二甲基甲酰胺对HL7702肝细胞毒性及线粒体膜电位的影响
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摘要
目的观察二甲基甲酰胺(DMF)对HL7702肝细胞的细胞活力、凋亡及线粒体膜电位的影响,探讨DMF引起肝细胞凋亡的可能机制。方法 HL7702肝细胞给予50、100、150、200和250 mmol/L DMF处理,于24 h后检测其细胞存活率,乳酸脱氢酶(LDH)释放量。根据细胞毒性测试结果设置对照组及DMF染毒组(200 mmol/L)。流式细胞术检测DMF对HL7702细胞凋亡以及对线粒体膜电位的影响。结果 DMF剂量依赖性地降低HL7702细胞的存活率,其中150、200和250 mmol/L DMF组与对照组相比,细胞存活率明显降低;并且,随着DMF染毒浓度增加,释放至细胞外的LDH的含量增加,与DMF浓度呈正相关。200 mmol/L DMF染毒24 h后,与对照组相比,细胞凋亡率增加,差异有统计学意义(P<0.001),线粒体膜电位降低,差异也有统计学意义(P<0.001)。结论 DMF对HL7702细胞的毒性作用具有剂量依赖性,DMF引起HL7702线粒体膜电位的改变与DMF导致细胞凋亡有关。
Objective To investigate the cytotoxicity and mitochondria damage of N,N-dimethylformamide( DMF) to HL7702 cells. Methods HL7702 cells were treated with 50,100,150,200 or 250 mmol/L DMF for 24 hours. Cell toxicities of DMF to HL7702 cells were tested by MTT and Lactate dehydrogenase( LDH) releasing assays. According to theresult of cytotoxicity,normal control group and DMF( 200 mmol/L) group were set in this study. Cell apoptosis and mitochondria membrane potential were detected using flow cytometry.Result The cell viability was inhibited in a dose dependent manner. Compared with control group,the cell growth in 150,200,and250 mmol/L DMF groups were significantly decreased. The level of LDH in medium was increased significantly with the concentration increasing,and it displayed a dose dependent manner. Compared with control group,cell apoptosis rate in 200 mmol/L DMF group was significantly increased( P < 0. 001),while the mitochondrial membrane potential was significantly decreased( P < 0. 001). Conclusion DMF resulted in liver cell toxicity in a dose dependent manner,and DMF caused cell apoptosis via reducing the mitochondrial membrane potential.
Objective To investigate the cytotoxicity and mitochondria damage of N,N-dimethylformamide( DMF) to HL7702 cells. Methods HL7702 cells were treated with 50,100,150,200 or 250 mmol/L DMF for 24 hours. Cell toxicities of DMF to HL7702 cells were tested by MTT and Lactate dehydrogenase( LDH) releasing assays. According to theresult of cytotoxicity,normal control group and DMF( 200 mmol/L) group were set in this study. Cell apoptosis and mitochondria membrane potential were detected using flow cytometry.Result The cell viability was inhibited in a dose dependent manner. Compared with control group,the cell growth in 150,200,and250 mmol/L DMF groups were significantly decreased. The level of LDH in medium was increased significantly with the concentration increasing,and it displayed a dose dependent manner. Compared with control group,cell apoptosis rate in 200 mmol/L DMF group was significantly increased( P < 0. 001),while the mitochondrial membrane potential was significantly decreased( P < 0. 001). Conclusion DMF resulted in liver cell toxicity in a dose dependent manner,and DMF caused cell apoptosis via reducing the mitochondrial membrane potential.
引文
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[11]房云,杨锦蓉,王菁,等.MTT法观察DMF致人肝细胞增殖抑制作用[J].浙江预防医学,2006,18(11):1-2,9.
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[14]路艳艳,宣志强,李雪芝,等.二甲基甲酰胺致人肝细胞凋亡及其对Bcl-2及Bax和Caspase-3表达的影响[J].中华劳动卫生职业病杂志,2008,26(5):276-279.
[15]Zhao CQ,Zhang YH,Jiang SD,et al.Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats[J].Age,2010,32(2):161-177.
[16]Colaco R,Moreno N,Feijo JA.On the fast lane:mitochondria structure,dynamics and function in growing pollen tubes[J].J microsc,2012,247(1):106-118.
[2]Kim TH,Kim SG.Clinical outcomes of occupational exposure to n,n-dimethylformamide:perspectives from experimental toxicology[J].Saf Health Work,2011,2(2):97-104.
[3]Miyauchi H,Tanaka S,Nomiyama T,et al.N,NDimethylformamide(DMF)vapor absorption through the skin in workers[J].J Occup Health,2001,43(2):92-94.
[4]钱亚玲,徐承敏,朱丽瑾,等.二甲基甲酰胺对接触工人肝功能的影响[J].中华劳动卫生职业病杂,2007,25(2):80-83.
[5]徐承敏,钱亚玲,朱丽瑾,等.GSTT1及GSTM1和CYP2E1基因多态性与二甲基甲酰胺所致肝脏损害的关系[J].中华劳动卫生职业病杂志,2009,27(6):333-337.
[6]Amato G,Grasso E,Longo V,et al.Oxidation of N,Ndimethylformamide and N,N-diethylformamide by human liver microsomes and human recombinant P450s[J].Toxicol Lett,2011,24(1-3):11-19.
[7]Nomiyama T,Haufroid V,Buchet JP,et al.Insertion polymorphism of CYP2E1 and urinary N-methylformamide after N,N-dimethylformamide exposure in Japanese workers[J].Int Arch Occup Environ Health,2001,74(7):519-522.
[8]Mraz J,Jheeta P,Gescher A,et al.Investigation of the mechanistic basis of N,N-dimethylformamide toxicity.Metabolism of N,N-dimethylformamide and its deuterated isotopomers by cytochrome P450 2E1[J].Chem Res Toxicol,1993,6(2):197-207.
[9]印倩,张文华,雷建华.二甲基甲酰胺(DMF)的亚急性毒性试验[J].基础医学与临床,1984,4(1):34.
[10]路艳艳,杨锦蓉,王菁,等.AO/EB染色法及流式细胞术检测DMF诱导人肝细胞凋亡[J].浙江预防医学,2007,19(6):11-14.
[11]房云,杨锦蓉,王菁,等.MTT法观察DMF致人肝细胞增殖抑制作用[J].浙江预防医学,2006,18(11):1-2,9.
[12]Savitskaya MA,Onishchenko GE.Mechanisms of apootosis[J].Biochemistry(Mosc),2015,8(11):1393-1405.
[13]石静,卿晨.线粒体膜电位改变与细胞凋亡[J].中国民族民间医药,2011,20(7):20-21.
[14]路艳艳,宣志强,李雪芝,等.二甲基甲酰胺致人肝细胞凋亡及其对Bcl-2及Bax和Caspase-3表达的影响[J].中华劳动卫生职业病杂志,2008,26(5):276-279.
[15]Zhao CQ,Zhang YH,Jiang SD,et al.Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats[J].Age,2010,32(2):161-177.
[16]Colaco R,Moreno N,Feijo JA.On the fast lane:mitochondria structure,dynamics and function in growing pollen tubes[J].J microsc,2012,247(1):106-118.