Randerath E, Avitts T A, Reddy M V, Miller R H, Everson R B, Randerath K
Cancer Res. 1986 Nov;46(11):5869-77.
Previous studies using a highly sensitive 32P-postlabeling assay for the analysis of carcinogen/mutagen-induced DNA damage have shown the presence of tobacco smoking-related DNA adducts in human placenta (Everson, R.B., Randerath, E., Santella, R.M., Cefalo, R.C., Avitts, T. A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The occurrence of such adducts in smokers' bronchus and larynx is reported here. Since the chemical nature of these adducts could not be characterized by direct methods due to the extremely low levels of individual adducts (less than 0.03 fmol per microgram DNA), we have sought an experimental animal model for studying the formation of tobacco-related DNA adducts. Because cigarette smoke condensate is known to initiate tumors in mouse skin, ICR mice were treated topically with cigarette tar equivalent to 1.5, 3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin DNA was isolated 1 day after the last treatment. When DNA from exposed mice was analyzed by the 32P-postlabeling assay, 12 distinct 32P-labeled DNA adduct spots, as well as a diagonal radioactive zone, which presumably reflected the presence of incompletely resolved adducts, were noted on polyethyleneimine-cellulose TLC fingerprints. One derivative in particular (adduct 1) was seen to increase rapidly during the early treatment phase and also to persist to 8 days after treatment. The prominent adduct 1 was observed in the same location on the fingerprints of DNA samples from smokers. Cochromatography experiments suggested identity of human and mouse DNA adduct 1. Similarly, several other human and mouse adducts (adducts 3, 5, 6, and 9) appeared identical, and the diagonal radioactive zone was also present on DNA adduct maps from smokers. While absolute levels of individual human adducts were too low to be accurately quantitated, semiquantitative estimation of total tobacco-related aromatic DNA adducts in the human specimens gave values of 1 adduct in (1.7-2.9) X 10(7) nucleotides (0.10-0.18 fmol per micrograms DNA), with adduct 1 constituting 8.5-14% of the total. On the basis of these results, it appears now feasible to determine the chemical origin of smoking-induced DNA adducts in human tissues by preparation of authentic 32P-labeled reference adducts from animals treated with characterized subfractions of cigarette tar, 32P-postlabeling, and cochromatography of 32P-labeled human and animal adducts.
此前使用高灵敏度的³²P后标记分析法来分析致癌物/诱变剂诱导的DNA损伤的研究表明，人胎盘中存在与吸烟相关的DNA加合物（埃弗森，R.B.，兰德拉斯，E.，桑泰拉，R.M.，塞法洛，R.C.，阿维茨，T.A.，以及兰德拉斯，K.，《科学》（华盛顿特区），231: 54 - 57，1986年）。本文报道了此类加合物在吸烟者支气管和喉部的出现情况。由于这些加合物的化学性质因单个加合物水平极低（每微克DNA少于0.03飞摩尔）而无法通过直接方法进行表征，我们一直在寻找一种实验动物模型来研究与烟草相关的DNA加合物的形成。因为已知香烟烟雾冷凝物可引发小鼠皮肤肿瘤，所以分别用相当于1.5、3、6和9支香烟的香烟焦油对ICR小鼠进行局部处理，处理时间分别为0.4、3、5和7天，在最后一次处理后1天分离皮肤DNA。当通过³²P后标记分析法分析暴露小鼠的DNA时，在聚乙烯亚胺 - 纤维素薄层层析指纹图谱上发现了12个不同的³²P标记的DNA加合物斑点，以及一个对角放射性区域，推测该区域反映了未完全分离的加合物的存在。特别是一种衍生物（加合物1）在处理早期阶段迅速增加，并且在处理后8天仍持续存在。在吸烟者DNA样本的指纹图谱上的相同位置也观察到了突出的加合物1。共色谱实验表明人和小鼠的DNA加合物1相同。同样，其他几种人和小鼠的加合物（加合物3、5、6和9）看起来也相同，并且吸烟者的DNA加合物图谱上也存在对角放射性区域。虽然单个人类加合物的绝对水平过低无法准确定量，但对人类样本中与烟草相关的总芳香族DNA加合物进行半定量估计得出的值为每（1.7 - 2.9）×10⁷个核苷酸中有1个加合物（每微克DNA为0.10 - 0.18飞摩尔），加合物1占总量的8.5 - 14%。基于这些结果，现在看来通过用经表征的香烟焦油水相馏分处理动物制备真实的³²P标记参考加合物、³²P后标记以及³²P标记的人和动物加合物的共色谱来确定人类组织中吸烟诱导的DNA加合物的化学来源是可行的。
