Randerath K, Randerath E
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Princess Takamatsu Symp. 1990;21:317-28.
Among several recently developed analytical methods, 32P-postlabeling analysis is a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications. Since the method does not require radioactive carcinogens, it is suitable for DNA of humans exposed to environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into monomeric or dimeric hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitation by scintillation spectrometry. Microgram amounts of DNA are analyzed; thus the assay is well suited for limited amounts of cells or tissue. Various versions of the assay afford different sensitivities of adduct detection. Under optimal conditions, one aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/microgram DNA or 0.1-10 nmol adduct/mol DNA-P). The assay has been successfully applied to a variety of mutagenic (genotoxic) as well as non-mutagenic carcinogens. In humans, the 32P-postlabeling assay has been applied to DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers. Estimation of total aromatic adduct levels in exposed individuals gave values of 1 adduct in 10(6)-10(8) DNA nucleotides. These values are similar to the total levels of persistent adducts in tissues of animals after exposure to initiating or carcinogenic doses of authentic aromatic genotoxicants. Among the non-mutagenic carcinogens investigated are estrogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), choline-devoid diet, carbon tetrachloride, and peroxisome proliferators. In addition, age-dependent DNA modifications (I-compounds) are being detected by 32P-postlabeling in animals that have not been knowingly exposed to mutagens/carcinogens. I-compound profiles and levels are dependent on species, tissue, sex, and diet. Reduced levels of I-compounds have been consistently noted in the target organ of carcinogen-exposed animals and in resulting neoplasms, suggesting that I-compound loss may play a role in carcinogenesis.
在最近开发的几种分析方法中，32P后标记分析是一种用于检测和测量共价致癌物-DNA加合物及其他DNA修饰的高灵敏度方法。由于该方法不需要放射性致癌物，因此适用于接触环境或职业性遗传毒性物质的人类DNA。基本步骤包括将32P标记酶促掺入DNA的单体或二聚体水解产物中，随后对32P标记的消化产物进行色谱图谱分析和放射自显影，并通过闪烁光谱法进行定量。分析的DNA量为微克级；因此该检测方法非常适合有限量的细胞或组织。该检测方法的不同版本提供了不同的加合物检测灵敏度。在最佳条件下，可以检测和测量10(8)-10(10)个核苷酸中的一个芳香族或大体积/疏水性加合物（相当于0.3-30 amol加合物/微克DNA或0.1-10 nmol加合物/摩尔DNA-P）。该检测方法已成功应用于多种诱变（遗传毒性）以及非诱变致癌物。在人类中，32P后标记检测已应用于吸烟者、铸铁工人和炼焦炉工人的DNA样本。对暴露个体中总芳香族加合物水平的估计得出每10(6)-10(8)个DNA核苷酸中有1个加合物的值。这些值与动物组织在接触引发剂量或致癌剂量的纯正芳香族遗传毒性物质后持久性加合物的总水平相似。在研究的非诱变致癌物中包括雌激素、2,3,7,8-四氯二苯并-p-二恶英（TCDD）、缺乏胆碱的饮食、四氯化碳和过氧化物酶体增殖剂。此外，在未有意接触诱变剂/致癌物的动物中，通过32P后标记检测到了与年龄相关的DNA修饰（I-化合物）。I-化合物的谱型和水平取决于物种、组织、性别和饮食。在接触致癌物的动物的靶器官和由此产生的肿瘤中，一直观察到I-化合物水平降低，这表明I-化合物的损失可能在致癌过程中起作用。
