Uncoupling-mediated generation of reactive oxygen by halogenated aromatic hydrocarbons in mouse liver microsomes

Abstract Studying liver microsomes from 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced or vehicle-treated (noninduced) mice, we evaluated the in vitro effects of added chemicals on the production of reactive oxygen due to substrate/P450-mediated uncoupling. The catalase-inhibited NADPH-dependent H 2 O 2 production (luminol assay) was lower in induced than noninduced microsomes. The effects of adding chemicals (2.5 μM) in vitro could be divided into three categories: Group 1, highly halogenated and coplanar compounds that increased H 2 O 2 production at least 5-fold in induced, but not in noninduced, microsomes; Group 2, non-coplanar halogenated biphenyls that did not affect H 2 O 2 production; Group 3, minimally halogenated biphenyls and benzo[ a ]pyrene that decreased H 2 O 2 production. Molar consumption of NADPH and O 2 and molar H 2 O 2 production ( o -dianisidine oxidation) revealed that Group 1 compounds mostly increased, Group 2 had no effect, and Group 3 decreased the H 2 O 2 /O 2 and H 2 O 2 /NADPH ratios. Microsomal lipid peroxidation (thiobarbituric acid-reactive substances) was proportional to H 2 O 2 production. Although TCDD induction decreased microsomal production of H 2 O 2 , addition of Group 1 compounds to TCDD-induced microsomes in vitro stimulated the second-electron reduction of cytochrome P450 and subsequent release of H 2 O 2 production. This pathway is likely to contribute to the oxidative stress response and associated toxicity produced by many of these environmental chemicals.