Nesnow Stephen, Davis Christine, Nelson Garret B, Lambert Guy, Padgett William, Pimentel Maria, Tennant Alan H, Kligerman Andrew D, Ross Jeffrey A
Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, MD-68, Research Triangle Park, NC 27711, USA.
Mutat Res. 2002 Nov 26;521(1-2):91-102. doi: 10.1016/s1383-5718(02)00218-8.
Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.
苯并[a]芘（B[a]P）是研究最为深入的多环芳烃（PAH）。人们提出了许多机制来解释其致癌活性，但仍存在许多问题。PAH的K区域二氢二醇是依赖特定细胞色素P450的代谢中间体，曾被认为是解毒产物。然而，最近有研究表明，几种PAH的K区域二氢二醇可使小鼠胚胎C3H10T1/2CL8细胞（C3H10T1/2细胞）发生形态转化。由于C3H10T1/2细胞不会通过代谢作用由PAH生成K区域二氢二醇，因此这些细胞为独立研究PAH及其K区域二氢二醇的作用机制提供了有用的工具。在此，我们比较了B[a]P的K区域二氢二醇反式-B[a]P-4,5-二醇与B[a]P在细胞形态转化、DNA损伤和DNA加合物形成方面的活性。反式-B[a]P-4,5-二醇和B[a]P均可通过产生II型和III型转化灶使C3H10T1/2细胞发生形态转化。反式-B[a]P-4,5-二醇和B[a]P的细胞形态转化和细胞毒性剂量反应曲线无法区分。由于细胞形态转化与C3H10T1/2细胞中的突变和/或大规模DNA损伤密切相关，因此我们试图确定反式-B[a]P-4,5-二醇在这些细胞中诱导的DNA损伤情况。使用彗星试验，反式-B[a]P-4,5-二醇和B[a]P均表现出显著的DNA损伤活性，且无明显的细胞毒性，但剂量反应和彗星尾分布不同。使用32P后标记技术和旨在分离极性DNA加合物的改进TLC洗脱系统，检测了反式-B[a]P-4,5-二醇或B[a]P处理后C3H10T1/2细胞的DNA加合物模式。虽然B[a]P处理产生了一种主要的DNA加合物，鉴定为反式-B[a]P-7,8-二醇-9,10-环氧化物-脱氧鸟苷，但在反式-B[a]P-4,5-二醇处理的细胞DNA中未检测到稳定的共价DNA加合物。总之，本研究提供了证据，证明在没有共价稳定DNA加合物的情况下，B[a]P的K区域二氢二醇具有DNA损伤和细胞形态转化活性。虽然反式-B[a]P-4,5-二醇和B[a]P均可诱导细胞形态转化，但它们作为DNA损伤剂的活性在定性和定量方面均有所不同。与PAH的其他K区域二氢二醇的细胞形态转化活性一致，这些数据提示了B[a]P及其代谢产物细胞形态转化活性的新机制/途径。
