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Exploitable results
The major objectives are - to define structural rules and biological action principles underlying the mutagenic and carcinogenic potency of agents capable of forming etheno- bases in DNA, - to assess the relative contribution to dietary factors on the formation of e-bridged DNA bases and the consequences of their formation to human health (malignant and neurodegenerative diseases). e-Adducts in human DNA are formed by environmental and endogenous genotoxins that are generated from oxidative stress, dietary factors and metal storage diseases.
1. e-DNA adducts as markers for lipid peroxidation-induced DNA damage: Increased oxidative stress and lipid peroxidation (LPO) are implicated in cancer and neurodegenerative diseases. Using a sensitive immunoaffinity-32P-postlabelling assay, we have demonstrated that e-addicts occur in normal rodent and unsymptomatic human tissues, reflecting their generation by physiological lipid peroxidation.
2. Development of immunohistochchemical method for detection of e-adducts in tissue sections: We have developed a quantitative immunohistochemical method to detect e-adducts at a celluar level and standardized optimal conditions for assaying histological sections. The method is now being applied to tissue sections and cells from humans for etiological and mechanistic studies.
3. The validation of e-adducts as biomarkers after w-6 fatty acid intake in target organs and surrogate tissue in rats: Rats were treated P.O. with coconut oil, linoleic acid (LA) and oleic acid. Males and females were sacrificed on day 30 and liver, colon, prostate, breast and blood were collected. The serum levels of free acids were compared to e-adducts in DNA isolated from these organs.
4. Relationship between etheno adducts and mutations in specific genes: e-adducts were induced in Raji cells by chloroacetaldehyde. The relationship between e-adducts and specific mutations in several mitochondrial genes was investigated. Using a restriction site mutation assay, evidence was obtained for the presence of six putative mutations in two mitochondrial genes. DNA containing these mutations is being sequenced and frequency of mutants verified.
5. Formation of e-adducts at nucleotide resolution in the p53 gene: Two methods were established to analyze the formation and repair of etheno adducts at the DNA sequence level, the ligiation-mediated polymerase chain reaction and the DNA polymerase fingerprint analysis.
6. Formation of e-adducts in tissues and cells from mice defficient of DNA repair: Two embryo fibroblasts cell lines from Msh2+/- and Msh -/- mice were used to investigate the effects of mismatch repair deficiency on DNA damage induced by hydroxynonenal. The results showed a greater sensitivity of the heterozygous cell line. e-Adducts were analyzed in tissues from ANPG -/- mice, deficient for the DNA glycosylase that exercises alkylpurines and e-adducts. The results suggested that involvement of both ANGP (base excision repair) and another pathway in the repair of eA in vivo.
7. Genetic effects of e-formating chemicals in Drosphila: After the synthesis of vinyl carbamate (VCA) and its epoxide, the determination of genetic activity profiles of e-adduct inducing carcinogens in postmeiotic germ cells of Drosphila was completed. Studies on the induction of multiple-loci and specific -locus mutations in pre-meiotic germ cells of Drosophila were initiated. The most interesting finding was e-adduct generating carcinogens from persistent lesions in both somatic tissues and in highly repair active premeiotic male germ cells. Further isolation and characterization of vermillion and rosy mutants from premeiotic germ cells exposed to VCA, and quantification of clastogenicity versus homologous mitotic recombination in somatic Drosophila tissue after exposure are in progress.
8. Identification of enzymes implicated in the removeal of e-cytosin and e-guanine: The eA residues are excised in humans and in E.coli by 3-methyladenine-DNA glycosylases(ANPG and AlkA proteins, resp). We have identified two homologous proteins present in E.coli and human cells that remove eC as the mismatch-specific thymine-DNA glycosylase (hTDG_ and the double-stranded uracil-DNA glycosylase (dsUDG), that is the homologue of the hTDG. We have constructed an E.coli strain that is deficient in the activity excising sC. It has been characterized as the genetic and enzymological level. Double mutants lacking, beside the base excision pathway, either the nucleotide excision or the mismatch repair pathway are being constructed.
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