In vitro induction of reactive oxygen species by 2,3,7,8-tetrachlorodibenzo- p-dioxin, endrin, and lindane in rat peritoneal macrophages, and hepatic mitochondria and microsomes

Abstract

Hepatic mitochondria and microsomes as well as peritoneal macrophages from female Sprague-Dawley rats were incubated for up to 30 min at 37°C in the presence of 0–200 ng/ml 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), endrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4α,5,6,7,8,8α-octahydroendo,endo-1,4:5,8-dimethanonaphthalene), and lindane (hexachlorocyclohexane). Production of reactive oxygen species was determined by chemiluminescence and cytochrome c reduction, while potential tissue damage was assessed by alterations in membrane fluidity. Chemiluminescence, a sensitive but nonspecific measure of free radical generation, increased 40–70% when macrophages (3 × 10 6 cells/ml), mitochondria and microsomes (1 mg/ml) were incubated with the three polyhalogenated cyclic hydrocarbons (PCH). Maximum increases in chemiluminescence occurred within 5–10 min of incubation and persisted for over 30 min. The cytochrome c reduction assay is most specific for superoxide anion production. When hepatic mitochondria were incubated with endrin and lindane for 15 min at 100 ng/ml, increases in cytochrome c reduction of 6.5- and 7.5-fold occurred, respectively, while when microsomes were incubated with these same two PCH, increases in cytochrome c reduction of 8.6- and 11.6-fold occurred, respectively. When mitochondria, microsomes, and macrophages were incubated with TCDD under identical conditions, small increases in superoxide anion production were detected. Changes in microsomal membrane fluidity were determined spectrofluorometrically following incubation with the three PCH using diphenyl-1,3,5-hexatriene as the fluorescent probe. TCDD, endrin, and lindane enhanced microsomal membrane apparent microviscosity by 2.3-, 2.1-, and 2.5-fold, respectively, indicating a significant decrease in membrane fluidity. The results clearly indicate that the three PCH induce free radical formation, but superoxide anion production is enhanced more in the presence of endrin and lindane than with TCDD.