While the effects of cytotoxic antimetabolites on DNA precursor synthesis and DNA replication have been extensively studied and in some cases related to the anti-tumor activities of these agents, the effects of antimetabolites on DNA structure and integrity have not been as well characterized. The focus of this project is to investigate the nature and extent of DNA damage produced by several antimetabolites (5-aza-2'-deoxycytidine, aza-dC; 5,6-dihydro-5-azacytidine, H2-aza-C; 6-thioquanine, TG) using the alkaline elution technique, and to determine the relationship of this damage with cytotoxicity. We have shown that aza-dC produces alkalilabile sites in L1210 DNA which has incorporated the drug. These lesions are removed slowly with no decrease in the first 24 h after drug washout. The DNA damage may arise through the chemical instability of the azacytosine ring, which, when incorporated into DNA, leads to strand scission in alkaline solution. In contrast, the stable analog H2-aza-C produces DNA single-strand breaks (SSB) with no alkali dependence. H2-aza-C was also shown to have a potent inhibitory effect on DNA elongation and/or ligation, while aza-dc does not. Substantial repair of H2-aza-C SSB occurs after 24 h. These observations suggest that aza-dC and H2-aaz-C have markedly different effects on DNA integrity and that the two agents should not be considered simple pharmacologic congeners. TG has been shown to produce a moderate level of SSB in cultured cells, an observation confirmed in our studies. We have shown, however, that TG induces DNA-protein crosslinks (DPC) at a frequency far in excess of SSB. DPC were produced in both the daughter DNA strand (i.e., the strand which has incorporated the drug) and in the parental strand. The lesions could be prevented by co-incubation with hydroxyurea and reversed by treatment with proteinase-K. It is proposed that TG in DNA becomes linked to protein(s), perhaps via a disulfide bond. If this occurs at or near the point of DNA replication, trapping of the template in a frozen replication complex may explain the parental lesions. These observations are consistent with the delayed cytotoxicity and unilateral chromatid damage which has been reported for TG.
