Stanley Venitt

The use of 32P-postlabelling in studies of the nature and origin of DNA adducts formed by bile from patients with familial adenomatous polyposis and from normal patients

32P-postlabelling is a highly sensitive technique for the detection of DNA adducts. It is unique in that it requires no prior knowledge of the nature of adducts or adduct-forming species under investigation. In the past, we have used this technique to investigate the role of bile in the production of foregut adenomas in patients with familial adenomatous polyposis (FAP). We have found that bile contains constituents that form DNA adducts directly, and after metabolic activation, and that the bile of FAP patients has an increased capacity for adduct formation with DNA in vitro, in human cell lines in culture, and in the gastrointestinal tract of rats given bile by gavage. The sensitivity of 32P-postlabelling is such that it is difficult to obtain sufficient quantities of DNA adducts for chemical analysis. The nature of the adducts produced by bile, or of the bile constituents that produce them is as yet undetermined. In the present studies, we have combined 32P-postlabelling with indirect methods to gain some insight into the nature of DNA adducts produced by bile and the properties of the reactive species that form them. Firstly, bile was incubated with synthetic monodeoxynucleotides or polydeoxynucleotides. Bile did not produce adducts when incubated with monodeoxynucleotides or single-stranded polydeoxynucleotides. However, it did produce adducts when incubated with double-stranded polydeoxynucleotides. The pattern of adduct formation suggested that human bile forms a mixture of adenine and guanine adducts. Secondly, bile was fractionated by extraction with blue cotton or with neutral, acid or alkaline organic solvent. Blue cotton, which efficiently and selectively absorbs mutagens having 3 or more fused aromatic rings, did not absorb biliary constituents that could form adducts with DNA in vitro or with DNA of MCL-5 cells, a metabolically competent human cell line. This suggests that biliary DNA adduct precursors are polar compounds that contain fewer than 3 aromatic rings or are non-aromatic. 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 or alkaline organic extracts, suggesting that constituents of bile that form DNA adducts are acidic in nature.