DISCRIMINATION BETWEEN GENOTOXICITY AND CYTOTOXICITY IN THE INDUCTIONOF DNA DOUBLE-STRAND BREAKS IN CELLS TREATED WITH ETOPOSIDE, MELPHALAN, CISPLATIN, POTASSIUM CYANIDE, TRITON X-100, AND GAMMA-IRRADIATION

The dose-response relationships for DNA fragmentation (assessed by pul sed-field gel electrophoresis, PFGE) and for viability (evaluated by m easuring the reduction of MTT dye which can be accomplished by viable cells only) were investigated in order to discriminate between genotox icity and cytotoxicity in the pathogenesis of DNA double-strand breaks (DSB). Cultured human lung epithelial cells (A549) were treated with the DNA-intrastrand crosslinker cisplatin, the DNA-interstrand crossli nker melphalan and the topoisomerase II inhibitor etoposide. The cytot oxic mode of DSB induction was investigated by using the mitochondrial respiratory chain toxin potassium cyanide (KCN) and the deter-gent Tr iton X-100. gamma-Irradiation induced a linens dose response for DSB w hich were efficiently repaired and did not cause reduction in cell sur vival over a period of 72 h. With etoposide and melphalan a significan t increase in DSB was seen 8 h after treatment initiation with concent rations that did not affect cell survival, implicating genotoxicity as the causal event. In contrast, induction of DSB by KCN and Triton X-1 00, and also by cisplatin, was seen only after cell viability was redu ced to less than about 60%, indicating that DSB were the consequence o f extragenomic damage. This mechanistic distinction of the two classes was supported by DNA fragment length analysis. In line with a genotox ic mechanism and absence of additional cytotoxic effects, the DNA frag ments generated by gamma-irradiation: as well as by etoposide and melp halan displayed a distribution between 1 and 4 Mbp with a peak around 2 Mbp. In contrast, DNA fragments induced by Triton X-100 and KCN peak ed below 0.5 Mbp, implicating activation of DNA-degrading enzymes. Thi s type of investigation is suggested for the study of chemicals for po tential DNA inter strand crosslinking, an important promutagenic type of DNA damage. To avoid false positive results in genetic toxicity tes ting it is suggested that all assays include a dose-response relations hip for both genotoxicity and viability. (C) 1998 Elsevier Science B.V .