Objective
Oxidative stress- and lipid per oxidation-induced DNA damage is implicated in human carcinogen sis. Ethno adducts can serve as exposure and risk markers due to ultra sensitive detection methods developed by us. According to existing EU guidelines the study of mechanisms of action is crucial for selecting appropriate models for high-to-low-dose risk extrapolation. This project aims to define the action principles underlying the formation and persistence of promutagenic ethno-DNA adduct. Exogenous factors and endogenous processes leading to their formation will be studied. Key parameters, I. e. repair functions, persistence of lesions and mutational specificity at the gene level, will be characterized in humans, rodents, Drosophilae and Ecolab, in particular at low-dose exposures. Thereof, assay kits for industrial application can be developed. This integrated approach will improve geotaxis risk assessment of oxidative stress-related DNA lesions.
Etheno-DNA adducts as biomarkers of oxidative stress induced by dietary fatty acids and altered estrogen metabolism were studied in relation to human breast cancer. The ratio of 4- to 2-hydroxyestradiol (E) in serum and the level of etheno-DNA-adducts in WBC in premenopausal women was found to be highly variable. 4-Hydroxy-E, a metabolite of E implicated in redox cycling, and arachidonic acid were found to be synergistic factors in the generation of etheno-adducts in calf thymus DNA. These results support a mechanism whereby metabolic redox-cycling of 4-hydroxy-E leads to ROS and DNA damage, which could play a role in breast cancer etiology. High individual variations in repair activities for etheno-adducts were observed in colon tissues from colon cancer patients. The malondialdehyde modified DNA adduct (M1dG) can serve as a biomarker for lipid peroxidation-induced DNA damage. We have developed an immuno-enriched 32P-postlabeling-HPLC method for M1dG in small amounts of DNA. Background levels of the M1dG could be reproducibly detected in human breast and WBC samples, making this method suitable for molecular epidemiology studies. Pathways of formation and repair of etheno-DNA adducts were investigated in transgenic rodents with modified repair genes.
Etheno-DNA adducts are repaired in mice through at least two pathways, base excision and mismatch repair. Single chain antibody Fragments (scFv) that specifically recognized etheno-A-, etheno-C- and etheno-G-containing oligos were produced. A dosimetry of etheno-DNA adducts was carried out on suckling and juvenile mice treated with vinyl carbamate. Linear dose-response relationships were obtained for the formation of etheno-A and etheno-C in liver DNA. Chemicals that produced etheno-DNA adducts also effected cell cycle, apoptosis, cell proliferation. 4-Hydroxy-nonenal (HNE) produces etheno-adducts, but also generated more complex adducts in DNA. In single-stranded phage the latter were replication blocking lesions of high recombinogenic potential. In E.coli long-chain HNE-adducts were eliminated from double-stranded DNA by nucleotide excision repair, and from single-stranded DNA by recombination. HNE interacts with cDNA of P53 gene in a sequence-dependent manner with preference for modification of C and G, also in mutational hot-spots. Known 8-oxoguanosine triphosphate pyrophosphohydrolases, bacterial and human MutT proteins involved insanitation of the nucleotide pool were inactive towards etheno-dCTP and etheno-dATP. The genetic activity profile and repair of etheno-adduct forming chemicals were investigated in germ and somatic cells of Drosophila. Some endpoints were correlated with å-adduct levels in DNA of larvae tissue. Etheno-adducts and not DNA cross-links are involved in mutation induction by the etheno-adduct forming chemicals, vinyl bromide and vinyl carbamate.
NER was not found to be the major DNA repair for etheno-adducts. DNA repair enzymes removing etheno-DNA adducts were identified. In bacteria and mammalian cells two structurally unrelated proteins, the E.coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N glycosylase (ANPG) can excise 1,N2-ethenoguanine. Identification of a novel nucleotide incision repair pathway for oxidative DNA damage in bacteria, yeast and human supported an alternative DNA glycosylase-independent repair pathways for oxidative DNA damage. Inhibition of DNA repair by a human 30 kDa-protein (p30) was characterized. p30 interacts specifically with ethenocytosine (etheno-C) residues inhibiting its excision by the human thymine-DNA glycosylase in vitro probably causing persistence of etheno-C in the genome and the replication fork arrest. Development of a high throughput screening assay for detection of various DNA repair activities was accomplished. This assay for DNA glycosylase and AP endonuclease activities can probe the cellular level of repair enzymes and be used for high throughput screening of specific inhibitors. Quantitative correlations between levels of etheno-adducts and their biological consequences, under DNA repair proficient- and deficient conditions in mice were explored for QSAR and risk assessment. In hepatic DNA rodents there was a quantitative linear correlation between the carcinogenic potency and covalent binding index for etheno-A of etheno adduct-forming chemicals. This strongly supports ethenobases to be critical initiating lesions in hepato-carcinogenesis. When overproduced by chronic oxidative stress and lipid peroxidation, these miscoding DNA lesions may act as a driving force to malignancy in other tissues.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- medical and health sciences health sciences public health epidemiology
- natural sciences biological sciences biochemistry biomolecules lipids
- medical and health sciences clinical medicine oncology breast cancer
- medical and health sciences clinical medicine oncology colorectal cancer
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Coordinator
69120 HEIDELBERG
Germany
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