Biological effects of bulky C8-deoxyguanosine adducts of nitroaromatic compounds by a site-specific approach

Date of Completion

January 2005


Chemistry, Biochemistry




Nitroaromatic compounds commonly found in the environment are well known mutagens in bacterial and mammalian cells. Nitropyrenes undergo a metabolic process by a nitroreductive pathway in biological systems. The major DNA adducts formed are N-deoxyguanosin-8-yl-1-aminopyrene (dG AP), N-deoxyguanosin-8-yl-1-amino-6-nitropyrene (dG 1,6-ANP), and N-deoxyguanosin-8-yl-1-amino-8-nitropyrene (dG1,8-ANP) by 1-nitropyrene (1-NP) , 1,6- and 1,8-dinitropyrene (1,6- and 1,8-DNPs), respectively. The structure-activity relationship of these adducts has been explored. In the Ames Salmonella typhimurium reversion assay, 1,6- and 1,8-dinitropyrenes are much more potent mutagens than 1-nitropyrene. It remained unclear, however, if the dinitropyrene adducts, which contain a nitro group on the pyrene covalently linked to the guanine C8, are more mutagenic than the major 1-nitropyrene adduct, even though dinitropyrenes are stronger mutagens than 1-nitropyrene. In order to address this issue, 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 repair-competent 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 mutS strain, which is impaired in the methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in the proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene at the 8-position can provide an 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 the CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP. ^ A single-stranded pMS2 vector containing dGAP adducted hexamer was constructed and replicated in COS7 cells. Analysis of the progeny showed that the dGAP containing pMS2 resulted in a significant percentage of base substitutions (G→T, 5.4%, G→A 1.3%, G→C 1.0%). Two base deletions were not detected. ^ Finally, translesion synthesis of the 26mer construct containing the major adduct derived from 1-nitropyrene was carried out using error-free and error-prone DNA polymerases. The studies indicated that although the 1-nitropyrene adduct is a replication-blocking lesion, error-prone polymerase bypass occurred to a limited extent, which could lead to mutagenic events in vivo . ^