Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNAEPA Grant Number: R825809
Title: Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNA
Investigators: Shank, Ronald C. , Said, Boctor
Institution: University of California - Irvine
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 1999 (Extended to September 30, 2001)
Project Amount: $505,497
RFA: Issues in Human Health Risk Assessment (1997) RFA Text | Recipients Lists
Research Category: Human Health , Health Effects , Human Health Risk Assessment , Health
Nucleophilic attack by DNA on an electrophilic chemical genotoxin in formation of a DNA adduct is regarded by most as an early and critical step in the multistage processes of reproductive and developmental toxicity as well as carcinogenesis. Much is known regarding metabolic activation of genotoxins to reactive electrophiles, and the chemical nature of various adducts have been elucidated. Little is known, however, about the mechanisms by which mixtures of genotoxins form adducts with DNA. Hypothesis: the presence of a chemical adduct in DNA can alter the site of formation of a second adduct upon exposure to a second genotoxin, and that the first adduct can block or shift the binding site for the second adduct, such that formation of multiple adducts can be other than additive; the modulation of adduct formation results from alterations in the nucleophilicity of the N7 atom in guanine, steric influences at neighboring binding sites and conformational changes in the helix at more distant binding sites.
Synthetic oligonucleotides will be used to allow exact positioning of single adducts to influence reaction at specific sites during a subsequent treatment with a second genotoxin. Combinations of the following genotoxic teratogens will be used: N-methyl-, ethyl-, propyl, and n-butyl-N-nitrosourea, aflatoxin B1-8,9-epoxide, (+) and (-) r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-amino- fluorene. The specific aims are: 1) using pairs of genotoxins, determine the distance of influence between modified and target guanines in a double stranded (ds) 27-mer oligonucleotide; 2) using chemical probes, determine changes in double helix conformation induced in the above oligonucleotide by the presence of the first guanine adduct and by formation of the second adduct; 3) determine the quantitative effect of a single modified guanine on teratogen binding to selected target guanines in 2 ds 60- and 62-mer DNA sequences equivalent to mutational hot spots in the human p53 gene; 4) using chemical probes, determine changes in double helix conformation of the above 60- and 62-mer polynucleotides induced by formation of the second adduct; and 5) determine the influence of 8-hydroxyguanine and 5-methylcytosine on formation of guanine adducts by the above genotoxins in the above 60- and 62-mer polynucleotides. Once understanding of model systems is achieved, more complex systems involving chromatin DNA, metabolic alteration of additional genotoxic teratogens, and DNA repair systems will be considered.
The proposed study is expected to provide quantitative information on the toxicodynamics of interactions between DNA and multiple chemical genotoxic teratogens. It will determine the influence small alkylating teratogens can have on the covalent binding of bulky teratogens to target cell DNA, and vice versa. It will contribute information on the mechanism(s) of action by which one adduct influences the formation of a second adduct by differentiating between nucleophilicity effects, steric effects and helix conformation effects. The primary benefit of such information will be its application to the understanding of how chemical teratogens interact with DNA as the first step in chemical teratogenesis. These will be model studies, using synthetic oligonucleotides that allow exact positioning of single adducts to influence reaction at specific sites during a subsequent treatment with a second teratogen. Once understanding of the model systems is achieved, more complex systems involving chromatin DNA, and, eventually, metabolic alteration of teratogens, DNA repair systems, etc., can be considered; such is a long-term goal of the proposed project.