidence for expression of FMO4 in mouse and human liver and kidney microsomes. FMO4 expression in human liver (6 males and 4 females) and kidney (7 males and 5 females) samples did not exhibit gender differences. However, interindividual variations of approximately 4- and 13-fold in FMO4 protein expression were observed in human liver and kidney microsomes, respectively. Finally, because alterations in mouse hepatic Fmo mRNA after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and castration were previously reported, we investigated the change in Fmo mRNA expression for up to 32 days after oral TCDD (10 or 64 microg/kg) exposure and the combined effects of TCDD and castration. The results exhibited isoform distinct dose- and time-dependent hepatic Fmo mRNA alterations, including significant increases in Fmo2 and Fmo3 mRNA at multiple time points. TCDD treatment in combination with castration resulted in a greater than additive effect on Fmo1, Fmo2, and Fmo3 mRNA expression. An increase in hepatic FMO3 protein in TCDD treated mice was observed by immunoblotting and assaying the methionine S-oxidase activity, a good indicator of FMO3 in liver microsomes from several species. Collectively, these studies elucidate the unique properties of FMO isoforms with regards to their metabolism, localization, expression, and regulation.