PHENOXYL RADICALS OF ETOPOSIDE (VP-16) CAN DIRECTLY OXIDIZE INTRACELLULAR THIOLS - PROTECTIVE VERSUS DAMAGING EFFECTS OF PHENOLIC ANTIOXIDANTS

Phenolic compounds can act as radical scavengers due to their ability to donate a mobile hydrogen to peroxyl radicals produc ing a phenoxyl radical if the phenoxyl radical formed in the radical scavenging react ion efficiently interacts with vitally important biomolecules, then th is interaction may result in cytotoxic effects rather than in antioxid ant protection. In the present work we have chosen two model compounds - a phenolic antitumor drug, VP-16, known to be highly cytotoxic, and a homolog of vitamin E, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC) -as typical representatives of phenoxyl radicals to study interactions of their phenoxyl radicals with intracellular thiols. Using a water-s oluble source of peroxyl radicals, the azo-initiator 2,2'-azobis(2-ami nodinopropane) (AAPH), we found that both PMC and VP-16 are very effic ient scavengers of peroxyl radicals as evidenced by their ability to i nhibit AAPH-induced chemiluminescence of luminol and oxidation of PnA incorporated into DOPC liposomes. Both PMC and VP-16 were also able to protect against AAPH-induced oxidative degradation of DNA in nuclei f rom human leukemic K562 cells. In contrast, there was a dramatic diffe rence in the ability of VP-16 and PMC to protect GSH against AAPH-indu ced oxidation: while PMC inhibited AAPH-induced oxidation of GSH in a concentration-dependent manner, VP-16 did not protect GSH against oxid ation. We hypothesized that this was due to different reactivities of the phenoxyl radicals formed by AAPH-derived peroxyl radicals from VP- 16 and PMC toward GSH. To substantiate this hypothesis, we compared in teractions of the phenoxyl radicals generated from VP-16 and PMC with intracellular thiols in K562 cell homogenates. While the PMC phenoxyl radicals were only slightly affected by thiols, the VP-16 phenoxyl rad icals were reduced by thiols. This is evidenced by (i) a significant i nhibition of the tyrosinase-induced VP-16 consumption upon addition of K562 cell homogenates, (ii) a depletion of endogenous thiols in K562 cell homogenates induced by VP-16+-tyrosinase, (iii) a transient disap pearance of the VP-16 phenoxyl radical signal from the ESR spectra and its reappearance after depletion of endogenous thiols, and (iv) elimi nation of the lag period for the appearance of the VP-16 phenoxyl radi cal ESR signal subsequent to depletion of thiols by mersalyl acid. To evaluate the contribution of GSH and protein thiols to reduction of th e VP-16 phenoxyl radical, we treated K562 cell homogenates with GSH-pe roxidase + cumene hydroperoxide to specifically de plete endogenous GS H. We found that GSH and protein thiols each account for about 50% of the reduction of the VP-16 phe noxyl radical by K562 homogenates. Simi larly, endogenous thiols in cell homogenates from several different cu ltured cell lines were oxidized by VP-16 phenoxyl radicals (but not PM C phenoxyl radicals). The results of this study suggest that the diffe r ential effects of PMC and VP-16 in intracellular environments, antio xidant protection or cytotoxicity, may be due, at least in part, to a striking difference in the reactivity of their respective phenoxyl rad icals toward endogenous thiols. In addition to their radical scavengin g activity, the reactivity of phenoxyl radicals toward critical biomol ecules should be carefully considered in the design and development of biomedical antioxidants. (C) 1995 Academic Press, Inc.