CHROMOSOME-ABERRATIONS - PERSISTENCE OF ALKYLATION DAMAGE AND MODULATION BY O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE

Alkylating agents produce a spectrum of DNA lesions alkylated at diffe rent sites on the molecule. These lesions differ in their propensities to cause effects such as cytotoxicity, mutations and sister-chromatid exchanges. We have used our observations that some methylating agents produce increasing levels of chromosome aberrations (abs) through suc cessive cell cycles in Chinese hamster ovary cells, but not in normal human cells, to begin a study of which alkylated products are most lik ely to lead to chromosome abs, and in particular which adducts persist in DNA and cause abs after the first cell cycle. We previously observ ed increasing yields of abs with successive cell cycles in CHO-WBL cel ls treated with dimethyl nitrosamine (DMN), e.g., at 10 mM DMN, 8.8% c ells with abs at first metaphase (M1) and 26.0% at third metaphase (M3 ) after treatment. Here we tested 4 methylating agents and their ethyl analogs in CHO cells, normal human fibroblasts (L136), and human lymp hocytes. We sampled cells at several times after treating for 3 h (CHO and lymphocytes) or 4.5 h (L136). S9 metabolic activation was used fo r DMN and diethyl nitrosamine. BrdUrd labeling was used to identify ce lls in M1, M2 and M3. The methylating agents were more potent aberrati on (ab) inducers than ethylating agents, on a molar basis. In CHO cell s, yields of abs were maintained or increased through up to 3 cell cyc les after treatment with DMN, methyl methanesulfonate, methyl nitrosou rea and 1-methyr-3-nitro-1-nitrosoguanidine (MNNG). With ethylating ag ents the ab yields in CHO cells were similar or lower in second and th ird cycles. In contrast, there was no evidence for persistence of lesi ons leading to abs in either human cell type; ab yields were markedly decreased with subsequent cell cycles for all agents. Normal human cel ls are proficient in repair of alkylation at the O-6 Site of guanine b y O-6-alkylguanine-DNA arkyltransferase (AGT), whereas CHO cells lack AGT activity. To explore the role of repair by AGT on the lesions invo lved in production of abs, we studied L136 cells, with and without O-6 -benzylguanine (BZG), a specific inhibitor of AGT. With MNNG, inhibiti on of AGT resulted in higher ab yield and production of abs through la ter cell cycles, so that human fibroblasts now behaved similarly to CH O cells. Preliminary data from the reciprocal experiment in CHO cells engineered to express high levels of AGT revealed a greatly decreased ab response to MNNG. In addition, the low ab yields observed were simi lar through later cycles or increased only slightly. Although multiple types of lesions and mechanisms of repair may be involved in ab produ ction, O-6-methylguanine is an important lesion, particularly in secon dary ab production in later cycles. The differences between CHO and no rmal human cells in response to methylating agents may be due largely to inherent levels of AGT.