Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences , University of Montana , Missoula , Montana 59812 , United States.
Chem Res Toxicol. 2019 Aug 19;32(8):1545-1553. doi: 10.1021/acs.chemrestox.9b00075. Epub 2019 Jul 16.
Diversity in physicochemical properties of engineered multiwalled carbon nanotubes (MWCNTs) increases the complexity involved in interpreting toxicity studies of these materials. Studies indicate that epigenetic changes could be at least partially involved in MWCNTs-induced pro-inflammatory and fibrotic lung pathology. Therefore, we examined distinct methylation changes in response to MWCNTs of varied sizes to identify potential epigenetic biomarkers of MWCNTs exposure and disease progression. C57BL/6 mice were exposed via oropharyngeal instillation to a single dose (50 μg) to one of three differently sized MWCNTs: "narrow short" (NS), "wide short" (WS), and "narrow long" (NL). Vehicle-treated control mice received dispersion media (DM) only. Whole lung lavage fluid (LLF) and lung tissue were collected 24 h and 7 days postexposure to evaluate pro-inflammatory cytokines, epigenetic, or histological responses at acute and subchronic intervals, respectively. Luminometric methylation assay and pyrosequencing were used to measure global DNA methylation as well as promoter methylation of inflammation and fibrosis-related genes, respectively. Pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, were measured using enzyme-linked immunosorbant assay, while airway thickening and interstitial collagen accumulation were measured in 7-day lung tissue using laser scanning cytometry. Distinct patterns of methylation (i.e., , , and ) among the different sized MWCNTs at 24 h postexposure corresponded to some pro-inflammatory cytokine measurements from whole LLF. Fibrosis-related gene, , was significantly hypermethylated after exposures to WS and NL MWCNTs, while only NL MWCNTs induced significantly lower global DNA methylation. After 7 days, a hierarchy in airway thickness and interstitial collagen deposition was observed: NS < WS < NL. However, only airway thickness was significantly greater in the WS and NL MWCNTs-exposed groups than the DM-exposed group. These data suggest that methylation changes could be involved in the initial immune response of inflammation and tissue remodeling that precedes lung disease in response to different MWCNTs sizes.
工程化多壁碳纳米管 (MWCNTs) 的物理化学性质多样性增加了解释这些材料毒性研究的复杂性。研究表明，表观遗传变化至少可能部分参与 MWCNTs 诱导的炎症和纤维化肺病理学。因此，我们研究了不同大小的 MWCNTs 引起的不同甲基化变化，以确定 MWCNTs 暴露和疾病进展的潜在表观遗传生物标志物。C57BL/6 小鼠通过口咽滴注暴露于三种不同大小的 MWCNTs 之一：“窄短”（NS）、“宽短”（WS）和“窄长”（NL），剂量为 50 μg。接受载剂（DM）处理的对照小鼠仅接受载剂。分别在暴露后 24 小时和 7 天收集全肺灌洗液（LLF）和肺组织，以评估急性和亚慢性间隔的促炎细胞因子、表观遗传或组织学反应。使用发光甲基化测定法和焦磷酸测序分别测量全基因组 DNA 甲基化和炎症和纤维化相关基因的启动子甲基化。使用酶联免疫吸附测定法测量促炎细胞因子，包括 IL-1ß、IL-6 和 TNF-α，而气道增厚和间质胶原积累在 7 天肺组织中使用激光扫描细胞仪测量。暴露后 24 小时不同大小的 MWCNTs 之间存在不同的甲基化模式（即 、 、和 ），与全 LLF 中的一些促炎细胞因子测量值相对应。纤维化相关基因 在 WS 和 NL MWCNTs 暴露后显著超甲基化，而仅 NL MWCNTs 诱导的全基因组 DNA 甲基化显著降低。7 天后，观察到气道厚度和间质胶原沉积的层次结构：NS<WS<NL。然而，仅 WS 和 NL MWCNTs 暴露组的气道厚度显著大于 DM 暴露组。这些数据表明，甲基化变化可能参与不同 MWCNTs 大小引起的炎症和组织重塑的初始免疫反应，这是导致肺部疾病的原因。
