Quantitative Analysis of Low Levels of Benzo(A)Pyrene Diol Epoxide Bound to DNA: Acid-Induced Liberation of Tetraols Followed by HPLC and Fluorometric Detection

摘要

Under normal environmental conditions, outside the laboratory, neither the DNA of an organism nor the carcinogens to which it may be exposed are radioactively labeled; hence, the conventional array of radiometric methods for analyzing DNA damage are not applicable. Because of the intrinsic fluorescence associated with B and its metabolites. The use of fluorescence detection for quantitating the amount of BPDE bound to the DNA of exposed animals was explored. Such techniques may have application to the detection of other fluorescent carcinogens as well. Assuming that 100 mu g of DNA can be isolated from the back of a single treated mouse, then a detection limit of 5 pg of tetraol represents a binding level of 5 molecules of BP per 10 exp 8 bases. This level of sensitivity is about a factor of 10 less than that calculated by Baird for 100 mu g of DNA employing a specific activity of 20 Ci per mmol tritiated BP. Additional improvements in the fluorescence detection system are planned, which could increase the sensitivity by another order of magnitude. (ERA citation 07:048226)