AT BASE-PAIRS ARE THE MAIN TARGET FOR MUTATIONS AT THE HPRT LOCUS OF RAT SKIN FIBROBLASTS EXPOSED IN-VITRO TO THE MONOFUNCTIONAL ALKYLATINGAGENT N-ETHYL-N-NITROSOUREA

Spectra of N-ethyl-N-nitrosourea (ENU)-induced mutations differ widely among various in vitro and in vivo mutational systems. To investigate possible reasons for these differences, a mutational system is needed in which the same target gene is used for comparison in the same type of cells in vitro and in vivo. In the present study, this was achieve d by analysing at the molecular level 35 hprt mutant rat fibroblast cl ones obtained from cell populations exposed in vitro to ENU and compar ing the mutational spectrum with the previously determined spectrum of ENU-induced hprt mutants in the same target cells exposed in vivo. Tw enty-eight mutants contained a single base pair alteration in the hprt coding sequence. Most of these changes were found at AT base pairs (1 9/28), the AT to TA transversion being the most frequent kind of mutat ion (12/19), which is probably caused by O-2-ethylthymine. Transversio ns at AT base pairs showed all mutated T's to be located in the nontra nscribed strand of the hprt gene, suggesting a strand specific fixatio n of mutations induced by O-2-ethylthymine, which appears to be a gene ral feature of ENU- and ENNG-induced hprt mutations in mammalian cells . GC to AT transitions, probably caused by O-6-ethylguanine, were dete cted at a lower frequency (7/28). This in vitro mutational spectrum wa s very similar to that of the same target cells exposed in vivo to ENU . A comparison of the mutational spectra in AGT-proficient and AGT-def icient rodent cells exposed to ethylating agents showed that in contra st to the situation in AGT-proficient rat fibroblasts, GC to AT base p air changes (and not AT to TA) are the predominant mutations in AGT-de ficient hamster cells.