Carcinogenesis, Vol. 21, No. 2, 235-242,
February 2000
© 2000 Oxford University Press
Carcinogenesis |
Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene
Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA–AAI and dA–AAII) and guanine adducts (dG–AAI and dG–AAII) by the 32P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC–AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI–DNA and AAII–DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the 32P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by 32P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.
Abbreviations: AA, aristolochic acid; AAI, aristolochic acid I (8-methoxy-6-nitrophen-anthro[3,4-d]-1,3-dioxolo-5-carboxylic acid); AAII, aristolochic acid II (6-nitrophen-anthro[3,4-d]-1,3-dioxolo-5-carboxylic acid); dA–AAI, 7-(deoxyadenosin-N6-yl)aristolactam I; dA–AAII, 7-(deoxyadenosin-N6-yl)aristolactam II; dG–AAI, 7-(deoxyguanosin-N2-yl)aristolactam I; dG–AAII, 7-(deoxyguanosin-N2-yl)aristolactam II.
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