Role of O-Alkylguanine-DNA Alkyltransferase in Protecting against Cyclophosphamide-induced Toxicity and Mutagenicity

Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHO) and pcDNA3 (CHO) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHO is 7or 20-fold less sensitive to the toxic effects of 30 mM 4-HC or 300 mM 4-HDC, respectively, than CHO cells as measured by cell survival using a colony-forming assay. CHO cells treated with 20 mM 4-HC or 200 mM 4-HDC produced 4or 7-fold lower mutation frequency as measured at the HPRT locus than CHO cells treated with the same dose of drugs. At 30 mM acrolein, the cell survival for CHO was 30% compared with 18.7% for CHO. The mutation frequency of acrolein at the same dose was 57 mutants/10 cells in CHO compared with no mutants in CHO. In contrast, CHO and CHO cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein.