Studies on detoxification mechanisms that protect cells against toxicity, mutagenicity and cancer due to exposure to environmental agents are highly significant for human health. Recently, the investigators found that dicoumarol sensitive cytosolic NAD(P)H: Quinone Oxidoreductases (NQO1 and NQO2) compete with P450 reductase and prevent the binding of metabolically activated quinones (semiquinones and reactive oxygen species) to the DNA and protect the cells from oxidative stress, cytotoxicity and mutagenicity of quinones. More recently, the investigators have identified and characterized two additional NQOs (29 and 18 kDa) in the microsomal membranes and designated them as microsomal NAD(P)H:Quinone Oxidoreductases (mNQOs). The mNQOs are unique proteins, dicoumarol insensitive, metabolize minadione and other quinones with high affinity and cross react with antibodies against cytosolic NQO1. The microsomal mNQO proteins have not been cloned and their role in prevention of quinone toxicity and mutagenicity is expected but remains unknown. The quinone carcinogenicity and role of various cytosolic and microsomal NQO proteins in prevention of quinone carcinogenecity also remains unknown. The major goals of this proposal are to clone the microsomal mNQOs and study the role of these proteins in reducing/increasing the quinone mutagenicity and cytotoxicity. In addition, generate a knock out mouse which does not express cytosolic NQO1 and determine the role of cytosolic NQO1 in quinone carcinogenicity. To this effects, the microsomal mNQOs will be purified by biochemical methods and by screening a cDNA library with antibodies against mNQOs and/or oligonucleotide probes designed from peptide sequences. The role of membrane bound mNQO proteins in cellular protection and/or damage due to exposure to quinones will be investigated by elevating or inhibiting the cellular levels of mNQOs and P450 reductase by transfecting mammalian cells with respective cDNAs and antisense oligonucleotides. The transfected cells will be analyzed for their capacity to reduce/increase quinones binding to the DNA by 32P-postlabeling assays, cause mutations by SupF tRNA model system and cytotoxicity by following the survival/death of cells in absence and presence of quinones. In addition, the investigators will generate a knockout mouse which does not express NQO1. The role of NQO1 in carcinogenicity will be determined by comparing the sensitivity of mice possessing the null genotype with that of mice expression NQO1 following exposure to quinones.
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