Scates D K, Spigelman A D, Venitt S
Section of Molecular Carcinogenesis, Royal Marsden Hospital, Sutton, Surrey, UK.
Carcinogenesis. 1995 Jul;16(7):1489-91. doi: 10.1093/carcin/16.7.1489.
We reported (Scates et al. Carcinogenesis 1994, 15, 2945-2948) that incubating a range of bile acids with DNA in vitro, with or without exogenous metabolic activation, gave no evidence of DNA adduct formation as judged by the nuclease P1 method of 32P-postlabelling. In contrast Hamada et al. (Carcinogenesis 1994, 15, 1911-1915), also using postlabelling, claimed that chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid bound covalently to DNA in vitro. To investigate this discordance we incubated solutions of salmon sperm DNA for 1 h at 37 degrees C with 1 mg/ml of cholic acid, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid or taurolithocholic acid. Each incubate was extracted extensively with diethyl ether after which a sample of DNA was taken and 32P-postlabelled using the nuclease P1 method. The DNA in the remaining incubate was precipitated from high salt solution with ethanol. Aliquots of this DNA were postlabelled. The remainder of the DNA was purified with proteinase-K, ribonuclease, phenol-chloroform, precipitated and postlabelled. Parallel incubates were made with the same bile acids, under the same conditions but in the absence of DNA and were then extracted, precipitated and postlabelled as described above. When DNA was present in the incubate but was not precipitated, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid, but not cholic acid, produced spots similar to those reported by Hamada et al. No such spots were seen when DNA was postlabelled after precipitation, or after precipitation and purification. These same bile acids produced spots when postlabelled in the absence of DNA, but spots were absent when these incubates were precipitated and purified before postlabelling. We conclude that the spots obtained when bile acids are incubated with DNA which is not precipitated from high salt before it is postlabelled are technical artefacts, and cannot be regarded as evidence that bile acids bind covalently to DNA to form adducts. We also confirm reports (Vulimiri et al. Carcinogenesis 1994, 15, 2061-2064) that bile acids alone can produce spots when incubated with T4 polynucleotide kinase and [gamma-32P]ATP.
我们曾报道过（Scates等人，《癌变》，1994年，第15卷，2945 - 2948页），在体外将一系列胆汁酸与DNA一起孵育，无论有无外源性代谢活化作用，通过32P后标记的核酸酶P1法判断，均未发现DNA加合物形成的证据。相比之下，Hamada等人（《癌变》，1994年，第15卷，1911 - 1915页）同样采用后标记法，声称鹅去氧胆酸、石胆酸、甘氨石胆酸和牛磺石胆酸在体外可与DNA共价结合。为研究这种差异，我们将鲑鱼精DNA溶液与1mg/ml的胆酸、鹅去氧胆酸、石胆酸、甘氨石胆酸或牛磺石胆酸在37℃孵育1小时。每个孵育物用乙醚进行充分萃取，之后取一份DNA样本，采用核酸酶P1法进行32P后标记。将剩余孵育物中的DNA从高盐溶液中用乙醇沉淀出来。取该DNA的等分试样进行后标记。其余DNA用蛋白酶K、核糖核酸酶、酚 - 氯仿进行纯化，沉淀后再进行后标记。用相同的胆汁酸在相同条件下但无DNA存在的情况下进行平行孵育，然后按上述方法进行萃取、沉淀和后标记。当孵育物中有DNA但未沉淀时，鹅去氧胆酸、石胆酸、甘氨石胆酸和牛磺石胆酸（而非胆酸）产生的斑点与Hamada等人报道的相似。当DNA在沉淀后或沉淀并纯化后进行后标记时，未观察到此类斑点。这些相同的胆汁酸在无DNA存在下进行后标记时会产生斑点，但在标记前对这些孵育物进行沉淀和纯化后则无斑点。我们得出结论，在对未从高盐溶液中沉淀就进行后标记的DNA与胆汁酸孵育时所获得的斑点是技术假象，不能视为胆汁酸与DNA共价结合形成加合物的证据。我们还证实了一些报道（Vulimiri等人，《癌变》，1994年，第15卷，2061 - 2064页），即胆汁酸单独与T4多核苷酸激酶和[γ - 32P]ATP孵育时可产生斑点。
