MARKED DIFFERENCES IN THE ROLE OF O-6-ALKYLGUANINE IN HPRT MUTAGENESIS IN T-LYMPHOCYTES OF RATS EXPOSED IN-VIVO TO ETHYLMETHANESULFONATE, N-(2-HYDROXYETHYL)-N-NITROSOUREA, OR N-ETHYL-N-NITROSOUREA

The role of DNA alkylation at the O-6 position of guanine in the induc tion of gene mutations in vivo was studied in the hprt gene of rat T-l ymphocytes from spleen exposed in vivo to the monofunctional ethylatin g agents ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU), or the hydroxyethylating agent N-(2-hydroxyethyl)-N-nitrosourea (HOENU ). Ah chemicals showed an exposure-dependent increase in hprt mutant f requency, HOENU and ENU, however, were much more mutagenic than EMS wh en compared at equimolar levels, DNA sequence analysis was performed o n PCR products of hprt cDNA from 40 EMS-, 35 HOENU-, and 46 ENU-induce d 6 thioguanine-resistant T-lymphocyte clones, Thirty EMS-induced muta nts contained a single base pair substitution with GC to AT transition s being the predominant type of mutation (26 of 30) which are probably caused by mispairing of O-6-ethylguanine with T during DNA replicatio n, No strand specificity of mutated G's among GC to AT transitions was observed, Twenty-three HOENU- and 42 ENU-induced mutants contained a single base pair substitution. In contrast to EMS, GC to AT transition s were found at a low frequency, 4 of 23 for HOENU and 5 of 42 for ENU , indicating that O-6-hydroxyethylguanine and O-6-ethylguanine are les s important in HOENU- and ENU-induced mutagenesis in vivo, respectivel y, Also here no strand bias for mutated G's was observed, although the number of this type of mutation was limited, The most frequently indu ced base pair alterations by HOENU and ENU were transversions at AT ba se pairs, 16 of 23 and 28 of 42, respectively, with AT to TA being the predominant type of mutation. In both ENU and HOENU mutational spectr a, an extreme strand bias for mutated T's toward the nontranscribed st rand was found, The results suggest that DNA damage induced in rat T-l ymphocytes in vivo by HOENU and ENU is processed in similar ways.