氧化应激在丙烯醛致人肝癌细胞HepG2 DNA损伤中的作用
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摘要
目的:丙烯醛广泛的存在于自然环境中,特别是吸烟所产生的烟雾中含有大量丙烯醛,丙烯醛还是抗癌药环磷酰胺的代谢产物,在脂质的氧化过程中也可以产生丙烯醛。
国际癌症研究机构(International Agency for Research on Cancer, IARC)认为,目前尚无充分的证据表明丙烯醛对实验动物具有致癌性。另外,越来越多的证据显示丙烯醛具有遗传毒性。大量研究显示,丙烯醛在多种细胞系中可以引起活性氧(ROS)的产生,同时体内体外实验均显示丙烯醛能导致谷胱甘肽(GSH)含量的下降。丙烯醛的遗传毒性已经在很多细胞系中得到证实,但迄今尚未见到关于丙烯醛致人肝癌细胞系(HepG2)DNA损伤的研究报道。HepG2细胞保留了生物转化代谢I相酶和II相酶的活性,它被认为是检测外来化合物遗传毒性的一个理想细胞系。
本研究选用HepG2细胞作为试验系统,探讨丙烯醛的DNA损伤作用及可能的机制,旨在为评估丙烯醛对人类健康的危害性提供有价值的实验室依据。
方法:试验系统为HepG2细胞系。通过标准的以及蛋白酶K改良的单细胞微凝胶电泳(SCGE)试验检测细胞DNA损伤情况。为了阐明HepG2细胞中的氧化性DNA损伤机制,通过2',7'—二氢二氯荧光素(DCFH)和苯二醛(OPT)分别测定细胞内ROS以及GSH水平。用免疫组化方法测定8-羟基脱氧鸟苷(8-OHdG)在细胞内的表达水平。我们还研究了抗氧化物质N-乙酰半胱氨酸(NAC)的保护作用以进一步评价ROS以及GSH在丙烯醛所致的DNA链断裂中的作用。
结果:当低浓度的丙烯醛(12.5-25μM)作用于细胞后,DNA的迁移距离明显增加,且呈剂量依赖关系。而当较高浓度的丙烯醛(50-100μM)作用于细胞后,DNA的迁移距离与丙烯醛在25μM时的最大迁移量相比明显缩短。暴露较高浓度丙烯醛(50-100μM)的细胞,经蛋白酶K处理后与未经蛋白酶K处理的细胞相比,DNA的迁移距离明显增加。这些结果提示,丙烯醛在较低浓度引起细胞DNA链断裂,而在较高浓度导致DNA-蛋白质交联(DPC)的形成。丙烯醛作用于HepG2细胞可引起细胞内ROS表达水平的明显增加以及GSH的耗竭,其作用剂量分别是50-100μM和25-100μM。此外,25 -100μM的丙烯醛可以明显增加HepG2细胞内8-OHdG水平。NAC(GSH的前体和细胞内ROS的清除剂)能够拮抗丙烯醛引起的DNA链断裂的形成。
结论:丙烯醛可致HepG2细胞DNA损伤,其作用机制可能是通过ROS的增高以及细胞内GSH的耗竭,进而导致氧化性DNA损伤、DNA链断裂以及DPC的形成。
Objective: Acrolein is found widely in the environment, particularly as a component of smoke. Acrolein can generate endogeneously as a metabolic product of the anticancer-drug cyclophosphamide and during conditions of lipid oxidation.
Acrolein is an intense irritant and displays a range of toxic effects. The International Agency for Research on Cancer (IARC) concluded that there was inadequate evidence for its carcinogenicity in experimental animals. In addition, there is increasing evidence that acrolein is genotoxic. Previous studies demonstrated that acrolein was able to induce the generation of reactive oxygen species (ROS) in some cell types. In addition, both in vitro and in vivo studies showed that acrolein caused a significant reduction of intracellular GSH. The genotoxic effects of acrolein have already been demonstrated in many cell lines. However, this is the first evidence of acrolein-induced DNA damage in human hepatoma line (HepG2). HepG2 cells retain the activities of several phase I and II xenobiotic metabolizing enzymes presented in human hepatocytes. It has been shown to be a suitable system for investigation of genotoxicity.
The overall object of present study is to explore whether acrolein causes DNA damage in HepG2 cells and to elucidate the underlying mechanism of acrolein-induced DNA damage. Thus it may provide some information for safety assessment to humans on acrolein.
Method: DNA damage induced by acrolein was assessed by standard and proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. To elucidate the oxidative DNA damage mechanism in HepG2 cells, we used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH). We analyzed the oxidative DNA damage in acrolein-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG). To further evaluate the involvements of ROS and GSH in the formation of acrolein-induced DNA strand breaks, we studied the protective effect of NAC.
Result: Using the standard SCGE assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein (12.5- 25μM); but at the higher tested concentrations (50-100μM), a reduction in the migration compared to the maximum migration at 25μM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein (50-100μM). These results indicated that acrolein caused DNA strand breaks at lower concentrations of acrolein and DNA-protein crosslinks (DPC) formation at higher concentrations. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 50-100μM and 25-100μM, respectively. Moreover, acrolein significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 25 to 100μM. N-acetylcysteine (NAC), a precursor of GSH and intracellular ROS scavenger, prevented the formation of DNA strand breaks caused by acrolein.
Conclusion: we conclude that the DNA damage of acrolein is mediated by the formation of ROS and depletion of GSH, which cause oxidative DNA damage, formation of DNA strand breaks and DPC.
国际癌症研究机构(International Agency for Research on Cancer, IARC)认为,目前尚无充分的证据表明丙烯醛对实验动物具有致癌性。另外,越来越多的证据显示丙烯醛具有遗传毒性。大量研究显示,丙烯醛在多种细胞系中可以引起活性氧(ROS)的产生,同时体内体外实验均显示丙烯醛能导致谷胱甘肽(GSH)含量的下降。丙烯醛的遗传毒性已经在很多细胞系中得到证实,但迄今尚未见到关于丙烯醛致人肝癌细胞系(HepG2)DNA损伤的研究报道。HepG2细胞保留了生物转化代谢I相酶和II相酶的活性,它被认为是检测外来化合物遗传毒性的一个理想细胞系。
本研究选用HepG2细胞作为试验系统,探讨丙烯醛的DNA损伤作用及可能的机制,旨在为评估丙烯醛对人类健康的危害性提供有价值的实验室依据。
方法:试验系统为HepG2细胞系。通过标准的以及蛋白酶K改良的单细胞微凝胶电泳(SCGE)试验检测细胞DNA损伤情况。为了阐明HepG2细胞中的氧化性DNA损伤机制,通过2',7'—二氢二氯荧光素(DCFH)和苯二醛(OPT)分别测定细胞内ROS以及GSH水平。用免疫组化方法测定8-羟基脱氧鸟苷(8-OHdG)在细胞内的表达水平。我们还研究了抗氧化物质N-乙酰半胱氨酸(NAC)的保护作用以进一步评价ROS以及GSH在丙烯醛所致的DNA链断裂中的作用。
结果:当低浓度的丙烯醛(12.5-25μM)作用于细胞后,DNA的迁移距离明显增加,且呈剂量依赖关系。而当较高浓度的丙烯醛(50-100μM)作用于细胞后,DNA的迁移距离与丙烯醛在25μM时的最大迁移量相比明显缩短。暴露较高浓度丙烯醛(50-100μM)的细胞,经蛋白酶K处理后与未经蛋白酶K处理的细胞相比,DNA的迁移距离明显增加。这些结果提示,丙烯醛在较低浓度引起细胞DNA链断裂,而在较高浓度导致DNA-蛋白质交联(DPC)的形成。丙烯醛作用于HepG2细胞可引起细胞内ROS表达水平的明显增加以及GSH的耗竭,其作用剂量分别是50-100μM和25-100μM。此外,25 -100μM的丙烯醛可以明显增加HepG2细胞内8-OHdG水平。NAC(GSH的前体和细胞内ROS的清除剂)能够拮抗丙烯醛引起的DNA链断裂的形成。
结论:丙烯醛可致HepG2细胞DNA损伤,其作用机制可能是通过ROS的增高以及细胞内GSH的耗竭,进而导致氧化性DNA损伤、DNA链断裂以及DPC的形成。
Objective: Acrolein is found widely in the environment, particularly as a component of smoke. Acrolein can generate endogeneously as a metabolic product of the anticancer-drug cyclophosphamide and during conditions of lipid oxidation.
Acrolein is an intense irritant and displays a range of toxic effects. The International Agency for Research on Cancer (IARC) concluded that there was inadequate evidence for its carcinogenicity in experimental animals. In addition, there is increasing evidence that acrolein is genotoxic. Previous studies demonstrated that acrolein was able to induce the generation of reactive oxygen species (ROS) in some cell types. In addition, both in vitro and in vivo studies showed that acrolein caused a significant reduction of intracellular GSH. The genotoxic effects of acrolein have already been demonstrated in many cell lines. However, this is the first evidence of acrolein-induced DNA damage in human hepatoma line (HepG2). HepG2 cells retain the activities of several phase I and II xenobiotic metabolizing enzymes presented in human hepatocytes. It has been shown to be a suitable system for investigation of genotoxicity.
The overall object of present study is to explore whether acrolein causes DNA damage in HepG2 cells and to elucidate the underlying mechanism of acrolein-induced DNA damage. Thus it may provide some information for safety assessment to humans on acrolein.
Method: DNA damage induced by acrolein was assessed by standard and proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. To elucidate the oxidative DNA damage mechanism in HepG2 cells, we used the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH). We analyzed the oxidative DNA damage in acrolein-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG). To further evaluate the involvements of ROS and GSH in the formation of acrolein-induced DNA strand breaks, we studied the protective effect of NAC.
Result: Using the standard SCGE assay, a significant dose-dependent increment in DNA migration was detected at lower concentrations of acrolein (12.5- 25μM); but at the higher tested concentrations (50-100μM), a reduction in the migration compared to the maximum migration at 25μM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of acrolein (50-100μM). These results indicated that acrolein caused DNA strand breaks at lower concentrations of acrolein and DNA-protein crosslinks (DPC) formation at higher concentrations. The present study showed that acrolein induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 50-100μM and 25-100μM, respectively. Moreover, acrolein significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 25 to 100μM. N-acetylcysteine (NAC), a precursor of GSH and intracellular ROS scavenger, prevented the formation of DNA strand breaks caused by acrolein.
Conclusion: we conclude that the DNA damage of acrolein is mediated by the formation of ROS and depletion of GSH, which cause oxidative DNA damage, formation of DNA strand breaks and DPC.
引文
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