DNA STRAND CLEAVAGE AS A SENSITIVE ASSAY FOR THE PRODUCTION OF HYDROXYL RADICALS BY MICROSOMES - ROLE OF CYTOCHROME P4502E1 IN THE INCREASED ACTIVITY AFTER ETHANOL TREATMENT

There is increasing interest in the role of reactive oxygen radicals i n the hepatotoxicity associated with ethanol consumption. Reactive oxy gen intermediates interact with DNA and can cause single-strand breaks of supercoiled DNA. Experiments were carried out to evaluate the util ity of this system as a sensitive assay for the detection of potent ox idants generated by rat liver microsomes isolated from pair-fed contro l rats and rats treated chronically with ethanol. DNA strand cleavage. was, assayed by monitoring the migration of the supercoiled and open circular forms in agarose. Microsomes catalysed DNA strand breakage wi th either NADPH or NADH as cofactors; iron was required to catalyse th e reaction and Various ferric complexes were effective in promoting th e reaction. DNA strand cleavage was prevented by catalase, superoxide dismutase, GSH and hydroxyl-radical-scavenging agents, suggesting that a hydroxyl-radical-like species was the oxidant responsible for the b reakage. This assay system proved to be much more sensitive in detecti ng hydroxyl radicals than are other methods, such as e.s.r. spectrosco py or oxidation of chemical scavenging agents with respect to the amou nt of microsomal protein and the nature and concentration of the iron catalyst required. Microsomes from ethanol-treated rats were more reac tive than control microsomes in catalysing the DNA strand cleavage wit h either NADPH or NADH; increased catalytic activity was observed with various ferric complexes and was sensitive to the above antioxidants. Compared with preimmune IgG, anti-(cytochrome P4502E1) IgG had no eff ect on DNA strand cleavage by the control microsomes, but completely p revented the NADPH- and the NADH-dependent increased activity found wi th microsomes from the ethanol-treated rats. Inhibitors of cytochrome P4502E1, such as diethyl dithiocarbamate and tryptamine, also lowered the extent of increase of DNA strand cleavage produced by microsomes f rom the ethanol-treated rats. These results indicate that DNA strand c leavage is a very sensitive assay for detecting the production of hydr oxyl radicals by microsomes and to demonstrate increased activity by m icrosomes after chronic ethanol treatment. This increased activity wit h NADPH and NADH is due, at least in part, to induction of cytochrome P4502E1.