Metabolic fate of chemical mixtures. I. "Shuttle oxidant" effect of lipoxygenase-generated radical of chlorpromazine and related phenothiazines on the oxidation of benzidine and other xenobiotics

Many carcinogens, mutagens, teratogens, and other toxicants are known to be oxidized by lipoxygenases to potentially deleterious free radical intermed iates. In this study, we tested for the first time the possibility that cer tain efficient substrates for lipoxygenase produce shuttle oxidants that st imulate the generation of reactive species from other chemicals. To evaluat e the hypothesis, we investigated the metabolic interaction of two well-kno wn substrates, chlorpromazine and benzidine, which have been shown to be ox idized by soybean lipoxygenase in the presence of hydrogen peroxide. The ev idence presented here clearly indicates that the chlorpromazine cation radi cal generated by the lipoxygenase triggers a rapid oxidation of benzidine t o benzidine diimine. Under the experimental conditions employed, the metabo lic interaction resulted in a 42-fold stimulation in the rate of benzidine oxidation. The magnitude of stimulation of benzidine oxidation exhibited a dependence on the pH of the reaction medium, amount of the enzyme, and conc entration of chlorpromazine, benzidine, and hydrogen peroxide. A number of other phenothiazines were also found to stimulate benzidine oxidation, albe it to a lesser degree. The chlorpromazine cation radical stimulated the oxi dation of all six other xenobiotics tested. The highest stimulation (94-fol d) was noted with tetramethyl phenylenediamine oxidation to the Wursters bl ue radical, while the lowest stimulatory response (2-fold) was observed wit h guaiacol. Preliminary data suggest that purified human term placental lip oxygenase also displays a similar stimulatory response in the benzidine oxi dation in the presence of chlorpromazine. Although the toxicological signif icance of these in vitro findings remains to be established, it is worth po ndering whether such a synergistic interaction occurs in humans in vivo. Te ratogenesis Carcinog. Mutagen. 20:195-208, 2000. (C) 2000 Wiley-Liss, Inc.