THE MAJOR, N-2-GUA ADDUCT OF THE (+)-ANTI-BENZO[A]PYRENE DIOL EPOXIDE IS CAPABLE OF INDUCING G-]A AND G-]C, IN ADDITION TO G-]T, MUTATIONS

Mutations induced by the (+)-anti-diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] were collected in the supF gene of the Escherichia coli plasmid pUB3. pUB3 was reacted with (+)-anti-B[a]PDE and then either (1) transformed immediately into E. cell or (2) heated at 80 degrees C for 10 min and then cooled prior to transformation-the latter to probe mechanism [Rodriguez gr Loechler (1993) Biochemistry 32, 1759]. Qualitatively, heating did not affect the mutagenic pattern, except at the major base substitution hotspot in supF, G115, where principally G-->T mutations were obtained prior to heating, while after heating, G-->A and G-->C mutations became statistically significantly more prevalent. Several studies have suggested that a heat-induced chemical transformation of a (+)-anti-B[a]PDE adduct at G115 (e.g., into an apurinic site) is not likely to explain the change in mutational pattern. The most likely model is that (+)-anti-B[a]P-N-2-Gua is initially trapped in a metastable conformation giving principally G-->T mutations, while heating induces a change to a stable conformation(s) resulting in G-->T, A, and C mutations. This suggests that adduct conformational complexity is at the root of adduct mutational complexity. To investigate this model, a plasmid (B[a]P-G115-pRE1) with (+)-anti-B [a]P-N-2-Gua in the G115 sequence context is constructed using adduct site-specific techniques. Following transformation of B[a]P-G115-pRE1 into E. coli (ES87) cells, targeted G115-->T (59%), A (22%), and C (19%) mutations are isolated from (+)-anti-B[a]P-N-2-Gua, which approximates the ratio obtained at G115 with (+)-anti-B[a]PDE itself. (+)-anti-B[a]P-N-2-Gua principally induced G-->T mutations in another sequence context [5'-T (G) under bar C-3'; Mackay et al. (1992) Carcinogenesis 13, 1415]. Collectively, these findings demonstrate that (+)-anti-B[a]P-N-2-Gua is able to induce all three base substitution mutations and that DNA sequence context can influence the qualitative pattern of mutations from (+)-anti-B[a]P-N-2-Gua. Finally, it appears that (+)-anti-B[a]P-N-2-Gua at G115 may be able to induce semitargeted G116-->A mutations as well, although this conclusion is more tentative.