Hc. Birnboim et Jk. Sandhu, LEVELS OF DNA STRAND BREAKS AND SUPEROXIDE IN PHORBOL ESTER-TREATED HUMAN GRANULOCYTES, Journal of cellular biochemistry, 66(2), 1997, pp. 219-228
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.