Stimulation of oxyradical production of hepatic microsomes of flounder (Platichthys flesus) and perch (Perca fluviatilis) by model and pollutant xenobiotics

Abstract

Stimulation of hepatic microsomal NAD(P)H-dependent hydroxyl radical (.OH) production by model compounds, viz. menadione (2-methyl-1,4-naphthoquinone) and nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-aminohydantoin), and pollutant xenobiotics, viz. benzo[a]pyrene (BaP) diones (products of microsomal BaP metabolism), duroquinone (tetramethyl-1,4-benzoquinone--present in pulp mill effluent), and the pesticide lindane (gamma-hexachlorocyclohexane), was examined in flounder Platichthys flesus. Duroquinone was also studied in perch Perca fluviatilis, a freshwater species used in studies of pulp mill effluents in the aquatic environment. Microsomal .OH production was detected by the oxidation of the scavenging agent 2-keto-4-methiolbutyric acid (KMBA), using FeCl3/EDTA as a promotor of the Haber-Weiss reaction (O2- + H2O2 = .OH+OH- + O2). All xenobiotics tested, except lindane, showed synergistic interactions with ferric/EDTA indicative of redox cycling of the xenobiotic. Inhibition of menadione- and nitrofurantoin-stimulated .OH production by superoxide dismutase (50% inhibition) and catalase (80%) indicated respectively the involvement of O2- and H2O2 in .OH production. Maximal rates of KMBA oxidation (Vmax in nmol ethylene/min/mg protein) were similar for NADH and NADPH for menadione (4.58-4.61) and duroquinone (0.26-0.3 [flounder] and 0.93-0.99 [perch]), higher for NADPH than NADH for nitrofurantoin (1.21 and 0.77), and higher for NADH than NADPH for BaP diones, decreasing in the order 1,6-dione (1.12 and 0.14), 3,6-dione (0.75 and 0.25), and 6,12-dione (0.31 and 0.09). Rates for lindane, lacking a redox cycling structure, were low (0.01-0.05). Apparent Km (app. Km) values for xenobiotic were 1-2 orders of magnitude lower for BaP diones than the other compounds. App. Km was lower for NADH than NADPH for 3,6-dione (1.23 and 1.66 microM) and 6,12-dione (0.85 and 1.81 microM), but the reverse of this was found for the 1,6-dione (1.41 and 0.78 microM). App. Km values were almost identical for menadione and duroquinone and lower for NADPH (32-44 microM) than NADH (346-382 microM). The reverse was seen for nitrofurantoin, viz., 76 microM (NADH) and 269 microM (NADPH). Hepatic 1000 g supernatants of P. flesus metabolized BaP to oxyradical-generating products, moreso for beta-naphthoflavone-induced than control fish, and production was reduced by UDP-glucuronic acid for the latter but not the former. The studies indicate a widespread potential for contaminant-stimulated oxyradical generation via redox cycling and other free radical interactions of xenobiotics and their metabolites.