Toxicity, interstrand cross-links and DNA fragmentation induced by ‘activated’ cyclophosphamide in yeast

Treatment of yeast cells with 4-hydroperoxy-cyclophosphamide (4-OOH-CP), the chemically activated form of cyclophosphamide, results in cell killing, induction of DNA interstrand cross-links and DNA fragmentation. Toxicity of 4-OOH-CP is greatly influenced by the cell's capacity of DNA dark-repair: genetic blocking of non-epistatic pathways of DNA repair results in an increase of sensitivity by several orders of magnitude. DNA primary lesions have been measured using a haploid, excision deficient, dTMP-uptaking mutant of S. cerevisiae. In this strain, a significant extent of DNA cross-linking can already be observed at a survival of 88%. At a concentration of 100 nmol/ml 4-OOH-CP, renaturability of DNA increases up to 12 h of drug exposure and drops to lower values upon further incubation. In contrast to the time course of renaturability, DNA double-strand breakage is seen at later stages of drug treatment and continuously increases as a function of incubation time. Whereas inactivation of cells and induction of strand breakage continue upon postincubation of cells, comparable effects are much less pronounced for DNA renaturability.