Enzymatic and immunohistochemical studies on the role of cytochrome P450 and the flavin-containing monooxygenase of mouse skin in the metabolism of pesticides and other xenobiotics

Abstract

The cytochrome P450 (P450) content, the cytochrome c reductase activity, the metabolism of a variety of P450 substrates, and the presence and role of flavin-containing monooxygenase (FMO) in xenobiotic metabolism were studied in skin microsomes and compared to those of liver. The cytochrome P450 content of skin as determined by CO-dithionite-reduced minus CO-oxidized spectra was approximately 6.8% of the liver P450 content. By comparison, cytochrome c reductase activity in skin microsomes was high, being equivalent to approximately one-third of the liver microsomal enzyme activity. Skin microsomes metabolized several known P450 substrates and, depending upon the substrate used, the specific activity ranged from 2.5 to 13.4% of the corresponding rates seen in liver microsomes. Skin microsomes exhibited the highest enzymatic activity with benzo[ a]pyrene and ethoxyresorufin, moderate activity with parathion and aldrin, and low activity with benzphetamine and ethoxycoumarin. Skin microsomes also metabolized the triazine herbicides atrazine, simazine, and terbutryn, with the activity being 2 to 5% of the liver microsomal activity. FMO activity in skin microsomes with thiobenzamide and methimazole as substrates ranged from 10 to 20% of the liver FMO activity. Immunohistochemical studies using antibodies to mouse liver FMO showed localization primarily in the epidermis. Additional studies using pig skin showed a similar distribution pattern. Antibodies developed to mouse liver FMO and the constitutive liver P450 isozyme, 1A2, showed cross-reactivity on Western blots with proteins in skin microsomes that appeared identical to the cross-reacting proteins present in liver microsomes. The relative contribution of P450 and FMO in mouse skin to the sulfoxidation of phorate was investigated and compared to that of liver microsomes. Several procedures were employed to selectively inhibit either P450 or FMO to determine the role of each monooxygenase system in the absence of the other system. In liver microsomes, P450 was responsible for 68 to 85% of the phorate sulfoxidation activity. In contrast, in skin microsomes 66 to 69% of the phorate sulfoxidation activity was due to FMO, while P450 was responsible for the remainder of the activity. Thus, although the overall phorate sulfoxidation rate in mouse skin microsomes was only 3 to 4% of the rate seen in liver, FMO appears to assume a greater relative role to P450 in the metabolic processes in skin.