DNA strand cleavage as a sensitive assay for the production of @entity3787 radicals by microsomes: role of cytochrome P4502E1 in the increased activity after XXXX treatment.

@entity2125 @entity7300 @entity26 @entity1110 @entity2197 @entity3787 @entity8584 @entity12609 @entity35 @entity224 @entity1068 @entity68
There is increasing interest in the role of reactive @entity26 radicals in the hepatotoxicity associated with @entity1068 consumption. Reactive @entity26 intermediates interact with DNA and can cause single-strand breaks of supercoiled DNA. Experiments were carried out to evaluate the utility of this system as a sensitive assay for the detection of potent oxidants generated by @entity35 liver microsomes isolated from pair-fed control @entity35 and @entity35 treated chronically with @entity1068 . DNA strand cleavage was assayed by monitoring the migration of the supercoiled and open circular forms in agarose. Microsomes catalysed DNA strand breakage with either @entity1110 or @entity2125 as cofactors; @entity68 was required to catalyse the reaction and various ferric complexes were effective in promoting the reaction. DNA strand cleavage was prevented by @entity12609 , @entity224 dismutase, @entity2197 and @entity3787 -radical-scavenging agents, suggesting that a @entity3787 -radical-like species was the oxidant responsible for the breakage. This assay system proved to be much more sensitive in detecting @entity3787 radicals than are other methods, such as e.s.r. spectroscopy or oxidation of chemical scavenging agents with respect to the amount of microsomal protein and the nature and concentration of the @entity68 catalyst required. Microsomes from @entity1068 -treated @entity35 were more reactive than control microsomes in catalysing the DNA strand cleavage with either @entity1110 or @entity2125 ; 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 effect on DNA strand cleavage by the control microsomes, but completely prevented the @entity1110 - and the @entity2125 -dependent increased activity found with microsomes from the @entity1068 -treated @entity35 . Inhibitors of cytochrome P4502E1, such as @entity8584 and @entity7300 , also lowered the extent of increase of DNA strand cleavage produced by microsomes from the @entity1068 -treated @entity35 . These results indicate that DNA strand cleavage is a very sensitive assay for detecting the production of @entity3787 radicals by microsomes and to demonstrate increased activity by microsomes after chronic @entity1068 treatment. This increased activity with @entity1110 and @entity2125 is due, at least in part, to induction of cytochrome P4502E1.
