THE USE OF P-32 POSTLABELING IN STUDIES OF THE NATURE AND ORIGIN OF DNA-ADDUCTS FORMED BY BILE FROM PATIENTS WITH FAMILIAL ADENOMATOUS POLYPOSIS AND FROM NORMAL-PATIENTS

P-32-postlabelling is a highly sensitive technique for the detection o f DNA adducts. It is unique in that it requires no prior knowledge of the nature of adducts or adduct-forming species under investigation. I n the past, we have used this technique to investigate the role of bil e in the production of foregut adenomas in patients with familial aden omatous polyposis (FAP). We have found that bile contains constituents that form DNA adducts directly, and after metabolic activation, and t hat the bile of FAP patients has an increased capacity for adduct form ation with DNA in vitro, in human cell lines in culture, and in the ga strointestinal tract of rats given bile by gavage. The sensitivity of P-32-postlabelling is such that it is difficult to obtain sufficient q uantities of DNA adducts for chemical analysis. The nature of the addu cts produced by bile, or of the bile constituents that produce them is as yet undetermined, In the present studies, we have combined P-32-po stlabelling with indirect methods to gain some insight into the nature of DNA adducts produced by bile and the properties of the reactive sp ecies that form them. Firstly, bile was incubated with synthetic monod eoxynucleotides or polydeoxynucleotides. Bile did not produce adducts when incubated with monodeoxynucleotides or single-stranded polydeoxyn ucleotides. However, it did produce adducts when incubated with double -stranded polydeoxynucleotides. The pattern of adduct formation sugges ted that human bile forms a mixture of adenine and guanine adducts. Se condly, bile was fractionated by extraction with blue cotton or with n eutral, acid or alkaline organic solvent. Blue cotton, which efficient ly and selectively absorbs mutagens having 3 or more fused aromatic ri ngs, did not absorb biliary constituents that could form adducts with DNA in vitro or with DNA of MCL-5 cells, a metabolically competent hum an cell line. This suggests that biliary DNA adduct precursors are pol ar compounds that contain fewer than 3 aromatic rings or are non-aroma tic. Acidic organic extracts of human bile produced much higher levels of DNA adducts in vitro or with DNA of MCL-5 cells than did neutral o r alkaline organic extracts, suggesting that constituents of bile that form DNA adducts are acidic in nature. (C) 1997 Elsevier Science B.V.