Mutagenic Potential of Benzo[a]pyrene-Derived DNA Adducts Positioned in Codon 273 of the Human P53 Gene

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Abstract
Codon 273 (5‘CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (−)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides (5‘GAGGTGCGBPDETGTTTGT) modified with (+)- or (−)-trans-dG-N2-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N2-BPDE and (−)-trans-dG-N2-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G → T transversions, with some G → A transitions. When the cytosine 5‘ to dG-N2-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N2-BPDE and (−)-trans-dG-N2-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational “hot spot” at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE−DNA adducts positioned at this site.