Effect of scavengers of active oxygen species on cell damage caused in CHO-K1 cells by phenylhydroquinone, an o-phenylphenol metabolite

Abstract

Phenylhydroquinone (PHQ), a metabolite of o-phenylphenol (OPP), is easily autoxidized to phenylbenzoquinone (PBQ) via the semiquinone (phenylsemiquinone, PSQ) with concomitant production of superoxide anion radicals (O2-.). We have used scavengers of active oxygen species to examine whether or not O2-. produced during oxidation of PHQ is related to cell damage in CHO-K1 cells. PHQ at 10 micrograms/ml (3-h treatment) induced sister-chromatid exchange (SCE), endoreduplication (ERD) and cell-cycle delay in CHO-K1 cells. These effects were inhibited by catalase (280 U/ml), a scavenger of hydrogen peroxide (H2O2), as well as by the reductants, ascorbate (3 mM) and GSH (1 mM). Mannitol (50 mM), a scavenger of hydroxyl radical (OH.), was ineffective and superoxide dismutase (SOD, 150 U/ml), a scavenger of O2-., or SOD plus catalase rather intensified the toxicity as did aminotriazole (20 mM), an inhibitor of catalase. Analyses of incubation solutions by HPLC showed that the extent of cell damage is correlated with PHQ loss; catalase suppressed PHQ loss, whereas SOD promoted it. The correlation was more clearly seen in the time courses of cell death and PHQ loss during incubation of PHQ with each of the scavengers of active oxygen species. These results show that neither O2-. nor OH. participates in the cell damage, but rather H2O2 generated via dismutation of O2-. may participate, probably by accelerating the autoxidation of PHQ and thus causing an increase in the production of toxic intermediates. In fact, conversion of PHQ to PBQ, a reactive product, was demonstrated during incubation with PHQ in phosphate-buffered saline by following the changes in UV-visible spectra of PHQ. Inclusion of H2O2 (0.2 or 1 mM) in the incubation mixture accelerated the PHQ loss. The present results can be explained in terms of the autoxidation mechanism of hydroquinone proposed by O'Brien (1991). Different from the results in the absence of S9 mix, the cell damage induced by 50 micrograms/ml OPP in the presence of S9 mix was not influenced by any of the scavengers of active oxygen species used. We conclude that PHQ causes cytotoxic and genotoxic effects through its autoxidation, both enzymatic and nonenzymatic, and that reactive intermediate(s) such as PSQ and/or PBQ may be ultimately responsible for the effects. H2O2 formed during the oxidation process participates in the damaging effects caused in the absence of S9 mix, probably by accelerating the autoxidation.