Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Autor: J K Sandhu, H C Birnboim
Rok vydání: 1997
Předmět:
Zdroj: Journal of Cellular Biochemistry. 66:219-228
ISSN: 1097-4644
0730-2312
DOI: 10.1002/(sici)1097-4644(19970801)66:2<219::aid-jcb9>3.0.co;2-t
Popis: Phorbol ester treatment of granulocytes triggers release of superoxide (O2.-) and a concomitant burst of DNA strand breaks. The relationship between the amount of O2.- and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O2.- generated over a 40-min period by 1 x 10(6) granulocytes/mL, a discontinuous "10-min pulse" method employing cytochrome c was used; 140 nmol O2.- per 1 x 10(6) cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O2.- released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1-10 mM) and staurosporine (2-10 nM) both inhibited O2.- production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O2.-. Zinc chloride (50-200 microM) inhibited both O2.- and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O2.- production more effectively than it inhibited DNA breaks. O2.- dismutes to H2O2, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H2O2 had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O2.- 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.
Databáze: OpenAIRE
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