Randerath E, Zhou G D, Donnelly K C, Safe S H, Randerath K
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Arch Toxicol. 1996;70(11):683-95. doi: 10.1007/s002040050329.
Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. 32P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with 32P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6 x 10(6) DNA nucleotides in skin (both extracts) and one adduct in 3.2 x 10(7) or 1.2 x 10(7) DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the 32P-labeled derivative of the reaction product of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N2 of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by 32P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts.
美国众多的木材防腐废料（WPW）场地存在基因毒性危害。WPW由复杂混合物构成，其中含有包括基因毒性化合物在内的有毒化合物，这些化合物源自防腐剂煤焦油杂酚油和五氯苯酚（PCP）以及其他多氯芳烃。由于可能存在化合物间的相互作用，WPW提取物的基因毒性尚未在哺乳动物中进行测试，因此无法根据单个成分的数据来评估。所以，需要对整个提取物进行检测。32P后标记法是一种强大的工具，可用于确定复杂基因毒性混合物（如香烟烟雾、柴油机废气以及焦炉和铸造厂排放物）在实验动物和人类中诱导的DNA加合物形成情况。在本研究中，将小鼠生物测定法与32P后标记法结合使用，以确定来自一个WPW场地的两个样品的己烷/丙酮提取物诱导的DNA加合物形成情况。雌性ICR小鼠每天经皮给予相当于3毫克残留物的提取物或赋形剂对照，连续2天，24小时后处死。通过核酸酶P1增强后标记法检测皮肤、肺、肝、肾和心脏的DNA制剂。加合物图谱具有组织特异性，显示出多种非极性DNA加合物，皮肤（两种提取物）中加合物水平相当于每1.6×10⁶个DNA核苷酸中有一个加合物，肝脏（提取物1或提取物2）中加合物水平相当于每3.2×10⁷或1.2×10⁷个DNA核苷酸中有一个加合物。根据其色谱特性，这些加合物似乎主要源自提取物中存在的多环芳烃（PAH）。其中一种主要加合物被鉴定为7β,8α - 二羟基 - 9α,10α - 环氧 - 7,8,9,10 - 四氢苯并[a]芘（BPDE I）与脱氧鸟苷的N²反应产物的32P标记衍生物。非极性DNA加合物总水平在皮肤和肺中最高，在肺中提取物1和提取物2分别相当于皮肤值的17.4%和24.0%，而肝脏中的相应水平分别为5.0%和12.6%。这些结果与PAH在这些器官中的致癌潜能一致。提取物2在内脏中诱导的加合物水平更高，尽管其PAH浓度低于提取物1，即肺、肝、肾和心脏中的总加合物水平分别高1.4、2.5、1.9和1.7倍，苯并[a]芘加合物水平分别高1.6、3.3、1.6和1.9倍。除了肺中的总加合物外，两种提取物之间的差异均具有显著性，提示存在化合物间的相互作用。五个组织中的苯并[a]芘加合物水平与总加合物水平呈线性相关，因此可作为后者的替代指标。总体而言，结果表明，通过32P后标记法分析的小鼠组织中的DNA加合物是WPW提取物基因毒性的合适生物标志物和剂量计。
