Adenine-DNA adduct derived from the nitroreduction of 6-nitrochrysene is more resistant to nucleotide excision repair than guanine-DNA adducts.

Previous studies in rats, mice, and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: (1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts, N-(dG-8-yl)-6-AC, 5-(dG-N(2)-yl)-6-AC, and N-(dA-8-yl)-6-AC, and (2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N(2)-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here, we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ∼2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ∼8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N(2)-yl)-6-AC ≃ N-(dG-8-yl)-6-AC ≃ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N(2)-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland.

Methylation of cytosine at C5 in a CpG sequence context causes a conformational switch of a benzo[a]pyrene diol epoxide-N2-guanine adduct in DNA from a minor groove alignment to intercalation with base displacement.

It is well known that CpG dinucleotide steps in DNA, which are highly methylated at the 5-position of cytosine (meC) in human tissues, exhibit a disproportionate number of mutations within certain codons of the p53 gene. There is ample published evidence indicating that the reactivity of guanine with anti-B[a]PDE (a metabolite of the environmental carcinogen benzo[a]pyrene) at CpG mutation hot spots is enhanced by the methylation of the cytosine residue flanking the target guanine residue on the 5'-side. In this work we demonstrate that such a methylation can also dramatically affect the conformational characteristics of an adduct derived from the reaction of one of the two enantiomers of anti-B[a]PDE with the exocyclic amino group of guanine ([BP]G adduct). A detailed NMR study indicates that the 10R (-)-trans-anti-[BP]G adduct undergoes a transition from a minor groove-binding alignment of the aromatic BP ring system in the unmethylated C-[BP]G sequence context, to an intercalative BP alignment with a concomitant displacement of the modified guanine residue into the minor groove in the methylated meC-[BP]G sequence context. By contrast, a minor groove-binding alignment was observed for the stereoisomeric 10S (+)-trans-anti-[BP]G adduct in both the C-[BP]G and meC-[BP]G sequence contexts. This remarkable conformational switch resulting from the presence of a single methyl group at the 5-position of the cytosine residue flanking the lesion on the 5'-side, is attributed to the hydrophobic effect of the methyl group that can stabilize intercalated adduct conformations in an adduct stereochemistry-dependent manner. Such conformational differences in methylated and unmethylated CpG sequences may be significant because of potential alterations in the cellular processing of the [BP]G adducts by DNA transcription, replication, and repair enzymes.

Conformational changes of a benzo[a]pyrene diol epoxide-N(2)-dG adduct induced by a 5'-flanking 5-methyl-substituted cytosine in a (Me)CG double-stranded oligonucleotide sequence context.

Mutations in p53 genes are one of the most common genetic alterations in human cancers. A disproportionate number of mutations are found in certain codons of the p53 gene, mostly at CpG dinucleotide sequences, which are highly methylated in human tissues. The reactivities of the mutagenic metabolite of benzo[a]pyrene, the bay region diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), to yield adducts with guanine at the exocyclic amino group (e.g., trans-anti-BPDE-N(2)-dG, or G*), are enhanced when the cytosine in CpG sequences in DNA is methylated at its 5-position ((Me)CpG). However, methylation may also affect the characteristics of these adducts, and we have therefore investigated whether adduct conformations are different in double-stranded DNA in methylated (Me)CpG* and in unmethylated CpG* sequence contexts in the oligonucleotide model system duplex 5'-d(CCAT[(5X)C]GCTACC).d(GGTAGCGATGG) with X = H or -CH(3). The (-)-trans-adduct exhibits a striking conformational change from a minor groove structure external to the DNA duplex in the unmethylated CpG* sequence, to an intercalative conformation in the (Me)CG* sequence context. In contrast, the conformation of the stereoisomeric (+)-trans-adduct is predominantly of the minor groove type in both the methylated and unmethylated sequences. These results indicate that methylation of CpG sequences may affect not only chemical reactivities of chemically reactive intermediates with DNA, but also the conformational properties of the DNA adducts formed. Thus, both factors must be considered in evaluating the effects of cytosine methylation in CpG sequences on the biological consequences of the DNA adducts formed.