比较不同长度及表面修饰的多壁碳纳米管的细胞毒性和遗传毒性

Comparative analysis for the cytotoxicity and genotoxicity of multi-walled carbon nanotubes with different lengths and surface modifications in A549 cells

目的:比较不同长度和表面修饰的多壁碳纳米管(multi-walled carbon nanotubes,MWCNTs)对人肺泡Ⅱ型上皮细胞(A549细胞)的细胞毒性和遗传毒性的影响。方法:选取长(515μm)、短(350700 nm)两种不同长度的MWCNTs,及羧基(carboxyl,COOH-)、氨基(amino,NH2-)和牛磺酸(taurine,Tau-)3种不同表面修饰的MWCNTs分别进行实验研究,其中短的MWCNTs作为未修饰的MWCNTs(Pristine-MWCNTs)与表面修饰的MWCNTs进行比较。细胞毒性实验采用cell counting kit-8(CCK-8)法,染毒浓度为2、8、32 mg/L,染毒时间分别为12、24、36、48 h;遗传毒性采用单细胞凝胶电泳实验检测DNA链断裂,染毒剂量为8 mg/L,染毒时间24 h。结果:两种长度的MWCNTs在所观察的1248 h染毒时间内,均表现出剂量依赖的细胞毒性;其中在2448 h处理时间组,MWCNTs的细胞毒性随长度增加而增大。经过表面修饰的3种MWCNTs与未修饰的MWCNTs相比,表面修饰过的MWCNTs在染毒时间12 h、染毒浓度为32 mg/L时相对细胞活性:COOH-MWCNTs为(86.55±1.80)%、NH2-MWCNTs为(84.67±1.32)%、Tau-MWCNTs为(80.15±3.53)%,均高于未修饰MWCNTs的细胞活性(71.44±5.58)%,差异具有统计学意义(P<0.05);在染毒时间24 h、染毒浓度为8 mg/L时修饰过的MWCNTs相对细胞活性:COOH-MWCNTs为(96.74±1.00)%、NH2-MWCNTs为(96.74±3.35)%、Tau-MWCNTs为(106.39±3.83)%,均高于未修饰MWCNTs的细胞活性(91.02±2.53)%,差异具有统计学意义(P<0.05);在染毒时间24 h、染毒浓度为32 mg/L时修饰过的MWCNTs相对细胞活性:COOH-MWCNTs为(80.88±2.67)%、NH2-MWCNTs为(82.90±3.25)%、Tau-MWCNTs为(82.55±3.32)%,均高于未修饰MWCNTs的细胞活性(76.08±4.27)%,差异具有统计学意义(P<0.05);在染毒时间36 h、染毒浓度为8 mg/L时修饰过的MWCNTs相对细胞活性:COOH-MWCNTs为(96.87±1.05)%、NH2-MWCNTs为(96.66±4.76)%、Tau-MWCNTs为(100.23±2.84)%,均高于未修饰MWCNTs的细胞活性(89.61±3.78)%,差异具有统计学意义(P<0.05)。在其他观察时间和染毒浓度下,经过表面修饰的MWCNTs与未修饰MWCNTs的细胞活性相比,差异无统计学意义(P>0.05)。经过表面修饰的3种MWCNTs DNA链断裂损伤情况:Olive尾距COOH-MWCNTs为1.56±0.22、NH2-MWCNTs为2.25±1.62、Tau-MWCNTs为2.23±0.94,尾部DNA含量COOH-MWCNTs为(3.96±0.60)%、NH2-MWCNTs为(6.16±4.68)%、Tau-MWCNTs为(6.05±2.31)%,均在不同程度上低于未修饰的MWCNTs[Olive尾距为3.00±0.64,尾部DNA含量为(8.23±2.27)%],差异具有统计学意义(P<0.05),其中COOH-MWCNTs所致DNA链断裂损伤最小。结论:上述材料在所观察时间和染毒剂量下,均引起了A549细胞不同程度的细胞毒性和DNA链断裂,相同染毒浓度下,不同长度的MWCNTs所致细胞毒性及DNA链断裂程度有所差别;表面修饰可在一定程度上降低MWCNTs对A549细胞的细胞毒性和DNA链断裂损伤。

Objective：To compare the cytotoxicity and DNA strand breakage induced by multi-walled carbon nanotubes （MWCNTs） with different lengths and different surface modifications in human alveolar type II cells （A549 cells）. Methods： Two different lengths （5 -15 nm, 350 -700 nm） of MWCNTs and three different kinds of surface modified MWCNTs （COOH-MWCNTs, NHE-MWCNTs, and Tau- MWCNTs） were used in the experiments. The short MWCNTs were used as pristine MWCNTs to compare with the 3 surface modified MWCNTs. The cytotoxicity was determined by cell counting kit-8 （CCK-8） assay at the concentrations of 2, 8, and 32 mg/L at hours 12, 24, 36, and 48 respectively. Single cell gel electrophoresis （SCGE） assay was performed to evaluate DNA strand breakage in A549 cells after 24 h treatment of 8 mg/L of each tested material. Results： Long multi-walled carbon nanotubes （Long- MWCNTs） and short multi-walled carbon nanotubes （Short-MWCNTs） showed a dose-dependent cytotoxicity within the exposure time 12 -48 h. Especially, Long-MWCNTs showed greater cytotoxicity than Short-MWCNTs from 24 to 48 h at the same concentration. The relative cell viability of the 3 surface modified MWCNTs was higher than that of the pristine MWCNTs at h 12 at the concentration of 32 mg/L [ COOH-MWCNTs （86.55 ±1.80） %, NH2-MWCNTs （ 84.67 ± 1.32 ） %, Tau-MWCNTs （ 80. 15 ± 3.53 ） % and Pristine-MWCNTs （71.44 ± 5.58） % ], at h 24 at the concentration of 8 mg/L [ COOH- MWCNTs （96.74 ± 1.00） %, NH2-MWCNTs （96.74 ± 3.35 ） %, Tau-MWCNTs （ 106.39 ± 3.83 ） % and Pristine-MWCNTs （91.02 ± 2.53） % ], at h 24 at the concentration of 32 mg/L [ COOH-MWCNTs （80.88 ± 2.67 ） %, NH2-MWCNTs （82.90 ± 3.25 ） %, Tau-MWCNTs （82.55 ± 3.32） % and Pristine- MWCNTs （76.08 ± 4.27）% ] and at h 36 at the concentration of 8 mg/L [ COOH-MWCNTs （96.87 ± 1.05 ） %, NH2-MWCNTs （96.66 ± 4.76） %, Tau-MWCNTs （ 100.23 ± 2.84 ） % and Pristine-MWC- NTs （ 89.61 ± 3.78 ） % ], and the differences were statistically significant （ P 〈 0.05 ）. Compared with the Pristine-MWCNTs, the relative cell viability of the 3 surface modified MWCNTs didnt demonstrate a statistically significant difference （P 〉 0.05 ） at other observation time and exposure concentrations. The DNA strand breakage of the 3 surface modified MWCNTs ： the Olive tail moment of COOH-MWCNTs was 1.56 ± 0.22, the Olive tail moment of NH2-MWCNTs 2.25 ± 1.62 and the Olive tail moment of Tau- MWCNTs 2.23 ± 0.94 ; the tail DNA% of COOH^MWCNTs was （3.96 ± 0.60） %, the tail DNA% of NH2-MWCNTs （6.16 ±4.68）% and the tail DNA% of Tau-MWCNTs （6.05±2.31 ）%, which were lower than that of the pristine MWCNTs （ P 〈 0.05 ）, whose Olive tail moment was 3.00 ± 0.64 and tail DNA% （8.23 ± 2.27）%. Moreover, the COOH-MWCNTs induced the lowest DNA damage among the three modified MWCNTs. Conclusion： Long-MWCNTs compared with Short-MWCNTs demonstrated a greater cytotoxicity and lower DNA strand breakage damage. The surface modifications of MWCNTs can reduce the cytotoxicity and DNA strand breakage in A549 cells.