LEVELS OF DNA STRAND BREAKS AND SUPEROXIDE IN PHORBOL ESTER-TREATED HUMAN GRANULOCYTES

Phorbol ester treatment of granulocytes triggers release of superoxide (O-2 radical anion) and a concomitant burst of DNA strand breaks. The relationship between the amount of O-2 radical anion and the number o f DNA breaks has not previously been explored. To quantify the relativ ely large amount of O-2 radical anion generated over a 40-min period b y 1 x 10(6) granulocytes/ml, a discontinuous ''10-min pulse'' method e mploying cytochrome c was used; 140 nmol O-2 radical anion per 1 x 10( 6) cells was detected. DNA strand breaks were quantified by fluorimetr ic analysis of DNA unwinding (FADU). To vary the level of O-2 radical anion released by cells, inhibitors of the respiratory burst were used . Sodium fluoride (1-10 mM) and staurosporine (2-10 nM) both inhibited O-2 radical anion production. In both cases, however, inhibition of s trand breakage was considerably more pronounced than inhibition of O-2 radical anion. Zinc chloride (50-200 mu M) inhibited both O-2 radical anion and DNA breaks, approximately equally. Dinophysistoxin-1 (okada ic acid) inhibited O-2 radical anion production more effectively than it inhibited DNA breaks. O-2 radical anion dismutes to H2O2, a reactiv e oxygen species known to cause DNA breaks. The addition of catalase t o remove extracellular H2O2 had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected c ompared to the number detected by FADU, indicating that about 95% of b reaks were single-stranded. The level of DNA breaks is not directly re lated to the amount of extracellular O-2 radical anion or H2O2 in PMA- stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. (C) 1 997 Wiley-Liss, Inc.