Environmental factors affecting monooxygenase activity of microsomal fractions of human liver biopsies [proceedings].

using lung and liver microsomes in the presence of paraquat, to generate superoxide and covalently bind a metabolite of DOPA. Male Wistar rats (150-250 g) were killed by decapitation and their lungs were perfused with ice cold normal saline. The lungs and livers were removed, homogenised and the microsomal fraction was isolated by differential centrifugation. The covalent binding of DOPA to washed microsomal protein was measured according to the method of Dybing et al. (1976). In the absence of paraquat the covalent binding ofradioactivity (expressed as nmol ofDOPA) to microsomal protein was 0.200 + 0.012 nmol mg protein 1 minm(mean + s.e. mean, n = 11) for lung and 0.401 + 0.012 nmol mg protein-' min-' (n = 7) for liver. However, the addition of paraquat (5 mM) caused a 132% increase in binding with lung microsomes with only a 22.4% increase with liver microsomes. Superoxide dismutase (30 jLg/ml) in the absence of paraquat completely inhibited the covalent binding while in the presence of paraquat (5 mm) significant superoxide dismutase insensitive covalent binding remained. Preliminary studies to ascertain the involvement of species other than superoxide in the covalent binding were therefore initiated. Benzoate, a hydroxyl radical scavenger and the singlet oxygen scavenger, 1,4-diazabicyclo-2,2,2-octane in the absence or presence of paraquat had no effect on the covalent binding. Catalase in the absence of paraquat significantly reduced the binding whereas in the presence of paraquat it had no effect. The addition of glutathione, which can form a conjugate with the reactive intermediate of DOPA, completely inhibited covalent binding in the absence and presence of paraquat. If glutathione plays a similar role in the intact cell then it may be necessary to deplete intracellular glutathione before this technique can be used in vivo to identify the cell type or types in the lung involved in paraquat toxicity.