Studies on the mechanisms that detoxify chemicals and protect cells against toxicity, mutagenicity and cancer are highly significant for human health. Two cytosolic proteins [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)], with the expected function of quinone detoxification, were cloned and sequenced. Cellular studies showed that NQO1 and NQO2 reduced the DNA binding and mutagenicity of quinones. Knockout NQO1 -/- mice were produced using target gene disruption. The NQO1 -/- mice were born normal and reproduced the same as the wild-type NQO1 +/+ mice. However, NQO1 -/- mice exhibited a significant reduction in abdominal adipose tissue, bone marrow hyperactivity and hyperplasia. The NQO1 -/- mice, upon exposure to benzo(a)pyrene, developed skin tumors at a higher frequency, when compared with the wild-type mice. NQO1 and NQO2 were found to be differentially expressed among various mouse tissues. The mouse NQO2 cDNA and gene were cloned and sequenced. A targeting construct to generate NQO2 -/- mice was prepared. One of the major goals of this proposal is to extend our studies on the NQO1 -/- mice. We will determine the susceptibility of NQO1 -/- mice to benzo(a)pyrene, benzene and their quinone metabolites induced carcinogenicity when compared with wild-type NQO +/+ mice and investigate the role of NQO1 in the accumulation of abdominal adipose tissue. In addition, we will generate knockout NQO2 -/- mice that do not express NQO2 and compare the sensitivity of these mice, to that of mice expressing NQO2, following carcinogen exposure. We also plan to generate double knockout mice that do not express both NQO1 and NQO2 and determine the susceptibility of double knockout mice to chemical carcinogens when compared with wild-type and single knockout mice. To this effect, we will expose the NQO1 -/- and NQO +/+ mice to chemicals and analyze the development skin and tissue specific tumors. To investigate the mechanism of reduced abdominal adipose tissue in NQO1 -/- mice, we plan to analyze the ratio of reduced and oxidized cytosolic NAD(P) from livers an kidneys of NQO1 -/- and NQO1 +/+ mice. This ratio plays a role in regulation of gluconeogenesis and fatty acid biosynthesis. NQO1 utilizes NAD(P)H and may participate in maintaining a balance between reduced and oxidized forms of NAD(P). We will use gene targeting and homologous recombination to generate NQO2 -/- mice deficient in NQO2. The NQO1 -/- mice will be bred with the NQO2 -/- mice to produce NQO1 -/-/NQO2 -/- double knockout mice. NQO2 -/- and double knockout mice, along with wild-type mice, will be exposed to quinones and precursors and analyzed for DNA/membrane damage and the development of skin and tissue specific tumors. Upon completion, these studies will provide significant information on the in vivo function of NQO1 and NQO2, as well as their role in chemical carcinogenesis.
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