Chromatographic assay and peptide substrate characterization of partially purified farnesyl- and geranylgeranyltransferases from rat brain cytosol

The dose-response relationships for DNA fragmentation (assessed by pulsed-field gel electrophoresis, PFGE) and for viability (evaluated by measuring the reduction of MTT dye which can be accomplished by viable cells only) were investigated in order to discriminate between genotoxicity 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 crosslinker melphalan and the topoisomerase II inhibitor etoposide. The cytotoxic mode of DSB induction was investigated by using the mitochondrial respiratory chain toxin potassium cyanide (KCN) and the detergent Triton X-100. gamma-Irradiation induced a linear dose response for DSB which were efficiently repaired and did not cause reduction in cell survival over a period of 72 h. With etoposide and melphalan a significant increase in DSB was seen 8 h after treatment initiation with concentrations that did not affect cell survival, implicating genotoxicity as the causal event. In contrast, induction of DSB by KCN and Triton X-100, and also by cisplatin, was seen only after cell viability was reduced to less than about 60%, indicating that DSB were the consequence of extragenomic damage. This mechanistic distinction of the two classes was supported by DNA fragment length analysis. In line with a genotoxic mechanism and absence of additional cytotoxic effects, the DNA fragments generated by gamma-irradiation as well as by etoposide and melphalan 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 peaked below 0.5 Mbp, implicating activation of DNA-degrading enzymes. This type of investigation is suggested for the study of chemicals for potential DNA interstrand crosslinking, an important promutagenic type of DNA damage. To avoid false positive results in genetic toxicity testing it is suggested that all assays include a dose-response relationship for both genotoxicity and viability.