Drug Metabolism Reviews, 38: 171–196, 2006 Copyright © Taylor & Francis Group, LLC ISSN: 0360-2532 print / 1097-9883 online DOI: 10.1080/03602530600570040 171 ANTIOXIDANT PROTECTIVE MECHANISMS AGAINST REACTIVE OXYGEN SPECIES (ROS) GENERATED BY MITOCHONDRIAL P450 SYSTEMS IN STEROIDOGENIC CELLS Israel Hanukoglu Department of Molecular Biology, College of Judea and Samaria, Ariel, Israel Mitochondrial P450 type enzymes catalyze central steps in steroid biosynthesis, including cholesterol conversion to pregnenolone, 11β and 18 hydroxylation in glucocorticoid and mineralocorticoid synthesis, C-27 hydroxylation of bile acids, and 1α and 24 hydroxylation of 25-OH-vitamin D. These monooxygenase reactions depend on electron transfer from NADPH via FAD adrenodoxin reductase and 2Fe-2S adrenodoxin. These systems can function as a futile NADPH oxidase, oxidizing NADPH in absence of substrate, and leak electrons via adrenodoxin and P450 to O 2 , producing superoxide and other reactive oxygen species (ROS). The degree of uncoupling depends on the P450 and steroid substrate. Stud- ies with purified proteins and overexpression in cultured cells show consistently that adren- odoxin, but not reductase, is responsible for ROS production that can lead to apoptosis. In the ovary and corpus luteum, antioxidant enzyme activities superoxide dismutase, catalase, and glutathione peroxidase parallel steroidogenesis. Antioxidant β-carotene, α-tocopherol, and ascorbate can protect against oxidative damages of P450 systems. In testis Leydig cells, steroidogenesis is associated with aging of the steroidogenic capacity. Key Words: Ascorbate; Adrenodoxin; Superoxide; Catalase; P450scc; Tocopherol; Vitamin C; Vitamin E. Electron Transfer in Mitochondrial P450 Systems Cytochrome P450 type enzymes represent the largest superfamily of enzymes that are involved in the metabolism of generally small hydrophobic molecules. These enzymes share a common structural topology with a heme group in their active site and derive their name from the absorption maximum of their reduced carbon monoxide complex at 450 nm (Coon, 2005; Estabrook, 2005). In eukaryotes there are two classes of P450s that are located in the mitochondria and the endoplasmic reticulum. The nomenclature for P450 genes starts with the root name CYP and follows the order CYP:family:subfamily:gene (Nelson et al., 1996). Mitochondrial P450 type enzymes are generally involved in the biosynthesis of cho- lesterol derived steroidal compounds (Hanukoglu, 1992; Miller, 2005). In mammals, the reactions catalyzed by these enzymes include cholesterol conversion to pregnenolone Address correspondence to Israel Hanukoglu, Department of Molecular Biology, College of Judea and Samaria, Ariel 44837, Israel; E-mail: hi@science.co.il