A6: DNA PHOTOCLEAVAGE & PHOTOINDUCED TOXICITY OF POLYCYCLIC AROMATIC HYDROCARBON

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In this project the light-induced DNA cleavage by the environmentally important carcinogen polycyclic aromatic hydrocarbons (PAH) and test their genotoxicity toward bacteria cell upon irradiation with visible and UVA light in the presence of various biologically important chemicals will be investigated. The carcinogenic metabolite 7,8dihydroxy.9,1 O-epoxy-7,8,9,1 0-tetrahydrobenzo[a]pyrene is found to cleave DNA when irradiated with 355 nm laser light. Our hypothesis is that other PAHs can also cleave DNA when photoichemically excited and the DNA cleavage may be a source of phototoxicity. Studies of DNA photocleavage will focus on various PAHs, especially the nitro, hydroxy, methyl, and aza derivatives and their metabolites such as the diol epoxides and diones. Photoreactivity of PAH derivatives toward DNA cleavage will be examined using plasmid DNA. The concentration (C50) at which 50% of DNA cleavage for each PAH will be determined. The effect on the photocleavage by biologically important chemicals, metal ions and coenzymes (NADH, Flavin), will also be examined. Genotoxicity and ecotoxicity of compounds exhibiting clear ability, to cleave DNA photochermically will be evaluated with a variety of microbial bioassays upon UV-Iight or outdoor sun-light irradiation. The bioassays include measurements of total bacterial numbers, ATP and total adenylate concentrations, DNA concentration, heterotrophic activity, chlorophyll a concentration, Microtox. and Mutatox Tests. A profile that relates the photoxicity and DNA photocleavage will be established. Finally, the effect of a variety of sensitizers on the photochemical and microbial degradation rates of PAHs will be studied. A protocol will be established to enhance the removal and detoxification rates of PAHs. Compounds that are strongest DNA photocleavers will be used further for studying the photocleavage products. Photoproducts of both cleaved DNA and PAHs will be identified using a combination of analytical techniques: HPLC. LC-MS, and NMR. Mechanisms for the photoinduced DNA cleavage will be deduced from the structure Of the photoproducts. The involvement of active oxygen species and free radicals will also be studied. Structure Activity- Relationship (SAR) will be studied using the 50 for the photocleavage and LC*/LOEC for the phototoxicit-v.