氢醌对HepG2细胞的遗传毒性及氧化性DNA损伤
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摘要
目的:氢醌(1,4-dihydroxybenzene, HQ)被广泛应用于各种工业领域,也被用于非处方药(OTC)中,还可作为皮肤美白剂的成分;它又是一种天然成分,存在于许多植物源性产品中。HQ可通过环境、职业、饮食和香烟烟雾等方式暴露于人体,也可来源于苯的暴露。2001年,欧盟国家禁止将HQ用于化妆品的美白配方中,而HQ的类似物熊果苷和植物源性的HQ仍可继续使用。
早在1999年,国际癌症研究机构(International Agency for Research on Cancer, IARC)就将HQ归为第三类致癌物,即现有证据尚不能就其对人类致癌性进行分类。体外实验表明,HQ可引起染色体突变。HQ通过多种途径代谢,肝脏是HQ代谢的最初部位,其代谢过程中可产生活性氧(ROS)。因此,我们试探论HQ的遗传毒性,是否与ROS引起的氧化性DNA损伤有关。本研究选用一种代谢完全的人类来源的肝肿瘤细胞株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。
本研究选用HepG2细胞,研究HQ的遗传毒性及氧化性DNA损伤的机制,为评估HQ对人类的危害提供实验室资料。
方法:以HepG2细胞作为试验系统。通过标准的以及改良的蛋白酶K单细胞凝胶电泳(SCGE)试验检测细胞DNA链损伤,微核试验(MNT)检测细胞染色体损伤情况。为探讨遗传毒性的可能机制,以2',7'—二氢二氯荧光素(DCFH)法和苯二醛(OPT)分别测定细胞内ROS以及谷胱甘肽(GSH)水平,还通过免疫组化方法测定8-羟脱氧鸟苷(8-OHdG)在细胞内的表达水平。
结果:6.25、12.5、25、50μM的HQ作用HepG2细胞1h后,DNA链断裂细胞形成彗星样拖尾,尾长、尾DNA含量及尾距增大,与未用HQ处理的细胞比较,其差异有统计学意义(P<0.01)。低浓度HQ(6.25～25μM)处理的细胞,DNA的迁移距离明显增加,且呈剂量依赖关系,而当较高浓度HQ(50μM)处理的细胞,DNA的迁移距离与HQ在25μM时的最大迁移量相比明显缩短。接受较高浓度HQ(50μM)作用的细胞,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,HQ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。HepG2细胞与12.5～50μM?的HQ接触24h后,细胞微核率明显高于未用HQ处理细胞(P<0.01)。HQ作用于HepG2细胞1h,其浓度为25～50μM和6.25～50μM,可分别引起细胞内ROS表达水平的明显增加以及细胞内GSH的耗竭。12.5～50μM?的HQ作用于HepG2细胞3h后,细胞内8-OHdG水平的表达增强,与对照组比较,其差别具有统计学意义。
结论:HQ可引起HepG2细胞DNA链断裂和染色体损伤,表明HQ对HepG2细胞具有遗传毒性。HQ对HepG2细胞的遗传毒效应,可能与ROS的增高,细胞内GSH的耗竭及8-OHdG形成的增加而造成的氧化性DNA损伤和DPC有关。
Objective: Hydroquinone (1, 4-dihydroxybenzene, HQ) is widely used in various industrial fields. HQ and products containing HQ are used in over-the-counter (OTC) drugs as an ingredient in skin lighteners and also a natural ingredient in many plant-derived products. Human exposure to HQ can occur by environmental, occupational, dietary and cigarette smoke exposure and from exposure to benzene, which can be metabolized to HQ. In 2001, HQ was banned in cosmetic skin lightening formulations in European Union countries although products containing arbutin being an analogue of HQ and botanicals including plants that naturally contain HQ and arbutin, continued to remain available in European countries.
In 1999 the International Agency for Research on Cancer (IARC), assessed Sudan I as a Group 3 carcinogen. HQ was mutagenic in many mammalian cells in vitro using a variety of end- points. HQ is metabolized by several routes, reactive oxygen species (ROS) are produced. The liver is the initial site of HQ metabolism. The aim of this study was to assess the genotoxic effects of HQ in vitro and to elucidate the mechanism of oxidative DNA damage. 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 metabolising enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.
The purpose of this study was to assess the genotoxic effects of HQ in vitro and to illuminate the mechanisms in HepG2 cells. Thus it may provide some information for occupational hazard assessment to humans on HQ.
Methods: HepG2 cells were selected as test system. Genotoxicity of HQ was assessed by standard and proteinase K-modified alkaline single cell gel electrophoresis (SCGE) and micronucleus test (MNT). To further investigate the mechanism of genotoxicity of HQ 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 HQ-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG).
Results: The damage to DNA-strand of HepG2 cells significantly increased after exposure to 6.25, 12.5, 25, 50μM HQ for 1h. A significant dose-dependent increment in DNA migration was detected at lower concentrations of HQ (6.25 - 25μM); but at the higher tested concentrations (50μM), 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 HQ (50μM). These results indicated that HQ caused DNA strand breaks at lower concentrations of HQ (6.25 - 25μM) and DNA-protein crosslinks (DPC) formation at higher concentrations. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 12.5-50μM ?HQ for 24h. The present study showed that ?HQ induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 25-50μM and 6.25-50μM, respectively. Moreover, HQ significantly caused 8-OHdG formation in HepG2 cells at concentrations from 12.5 to 50μM.
Conclusion: The data suggest that HQ caused DNA strand breaks and chromosome breaks, which indicate that HQ induced genotoxic effects in HepG2 cells. HQ exerts genotoxic effects in HepG2 cells, probably through the formation of ROS, depletion of GSH and increase of 8-OHdG formation, which cause oxidative DNA damage and DPC.
早在1999年,国际癌症研究机构(International Agency for Research on Cancer, IARC)就将HQ归为第三类致癌物,即现有证据尚不能就其对人类致癌性进行分类。体外实验表明,HQ可引起染色体突变。HQ通过多种途径代谢,肝脏是HQ代谢的最初部位,其代谢过程中可产生活性氧(ROS)。因此,我们试探论HQ的遗传毒性,是否与ROS引起的氧化性DNA损伤有关。本研究选用一种代谢完全的人类来源的肝肿瘤细胞株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。
本研究选用HepG2细胞,研究HQ的遗传毒性及氧化性DNA损伤的机制,为评估HQ对人类的危害提供实验室资料。
方法:以HepG2细胞作为试验系统。通过标准的以及改良的蛋白酶K单细胞凝胶电泳(SCGE)试验检测细胞DNA链损伤,微核试验(MNT)检测细胞染色体损伤情况。为探讨遗传毒性的可能机制,以2',7'—二氢二氯荧光素(DCFH)法和苯二醛(OPT)分别测定细胞内ROS以及谷胱甘肽(GSH)水平,还通过免疫组化方法测定8-羟脱氧鸟苷(8-OHdG)在细胞内的表达水平。
结果:6.25、12.5、25、50μM的HQ作用HepG2细胞1h后,DNA链断裂细胞形成彗星样拖尾,尾长、尾DNA含量及尾距增大,与未用HQ处理的细胞比较,其差异有统计学意义(P<0.01)。低浓度HQ(6.25～25μM)处理的细胞,DNA的迁移距离明显增加,且呈剂量依赖关系,而当较高浓度HQ(50μM)处理的细胞,DNA的迁移距离与HQ在25μM时的最大迁移量相比明显缩短。接受较高浓度HQ(50μM)作用的细胞,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,HQ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。HepG2细胞与12.5～50μM?的HQ接触24h后,细胞微核率明显高于未用HQ处理细胞(P<0.01)。HQ作用于HepG2细胞1h,其浓度为25～50μM和6.25～50μM,可分别引起细胞内ROS表达水平的明显增加以及细胞内GSH的耗竭。12.5～50μM?的HQ作用于HepG2细胞3h后,细胞内8-OHdG水平的表达增强,与对照组比较,其差别具有统计学意义。
结论:HQ可引起HepG2细胞DNA链断裂和染色体损伤,表明HQ对HepG2细胞具有遗传毒性。HQ对HepG2细胞的遗传毒效应,可能与ROS的增高,细胞内GSH的耗竭及8-OHdG形成的增加而造成的氧化性DNA损伤和DPC有关。
Objective: Hydroquinone (1, 4-dihydroxybenzene, HQ) is widely used in various industrial fields. HQ and products containing HQ are used in over-the-counter (OTC) drugs as an ingredient in skin lighteners and also a natural ingredient in many plant-derived products. Human exposure to HQ can occur by environmental, occupational, dietary and cigarette smoke exposure and from exposure to benzene, which can be metabolized to HQ. In 2001, HQ was banned in cosmetic skin lightening formulations in European Union countries although products containing arbutin being an analogue of HQ and botanicals including plants that naturally contain HQ and arbutin, continued to remain available in European countries.
In 1999 the International Agency for Research on Cancer (IARC), assessed Sudan I as a Group 3 carcinogen. HQ was mutagenic in many mammalian cells in vitro using a variety of end- points. HQ is metabolized by several routes, reactive oxygen species (ROS) are produced. The liver is the initial site of HQ metabolism. The aim of this study was to assess the genotoxic effects of HQ in vitro and to elucidate the mechanism of oxidative DNA damage. 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 metabolising enzymes. HepG2 cells have been shown to be a suitable system for genotoxicity testing.
The purpose of this study was to assess the genotoxic effects of HQ in vitro and to illuminate the mechanisms in HepG2 cells. Thus it may provide some information for occupational hazard assessment to humans on HQ.
Methods: HepG2 cells were selected as test system. Genotoxicity of HQ was assessed by standard and proteinase K-modified alkaline single cell gel electrophoresis (SCGE) and micronucleus test (MNT). To further investigate the mechanism of genotoxicity of HQ 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 HQ-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG).
Results: The damage to DNA-strand of HepG2 cells significantly increased after exposure to 6.25, 12.5, 25, 50μM HQ for 1h. A significant dose-dependent increment in DNA migration was detected at lower concentrations of HQ (6.25 - 25μM); but at the higher tested concentrations (50μM), 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 HQ (50μM). These results indicated that HQ caused DNA strand breaks at lower concentrations of HQ (6.25 - 25μM) and DNA-protein crosslinks (DPC) formation at higher concentrations. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 12.5-50μM ?HQ for 24h. The present study showed that ?HQ induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 25-50μM and 6.25-50μM, respectively. Moreover, HQ significantly caused 8-OHdG formation in HepG2 cells at concentrations from 12.5 to 50μM.
Conclusion: The data suggest that HQ caused DNA strand breaks and chromosome breaks, which indicate that HQ induced genotoxic effects in HepG2 cells. HQ exerts genotoxic effects in HepG2 cells, probably through the formation of ROS, depletion of GSH and increase of 8-OHdG formation, which cause oxidative DNA damage and DPC.
引文
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20. Leanderson P, Tagesson C. Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation. Chem. Biol. Interact. 1992, 81(1-2):197-208.
21. Blacker AM, Schroeder RE, English JC, Murphy SJ, Krasavage WJ, Simon GS. A two-generation reproduction study with hydroquinone in rats. Fundam. Appl. Toxicol. 1993, 21(4):420-424.
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28. Smith MT, Zhang L, Jeng M, Wang Y, Guo W, Duramad P, Hubbard AE, Hofstadler G, Holland NT. Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive bloodprogenitor cells. Carcinogenesis 2000, 21(8):1485-1490.
29. Stillman WS, Varella-Garcia M, Irons RD. The benzene metabolite, hydroquinone, selectively induces 5q31- and -7 in human CD34+CD19- bone marrow cells. Exp. Hematol. 2000, 28(2):169-176.
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32.李芳红,杨杏芬。氢醌/ Cu2对小鼠骨髓细胞线粒体氧化损伤的研究。中华劳动卫生职业病杂志,2001,19(1):53-55。
33. Soucek P, Ivan G, Pavel S. Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation. Chem. Biol. Interact. 2000, 126(1):45-61.
34.申东晓,史须,王应。硫氧还蛋白对氢醌细胞毒性的抑制。中国药理学与毒理学杂志,2003,17(1):55-60。
35. Silva M do C, Gaspar J, Duarte Silva I, Faber A, Rueff J. GSTM1, GSTT1, and GSTP1 genotypes and the genotoxicity of hydroquinone in human lymphocytes. Environ. Mol. Mutagen. 2004, (4):258-264.
36. Lindsey RH Jr, Bender RP, Osheroff N. Effects of benzene metabolites on DNA cleavage mediated by human topoisomerase II alpha: 1,4-hydroquinone is a topoisomerase II poison. Chem. Res. Toxicol. 2005, 18(4):761-770.
37. Turteltaub KW, Mani C. Benzene metabolism in rodents at doses relevant to human exposure from urban air. Res. Rep. Health. Eff. Inst. 2003, (113):1-26.
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40. Zheng JH, Pyatt DW, Gross SA, Le AT, Kerzic PJ, Irons RD. Hydroquinone modulates the GM-CSF signaling pathway in TF-1 cells. Leukemia 2004, 18(7):1296-1304.
41.陈怡,俞康,吴建波。体外培养下氢醌诱导骨髓单个核细胞凋亡的研究。中华劳动卫生职业病杂志,2004,22(3):161-164。
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44.李习艺,庄志熊,刘建军。利用蛋白质组学技术研究氢醌刺激后人胚肺成纤维细胞蛋白质表达改变。卫生研究,2004;6(33):654-656。
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18. Florin I, Rutberg L, Curvall M, Enzell CR. Screening of tobacco smoke constituents for mutagenicity using the Ames' test, Toxicology 1980 ; 15(3):219-232.
19. Ciranni R, Adler ID. Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells. Mutat. Res. 1991, 263(4) : 223-229.
20. Leanderson P, Tagesson C. Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation. Chem. Biol. Interact. 1992, 81(1-2):197-208.
21. Blacker AM, Schroeder RE, English JC, Murphy SJ, Krasavage WJ, Simon GS. A two-generation reproduction study with hydroquinone in rats. Fundam. Appl. Toxicol. 1993, 21(4):420-424.
22. Ames SR, Harris PL, Ludwig MI, Swanson WJ. Effect of DPPD, methylene blue, BHT, and hydroquinone on reproductive process in the rat. Proc. Soc. Exp. Biol. Med. 1956, 93(1):39-42.
23. Krasavage WJ, Blacker AM, English JC, Murphy SJ. Hydroquinone: a developmental toxicity study in rats. Fundam. Appl. Toxicol. 1992, 18(3):370-375.
24. Murphy SJ, Schroeder RE, Blacker AM, Krasavage WJ, English JC. A study of developmental toxicity of hydroquinone in the rabbit. Fundam. Appl. Toxicol. 1992, 19(2):214-221.
25. Frenk E, Loi-Zedda P. Occupational depigmentation due to a hydroquinone-containing photographic developer , Contact Dermatitis 1980 , 6(3) : 238-239.
26. GB7919—87(化妆品安全性评价程序与方法),1987:3-9。
27.吴小芹,吴向东。氢醌对作业接触工人健康影响。劳动医学,1995,12(1):6。
28. Smith MT, Zhang L, Jeng M, Wang Y, Guo W, Duramad P, Hubbard AE, Hofstadler G, Holland NT. Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive bloodprogenitor cells. Carcinogenesis 2000, 21(8):1485-1490.
29. Stillman WS, Varella-Garcia M, Irons RD. The benzene metabolite, hydroquinone, selectively induces 5q31- and -7 in human CD34+CD19- bone marrow cells. Exp. Hematol. 2000, 28(2):169-176.
30. Andreoli C, Leopardi P, Crebelli R. Detection of DNA damage in human lymphocytes by alkaline single cell gel electrophoresis after exposure to benzene or benzene metabolites. Mutat. Res. 1997, 377(1):95-104.
31. Andreoli C, Rossi S, Leopardi P, Crebelli R. DNA damage by hydroquinone in human white blood cells: analysis by alkaline single-cell gel electrophoresis. Mutat. Res. 1999, 438(1):37-45.
32.李芳红,杨杏芬。氢醌/ Cu2对小鼠骨髓细胞线粒体氧化损伤的研究。中华劳动卫生职业病杂志,2001,19(1):53-55。
33. Soucek P, Ivan G, Pavel S. Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation. Chem. Biol. Interact. 2000, 126(1):45-61.
34.申东晓,史须,王应。硫氧还蛋白对氢醌细胞毒性的抑制。中国药理学与毒理学杂志,2003,17(1):55-60。
35. Silva M do C, Gaspar J, Duarte Silva I, Faber A, Rueff J. GSTM1, GSTT1, and GSTP1 genotypes and the genotoxicity of hydroquinone in human lymphocytes. Environ. Mol. Mutagen. 2004, (4):258-264.
36. Lindsey RH Jr, Bender RP, Osheroff N. Effects of benzene metabolites on DNA cleavage mediated by human topoisomerase II alpha: 1,4-hydroquinone is a topoisomerase II poison. Chem. Res. Toxicol. 2005, 18(4):761-770.
37. Turteltaub KW, Mani C. Benzene metabolism in rodents at doses relevant to human exposure from urban air. Res. Rep. Health. Eff. Inst. 2003, (113):1-26.
38. Amin RP, Witz G. DNA-protein crosslink and DNA strand break formation in HL-60 cells treated with trans, trans-muconaldehyde, hydroquinone and their mixtures. Int. J. Toxicol. 2001, 20(2):69-80.
39.龚梓初。氢醌在体内的代谢活化及其骨髓毒性。职业卫生与应急救援,1996,14(1):28-30。
40. Zheng JH, Pyatt DW, Gross SA, Le AT, Kerzic PJ, Irons RD. Hydroquinone modulates the GM-CSF signaling pathway in TF-1 cells. Leukemia 2004, 18(7):1296-1304.
41.陈怡,俞康,吴建波。体外培养下氢醌诱导骨髓单个核细胞凋亡的研究。中华劳动卫生职业病杂志,2004,22(3):161-164。
42.张玲。苯致白血病的分子机制。湖北预防医学杂志,2002,13(3):21-22。
43. Li Q, Geiselhart L, Mittler JN, Mudzinski SP, Lawrence DA, Freed BM. Inhibition of human T lymphoblast proliferation by hydroquinone. Toxicol. Appl. Pharmacol. 1996, 139(2):317-323.
44.李习艺,庄志熊,刘建军。利用蛋白质组学技术研究氢醌刺激后人胚肺成纤维细胞蛋白质表达改变。卫生研究,2004;6(33):654-656。