Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat

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Abstract
The metabolic activation of CCl4 to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury. ESR spin-trapping techniques were used to characterize the free-radical species involved. Spin trapping was applied to the activation of CCl4 by liver microsomal fractions in the presence of NADPH and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide (phenyl tert-butyl nitrone) (PBN) and [13C]CCl4 provided unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. With the spin trap 2-methyl-2-nitrosopropane, the major free-radical species trapped were unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. Although pulse radiolysis and other evidence supported the rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and O2, no clear evidence for the trapping of the peroxy radical was obtainable. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. High concentrations of the spin traps had significant effects on cytochrome P-450-mediated reactions; this required caution in interpreting results of experiments done in the presence of PBN at concentrations > 50 mM.