Öner Deniz, Ghosh Manosij, Coorens Robin, Bové Hannelore, Moisse Matthieu, Lambrechts Diether, Ameloot Marcel, Godderis Lode, Hoet Peter H M
KU Leuven, Department of Public Health and Primary Care, Unit of Environment and Health, Laboratory of Toxicology, 3000 Leuven, Belgium.
Hasselt University, Biomedical Research Institute, Agoralaan Building C, 3590 Diepenbeek, Belgium.
Environ Int. 2020 Apr;137:105530. doi: 10.1016/j.envint.2020.105530. Epub 2020 Feb 18.
Inhalation of asbestos induces lung cancer via different cellular mechanisms. Together with the increased production of carbon nanotubes (CNTs) grows the concern about adverse effects on the lungs given the similarities with asbestos. While it has been established that CNT and asbestos induce epigenetic alterations, it is currently not known whether alterations at epigenetic level remain stable after withdrawal of the exposure. Identification of DNA methylation changes after a low dose of CNT and asbestos exposure and recovery can be useful to determine the fibre/particle toxicity and adverse outcome.
Human bronchial epithelial cells (16HBE) were treated with a low and non-cytotoxic dose (0.25 µg/ml) of multi-walled carbon nanotubes (MWCNTs-NM400) or single-walled carbon nanotubes (SWCNTs-SRM2483) and 0.05 µg/ml amosite (brown) asbestos for the course of four weeks (sub-chronic exposure). After this treatment, the cells were further incubated (without particle/fibre) for two weeks, allowing recovery from the exposure (recovery period). Nuclear depositions of the CNTs were assessed using femtosecond pulsed laser microscopy in a label-free manner. DNA methylation alterations were analysed using microarrays that assess more than 850 thousand CpG sites in the whole genome.
At non-cytotoxic doses, CNTs were noted to be incorporated with in the nucleus after a four weeks period. Exposure to MWCNTs induced a single hypomethylation at a CpG site and gene promoter region. No change in DNA methylation was observed after the recovery period for MWCNTs. Exposure to SWCNTs or amosite induced hypermethylation at CpG sites after sub-chronic exposure which may involve in 'transcription factor activity' and 'sequence-specific DNA binding' gene ontologies. After the recovery period, hypermethylation and hypomethylation were noted for both SWCNTs and amosite. Hippocalcinlike 1 (HPCAL1), protease serine 3 (PRSS3), kallikrein-related peptidase 3 (KLK3), kruppel like factor 3 (KLF3) genes were hypermethylated at different time points in either SWCNT-exposed or amosite-exposed cells.
These results suggest that the specific SWCNT (SRM2483) and amosite fibres studied induce hypo- or hypermethylation on CpG sites in DNA after very low-dose exposure and recovery period. This effect was not seen for the studied MWCNT (NM400).
吸入石棉可通过不同的细胞机制诱发肺癌。随着碳纳米管（CNT）产量的增加，鉴于其与石棉的相似性，人们对其对肺部的不良影响愈发担忧。虽然已经确定碳纳米管和石棉会诱导表观遗传改变，但目前尚不清楚暴露停止后表观遗传水平的改变是否仍然稳定。识别低剂量碳纳米管和石棉暴露及恢复后的DNA甲基化变化，有助于确定纤维/颗粒的毒性和不良后果。
用低剂量且无细胞毒性（0.25μg/ml）的多壁碳纳米管（MWCNTs-NM400）或单壁碳纳米管（SWCNTs-SRM2483）以及0.05μg/ml铁石棉（棕色）石棉处理人支气管上皮细胞（16HBE）四周（亚慢性暴露）。处理后，将细胞进一步培养（不接触颗粒/纤维）两周，使其从暴露中恢复（恢复期）。使用飞秒脉冲激光显微镜以无标记方式评估碳纳米管的核沉积。使用可评估全基因组超过85万个CpG位点的微阵列分析DNA甲基化改变。
在无细胞毒性剂量下，四周后发现碳纳米管被纳入细胞核。暴露于多壁碳纳米管会在一个CpG位点和基因启动子区域诱导单个低甲基化。多壁碳纳米管恢复期后未观察到DNA甲基化变化。亚慢性暴露后，暴露于单壁碳纳米管或铁石棉会在CpG位点诱导高甲基化，这可能涉及“转录因子活性”和“序列特异性DNA结合”基因本体。恢复期后，单壁碳纳米管和铁石棉均出现高甲基化和低甲基化。在暴露于单壁碳纳米管或铁石棉的细胞中，类海马钙蛋白1（HPCAL1）、丝氨酸蛋白酶3（PRSS3）、激肽释放酶相关肽酶3（KLK3）、 Kruppel样因子3（KLF3）基因在不同时间点出现高甲基化。
这些结果表明，所研究的特定单壁碳纳米管（SRM2483）和铁石棉纤维在极低剂量暴露和恢复期后会诱导DNA中CpG位点的低甲基化或高甲基化。所研究的多壁碳纳米管（NM400）未出现这种效应。
