Benzene is one of the few environmental agents which is unequivocally established as initiating huma cancer. This is a important public health issue due to the fact that virtually everyone is exposed to varying levels of benzene. In contrast to its biologica importance, relatively little is known about the molecular mechanisms by which benzene induces its genotoxic effects. To address this, they proposed the development of a human cell line which can be used to investigate the mutageni properties of benzene's metabolites and related environmental agents. They proposed: (1) To isolate HL-60 cell line containing stably integrated copies o a lambda/lacI shuttle vector. This cell line will be developed by transfection of HL-60 cells with a lambda/lacI shuttle vector and a plasmid encoding resistance to neomycin. The clone selected for this study will have the following properties: (a) a high rescue efficiency for lambda/lacI shuttle vector, (b) contain significant levels of peroxidase enzyme and activate hydroquinone to form DNA adducts, and (c) shows a low spontaneous and a significant increase in lacI mutation frequency in treated cells. (2) To measure the frequency of lacI mutations induced by treatment of HL-60/lacI cells with varying concentrations of either p-benzouione or hydroquinone. They will determine if treatment of HL-60/lacI cells with these metabolites significantly increases the lacI mutation frequency compared to treatment with solvent alone. The formation of DNA adducts in treated cells will be determine by 32P-postlabeling; allowing us to estimate the levels of DNA adducts formed under the conditions used for analysis of lacI mutations. (3) To sequence individual lacI mutants from the control and the treatment groups. This analysis will allow them to determine if the mutation spectrum in the treatmen groups are different than the mutation spectrum in the control group. They wil determine if the spectrum of mutations observed in the para-benzoquione and hydroquione treatment groups are consistent with being derived from N2-(4-hydroxy phenyl) deoxyguanosine the DNA adduct identified as being formed by these two metabolites. These studies will provide insights as to the nature of mutations induced by these benzene metabolites. Although this proposal uses established procedures, it is innovative for the following reasons: (1) it wil use cellular peroxidase enzymes for activation of the metabolites, (2) it will allow for comparison of mutation induction with adduct formation, (3) the cell line developed can be applied to the analysis of the mutagenic potential of a wide range of environmental, occupational and medicinal agents.
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| Gaikwad, N W; Bodell, W J (2001) Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: role of quinone methide in DNA adduct formation. Chem Biol Interact 138:217-29 |
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