氟对斑马鱼甲状腺内分泌功能的干扰效应
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摘要
为探究氟对斑马鱼甲状腺内分泌功能的干扰效应,本研究以雌性斑马鱼成鱼为研究对象,分别以不同浓度氟(0、20、 40、80 mg·L~(-1))暴露45 d和90 d,对斑马鱼的生长发育指数进行测定,用组织学方法对斑马鱼甲状腺组织结构进行显微观察,用酶联免疫吸附法检测血浆中T3和T4激素水平,并用实时荧光定量PCR方法检测HPT轴上内分泌相关基因的表达.结果显示,与对照组相比,氟暴露组雌性斑马鱼的生长发育指数随着暴露浓度和时间的增加而呈下降趋势(p<0.05);甲状腺组织病变程度随着氟暴露浓度的增大和时间的延长而加重;氟暴露45 d时,T3和T4水平呈升高趋势,90 d时,T4水平呈下降趋势且在80 mg·L~(-1)组下降显著(p<0.05);氟暴露45 d时,40 mg·L~(-1)组除crh处,其他mRNA表达均显著下降(p<0.05),80 mg·L~(-1)组除tg、nis、ttr外其他mRNA表达均显著下降(p<0.05);暴露90 d时tg、dio1、dio2的mRNA表达水平在40 mg·L~(-1)和80 mg·L~(-1)组显著上升(p<0.05),ugt1ab mRNA表达在80 mg·L~(-1)组显著下降(p<0.05).综上,氟可通过影响雌性斑马鱼的生长发育、甲状腺组织结构、激素水平及HPT轴上内分泌相关基因的表达,从而对其甲状腺内分泌功能产生一定程度的干扰,进而影响机体的生长发育.本研究结果对水环境中氟的生态毒性效应及风险评估提供了理论资料.
In order to investigate the interference effects of fluoride on thyroid endocrine function in female zebrafish, healthy female zebrafish were exposed to different concentrations of fluoride(0, 20, 40, 80 mg·L~(-1)) for 45 and 90 days to measure the growth and development index, the thyroid microstructure by histological method, the levels of T3 and T4 in plasma by enzyme-linked immunosorbent assay(ELISA), and the expressions of endocrine-related mRNA in hypothalamus-pituitary-thyroid(HPT) axis by Quantitative Real-time PCR. The results showed that the growth and development index had a decreasing trend in fluoride-exposed groups with the increase of exposure concentration and time compared with the control group(p<0.05). The lesion degree of thyroid tissue was aggravated with the increase of exposure dose and time. The levels of T3 and T4 had an increasing trend after exposure for 45 days, while the T4 level had a decreasing trend and was significantly reduced in 80 mg·L~(-1) fluoride group after exposure for 90 days(p<0.05). After exposure for 45 days, all detected mRNA levels were significantly decreased in 40 mg·L~(-1) fluoride group except crh mRNA(p<0.05), while the mRNA levels were remarkably reduced in 80 mg·L~(-1) fluoride group except mRNA of tg, nis, ttr(p<0.05). After exposure for 90 days, the expressions of tg, dio1, dio2 mRNA were obviously elevated in 40 and 80 mg·L~(-1) fluoride groups(p<0.05), while the ugt1ab mRNA levels were significantly decreased in 80 mg·L~(-1) fluoride group(p<0.05). These results demonstrated that fluoride could disturb the thyroid endocrine function of female zebrafish by affecting the tissue structure of thyroid, the hormone levels, and the mRNA expressions in HPT axis, which inhibited the growth and development. All above provides the theoretical data for the ecotoxic effects and risk assessment of fluoride in aquatic environment.
In order to investigate the interference effects of fluoride on thyroid endocrine function in female zebrafish, healthy female zebrafish were exposed to different concentrations of fluoride(0, 20, 40, 80 mg·L~(-1)) for 45 and 90 days to measure the growth and development index, the thyroid microstructure by histological method, the levels of T3 and T4 in plasma by enzyme-linked immunosorbent assay(ELISA), and the expressions of endocrine-related mRNA in hypothalamus-pituitary-thyroid(HPT) axis by Quantitative Real-time PCR. The results showed that the growth and development index had a decreasing trend in fluoride-exposed groups with the increase of exposure concentration and time compared with the control group(p<0.05). The lesion degree of thyroid tissue was aggravated with the increase of exposure dose and time. The levels of T3 and T4 had an increasing trend after exposure for 45 days, while the T4 level had a decreasing trend and was significantly reduced in 80 mg·L~(-1) fluoride group after exposure for 90 days(p<0.05). After exposure for 45 days, all detected mRNA levels were significantly decreased in 40 mg·L~(-1) fluoride group except crh mRNA(p<0.05), while the mRNA levels were remarkably reduced in 80 mg·L~(-1) fluoride group except mRNA of tg, nis, ttr(p<0.05). After exposure for 90 days, the expressions of tg, dio1, dio2 mRNA were obviously elevated in 40 and 80 mg·L~(-1) fluoride groups(p<0.05), while the ugt1ab mRNA levels were significantly decreased in 80 mg·L~(-1) fluoride group(p<0.05). These results demonstrated that fluoride could disturb the thyroid endocrine function of female zebrafish by affecting the tissue structure of thyroid, the hormone levels, and the mRNA expressions in HPT axis, which inhibited the growth and development. All above provides the theoretical data for the ecotoxic effects and risk assessment of fluoride in aquatic environment.
引文
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Cao J L, Chen J J, Wang J D, et al. 2013. Effects of fluoride on liver apoptosis and Bcl-2, Bax protein expression in freshwater teleost, Cyprinus carpio[J]. Chemosphere, 91(8): 1203-1212
曹谨玲, 陈剑杰, 王俊东, 等. 2013. 氟对鲤鱼脑抗氧化系统及细胞凋亡的影响[J]. 环境科学学报, 33(3): 861-866
Chen J J, Cao J L, Wang J D, et al. 2013. Effects of fluoride on growth, body composition and serum biochemical profile in a freshwater teleost, Cyprinus carpio[J]. Environmental Toxicology & Chemistry, 32(10): 2315-2321
Chen J J, Xue W J, Cao J L, et al. 2016.Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio) [J]. Aquatic Toxicology, 171: 48-58
常菊花. 2012.丁草胺对斑马鱼的内分泌干扰效应研究[D]. 南京:南京农业大学. 1-126
崔玉山, 赵亮, 曾强, 等. 2016. 氧化应激在高碘高氟致甲状腺损伤中的作用[J]. 环境与职业医学, 33(1): 7-12
Davey J C, Nomikos A P, Manida W, et al. 2008. Arsenic as an endocrine disruptor: arsenic disrupts retinoic acid receptor-and thyroid hormone receptor mediated gene regulation and thyroid hormone mediated amphibian tail metamorphosis [J]. Environmental Health Perspectives, 116(2): 165-172
De Groef B, Van der Geyten S, Darras V M, et al. 2006. Role of corticotropin-releasing hormone as a thyrotropin-releasing factor in non-mammalian vertebrates[J]. Gen Comp Endocrinol, 146(1): 62-68
丁坚平, 毛健全, 顾尚义. 1998. 贵阳大坝地区熔岩地下水氟污染及其防治[J]. 环保科技, 3: 14-15
韩颖, 张宏民, 张永峰, 等. 2017. 大同盆地地下水高砷、氟、碘分布规律与成因分析及质量区划[J].中国地质调查,4(1): 57-68
Hood A, Klaassen C D. 2000. Differential effects of microsomal enzyme inducers on in vitro thyroxine (T4) and triiodothyronine (T3) glucuronidation [J]. Toxicological Sciences,55(1): 78-84
扈丽, 王正蓉, 喻茂娟. 2018. 氟中毒对仔鼠生长和神经行为发育的影响[J]. 贵州医科大学学报, 7: 757-761
江鹏, 张维东, 柴春彦, 等. 2009. 氟化物对FRTL细胞甲状腺激素代谢相关基因表达的影响[J]. 中国兽医学报, 29(7): 885-888
Jin Y, Chen R, Wang L, et al. 2011. Effects of metolachlor on transcription of thyroid system-related genes in juvenile and adult Japanese medaka (Oryzias latipes)[J]. General & Comparative Endocrinology, 170 (3): 487-493
Jugan M L,Levi Y,Blondeau J P. 2010. Endocrine disruptors and thyroid hormone physiology[J]. Biochemical Pharmacology, 79(7): 939-947
瞿璟琰, 施华宏, 刘青坡, 等. 2008. 四溴双酚-A和五溴酚对红鲫甲状腺激素和脱碘酶的影响[J]. 环境科学学报, 28(8): 1625-1630
Liu S, Chang J, Zhao Y, et al.2011.Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon[J]. Environ Toxicol Pharmacol, 32(3): 472-477
刘春华,王威,卫政润, 等. 2018. 微山湖流域高氟地下水的成因分析[J]. 地球学报, 39(3): 351-357
刘小燕, 刘珊, 张丽娟, 等. 2017. 六溴环十二烷对斑马鱼的甲状腺激素干扰效应研究[J]. 农业环境科学学报, 36(11): 2192-2198
刘忠慧, 王洋, 李文凤, 等. 2018.天津市儿童氟斑牙现状与水氟健康风险评价[J]. 公共卫生与预防医学, 3: 22-25
刘茂, 夏玉婷, 汪旸, 等. 2018. 江苏省17个病区县饮水型氟中毒监测结果分析[J]. 中华地方病学杂志,37(1): 40-44
刘洪亮, 于林玉, 崔玉山, 等. 2013. 长期摄入过量氟对大鼠甲状腺形态和功能的影响[J]. 环境卫生学杂志, 5: 390-393
刘莉云. 2017. 离子液体对人肝癌细胞HepG2活性氧水平和斑马鱼的毒性影响及机理研究[D]. 镇江:江苏大学.37-52
李晓岚, 张复兰. 2016.氟中毒对儿童智力、体格发育及血清骨形态发生蛋白水平的影响[J]. 中国地方病防治杂志, (8): 895-895
马燕燕, 周月红, 王敬花. 2018. 高氟水源对妇女体内性激素结合蛋白与雌二醇水平的影响[J]. 中国医药指南, 16 (12): 163-164
Manchado M, Infante C, Asensio E, et al. 2008. Thyroid hormones down-regulate thyrotropin β subunit and thyroglobulin during metamorphosis in the flatfish Senegalese sole (Solea senegalensis Kaup)[J]. General and Comparative Endocrinology, 155(2): 447-455
米智,柳照应, 朱勇. 2009. 氟化物对家蚕生长发育的影响[J]. 蚕学通讯, 29 (3): 42-46
木伟娜. 2017. 低剂量三丁基锡和镉联合暴露对鲤鱼甲状腺轴及抗氧化指标的影响[D]. 武汉: 华中农业大学, 22-39
Mukhopadhyay D, Priya P, Chattopadhyay A. 2015. Sodium fluoride affects zebrafish behaviour and alters mRNA expressions of biomarker genes in the brain: Role of Nrf2/Keap1[J]. Environmental Toxicology & Pharmacology, 40 (2): 352-359
Mukhopadhyay D, Chattopadhyay A. 2014. Induction of oxidative stress and related transcriptional effects of sodium fluoride in female zebrafish liver[J]. Bull Environ Contam Toxicol, 93(1): 64-70
秦晓飞, 秦占芬, 徐晓白. 2009. 非洲爪蟾在生态毒理学研究中的应用:甲状腺干扰作用评价[J]. 环境科学学报, 29(8): 1589-159
Ren F H, Jiao S Q. 1988. Distribution and formation of high-fluorinegroundwater in China[J]. Environmental Geology, 12(1): 3-10
Schreiber G. 2002. The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis[J]. Journal of Endocrinology, 175(1): 61-73
石小涛, 庄平, 章龙珍, 等. 2009. 水暴露下氟在西伯利亚鲟稚鱼硬骨和软骨中的积累和消除[J].生态毒理学报, 4(2): 218-223
Shi X, Zhuang P, Zhang L, et al. 2009. The bioaccumulation of fluoride ion (F-) in Siberian sturgeon (Acipenser baerii) under laboratory conditions[J]. Chemosphere, 75(3): 376-380
Singh R, Khatri P, Srivastava N, et al. 2017. Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections[J]. Fish & Shellfish Immunology, 63(4): 314-321
涂文清. 2014. 几种典型污染物对斑马鱼的甲状腺干扰效应[D]. 杭州: 浙江工业大学. 59-71
王明. 2016. 氟化物对中华大蟾蜍和中国林蛙生长发育的毒理效应研究[D]. 西安: 陕西师范大学. 1-78
解雨春, 李文, 汤金梅, 等. 2012. Aroclor1254 对大鼠甲状腺结构及功能的影响[J]. 环境科学学报, 32(11): 2891-2897
谢鹏, 李婷婷, 季晖. 2016. 尿苷二磷酸葡萄糖醛酸转移酶的转录调节与疾病相关性的研究进展[J].药学研究, 35(5): 295-299
Zhang X, Tian H, Wang W, et al. 2013. Exposure to monocrotophos pesticide causes disruption of the hypothalamic-pituitary-thyroid axis in adult male goldfish (Carassius auratus)[J]. Gen Comp Endocrinol, 193(4): 158-166
Zheng L C, Luo H J, Kazurou B, et al. 2006. High fluoridegroundwater with high salinity and fluorite in aquifer sediments ininner Mongolia, China[J]. Chinese Journal of Geochemistry, 25(supplement 1): 103-103
张维东. 2008. 氟诱发甲状腺肿的分子病理学研究[D]. 兰州: 甘肃农业大学. 1-84
Zhang J, Zhu Y, Shi Y, et al. 2017. Fluoride-induced autophagy via the regulation of phosphorylation of mammalian targets of rapamycin in mice leydig cells[J]. Journal of Agricultural & Food Chemistry,65(40): 8966-8976
朱莉萍, 胡秀, 杨林, 等. 2016. 芪参二术汤对氟所致大鼠甲状腺损伤的保护作用研究[J]. 世界中西医结合杂志, 11 (11): 1536-1539
Bhatnagar C, Bhatnagar M, Regar B C. 2007. Fluoride-induced histopathological changes in gill, kidney, and intestine of fresh water teleost, Labeo rohita[J]. Fluoride, 40(1): 55-61
Cao J L, Chen J J, Wang J D, et al. 2013. Effects of fluoride on liver apoptosis and Bcl-2, Bax protein expression in freshwater teleost, Cyprinus carpio[J]. Chemosphere, 91(8): 1203-1212
曹谨玲, 陈剑杰, 王俊东, 等. 2013. 氟对鲤鱼脑抗氧化系统及细胞凋亡的影响[J]. 环境科学学报, 33(3): 861-866
Chen J J, Cao J L, Wang J D, et al. 2013. Effects of fluoride on growth, body composition and serum biochemical profile in a freshwater teleost, Cyprinus carpio[J]. Environmental Toxicology & Chemistry, 32(10): 2315-2321
Chen J J, Xue W J, Cao J L, et al. 2016.Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio) [J]. Aquatic Toxicology, 171: 48-58
常菊花. 2012.丁草胺对斑马鱼的内分泌干扰效应研究[D]. 南京:南京农业大学. 1-126
崔玉山, 赵亮, 曾强, 等. 2016. 氧化应激在高碘高氟致甲状腺损伤中的作用[J]. 环境与职业医学, 33(1): 7-12
Davey J C, Nomikos A P, Manida W, et al. 2008. Arsenic as an endocrine disruptor: arsenic disrupts retinoic acid receptor-and thyroid hormone receptor mediated gene regulation and thyroid hormone mediated amphibian tail metamorphosis [J]. Environmental Health Perspectives, 116(2): 165-172
De Groef B, Van der Geyten S, Darras V M, et al. 2006. Role of corticotropin-releasing hormone as a thyrotropin-releasing factor in non-mammalian vertebrates[J]. Gen Comp Endocrinol, 146(1): 62-68
丁坚平, 毛健全, 顾尚义. 1998. 贵阳大坝地区熔岩地下水氟污染及其防治[J]. 环保科技, 3: 14-15
韩颖, 张宏民, 张永峰, 等. 2017. 大同盆地地下水高砷、氟、碘分布规律与成因分析及质量区划[J].中国地质调查,4(1): 57-68
Hood A, Klaassen C D. 2000. Differential effects of microsomal enzyme inducers on in vitro thyroxine (T4) and triiodothyronine (T3) glucuronidation [J]. Toxicological Sciences,55(1): 78-84
扈丽, 王正蓉, 喻茂娟. 2018. 氟中毒对仔鼠生长和神经行为发育的影响[J]. 贵州医科大学学报, 7: 757-761
江鹏, 张维东, 柴春彦, 等. 2009. 氟化物对FRTL细胞甲状腺激素代谢相关基因表达的影响[J]. 中国兽医学报, 29(7): 885-888
Jin Y, Chen R, Wang L, et al. 2011. Effects of metolachlor on transcription of thyroid system-related genes in juvenile and adult Japanese medaka (Oryzias latipes)[J]. General & Comparative Endocrinology, 170 (3): 487-493
Jugan M L,Levi Y,Blondeau J P. 2010. Endocrine disruptors and thyroid hormone physiology[J]. Biochemical Pharmacology, 79(7): 939-947
瞿璟琰, 施华宏, 刘青坡, 等. 2008. 四溴双酚-A和五溴酚对红鲫甲状腺激素和脱碘酶的影响[J]. 环境科学学报, 28(8): 1625-1630
Liu S, Chang J, Zhao Y, et al.2011.Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon[J]. Environ Toxicol Pharmacol, 32(3): 472-477
刘春华,王威,卫政润, 等. 2018. 微山湖流域高氟地下水的成因分析[J]. 地球学报, 39(3): 351-357
刘小燕, 刘珊, 张丽娟, 等. 2017. 六溴环十二烷对斑马鱼的甲状腺激素干扰效应研究[J]. 农业环境科学学报, 36(11): 2192-2198
刘忠慧, 王洋, 李文凤, 等. 2018.天津市儿童氟斑牙现状与水氟健康风险评价[J]. 公共卫生与预防医学, 3: 22-25
刘茂, 夏玉婷, 汪旸, 等. 2018. 江苏省17个病区县饮水型氟中毒监测结果分析[J]. 中华地方病学杂志,37(1): 40-44
刘洪亮, 于林玉, 崔玉山, 等. 2013. 长期摄入过量氟对大鼠甲状腺形态和功能的影响[J]. 环境卫生学杂志, 5: 390-393
刘莉云. 2017. 离子液体对人肝癌细胞HepG2活性氧水平和斑马鱼的毒性影响及机理研究[D]. 镇江:江苏大学.37-52
李晓岚, 张复兰. 2016.氟中毒对儿童智力、体格发育及血清骨形态发生蛋白水平的影响[J]. 中国地方病防治杂志, (8): 895-895
马燕燕, 周月红, 王敬花. 2018. 高氟水源对妇女体内性激素结合蛋白与雌二醇水平的影响[J]. 中国医药指南, 16 (12): 163-164
Manchado M, Infante C, Asensio E, et al. 2008. Thyroid hormones down-regulate thyrotropin β subunit and thyroglobulin during metamorphosis in the flatfish Senegalese sole (Solea senegalensis Kaup)[J]. General and Comparative Endocrinology, 155(2): 447-455
米智,柳照应, 朱勇. 2009. 氟化物对家蚕生长发育的影响[J]. 蚕学通讯, 29 (3): 42-46
木伟娜. 2017. 低剂量三丁基锡和镉联合暴露对鲤鱼甲状腺轴及抗氧化指标的影响[D]. 武汉: 华中农业大学, 22-39
Mukhopadhyay D, Priya P, Chattopadhyay A. 2015. Sodium fluoride affects zebrafish behaviour and alters mRNA expressions of biomarker genes in the brain: Role of Nrf2/Keap1[J]. Environmental Toxicology & Pharmacology, 40 (2): 352-359
Mukhopadhyay D, Chattopadhyay A. 2014. Induction of oxidative stress and related transcriptional effects of sodium fluoride in female zebrafish liver[J]. Bull Environ Contam Toxicol, 93(1): 64-70
秦晓飞, 秦占芬, 徐晓白. 2009. 非洲爪蟾在生态毒理学研究中的应用:甲状腺干扰作用评价[J]. 环境科学学报, 29(8): 1589-159
Ren F H, Jiao S Q. 1988. Distribution and formation of high-fluorinegroundwater in China[J]. Environmental Geology, 12(1): 3-10
Schreiber G. 2002. The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis[J]. Journal of Endocrinology, 175(1): 61-73
石小涛, 庄平, 章龙珍, 等. 2009. 水暴露下氟在西伯利亚鲟稚鱼硬骨和软骨中的积累和消除[J].生态毒理学报, 4(2): 218-223
Shi X, Zhuang P, Zhang L, et al. 2009. The bioaccumulation of fluoride ion (F-) in Siberian sturgeon (Acipenser baerii) under laboratory conditions[J]. Chemosphere, 75(3): 376-380
Singh R, Khatri P, Srivastava N, et al. 2017. Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections[J]. Fish & Shellfish Immunology, 63(4): 314-321
涂文清. 2014. 几种典型污染物对斑马鱼的甲状腺干扰效应[D]. 杭州: 浙江工业大学. 59-71
王明. 2016. 氟化物对中华大蟾蜍和中国林蛙生长发育的毒理效应研究[D]. 西安: 陕西师范大学. 1-78
解雨春, 李文, 汤金梅, 等. 2012. Aroclor1254 对大鼠甲状腺结构及功能的影响[J]. 环境科学学报, 32(11): 2891-2897
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