In vivo models for studying mutagenesis and carcinogenesis by environmental pollutants

Date of Completion

January 2001

Keywords

Chemistry, Biochemistry|Environmental Sciences

Degree

Ph.D.

Abstract

The goals of this study were: (a) Synthesize a site-specific lesion in an oligonucleotide containing 2-amino-3-methylimidazo[4.5-f]quinoline (IQ)—a food mutagen. (b) study the mechanism of mutagenesis and repair of the DNA damage caused by the environmental mutagens 1-nitropyrene(1-NP) using specific oligonucleotide probes and (c) examine the feasibility of developing a sensitive and reliable fish model for detecting environmental pollutants. ^ Oligonucleotides containing the major adduct of IQ: N-(deoxyguanosin-8-yl)-2-Amino-3-methylimidazo-[4,5,f]quinoline {dG[IQ]} was accomplished by the reaction of N-hydroxy IQ with a self-complementary 10 base long oligonucleotide. Despite various attempts the synthesized oligonucleotide containing the modified base was found to be extremely unstable. Oligonucleotides containing the major adduct of 1-nitropyrene(1-NP): N-(deoxyguanosin-8-yl)-1-aminopyrene {dG[AP]} was used to investigate the effect of dG[AP] on DNA structure, the mechanism of repair by E. coli UvrABC proteins and the mutagenesis in simian kidney cells using an SV-40 shuttle vector. Several other DNA adducts in the same sequence were used for comparison for all of the studies. Biophysical studies of a duplex containing the most prominent mutational event (G→T) in E. coli revealed the aminopyrene moiety of dG[AP] to be intercalated into the DNA helix and the adducted nucleoside rotated to syn orientation, which disrupted the hydrogen bonding, with the complementary dA. Excision repair studies with the E. coli UvrABC system showed that both recognition and repair of dG[AP], despite significant perturbation, is less efficient than structurally similar C8-guanine adducts of 2-aminofluorene and 2-acetylaminofluorene. ^ In our attempt to develop Japanese medaka (Oryzias latipes) as a sensitive and reliable fish model to detect environmental carcinogens, we first set out to investigate the p53 response machinery in OL-32 medaka cells to UV-radiation. A semi-comparative RT-PCR assay was developed and used to quantitate the level of p53 mRNA in response to UV radiation. Significant cell death was observed in the cell populations receiving radiation at the 200 J/m2 level (high dosage), while cells receiving lower levels of UV treatment showed an increase in the amount of p53 mRNA and grew normally. These observations suggest that in medaka cells the p53 gene performs an important role in the response to UV irradiation as observed in other animal species. Despite of the lack of success in establishing single cell clones with altered level of p53 protein, our preliminary data offered some insight at our current strategy to establish transgenic cell lines as well as the sturdy nature of the OL-32 cells and the cells derived from it. ^

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