This project aims to elucidate the cellular mechanisms by which damage to DNA leads to a cytotoxic response--cell death and reproductive impairment. Our previous work on this project has shown that cytotoxicity, as well as mutation and neoplastic transformation, from exposure of cells to the mutagenic/carcinogenic chemicals MNNG, BPDE, and Ac-AAF results from the damage they produce in genomic DNA. Our work suggests that while chemical damage to DNA results in mutagenesis and carcinogensis by interfering with DNA replication, the mechanism connecting cytotoxicity induced by these agents with the damage they incur in DNA is more complex. MNNG and BPDE are potent mutagens at the Na+/K+ ATPase locus, and the induction of both cytotoxicity and mutagenesis are strongly cell cycle-related, occuring with maximal sensitivity after chemical exposure in S phase. 4-NQO and Ac-AAF are potent mutagens at the HGPRT locus, but relatively weak mutagens at the Na+/K+ ATPase locus; they are also potent cytotoxins, but induce cell death independently of the cell cycle stage at exposure. These observations suggest that chemicals, such as MNNG and BPDE, which cause mutagenesis by misreplication, also may cause cytotoxicity through a mechanism involving perturbation of DNA replication. In contrast, 4-NQO and Ac-AAF, which induce mutation by producing deletions and strand discontinuities through a perturbation of DNA replication, rapidly kill cells whether or not they are in S phase at the time of exposure. Specifically, the goal of this continuing project is to investigate why DNA damage induced by some potent mutagens/carcinogens requires DNA replication to produce cytotoxic lesions while the DNA damage induced by other chemicals is directly cytotoxic. Flow microfluorimetric, multiparameter analysis of the response of individual cells to damage by these four mutagens/carcinogens will be performed to investigate the differential mechanisms by which these agents will kill mammalian cells. Analysis of the rates of transit between cell cycle compartments, DNA synthetic capability, DNA excision repair capacity, points of inhibition of cell cycle progression, and the correlation of these characteristics with the population fraction at highest risk of cell death will be performed on a cell by cell basis to further elucidate the mechanisms by which cells are killed in response to chemically-induced damage to DNA. In addition, the breakdown of DNA in affected cells will be examined by pulsed field and conventional gel electrophoresis.
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