Title

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

Date of Completion

January 2005

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

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 . ^

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