苏丹IV对HepG2细胞的遗传毒性及氧化性DNA损伤
摘要
前言:苏丹IV,又称猩红(Scarlet Red),是一种人工合成的偶氮类、油溶性的化工染色剂,被大量地应用在生物、化学等领域。虽然苏丹IV是一种工业染色剂,但由于其鲜艳的颜色和不易褪色的特性,印度等一些国家还允许将苏丹IV添加到辣椒粉中。
目前,国际癌症研究机构(IARC)将苏丹IV归为第三类可致癌物质,即现存证据不能就其对人类致癌性进行分类。但其潜在致癌性引起食用苏丹IV污染食物人群的恐慌。进入体内的苏丹IV主要通过胃肠道微生物还原酶、肝和肝外组织微粒体酶和细胞质的还原酶进行代谢,在体内生成相应的胺类物质。多项体外致突变试验和动物致癌试验中发现苏丹IV的致突变性和致癌性与代谢生成的胺类物质有关。苏丹IV作为一种动物致癌物,其致癌性已经在兔、大鼠身上得到了证实。但其对人类的致癌性和诱导肿瘤的机制至今仍然是不明确的。
本研究选用的是一种代谢完全的人类来源的肝肿瘤细胞系HepG2细胞,它不仅保持了人正常肝实质细胞的许多功能,还保留了一系列生物转化过程中的I相和II相酶,是检测外来化合物遗传毒性的理想细胞系。
本研究以HepG2细胞为试验系统,研究苏丹IV的遗传毒性及氧化性DNA损伤机制,以期为评价苏丹IV对人类的致癌危险性提供实验室依据。
方法:以HepG2细胞为检测系统。用单细胞凝胶电泳(SCGE)检测DNA链断裂,微核试验(MNT)检测细胞染色体损伤。为了阐明苏丹IV对HepG2细胞的遗传毒性机制,我们用细胞内2’,7-二氢二氯荧光素(DCFH-DA)法测定细胞内活性氧(ROS)的浓度,用免疫过氧化酶染色的方法检测细胞内8-羟基脱氧鸟苷(8-OHdG)的水平。通过加入谷胱苷肽(GSH)合成抑制剂2-氨基4-(S-丁基磺酰亚氨)(BSO)和GSH合成前体物N-乙酰半胱氨酸(NAC),来调节细胞内GSH的水平,再经苏丹IV染毒,用SCGE技术测定此时HepG2细胞彗星尾部DNA%。最后,我们还用MTT试验检测BSO预处理后苏丹IV肝细胞毒性。
结果:25-100μM苏丹IV与HepG2细胞接触1h后,引起细胞内DNA链的断裂,荧光显微镜下显示细胞呈现彗星样拖尾,其尾长和尾部DNA的百分含量明显增加,且呈剂量依赖关系。细胞内的微核发生率随着苏丹IV剂量的增加而增多,细胞内微核发生率与对照组相比其差异具有统计学意义(P<0.01)。50-100μM的苏丹IV作用于HepG2细胞1h后,细胞内ROS水平与对照组相比其差异具有统计学意义(P<0.05)。HepG2细胞与苏丹红IV接触3h后,细胞内8-OHdG表达水平在所有剂量组均比对照组明显(P<0.05或P<0.01),且呈剂量依赖关系。BSO预处理后可使苏丹IV对肝细胞DNA链断裂更明显,预处理后的各个剂量组尾部DNA (%)均比未预处理组的数值明显增高,其差别具有统计学意义(P<0.05)。而用NAC处理的HepG2细胞各剂量组尾部DNA(%)则明显低于未处理组(P<0.05)。MTT试验显示BSO预处理后可使苏丹IV的肝细胞毒性明显增强(P<0.05)。
结论:
苏丹IV(25-100μM)可引起HepG2细胞DNA链断裂和微核发生率增高,表明苏丹红IV对HepG2有明显的遗传毒性。本研究还发现苏丹IV细胞内ROS的大量生成和GSH的减少,破坏细胞内氧化还原平衡,引起HepG2细胞氧化应激,从而导致细胞内DNA的氧化性损伤,这可能是苏丹IV致HepG2细胞遗传毒性的重要机制。
Introduction: Sudan IV (C24H20N4O), a synthetic lipid soluble azo pigment, is widely used in various industrial fields. They had also been adopted for coloring various food- stuffs, particularly in those containing chilli powders, because of their intense dark red color.
In 1975, the international agency for research on cancer (IARC) assessed Sudan IV as group 3 carcinogen, for which the evidence of carcinogenicity was inadequate in humans and inadequate or limited in experimental animals. Sudan IV is mutagenic, but only after chemical reduction and metabolic activation. Reduction can take place by means of reductase of the gastrointestinal microflora and also through microsomal and cytosolic reductase of the liver and extra-hepatic tissues. The toxic and/or carcinogenic effects of Sudan IV in the gut or liver are possibly due to the products into which they are degrades. Sudan IV is a liver-specific toxin known as a carcinogen in experimental animals. but neither the relevance of these tumours to humans nor their mechanisms of induction is clear.
In this study, we selected a metabolically competent human hepatoma line (HepG2), which retains many of the functions of normal liver cells and expresses the activities of several phase I and phase II xenobiotic metabolizing enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.
The objective of the study was to assess the genotoxic effects of Sudan IV in vitro and to elucidate the mechanisms in these cells. Thus it may provide some information for safety assessment to humans on Sudan IV.
Methods: HepG2 cells were selected as test system. DNA-strand breaks in HepG2 cells were evaluated by single cell gel electrophoresis assay (SCGE). Micronucleus test (MNT) which reflects chromosome breakage and/or chromosome loss was applied. The level of the oxidative production 8-Hydroxydexyguanosine (8-OHdG) was measured using immunoperoxidase staining. The level of intracellular ROS was monitored with 2’, 7-dichlorofluorescein diacetate (DCFH-DA) assay. To explore the role of GSH in Sudan IV-induced DNA damage, the intracellular GSH level in HepG2 cells was modulated with buthionine-(S,R)-sulfoximine(BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor ,and the effects of GSH on Sudan IV-induced DNA damage were determined by the comet assay. Furthermore, the effect of GSH depletion on cytotoxicity of Sudan IV in HepG2 cells was examined by the cell viability assay.
Results: In the SCGE and MNT, a dose-dependent increase of DNA migration and of the MN frequencies was found after treatment with the test compound. Sudan IV (25-100μM) causes a significant increase in DNA damage of HepG2 cells for 1h. Twenty-four hours exposure of the cells to Sudan IV (50-100μM) results in a significant increase in the MN frequencies. The formation of intracellular ROS was significantly increased in Sudan IV-treated cells exposed to concentration (50 and 100μM) for 1 h. Sudan IV at the dose (12.5-100μM) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells for 3 h. It was also found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to Sudan IV-induced DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by Sudan IV was almost completely prevented.
Conclusion:
Based on these data we believe that Sudan IV exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.
目前,国际癌症研究机构(IARC)将苏丹IV归为第三类可致癌物质,即现存证据不能就其对人类致癌性进行分类。但其潜在致癌性引起食用苏丹IV污染食物人群的恐慌。进入体内的苏丹IV主要通过胃肠道微生物还原酶、肝和肝外组织微粒体酶和细胞质的还原酶进行代谢,在体内生成相应的胺类物质。多项体外致突变试验和动物致癌试验中发现苏丹IV的致突变性和致癌性与代谢生成的胺类物质有关。苏丹IV作为一种动物致癌物,其致癌性已经在兔、大鼠身上得到了证实。但其对人类的致癌性和诱导肿瘤的机制至今仍然是不明确的。
本研究选用的是一种代谢完全的人类来源的肝肿瘤细胞系HepG2细胞,它不仅保持了人正常肝实质细胞的许多功能,还保留了一系列生物转化过程中的I相和II相酶,是检测外来化合物遗传毒性的理想细胞系。
本研究以HepG2细胞为试验系统,研究苏丹IV的遗传毒性及氧化性DNA损伤机制,以期为评价苏丹IV对人类的致癌危险性提供实验室依据。
方法:以HepG2细胞为检测系统。用单细胞凝胶电泳(SCGE)检测DNA链断裂,微核试验(MNT)检测细胞染色体损伤。为了阐明苏丹IV对HepG2细胞的遗传毒性机制,我们用细胞内2’,7-二氢二氯荧光素(DCFH-DA)法测定细胞内活性氧(ROS)的浓度,用免疫过氧化酶染色的方法检测细胞内8-羟基脱氧鸟苷(8-OHdG)的水平。通过加入谷胱苷肽(GSH)合成抑制剂2-氨基4-(S-丁基磺酰亚氨)(BSO)和GSH合成前体物N-乙酰半胱氨酸(NAC),来调节细胞内GSH的水平,再经苏丹IV染毒,用SCGE技术测定此时HepG2细胞彗星尾部DNA%。最后,我们还用MTT试验检测BSO预处理后苏丹IV肝细胞毒性。
结果:25-100μM苏丹IV与HepG2细胞接触1h后,引起细胞内DNA链的断裂,荧光显微镜下显示细胞呈现彗星样拖尾,其尾长和尾部DNA的百分含量明显增加,且呈剂量依赖关系。细胞内的微核发生率随着苏丹IV剂量的增加而增多,细胞内微核发生率与对照组相比其差异具有统计学意义(P<0.01)。50-100μM的苏丹IV作用于HepG2细胞1h后,细胞内ROS水平与对照组相比其差异具有统计学意义(P<0.05)。HepG2细胞与苏丹红IV接触3h后,细胞内8-OHdG表达水平在所有剂量组均比对照组明显(P<0.05或P<0.01),且呈剂量依赖关系。BSO预处理后可使苏丹IV对肝细胞DNA链断裂更明显,预处理后的各个剂量组尾部DNA (%)均比未预处理组的数值明显增高,其差别具有统计学意义(P<0.05)。而用NAC处理的HepG2细胞各剂量组尾部DNA(%)则明显低于未处理组(P<0.05)。MTT试验显示BSO预处理后可使苏丹IV的肝细胞毒性明显增强(P<0.05)。
结论:
苏丹IV(25-100μM)可引起HepG2细胞DNA链断裂和微核发生率增高,表明苏丹红IV对HepG2有明显的遗传毒性。本研究还发现苏丹IV细胞内ROS的大量生成和GSH的减少,破坏细胞内氧化还原平衡,引起HepG2细胞氧化应激,从而导致细胞内DNA的氧化性损伤,这可能是苏丹IV致HepG2细胞遗传毒性的重要机制。
Introduction: Sudan IV (C24H20N4O), a synthetic lipid soluble azo pigment, is widely used in various industrial fields. They had also been adopted for coloring various food- stuffs, particularly in those containing chilli powders, because of their intense dark red color.
In 1975, the international agency for research on cancer (IARC) assessed Sudan IV as group 3 carcinogen, for which the evidence of carcinogenicity was inadequate in humans and inadequate or limited in experimental animals. Sudan IV is mutagenic, but only after chemical reduction and metabolic activation. Reduction can take place by means of reductase of the gastrointestinal microflora and also through microsomal and cytosolic reductase of the liver and extra-hepatic tissues. The toxic and/or carcinogenic effects of Sudan IV in the gut or liver are possibly due to the products into which they are degrades. Sudan IV is a liver-specific toxin known as a carcinogen in experimental animals. but neither the relevance of these tumours to humans nor their mechanisms of induction is clear.
In this study, we selected a metabolically competent human hepatoma line (HepG2), which retains many of the functions of normal liver cells and expresses the activities of several phase I and phase II xenobiotic metabolizing enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.
The objective of the study was to assess the genotoxic effects of Sudan IV in vitro and to elucidate the mechanisms in these cells. Thus it may provide some information for safety assessment to humans on Sudan IV.
Methods: HepG2 cells were selected as test system. DNA-strand breaks in HepG2 cells were evaluated by single cell gel electrophoresis assay (SCGE). Micronucleus test (MNT) which reflects chromosome breakage and/or chromosome loss was applied. The level of the oxidative production 8-Hydroxydexyguanosine (8-OHdG) was measured using immunoperoxidase staining. The level of intracellular ROS was monitored with 2’, 7-dichlorofluorescein diacetate (DCFH-DA) assay. To explore the role of GSH in Sudan IV-induced DNA damage, the intracellular GSH level in HepG2 cells was modulated with buthionine-(S,R)-sulfoximine(BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor ,and the effects of GSH on Sudan IV-induced DNA damage were determined by the comet assay. Furthermore, the effect of GSH depletion on cytotoxicity of Sudan IV in HepG2 cells was examined by the cell viability assay.
Results: In the SCGE and MNT, a dose-dependent increase of DNA migration and of the MN frequencies was found after treatment with the test compound. Sudan IV (25-100μM) causes a significant increase in DNA damage of HepG2 cells for 1h. Twenty-four hours exposure of the cells to Sudan IV (50-100μM) results in a significant increase in the MN frequencies. The formation of intracellular ROS was significantly increased in Sudan IV-treated cells exposed to concentration (50 and 100μM) for 1 h. Sudan IV at the dose (12.5-100μM) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells for 3 h. It was also found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to Sudan IV-induced DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by Sudan IV was almost completely prevented.
Conclusion:
Based on these data we believe that Sudan IV exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.
引文
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20.王捷忠,李云英,郑步宏.安多霖对食管癌放疗中扶正、升白作用临床观察.海峡药学,1999, 11(3):78
21.刘倩,秦莲,安一鹏.阿多拉胶囊降低飞行员高微核率临床研究.中华航空医学杂志,1997,8(4):238
22.洪振丰,刘凌冰.乌龙茶降低氟化钠和三氧化二砷诱发的小鼠骨髓细胞微核率的研究.遗传, 1990, 12(3):25-26
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26. Bolognesi C, Landini E, Perrone E ,Roqqieri P, Cytogenetic biomonitoring of a floriculturist population in Italy: micronucleus analysis by fluorescence in situ hybridization (FISH) with an all-chromosome centromeric probe. Mut. Res.,2004, 557(2): 109-l17
27. Benova D, Hadjidekova V, Hristovar R, Cytogenetic effects of hexavalent chromium in Bulgarian hromium platers[J].Mut. Res., 2002,514 (122): 29-38
28. Schmit TE, Lehmann L, Metaler M, et Al. Hormonal and genotoxic activity of resveratrol . Toxicol. Lett., 2002,136: 13-32
29. Their R, Bonacker D,Stoiber T, Bohm KJ, Wang M, Unqer E, Bolt HM, Deqen G .Interaction of metal salts with cytoskeletal motor protein systems. Toxicol. Lett., 2003, 140-141(11): 75-81
30. Hayashi M, MacGreqor JT, Gatehouse DG, Adler ID, Blakey DH, Dertinqer SD, Krishna G, Morita T, Russo A, Sutou S. In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. Environ. Mol. Mutagen,2000,35(3):234-252
31.李欣,黄俊明. Crest染色法在毒理学检测中的应用.毒理学杂志,2005, 19(3): 229-234
32. Ma TH, Xu J, Xia W, Jong X, Sun W, Lin G, Proficiency of the Tradescantia-micronucleus image analysis system for scoring micronucleus frequencies and data analysis. Mutat. Res., 1992, 270(1): 39-40
33. Weaver JL, Torous D, Flow cytometry assay for counting micronucleated erythrocytes: development process. Methods, 2000, 21(3):281-287
34. Kawamura K, Fujikawa-Yamamoto K, Ozaki M, Iwabuchi K, Nakashima H, Domiki C,Morita N,Inoue M,Tokunaga K,Shiba N, Ikeda R,Suzuki K, Centrosome hyperamplification and chromosomal damage after exposure to radiation. Oncol.,2004, 67(5-6): 460-470
35. Pozarowski P, Holden E, Darzynkiewicz Z , Laser scanning cytometry: principles and applications. Methods Mol. Biol., 2006, 319: 165-192
36. Sadowska A, Pluygers E, Narkiewicz M, Pawelczak A, Lata B. Eur J Cancer Prev., 1994, 3(1): 69-78
37. Banduhn N, Obe G, Mutagenicity of methyl 2-benzimidazolecarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies and micronuclei. Mutat.Res.,1985, 156(3): 199-218
38. Prosser JS, Moquet JE, Liovd DC, Edwards AA, Radiation induction of micronuclei in human lymphocytes. Mutat. Res., 1988 199(1): 37-45
39. Waters MD, Brady AL, Stack HF, Brockman HE. Antimutagenicity profiles for some model compounds. Mutat.Res., 1990, 238(1): 57-85