<i>In vitro</i> and <i>in vivo</i> genotoxic effects of straight <i>versus</i> tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) <i>in vitro</i> (primary genotoxicity) and <i>in vivo</i> (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1-200 µg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2-10.8 mg/m³) for 4 days, 4 h/day. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related <i>decrease</i> in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1-200 µg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m³). <i>In vitro</i> in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 µg/cm²), while MWCNT-T increased strand breakage only at 200 µg/cm². Neither of the MWCNTs was able to induce micronuclei <i>in vitro</i>. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.部分多壁碳纳米管（MWCNTs）可在啮齿类动物体内诱导间皮瘤，直链型多壁碳纳米管（MWCNT-S）相较于缠结型多壁碳纳米管（MWCNT-T）表现出更为显著的致癌效应。鉴于原发与继发遗传毒性可能在多壁碳纳米管致癌过程中发挥作用，本研究采用一系列DNA损伤与微核检测手段，在体外（in vitro）条件下评估原发遗传毒性、体内（in vivo）条件下评估原发或继发遗传毒性，以此对比直链型与缠结型多壁碳纳米管的遗传毒性。单次经咽染毒直链型多壁碳纳米管（1~200 μg/只小鼠）24小时后，通过彗星试验（comet assay）检测发现，C57Bl/6小鼠肺细胞中的DNA链断裂呈剂量依赖性升高。在以气溶胶形式暴露于直链型多壁碳纳米管（8.2~10.8 mg/m³）、每日暴露4小时、连续暴露4天的小鼠中，其肺与支气管肺泡灌洗（BAL）细胞的DNA链断裂水平，以及微核化肺泡II型细胞数量均有所增加。通过对血液单个核白细胞进行γ-H2AX检测（γ-H2AX assay），或对骨髓与血液中的微核化多染性红细胞（MNPCEs）进行检测，未在两种暴露方式下观察到直链型多壁碳纳米管引发的全身遗传毒性效应。单次经咽染毒（1~200 μg/只小鼠）后，缠结型多壁碳纳米管在小鼠支气管肺泡灌洗液细胞与肺细胞中引发的DNA损伤呈剂量依赖性降低；吸入暴露后（17.5 mg/m³），其诱导的微核化多染性红细胞数量也有所减少。在体外培养的人支气管上皮BEAS-2B细胞中，直链型多壁碳纳米管在低剂量（5和10 μg/cm²）下即可诱导DNA链断裂，而缠结型多壁碳纳米管仅在200 μg/cm²剂量下才会提升DNA链断裂水平。两种多壁碳纳米管均未在体外诱导微核形成。本研究结果表明，直链型多壁碳纳米管的遗传毒性可能同时涉及原发与继发两种作用机制。