The mutagenesis of the major DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG) formed by 1-nitropyrene was compared with the analogous C8-dG adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) in simian kidney (COS-7) cells. The DNA sequence chosen for this comparison contained 5′-CCATCGCTACC-3′ that has been used for solution NMR investigations. The structural and conformational differences among these lesions are wellestablished (Patel et al., 1998). Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and the AF adduct was least toxic with the AP adduct exhibiting intermediate toxicity. However, analysis of the progeny showed that translesion synthesis was predominantly error-free. Only low level mutations (<3%) were detected with G→T as the dominant type of mutation by all three DNA adducts. When C8-AP-dG was evaluated in a repetitive 5′-CGCGCG-3′ sequence, higher mutational frequency (~8%) was observed. Again, G→T was the major type of mutations in simian kidney cells, even though in bacteria CpG deletions predominate in this sequence (Hilario et al, 2002). Mutagenesis of C8-AP-dG in a 12-mer containing the local DNA sequence around codon 273 of the p53 tumor suppressor gene, where the adduct was located at the second base of this codon, was also investigated. In this 5′-GTGCGTGTTTGT-3′ site, the mutations were slightly lower but not very different from the progeny derived from the 5′-CGCGCG-3′ sequence. However, the mutational frequency increased by more than 50% when the 5′ C to the adduct was replaced with a 5-methylcytosine (5-MeC). With a 5-MeC, the most notable change in mutation was the enhancement of G→A, which occurred 2.5-times relative to a 5′ C. The C8-AP-dG adduct in codon 273 dodecamer sequence with a 5′ C or 5-MeC was also evaluated in human embryonic kidney (293T) cells. Similar to COS cells, targeted mutations doubled with a 5-MeC 5′ to the adduct. Except for an increase in G→C transversions, the results in 293T were similar to that in COS cells. We conclude that C8-AP-dG mutagenesis depends on the type of cell in which it is replicated, the neighboring DNA sequence, and the methylation status of the 5′ C.

In the Ames Salmonella typhimurium reversion assay 1,6-and 1,8-dinitropyrenes (1,6-and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 (8)-nitropyrene (Gua-C8 -1,6-ANP and Gua-C8 -1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8 -1,6-ANP, and Gua-C8 -1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repaircompetent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6-and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself. 1-Nitropyrene (1-NP)1 and the dinitropyrenes are common environmental pollutants (1-3). Most of the nitropyrenes are mutagenic (for a review see Ref. 4) and tumorigenic (5-8), but their potency sometimes differs by more than an order of magnitude. Nitroreduction is a major pathway of bioactivation of all nitropyrenes, whereas O-esterification enzymes, in addition, play a crucial role in the mutagenicity of DNPs.1-NP and the DNPs revert Salmonella typhimurium frameshift tester strains TA98 and TA1538 more efficiently than the strains TA100 and TA1535 that detect base pair substitutions (9). The most frequent mutation among the revertants in TA98 is a two-base deletion of a GpC or CpG pair within a CGCGCGCG hotspot sequence upstream of the hisD3052 mutation (10). The frequency of reversion induced by 1-NP in TA98 drops sharply in TA98NR that lacks the classical nitroreductase (11). By contrast, the frequency of reversion by 1,6-and 1,8-DNP is only slightly lower in TA98NR but is significantly reduced in TA98...

Two different approaches to synthesize oligonucleotides containing the 2′-deoxyguanosine adducts formed by nitropyrenes are described. A direct reaction of an unmodified oligonucleotide with an activated nitropyrene derivative is a convenient biomimetic approach for generating the major adducts in DNA. A total synthetic approach, by contrast, involves several synthetic steps, including Buchwald-Hartwig Pd-catalyzed coupling, but can be used for incorporating both the major and minor adducts in DNA in high yield.