Phillips Jennifer M, Goodman Jay I
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Toxicology. 2009 Jun 16;260(1-3):7-15. doi: 10.1016/j.tox.2009.03.001. Epub 2009 Mar 14.
The development of early biomarkers, both of exposure and effect, would substantially improve science-based risk assessment with regard to cigarette smoke (CS)-associated toxicity. Altered DNA methylation, an epigenetic mechanism, is linked to CS-induced lung tumorigenesis. We have taken an unbiased approach (i.e. genomic regions are not pre-selected) to assess early methylation changes within lung DNA from female SENCAR mice treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), and then exposed to air alone, or CS for 4 or 8 weeks. Regions of altered DNA methylation (RAMs) were detected in mice treated with DMBA alone, or DMBA+0.16, 0.32 or 0.48 mg wet total particulate matter per liter (WTPM/L) CS, using methylation-sensitive restriction digestion, arbitrarily primed PCR and capillary electrophoresis. Comparison of the RAMs that formed in different treatment groups revealed: (1) RAMs which "carried forward" across time (i.e. occurred at both 4 and 8 weeks) in a particular dose group, in addition to unique RAMs observed only at 8 weeks, and (2) RAMs which "carried forward" across dose (i.e. occurred in at least 2 dose groups at a particular time point), in addition to unique RAMs observed only in 1 dose group. Furthermore, a subset of RAMs was observed, at both 4 and 8 weeks, in DMBA-treated and DMBA+CS-exposed groups; the presence of unique RAMs in the latter suggest that combined DMBA+CS treatment more than just "magnifies" a subset of cell populations bearing the methylation changes induced by DMBA alone. Importantly, only minimal histopathological changes were observed in the lungs of CS-treated mice. This study is the first to demonstrate changes in lung DNA methylation at early times following exposure to CS, e.g., prior to overt histopathology. Thus, altered methylation might serve as a biomarker of CS exposure, and, in light of the fact that methylation changes are linked to CS-induced lung tumorigenesis, might also be useful as biomarkers of effect.
开发早期生物标志物，包括暴露和效应的生物标志物，将显著改善基于科学的香烟烟雾（CS）相关毒性风险评估。DNA甲基化改变作为一种表观遗传机制，与CS诱导的肺癌发生有关。我们采用了一种无偏倚的方法（即不预先选择基因组区域）来评估单次给予7,12 - 二甲基苯并[a]蒽（DMBA）处理后，再单独暴露于空气或CS中4周或8周的雌性SENCAR小鼠肺DNA中的早期甲基化变化。使用甲基化敏感限制性消化、任意引物PCR和毛细管电泳，在单独用DMBA处理的小鼠，或用DMBA + 0.16、0.32或0.48毫克每升湿总颗粒物（WTPM/L）CS处理的小鼠中检测到DNA甲基化改变区域（RAMs）。不同处理组中形成的RAMs比较显示：（1）在特定剂量组中随时间“延续”（即在4周和8周均出现）的RAMs，以及仅在8周观察到的独特RAMs；（2）在特定时间点随剂量“延续”（即在至少2个剂量组中出现）的RAMs，以及仅在1个剂量组中观察到的独特RAMs。此外，在DMBA处理组和DMBA + CS暴露组中，在4周和8周均观察到一部分RAMs；后者中独特RAMs的存在表明，DMBA + CS联合处理不仅仅是“放大”了仅由DMBA诱导甲基化变化的一部分细胞群体。重要的是，在CS处理小鼠的肺中仅观察到最小程度的组织病理学变化。本研究首次证明了在暴露于CS后的早期，例如在明显的组织病理学变化之前，肺DNA甲基化的改变。因此，甲基化改变可能作为CS暴露的生物标志物，并且鉴于甲基化变化与CS诱导的肺癌发生有关，也可能作为效应的生物标志物。
