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the same properties as MNNG. E N N G and M N N G are not only activated by naturally occur- ring thiols such as glutathione but also by both the D and the z form of N-acetylcysteine. In contrast to MNNG and ENNG, our results indicate that the activation of NC by thiols can be modified by glutathione-S-transferase. The present investiga- tion clearly demonstrates that intracellular reac- tions with thiols may lead to an activation mecha- nism, producing highly mutagenic intermediates.

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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 func- tion of gene expression in an EBV-derived shuttle vector

We have constructed an EBV-derived shuttle vector, pF1-EBV, which allows the mutation in- duction by DNA-damaging agents at a specific gene to be studied as a function of the transcrip- tional activity of that locus. The mouse metal- lothionein I promoter has been used to confer metal inducibility on the bacterial gpt gene which is the target gene for mutagenicity studies. Human 293 cells transformed with the recombinant plas- mid synthesize gpt mRNA and the expression of the gene is inducible by zinc. We isolated a clonal cell line created by the establishment of the pF1- EBV shuttle vector which shows a spontaneous gpt- mutation frequency of 2 × 10 -5. A 10-fold increase over background was induced by mutage- nizing this cell line with methylnitrosourea (MNU). The treatment with MNU was also per- formed 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 MNU 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

analysis of the mutants obtained by exposing the vector after induction of gpt transcription is cur- rently 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)

Evidence for AP site formation related to DNA- oxygen aikylation in CHO cells treated with ethyl- ating agents

CHO cells, which are unable to repair O6-ethyl - guanine (O6-etGua), respond to ENU-induced damage with a burst of DNA ssb that does not arise via chemical or enzymatic hydrolysis of the main ethylpurines. This subset of lesions is in- duced 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 DNA is treated in vitro with ENU. Interestingly enough a fast repair of DNA ssb has also been observed with another SNl-type ethylating agent, ENNG, but not with DES suggesting a relation- ship between this phenomenon and DNA-oxygen modifications. Our data would be consistent with a model where mer - CHO cells treated with ENU respond to the persistence of O6-etGua in their DNA by expressing gene product(s) which recog- nize O6-etGua paired with cytosine as a substrate for a repair mechanism leading to AP site forma- tion. A 300-bp fragment containing a single 06- etGua (kindly provided by J.M. Essigmann) is currently used as a probe for detecting such repair activity.