DNA adducts, detected by 32P-postlabelling, in DNA treated in vitro with bile from patients with familial adenomatous polyposis and from unaffected controls.
Patients with familial adenomatous polyposis (FAP) have a high risk of developing duodenal adenomas and carcinomas. The distribution of these neoplasms resembles mucosal exposure to bile, suggesting that bile may play a role in adenoma development. Our previous results, using DNA adducts detected by 32P-postlabelling as an index of genotoxicity, have supported this hypothesis. We found significantly higher adduct labelling in the duodenum of FAP patients than in the duodenum of control patients and significantly higher labelling in the small bowel of rats gavaged with FAP bile than in rats given control bile. We have now investigated the ability of human bile to form adducts with DNA in vitro. Bile obtained from the gallbladder of 18 FAP patients immediately before colectomy, and from 18 control patients, was incubated with salmon sperm DNA in solution at 37 degrees C for 1 h, after which the purified DNA was analysed for DNA adducts, using the nuclease P1 method of 32P-postlabelling. Relative adduct labelling values (RAL, adducts per 10(9) nucleotides) produced by FAP bile samples were significantly higher than RAL values produced by control bile samples (medians 197 versus 86, P = 0.0016, Mann-Whitney test). We found a consistent pattern of adduct labelling, varying in intensity between samples. Adduct spots were eluted from TLC plates and analyzed by reverse-phase HPLC. Each major spot gave several peaks that were consistent between bile samples from different patients and were similar in FAP and control bile. These results indicate that bile from FAP and control patients contains similar, directly acting genotoxic compounds but that levels are higher in FAP than in control patients. This suggests that bile from FAP patients is more genotoxic than bile from control patients. Incubation of bile with free-radical scavengers and deconjugating enzymes failed to influence adduct labelling in this system.
Section of Molecular Carcinogenesis, Royal Marsden Hospital, Sutton, Surrey, UK.
D K
DK
Scates
A D
AD
Spigelman
K P
KP
Nugent
R K
RK
Phillips
S
S
Venitt
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D001345:Autoradiography; D001646:Bile / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D005704:Gallbladder / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; D004247:DNA