MOLECULAR ANALYSIS OF HPRT GENE-MUTATIONS IN SKIN FIBROBLASTS OF RATSEXPOSED IN-VIVO TO N-METHYL-N-NITROSOUREA OR N-ETHYL-N-NITROSOUREA

The granuloma pouch assay in the rat is a model system in which relati ve frequencies of genetic and (pre-) neoplastic changes induced in viv o by carcinogenic agents can be determined within the same target tiss ue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitr o. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl- N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-s even MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell c lones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequ ences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O-6-methylguanine lesions. For most of the GC to AT transitions (1 6 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O-6-methylguanine lesions. ENU-induced m utations occurred predominantly at AT base pairs (28 of 36), being mos tly AT to TA and AT to CG transversions, and are probably caused by O- 2-ethylthymidine. Also here, DNA repair processes seem to act with dif ferent rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, sugg esting that O-6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU - and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair syste ms remove O-6-methylguanine, O-2, and/or O-4-ethylthymidine much faste r from the transcribed strand than the nontranscribed strand of the hp rt gene in these rat fibroblasts.