The proposed studies with various mammalian activator cells will relate the metabolic activation and DNA binding of benzo(a)pyrene [B(a)P] and dimethylbenza(a)anthracene (DMBA).
The specific aims are: 1) To continue studies of the role of specific cytochrome P-450 induction in the binding of B(a)P and DMBA to DNA in mammalian cells through the use of antibodies against specific cytochrome P-450 isozymes; 2) To continue the development of a lambda35S-ATP postlabeling procedure to allow detection of unlabeled hydrocarbon-DNA adducts at low levels with the advantages of analysis at the mononucleotide level by HPLC and the use of 35S rather than 32P. This assay will be used to facilitate studies in activator and target cells under conditions of mutation assays; 3) To investigate the binding of DMBA and B(a)P to specific regions of chromatin and DNA in cells from various species through the use of immunoaffinity chromatography on columns prepared with antibodies against DMBADE-DNA adducts and B(a)P-DNA adducts; 4) To determine the role of base sequence in the binding of DMBA and B(a)P to DNA through the use of defined sequence oligonucleotides produced by PCR; 5) To determine the relationship of DMBA and B(a)P adduct formation to the formation of oxidative damage as measured by formation of 8-hydroxy-dG in DNA in mammalian cells; 6) To relate the binding of B(a)P and DMBA to DNA to produce specific adducts or to specific regions of DNA and chromatin to mutation induction in cell-mediated V79 cell assays.
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