Mutation induction by methylnitrosourea as a function of gene expression in an EBV-derived shuttle vector

208 the same properties as M N N G . E N N G and M N N G are not only activated by naturally occurring thiols such as glutathione but also by both the D and the z form of N-acetylcysteine. In contrast to MNNG and E N N G , our results indicate that the activation of N C by thiols can be modified by glutathione-S-transferase. The present investigation clearly demonstrates that intracellular reactions with thiols may lead to an activation mechanism, producing highly mutagenic intermediates.

analysis of the mutants obtained by exposing the vector after induction of gpt transcription is currently in progress.

84 Fortini, P., S. Rosa, M. Bignami and E. Doghotti, Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanit~t, Rome (Italy)

83

Palombo, F. 1, E. Kohfeldt 2, A. Calcagnile t, p. Nehls 2 and E. Dogliotti 1, 1 Istituto Superiore di Sanith, Rome (Italy) and 2 University of Ulm, Munich (F.R.G.) Mutation induction by methylnitrosourea as a function of gene expression in an EBV-derived shuttle vector We have constructed an EBV-derived shuttle vector, pF1-EBV, which allows the mutation induction by DNA-damaging agents at a specific gene to be studied as a function of the transcriptional activity of that locus. The mouse metallothionein I promoter has been used to confer metal inducibility on the bacterial gpt gene which is the target gene for mutagenicity studies. H u m a n 293 cells transformed with the recombinant plasmid synthesize gpt m R N A and the expression of the gene is inducible by zinc. We isolated a clonal cell line created by the establishment of the pF1EBV shuttle vector which shows a spontaneous gpt- mutation frequency of 2 × 10 -5. A 10-fold increase over background was induced by mutagenizing this cell line with methylnitrosourea (MNU). The treatment with M N U was also performed after induction of gpt transcription by 5-h exposure to 100/~M zinc acetate. No difference in the frequency of gpt- mutanfs was observed. The analysis of 10 mutants induced by M N U showed that the base changes induced by this chemical are exclusively GC to AT transitions. If it is assumed that guanine is the target base, then all of these mutations are in the non-transcribed strand. The

Evidence for AP site formation related to DNAoxygen aikylation in C H O cells treated with ethylating agents CHO cells, which are unable to repair O6-ethyl guanine (O6-etGua), respond to ENU-induced damage with a burst of D N A ssb that does not arise via chemical or enzymatic hydrolysis of the main ethylpurines. This subset of lesions is induced and repaired very quickly. We explored the potential use of methoxyamine (MX), a chemical that reacts with AP sites in vitro, to detect AP site formation in vivo. We show that MX is able to inhibit specifically the repair of AP sites in cells in vivo and that the fast resealed ENU-induced ssb are due to AP sites. These AP sites are formed as intermediates in enzymatic processes as shown by the lack of appearance of AP sites when plasmid D N A is treated in vitro with ENU. Interestingly enough a fast repair of D N A ssb has also been observed with another SNl-type ethylating agent, ENNG, but not with DES suggesting a relationship between this phenomenon and DNA-oxygen modifications. Our data would be consistent with a model where m e r - C H O cells treated with ENU respond to the persistence of O6-etGua in their D N A by expressing gene product(s) which recognize O6-etGua paired with cytosine as a substrate for a repair mechanism leading to AP site formation. A 300-bp fragment containing a single 06etGua (kindly provided by J.M. Essigmann) is currently used as a probe for detecting such repair activity.