title: Chromosome aberrations: persistence of alkylation damage and modulation by O6-alkylguanine-DNA alkyltransferase.
abstract: Alkylating agents produce a spectrum of DNA lesions alkylated at different 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 successive cell cycles in Chinese hamster ovary cells, but not in normal human cells, to begin a study of which alkylated products are most likely to lead to chromosome abs, and in particular which adducts persist in DNA and cause abs after the first cell cycle. We previously observed increasing yields of abs with successive cell cycles in CHO-WBL cells treated with dimethyl nitrosamine (DMN), e.g., at 10 mM DMN, 8.8% cells 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 lymphocytes. We sampled cells at several times after treating for 3 h (CHO and lymphocytes) or 4.5 h (L136). S9 metabolic activation was used for DMN and diethyl nitrosamine. BrdUrd labeling was used to identify cells in M1, M2 and M3. The methylating agents were more potent aberration (ab) inducers than ethylating agents, on a molar basis. In CHO cells, yields of abs were maintained or increased through up to 3 cell cycles after treatment with DMN, methyl methanesulfonate, methyl nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). With ethylating agents the ab yields in CHO cells were similar or lower in second and third cycles. In contrast, there was no evidence for persistence of lesions leading to abs in either human cell type; ab yields were markedly decreased with subsequent cell cycles for all agents. Normal human cells are proficient in repair of alkylation at the O6 site of guanine by O6-alkylguanine-DNA alkyltransferase (AGT), whereas CHO cells lack AGT activity. To explore the role of repair by AGT on the lesions involved in production of abs, we studied L136 cells, with and without O6-benzylguanine (BZG), a specific inhibitor of AGT. With MNNG, inhibition of AGT resulted in higher ab yield and production of abs through later cell cycles, so that human fibroblasts now behaved similarly to CHO 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 similar through later cycles or increased only slightly.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Merck Research Laboratories, West Point, PA 19486.
authors.forename: C L
authors.initials: CL
authors.lastname: Bean
authors.forename: C I
authors.initials: CI
authors.lastname: Bradt
authors.forename: R
authors.initials: R
authors.lastname: Hill
authors.forename: T E
authors.initials: TE
authors.lastname: Johnson
authors.forename: M
authors.initials: M
authors.lastname: Stallworth
authors.forename: S M
authors.initials: SM
authors.lastname: Galloway
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D000478:Alkylation; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured / Q000187:drug effects; D002869:Chromosome Aberrations; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects; D008297:Male; D008780:Methyltransferases / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase
chemical_list: D000477:Alkylating Agents; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase