Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding

We reported (Scates et al. Carcinogenesis 1994, 15, 2945–2948)that incubating a range of bile acids with DNA in vitro, withor without exogenous metabolic activation, gave no evidenceof DNA adduct formation as judged by the nuclease P1 methodof ³²P-postlabelling. In contrast Hamada et al. (Carcinogenesis1994, 15, 1911–1915), also using postlabelling, claimedthat chenodeoxycholic acid, lithocholic acid, glycolithocholicacid and taurolithocholic acid bound covalently to DNA in vitro.To investigate this discordance we incubated solutions of salmonsperm DNA for 1 h at 37°C with 1 mg/ml of cholic acid, chenodeoxycholicacid, lithocholic acid, glycolithocholic acid or taurolithocholicacid. Each incubate was extracted extensively with diethyl etherafter which a sample of DNA was taken and ³²P-postlabelled usingthe nuclease P1 method. The DNA in the remaining incubate wasprecipitated from high salt solution with ethanol. Aliquotsof this DNA were postlabelled. The remainder of the DNA waspurified with proteinase-K, ribonuclease, phenol-chloroform,precipitated and postlabelled. Parallel incubates were madewith the same bile acids, under the same conditions but in theabsence of DNA and were then extracted, precipitated and postlabelledas described above. When DNA was present in the incubate butwas not precipitated, chenodeoxycholic acid, lithocholic acid,glycolithocholic acid and taurolithocholic acid, but not cholicacid, produced spots similar to those reported by Hamada etal. No such spots were seen when DNA was postlabelled afterprecipitation, or after precipitation and purification. Thesesame bile acids produced spots when postlabelled in the absenceof DNA, but spots were absent when these incubates were precipitatedand purified before postlabelling. We conclude that the spotsobtained when bile acids are incubated with DNA which is notprecipitated from high salt before it is postlabelled are technicalartefacts, and cannot be regarded as evidence that bile acidsbind covalently to DNA to form adducts. We also confirm reports(Vulimiri et al. Carcinogenesis 1994, 15, 2061–2064) thatbile acids alone can produce spots when incubated with T4 polynucleotidekinase and [