Beland F A, Doerge D R, Churchwell M I, Poirier M C, Schoket B, Marques M M
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Chem Res Toxicol. 1999 Jan;12(1):68-77. doi: 10.1021/tx980172y.
32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P-postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N-Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 +/- 0.8 adducts/10(8) nucleotides (mean +/- SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 +/- 1.7 dG-C8-4-ABP/10(8) nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG-C8-4-ABP/10(8) nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 +/- 26 and 63 +/- 20 dG-C8-4-ABP/10(8) nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.
32P后标记法是检测DNA加合物的一种强大技术；然而，由于加合核苷酸与正常核苷酸在水解和标记效率上存在差异，用这种方法对DNA加合物进行定量可能会导致误差。我们合成了一种用4-氨基联苯修饰的DNA样品，用作32P后标记法及其他DNA加合物检测方法的定量标准品。[2,2'-3H]-N-羟基-4-氨基联苯在pH 5条件下与小牛胸腺DNA反应，根据3H含量得到62±0.8个加合物/10(8)个核苷酸（平均值±标准差）。酶促水解为核苷后的HPLC分析表明有一个主要加合物，即N-(脱氧鸟苷-8-基)-4-氨基联苯（dG-C8-4-ABP）。通过HPLC/电喷雾电离质谱法确认了加合物的身份，结果表明修饰水平为19±1.7个dG-C8-4-ABP/10(8)个核苷酸。32P后标记法分析得到的值为0.84个dG-C8-4-ABP/10(8)个核苷酸，而解离增强镧系荧光免疫分析（DELFIA）表明，酶促水解为核苷酸和核苷后，dG-C8-4-ABP的水平分别为82±26和63±20个/10(8)个核苷酸。通过评估用[2,2'-3H]-4-氨基联苯处理的小鼠肝脏DNA中dG-C8-4-ABP的水平，确定了该DNA加合物标准品的实用性。基于测量32P掺入程度的32P后标记法分析低估了dG-C8-4-ABP的水平，而使用G-C8-4-ABP定量标准品的DELFIA高估了加合物水平。通过HPLC/电喷雾电离质谱法测定的加合物水平最能反映从3H掺入得到的结果。当使用体外经dG-C8-4-ABP修饰的DNA作为定量标准品进行32P后标记法分析和DELFIA时，能准确估计dG-C8-4-ABP在体内的形成程度。
