纳米银和PVP包被纳米银对HepG2细胞遗传毒性的比较研究
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摘要
为了探讨纳米银对HepG2细胞DNA损伤、染色体畸变等遗传毒性指标的影响,以期为纳米银体外遗传毒性评价提供参考依据,本文采用2种纳米银材料(20 nm-PVP包被纳米银、20 nm-无包被纳米银),分别以20μg·mL~(-1)、40μg·mL~(-1)、80μg·mL~(-1)、160μg·mL~(-1)的剂量对HepG2细胞染毒24 h,用Hoechst-33258染色法检测细胞凋亡,彗星实验检测DNA损伤,胞质分裂阻滞微核细胞组学试验法检测染色体畸变。结果表明,20 nm Ag NPs组在160μg·mL~(-1)时引起细胞凋亡数显著增多(P<0.05);20 nm PVP-Ag NPs组在80μg·mL~(-1)和160μg·mL~(-1)剂量组中细胞凋亡数显著增多(P<0.01)。2种纳米银引起HepG2细胞发生细胞凋亡,并呈剂量效应关系。彗星试验结果表明,20 nm Ag NPs和20 nm PVP-Ag NPs在40μg·mL~(-1)、80μg·mL~(-1)、160μg·mL~(-1)剂量组中,Olive尾矩、尾长和尾部DNA百分比与空白对照组相比均有显著差异(P<0.05)。2种纳米银对HepG2细胞DNA损伤程度为:20 nm Ag NPs>20 nm PVP-Ag NPs。胞质分裂阻滞微核细胞组学试验结果表明,2种纳米银均不会引起核质桥数发生明显改变(P>0.05),20 nm Ag NPs在高染毒剂量下引起微核总数、I型微核、II型微核、核芽数明显升高(P<0.05);20 nm PVP-Ag NPs在各染毒剂量下均会引起微核总数及I型微核数量升高(P<0.01),II型微核数在160μg·mL~(-1)剂量下升高明显(P<0.01),剂量大于20μg·mL~(-1)时核芽数升高(P<0.01)。20 nm PVP-Ag NPs对细胞核的影响大于20 nm Ag NPs(P<0.05)。总之,2种纳米银材料均会引起HepG2细胞DNA损伤及染色体畸变等遗传毒性效应的改变,无包被纳米银比PVP包被纳米银更容易引起DNA损伤,PVP包被纳米银比无包被纳米银更容易引起细胞染色体畸变相关效应;2种材料对HepG2细胞的损伤存在浓度-效应关系,浓度越高遗传毒性损伤越严重。
The aim of this study was to explore the genetic toxicity of nano silver, such as DNA damage and chromosomal aberration, in HepG2 cells and provide a reference for the in vitro evaluation of genetic toxicity. The HepG2 cells were infected with nano silver(20 nm Ag NPs and 20 nm PVP-Ag NPs) with a series of doses of 20μg·mL~(-1), 40 μg·mL~(-1), 80 μg·mL~(-1) and 160 μg·mL~(-1) for 24 hours. The cell apoptosis was detected by the hoechst-33258 staining, the DNA damage was detected by the comet assay, and the chromosome aberration was detected by the cytoplasmic block micronuclear assay. Results showed that the number of apoptosis was significantly increased in the 160 μg·mL~(-1)20 nm Ag NPs group compared with the control(P <0.05), while the numbers of apoptotic cells in the 80 μg·mL~(-1) and 160 μg·mL~(-1)20 nm PVP-Ag NPs group were significantly increased compared to the control(P <0.05,P <0.01). Two kinds of silver nanoparticles induced apoptosis in HepG2 cells in a concentration dependent manner. In the comet test, olive tail moment, tail length and tail DNA percentage in the 40 μg·mL~(-1), 80 μg·mL~(-1) and 160 μg·mL~(-1)20 nm Ag NPs and 20 nm PVP-Ag NPs were all significantly different from those in the blank control group(P <0.05). The DNA damage degree of HepG2 cells in 20 nm Ag NPs was higher than that in 20 nm PVP-Ag NPs. In the cytoplasmic block micronucleus assay, neither of silver nanoparticles caused any significant change in the number of cytoplasmic bridge(P >0.05). However, the number of micronucleus, type I micronucleus, type II micronucleus and nucleation buds were significantly increased when cells were exposed to20 nm Ag NPs with the high exposure dose(P <0.05). For the PVP-Ag NPs exposure, the total number of micronucleus and type I micronucleus count were all increased when cells were exposed to 20 nm PVP-Ag NPs with the three doses(P <0.01). The type II micronucleus count increased significantly in 160 μg·mL~(-1)20 nm PVP-Ag NPs group(P <0.01), and the nuclear buds number increased obviously in cells treated with PVP-Ag NPs at more than20 μg·mL~(-1)(P <0.01). Generally, The toxic effects of 20 nm PVP-Ag NPs on the nucleus were severer than 20 nm Ag NPs(P <0.05). We concluded that the two types of silver nanoparticles could cause genetic toxicity to HepG2 cells, such as DNA damage and chromosomal aberration. The ability of 20 nm Ag NPs causing DNA damage was stronger than 20 nm PVP-Ag NPs, while the ability of 20 nm PVP-Ag NPs causing chromosomal aberration was stronger than 20 nm Ag NPs. Overall, the toxic effects induced by both of Ag NPs was in a dose dependent manner,which means higher concentration of nanoparticles induces severer genotoxicity damage in HepG2 cells.
The aim of this study was to explore the genetic toxicity of nano silver, such as DNA damage and chromosomal aberration, in HepG2 cells and provide a reference for the in vitro evaluation of genetic toxicity. The HepG2 cells were infected with nano silver(20 nm Ag NPs and 20 nm PVP-Ag NPs) with a series of doses of 20μg·mL~(-1), 40 μg·mL~(-1), 80 μg·mL~(-1) and 160 μg·mL~(-1) for 24 hours. The cell apoptosis was detected by the hoechst-33258 staining, the DNA damage was detected by the comet assay, and the chromosome aberration was detected by the cytoplasmic block micronuclear assay. Results showed that the number of apoptosis was significantly increased in the 160 μg·mL~(-1)20 nm Ag NPs group compared with the control(P <0.05), while the numbers of apoptotic cells in the 80 μg·mL~(-1) and 160 μg·mL~(-1)20 nm PVP-Ag NPs group were significantly increased compared to the control(P <0.05,P <0.01). Two kinds of silver nanoparticles induced apoptosis in HepG2 cells in a concentration dependent manner. In the comet test, olive tail moment, tail length and tail DNA percentage in the 40 μg·mL~(-1), 80 μg·mL~(-1) and 160 μg·mL~(-1)20 nm Ag NPs and 20 nm PVP-Ag NPs were all significantly different from those in the blank control group(P <0.05). The DNA damage degree of HepG2 cells in 20 nm Ag NPs was higher than that in 20 nm PVP-Ag NPs. In the cytoplasmic block micronucleus assay, neither of silver nanoparticles caused any significant change in the number of cytoplasmic bridge(P >0.05). However, the number of micronucleus, type I micronucleus, type II micronucleus and nucleation buds were significantly increased when cells were exposed to20 nm Ag NPs with the high exposure dose(P <0.05). For the PVP-Ag NPs exposure, the total number of micronucleus and type I micronucleus count were all increased when cells were exposed to 20 nm PVP-Ag NPs with the three doses(P <0.01). The type II micronucleus count increased significantly in 160 μg·mL~(-1)20 nm PVP-Ag NPs group(P <0.01), and the nuclear buds number increased obviously in cells treated with PVP-Ag NPs at more than20 μg·mL~(-1)(P <0.01). Generally, The toxic effects of 20 nm PVP-Ag NPs on the nucleus were severer than 20 nm Ag NPs(P <0.05). We concluded that the two types of silver nanoparticles could cause genetic toxicity to HepG2 cells, such as DNA damage and chromosomal aberration. The ability of 20 nm Ag NPs causing DNA damage was stronger than 20 nm PVP-Ag NPs, while the ability of 20 nm PVP-Ag NPs causing chromosomal aberration was stronger than 20 nm Ag NPs. Overall, the toxic effects induced by both of Ag NPs was in a dose dependent manner,which means higher concentration of nanoparticles induces severer genotoxicity damage in HepG2 cells.
引文
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[2]Li Y,Bhalli J A,Ding W,et al.Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse[J].Nanotoxicology,2014,8(Suppl 1):36-45
[3]Nallanthighal S,Chan C,Bharali J,et al.Particle coatings but not silver ions mediate genotoxicity of ingested silver nanoparticles in a mouse model[J].Nano Impact,2017,5:92-100
[4]Reed R B,Zaikova T,Barber A,et al.Potential environmental impacts and antimicrobial efficacy of silver-and nanosilver-containing textiles[J].Environmental Science&Technology,2016,50(7):4018-4026
[5]Cattaneo A G,Gornati R,Sabbioni E,et al.Nanotechnology and human health:Risks and benefits[J].Journal of Applied Toxicology,2010,30(8):730-744
[6]Charehsaz M,Hougaard K S,Sipahi H,et al.Effects of developmental exposure to silver in ionic and nanoparticle form:A study in rats[J].DARU Journal of Pharmaceutical Sciences,2016,24:24
[7]Wen H,Dan M,Yang Y,et al.Acute toxicity and genotoxicity of silver nanoparticle in rats[J].PLo S ONE,2017,12(9):1-16
[8]Tavares P,Balbinot F,De Oliveria H M,et al.Evaluation of genotoxic effect of silver nanoparticles(Ag-Nps)invitro and in vivo[J].Journal of Nanoparticle Research,2012,14(4):1-7
[9]Kwok K W,Dong W,Marinakos S M,et al.Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation[J].Nanotoxicology,2016,10(9):1306-1317
[10]Ahamed M,Karns M,Goodson M,et al.DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells[J].Toxicology and Applied Pharmacology,2008,233(3):404-410
[11]Nguyen K C,Seligy V L,Massarsky A,et al.Comparison of toxicity of uncoated and coated silver nanoparticles[J].Journal of Physics:Conference Series,2013,429:012025
[12]Hadrup N,Lam H R.Oral toxicity of silver ions,silver nanoparticles and colloidal silver—A review[J].Regulatory Toxicology and Pharmacology,2014,68:1-7
[14]Bastos V,Duarte I F,Santos C,et al.Genotoxicity of citrate-coated silver nanoparticles to human keratinocytes assessed by the comet assay and cytokinesis blocked micronucleus assay[J].Environmental Science and Pollution Researh,2017,24(5):5039-5048
[15]Souza T A,Franchi L P,Rosa L R,et al.Cytotoxicity and genotoxicity of silver nanoparticles of different sizes in CHO-K1 and CHO-XRS5 cell lines[J].Mutation Research Genetic Toxicology and Environmental Mutagenesis,2016,795:70-83
[16]Juarez-Moreno K,Gonzalez E B,Girónvazquez N,et al.Comparison of cytotoxicity and genotoxicity effects of silver nanoparticles on human cervix and breast cancer cell lines[J].Human and Experimental Toxicology,2017,36(9):931-948
[17]Sahu S C,Roy S,Zheng J,et al.Contribution of ionic silver to genotoxic potential of nanosilver in human liver Hep G2 and colon Caco2 cell evaluated by the cytokinesisblock micronucleus assay[J].Journal of Applied Toxicology,2016,36(4):532-542
[18]Papageorgiou I,Brown C,Schins R,et al.The effect of nano-and micron-sized particles of cobalt-chromium alloy on human fibroblasts in vitro[J].Biomaterials,2007,28(19):2946-2958