The objectives are: (i) the qualitative and quantitative measurement of critical DNA base damage within specifically defined human cells and target genes, (ii) the assessment of the nature and extent of differential repair within selective gene sequences and overall cellular genome, and (iii) the establishment of a causal relationship for the patterns of intragenomic heterogeneity of DNA damage and repair to that of cellular genotoxicity. The proposed study is based on the hypothesis, postulating that molecular and cellular malfunction in genotoxin exposed mammalian cells results from inefficient repair of critical DNA damage within vital genes. Studies to date have focused on the measurement of overall cellular repair and its role in a particular biological consequence. However, the cells that appear to be fully proficient in DNA repair may actually be deficient in the repair of specific genes and vice versa. We propose to combine the microanalytical immunotechniques with molecular and cellular approaches to study the processing and biological consequences of the most critical promutagenic and precarcinogenic DNA base alterations in defined genes, stably established in human cells. The methodology to be utilized has been established during the period of previous grant. The basic approach to the accomplishment of our specific objectives is as follows. Two distinct genes, thymidine kinase (tk) and normal protooncogene (c-H-ras) will be incorporated into uniquely constructed EBV shuttle vector p220.2, developed recently for transporting genes in normal mammalian cells. This vector shows insignificant background mutation upon transfection and expression in human cells. The reconstructed plasmid DNA, harboring the target gene sequences, will be established and expressed in replication competent human kidney epithelial and skin fibroblast cells by electroporation and hygromycin B selection. Immunoanalysis on the frequency of formation and relative repair tics of specific adducts, induced by UV irradiation (thymine dimers) and ethylnitrosourea (O6-EtdGuo and O4-EtThy) will be performed with cellular chromosomal DNA and fractionated segments of target genes retrieved from the exposed transfectants. Sequence alterations, resulting from the host cell processing of DNA adducts in the target gene tk and ras, will be determined by functional analysis of the plasmid isolates shuttled in bacterial host E coli. Mutation spectra of the target loci, resulting from contrasting genotoxic insults and damage processing within host cells of varying repair potential, will be ascertained by direct sequence analysis of wild type and mutated target gene fragments. Results comparing the genotoxin induced mutation frequency with persistent damage within target genes will provide the test of above hypothesis. This novel approach based on the use of normal human cells, critical base alterations and target gene containing vectors, is of ultimate significance in the risk assessment of environmental hazards confronting human health.

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National Institute of Environmental Health Sciences (NIEHS)
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Toxicology Subcommittee 2 (TOX)
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Ohio State University
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