Aromatic hydrocarbon quinone-mediated reactive oxygen species production on hepatic microsomes of the flounder (Platichthys flesus L.).

The NAD(P)H-dependent redox cycling of a range of eight 1 ring to 5 ring aromatic hydrocarbon (AH) quinones by hepatic microsomes of flounder (Platichthys flesus) was studied in terms of oxygen consumption (Clark electrode) and reactive oxygen species (ROS) production (detection of hydroxyl radical by iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid). Stimulated oxygen consumption was detectable for only five AH-quinones (duroquinone, 1,2- and 1,4-naphthoquinones, menadione, 9,10-phenanthrenequinone), whereas stimulated ROS production was seen, or is known, for all eight (others plus 1,4-benzoquinone, anthraquinone, benzo[a]pyrene-3,6-dione), indicating that the former measurement is a more sensitive assay of redox cycling. Both processes showed Michaelis-Menten kinetics with respect to AH-quinone concentrations, with values for Vmax and apparent Km being, respectively, 146- to 9895-fold and 3- to 344-fold higher for stimulated oxygen consumption than ROS production. Marked correlation in values for both Vmax and apparent Km was seen between stimulated oxygen consumption and ROS production for 1,2-naphthoquinone, 1,4-naphthoquinone and 9,10-phenanthrenequinone, indicative of redox cycling and the univalent reduction of O2 to superoxide anion radical. Rates of stimulated oxygen consumption and ROS production were up to 10-fold higher for NADH- than for NADPH-dependent reactions and were highest for the naphthoquinones and 9,10-phenanthrenequinone. Comparison of the results for different AH-quinones indicates that enzyme substrate specificity is an important factor in determining redox cycling potential. Under the assay conditions used (0.1-2.0 mM AH-quinone), mutagenicity of the AH-quinone mediated processes could not be demonstrated using the Salmonella typhimurium umu assay. Overall, the results indicate a widespread potential for AH-quinone stimulated ROS production.