草鱼幼鱼肝胰脏抗氧化系统对MC-LR胁迫的响应
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摘要
为了研究微囊藻毒素(MC-LR)对草鱼(Ctenopharygodon idella)幼鱼肝胰脏抗氧化防御系统的影响,本研究通过腹腔注射的方法(剂量为25、100μg MC-LR·kg-1,分别称为低剂量和高剂量),对草鱼幼鱼进行染毒,于胁迫24、48 h和72 h后分离其肝胰脏,随后采用分光光度法检测了草鱼幼鱼肝胰脏中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性;同时采用荧光定量PCR方法分析了SOD、CAT、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)基因相对表达量的变化。结果显示:高剂量MC-LR诱导24 h和48 h后,草鱼幼鱼肝胰脏SOD活性显著增加(P<0.05),而SOD在低剂量组中活性没有显著变化(P>0.05);低剂量MC-LR对CAT活性没有显著影响(P>0.05),而高剂量MC-LR诱导24 h后可使草鱼幼鱼肝胰脏CAT活性显著增加(P<0.05),随后其活性又下降,但差异不显著(P>0.05)。在MC-LR胁迫过程中,SOD、CAT、GPx基因表达在两个剂量组中均显著低于对照组(P<0.05),而GR基因在低剂量MC-LR胁迫24 h后相对表达量显著上调(P<0.05),尽管在72 h相对表达量上调,但差异不显著(P>0.05),而高剂量组中GR基因在胁迫24 h和48 h后,其表达量被抑制,但不存在显著差异(P>0.05)。进行抗氧化酶活性和基因表达相关性分析发现,SOD和CAT酶活性与SOD和CAT基因表达不相关。上述研究表明MC-LR对草鱼幼鱼肝胰脏抗氧化系统产生了明显的胁迫效应,但MC-LR对机体抗氧化酶活性与基因编码调控之间的相互作用机制还有待更深入的研究。
To better understand the influence of microcystin-LR(MC-LR)on the oxidative stress response in the hepatopancreas of juvenile grass carp(Ctenopharyngodon idella), the present study was conducted injecting MC-LR intraperitoneally at doses of 25 μg MC-LR·kg-1(low-dose group)and 100 μg MC-LR·kg-1(high-dose group). Samples of hepatopancreases from juvenile grass carp were collected at24, 48, and 72 h after the initial injection. The activities and transcriptional levels of antioxidant enzymes, including superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GPx), and glutathione reductase(GR), were analyzed using spectrophotometry and quantitative real-time PCR, respectively. The enzymatic activity of SOD in the low-dose group was not significantly changed, but its content in the high-dose group was significantly enhanced at 24 and 48 h(P<0.05), while its content began to decrease at 72 h. The enzymatic activity of CAT just in the high-dose groups was significantly enhanced at 24 h(P<0.05), but the activities in the low-and high-dose groups all decreased at 48 h and 72 h with insignificant differences(P>0.05). The expressions of the SOD, CAT, and GPx genes were all significantly down-regulated during the whole experiment(P<0.05), but the expressions of the GR gene in the low-dose group were significantly upregulated just at 24 h(P<0.05); another, its expression in two treated group were also up-regulated at 72 h, but the differences were not significant(P>0.05). However, the expressions of GR gene in the high dose group were all down-regulated with insignificant differences at 24 h and 48 h. In addition, correlation analysis of the enzymatic activities and gene expressions showed that the enzymatic activities of SOD and CAT were not correlated with their gene expressions. This study demonstrated that MC-LR could induce oxidative stress in juvenile grass carp by adjusting enzymatic activity and gene expression, but the mechanism of interaction between these needs to be further studied.
To better understand the influence of microcystin-LR(MC-LR)on the oxidative stress response in the hepatopancreas of juvenile grass carp(Ctenopharyngodon idella), the present study was conducted injecting MC-LR intraperitoneally at doses of 25 μg MC-LR·kg-1(low-dose group)and 100 μg MC-LR·kg-1(high-dose group). Samples of hepatopancreases from juvenile grass carp were collected at24, 48, and 72 h after the initial injection. The activities and transcriptional levels of antioxidant enzymes, including superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GPx), and glutathione reductase(GR), were analyzed using spectrophotometry and quantitative real-time PCR, respectively. The enzymatic activity of SOD in the low-dose group was not significantly changed, but its content in the high-dose group was significantly enhanced at 24 and 48 h(P<0.05), while its content began to decrease at 72 h. The enzymatic activity of CAT just in the high-dose groups was significantly enhanced at 24 h(P<0.05), but the activities in the low-and high-dose groups all decreased at 48 h and 72 h with insignificant differences(P>0.05). The expressions of the SOD, CAT, and GPx genes were all significantly down-regulated during the whole experiment(P<0.05), but the expressions of the GR gene in the low-dose group were significantly upregulated just at 24 h(P<0.05); another, its expression in two treated group were also up-regulated at 72 h, but the differences were not significant(P>0.05). However, the expressions of GR gene in the high dose group were all down-regulated with insignificant differences at 24 h and 48 h. In addition, correlation analysis of the enzymatic activities and gene expressions showed that the enzymatic activities of SOD and CAT were not correlated with their gene expressions. This study demonstrated that MC-LR could induce oxidative stress in juvenile grass carp by adjusting enzymatic activity and gene expression, but the mechanism of interaction between these needs to be further studied.
引文
[1] Smith V H, Schindler D W. Eutrophication science:Where do we go from here[J]. Trends in Ecology and Evolution, 2009, 24(4):201-207.
[2] Dawson R M. The toxicology of microcystins[J]. Toxicon, 1998, 36(7):953-962.
[3] Pereira S R, Vasconcelos V M, Antunes A. Computational study of the covalent bonding of microcystins to cysteine residues-a reaction involved in the inhibition of the PPP family of protein phosphatases[J].FEBS Journal, 2013, 280(2):674-680.
[4] MacKintosh C, Beattie K A, Klumpp S, et al. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and2A from both mammals and higher plants[J]. FEBS Letters, 1990, 264(2):187-192.
[5] Runnegar M, Berndt N, Kong S M, et al. In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A[J]. Biochemical and Biophysical Research Communications, 1995, 216(1):162-169.
[6] Guzman R E, Solter P F. Hepatic oxidative stress following prolonged sublethal microcystin LR exposure[J]. Toxicologic Pathology, 1999, 27(5):582-588.
[7] Jayaraj R, Anand T, Rao P V. Activity and gene expression profile of certain antioxidant enzymes to microcystin-LR induced oxidative stress in mice[J]. Toxicology, 2006, 220(2/3):136-146.
[8] Pavagadhi S, Gong Z, Hande M P, et al. Biochemical response of diverse organs in adult Danio rerio(zebrafish)exposed to sublethal concentrations of microcystin-LR and microcystin-RR:A balneation study[J]. Aquatic Toxicology, 2012, 109:1-10.
[9] Hou J, Li L, Xue T, et al. Hepatic positive and negative antioxidant responses in zebrafish after intraperitoneal administration of toxic microcystin-LR[J]. Chemosphere, 2015, 120:729-736.
[10] Le?o J C, Geracitano L A, Monserrat J M, et al. Microcystin-induced oxidative stress in Laeonereis acuta(Polychaeta, Nereididae)[J]. Marine Environmental Research, 2008, 66(1):92-94.
[11] Amado L L, Monserrat J M. Oxidative stress generation by microcystins in aquatic animals:Why and how[J]. Environment International,2010, 6:226-235.
[12] Stara A, Kristan J, Zuskova E, et al. Effect of chronic exposure to prometryne on oxidative stress and antioxidant response in common carp(Cyprinus carpio L.)[J]. Pesticide Biochemmistry Physiology,2013, 105(1):18-23.
[13]杨静东,胡梁斌,周威,等.微囊藻毒素在鲋鱼体内生物富集及其体内的抗氧化反应[J].生态环境学报, 2009, 6:2044-2050.YANG Jing-dong, HU Liang-bin, ZHOU Wei, et al. Bioaccumulation of microcystin and antioxidative response in Carassius auratus L.[J].Ecology and Environmental Sciences, 2009, 18(6):2044-2050.
[14]任平,王玉军.微囊藻毒素慢性毒性染毒对斑马鱼卵巢组织抗氧化酶的影响[J].洛阳理工学院学报(自然科学版), 2010, 20(2):12-14, 36.REN Ping, WANG Yu-jun. Effects of sub-chronic exposure to microcystic toxins on antioxidant of zebrafish ovary organization[J]. Journal of Luoyang Institute of Science and Technology(Natural Science Edition), 2010, 20(2):12-14, 36.
[15]虞聪聪.淮河流域X县水体藻类及其毒素污染状况及微囊藻毒素LR与黄曲霉毒素B1的联合毒性作用研究[D].上海:复旦大学, 2014.YU Cong-cong. Pollutions by algae and microcystins in water bodies of X county in Huai river basin and the toxicity induced by the combination effects of microcystin-LR and aflatoxin-B1[D]. Shanghai:Fudan University, 2014.
[16] Gehringer M. Microcystin-LR and okadaic acid-induced cellular effects:A dualistic response[J]. FEBS Letters, 2004, 557:1-8.
[17]隗黎丽,刘毅,王自蕊,等. MC-LR急性胁迫对草鱼脾脏、头肾显微结构及BAFF和APRIL基因表达的影响[J].农业环境科学学报,2017, 36(1):2379-2387.WEI Li-li, LIU Yi, WANG Zi-rui, et al. Acute effects of microcystinLR on the microstructure of spleen and head kidney and the expression of BAFF and APRIL genes in grass carp[J]. Journal of Agro-Environment Science, 2017, 36(1):2379-2387.
[18] Zhao H, Jiang W, Liu Y, et al. Dietary choline regulates antibacterial activity, inflammatory response and barrier function in the gills of grass carp(Ctenopharyngodon idella)[J]. Fish and Shellfish Immunology, 2016, 52:139-150.
[19] Yuan J, Gu Z, Zheng Y, et al. Accumulation and detoxification dynamics of microcystin-LR and antioxidant responses in male red swamp crayfish Procambarus clarkii[J]. Aquatic Toxicology, 2016, 177:8-18.
[20]陈华,陈昱,王志红,等.颤藻粗提物对小鼠肝毒性及SOD活性影响研究[J].中国公共卫生, 2002, 18(2):133-134.CHEN Hua, CHEN Yu, WANG Zhi-hong, et al. Algae hepatotoxicity and superoxide dismutase(SOD)activity induced by Oscillatoria-extract in mouse[J]. China Public Health, 2002, 18(2):133-134.
[21]朱枫,钱晨,卢彦.微囊藻毒素诱导细胞氧化损伤胁迫的研究进展[J].生态毒理学报, 2010, 5(6):769-775.ZHU Feng, QIAN Chen, LU Yan. Progress in oxidative stress on cell induced by microcystin[J]. Asian Journal of Ecotoxicology, 2010, 5(6):769-775.
[22]许川,舒为群,曹佳,等.绿茶对微囊藻毒素诱导肝肾氧化损伤的拮抗效应[J].中华预防医学杂志, 2007, 41(1):8-12.XU Chuan, SHU Wei-qun, CAO Jia, et al. Antagonism effects of green tea against microcystin induced oxidant damage on liver and kidney[J]. China Journal Prevent Medicine, 2007, 41(1):8-12.
[23] Xiong Q, Xie P, Li H, et al. Acute effects of microcystins exposure on the transcription of antioxidant enzyme genes in three organs(liver,kidney, and testis)of male Wistar rats[J]. Journal of Biochemical Molecular Toxicology, 2010, 24(6):361-367.
[24] Sun Y, Tang R, Li D, et al. Acute effects of microcystins on the transcription of antioxidant enzyme genes in crucian carp Carassius auratus[J]. Environmental Toxicology, 2008, 23(2):145-152.
[25] Sicińska P, Bukowska B, Micha?owicz J, et al. Damage of cell membrane and antioxidative system in human erythrocytes incubated with microcystin-LR in vitro[J]. Toxicon, 2006, 47(4):387-397.
[26] Dimitrova M S T, Tsinova V, Velcheva V. Combined effects of zinc and lead on the hepatic superoxide dismutase-catalase system in carp(Cyprinus carpio)[J]. Comparative Biochemistry and Physiology:Part C, 1994, 108:43-46.
[27] Shi Y, Jiang J, Shan Z, et al. Oxidative stress and histopathological alterations in liver of Cyprinus carpio L. induced by intraperitoneal injection of microcystin-LR[J]. Ecotoxicology, 2015, 24:511-519.
[28] Galanti L N, AméM V, Wunderlin D A. Accumulation and detoxification dynamic of cyanotoxins in the freshwater shrimp Palaemonetes argentinus[J]. Harmful Algae, 2013, 27:88-97.
[29] Yin L, Huang J, Huang W, et al. Responses of antioxidant system in Arabidopsis thaliana suspension cells to the toxicity of microcystin-RR[J]. Toxicon, 2005, 46(8):859-864.
[2] Dawson R M. The toxicology of microcystins[J]. Toxicon, 1998, 36(7):953-962.
[3] Pereira S R, Vasconcelos V M, Antunes A. Computational study of the covalent bonding of microcystins to cysteine residues-a reaction involved in the inhibition of the PPP family of protein phosphatases[J].FEBS Journal, 2013, 280(2):674-680.
[4] MacKintosh C, Beattie K A, Klumpp S, et al. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and2A from both mammals and higher plants[J]. FEBS Letters, 1990, 264(2):187-192.
[5] Runnegar M, Berndt N, Kong S M, et al. In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A[J]. Biochemical and Biophysical Research Communications, 1995, 216(1):162-169.
[6] Guzman R E, Solter P F. Hepatic oxidative stress following prolonged sublethal microcystin LR exposure[J]. Toxicologic Pathology, 1999, 27(5):582-588.
[7] Jayaraj R, Anand T, Rao P V. Activity and gene expression profile of certain antioxidant enzymes to microcystin-LR induced oxidative stress in mice[J]. Toxicology, 2006, 220(2/3):136-146.
[8] Pavagadhi S, Gong Z, Hande M P, et al. Biochemical response of diverse organs in adult Danio rerio(zebrafish)exposed to sublethal concentrations of microcystin-LR and microcystin-RR:A balneation study[J]. Aquatic Toxicology, 2012, 109:1-10.
[9] Hou J, Li L, Xue T, et al. Hepatic positive and negative antioxidant responses in zebrafish after intraperitoneal administration of toxic microcystin-LR[J]. Chemosphere, 2015, 120:729-736.
[10] Le?o J C, Geracitano L A, Monserrat J M, et al. Microcystin-induced oxidative stress in Laeonereis acuta(Polychaeta, Nereididae)[J]. Marine Environmental Research, 2008, 66(1):92-94.
[11] Amado L L, Monserrat J M. Oxidative stress generation by microcystins in aquatic animals:Why and how[J]. Environment International,2010, 6:226-235.
[12] Stara A, Kristan J, Zuskova E, et al. Effect of chronic exposure to prometryne on oxidative stress and antioxidant response in common carp(Cyprinus carpio L.)[J]. Pesticide Biochemmistry Physiology,2013, 105(1):18-23.
[13]杨静东,胡梁斌,周威,等.微囊藻毒素在鲋鱼体内生物富集及其体内的抗氧化反应[J].生态环境学报, 2009, 6:2044-2050.YANG Jing-dong, HU Liang-bin, ZHOU Wei, et al. Bioaccumulation of microcystin and antioxidative response in Carassius auratus L.[J].Ecology and Environmental Sciences, 2009, 18(6):2044-2050.
[14]任平,王玉军.微囊藻毒素慢性毒性染毒对斑马鱼卵巢组织抗氧化酶的影响[J].洛阳理工学院学报(自然科学版), 2010, 20(2):12-14, 36.REN Ping, WANG Yu-jun. Effects of sub-chronic exposure to microcystic toxins on antioxidant of zebrafish ovary organization[J]. Journal of Luoyang Institute of Science and Technology(Natural Science Edition), 2010, 20(2):12-14, 36.
[15]虞聪聪.淮河流域X县水体藻类及其毒素污染状况及微囊藻毒素LR与黄曲霉毒素B1的联合毒性作用研究[D].上海:复旦大学, 2014.YU Cong-cong. Pollutions by algae and microcystins in water bodies of X county in Huai river basin and the toxicity induced by the combination effects of microcystin-LR and aflatoxin-B1[D]. Shanghai:Fudan University, 2014.
[16] Gehringer M. Microcystin-LR and okadaic acid-induced cellular effects:A dualistic response[J]. FEBS Letters, 2004, 557:1-8.
[17]隗黎丽,刘毅,王自蕊,等. MC-LR急性胁迫对草鱼脾脏、头肾显微结构及BAFF和APRIL基因表达的影响[J].农业环境科学学报,2017, 36(1):2379-2387.WEI Li-li, LIU Yi, WANG Zi-rui, et al. Acute effects of microcystinLR on the microstructure of spleen and head kidney and the expression of BAFF and APRIL genes in grass carp[J]. Journal of Agro-Environment Science, 2017, 36(1):2379-2387.
[18] Zhao H, Jiang W, Liu Y, et al. Dietary choline regulates antibacterial activity, inflammatory response and barrier function in the gills of grass carp(Ctenopharyngodon idella)[J]. Fish and Shellfish Immunology, 2016, 52:139-150.
[19] Yuan J, Gu Z, Zheng Y, et al. Accumulation and detoxification dynamics of microcystin-LR and antioxidant responses in male red swamp crayfish Procambarus clarkii[J]. Aquatic Toxicology, 2016, 177:8-18.
[20]陈华,陈昱,王志红,等.颤藻粗提物对小鼠肝毒性及SOD活性影响研究[J].中国公共卫生, 2002, 18(2):133-134.CHEN Hua, CHEN Yu, WANG Zhi-hong, et al. Algae hepatotoxicity and superoxide dismutase(SOD)activity induced by Oscillatoria-extract in mouse[J]. China Public Health, 2002, 18(2):133-134.
[21]朱枫,钱晨,卢彦.微囊藻毒素诱导细胞氧化损伤胁迫的研究进展[J].生态毒理学报, 2010, 5(6):769-775.ZHU Feng, QIAN Chen, LU Yan. Progress in oxidative stress on cell induced by microcystin[J]. Asian Journal of Ecotoxicology, 2010, 5(6):769-775.
[22]许川,舒为群,曹佳,等.绿茶对微囊藻毒素诱导肝肾氧化损伤的拮抗效应[J].中华预防医学杂志, 2007, 41(1):8-12.XU Chuan, SHU Wei-qun, CAO Jia, et al. Antagonism effects of green tea against microcystin induced oxidant damage on liver and kidney[J]. China Journal Prevent Medicine, 2007, 41(1):8-12.
[23] Xiong Q, Xie P, Li H, et al. Acute effects of microcystins exposure on the transcription of antioxidant enzyme genes in three organs(liver,kidney, and testis)of male Wistar rats[J]. Journal of Biochemical Molecular Toxicology, 2010, 24(6):361-367.
[24] Sun Y, Tang R, Li D, et al. Acute effects of microcystins on the transcription of antioxidant enzyme genes in crucian carp Carassius auratus[J]. Environmental Toxicology, 2008, 23(2):145-152.
[25] Sicińska P, Bukowska B, Micha?owicz J, et al. Damage of cell membrane and antioxidative system in human erythrocytes incubated with microcystin-LR in vitro[J]. Toxicon, 2006, 47(4):387-397.
[26] Dimitrova M S T, Tsinova V, Velcheva V. Combined effects of zinc and lead on the hepatic superoxide dismutase-catalase system in carp(Cyprinus carpio)[J]. Comparative Biochemistry and Physiology:Part C, 1994, 108:43-46.
[27] Shi Y, Jiang J, Shan Z, et al. Oxidative stress and histopathological alterations in liver of Cyprinus carpio L. induced by intraperitoneal injection of microcystin-LR[J]. Ecotoxicology, 2015, 24:511-519.
[28] Galanti L N, AméM V, Wunderlin D A. Accumulation and detoxification dynamic of cyanotoxins in the freshwater shrimp Palaemonetes argentinus[J]. Harmful Algae, 2013, 27:88-97.
[29] Yin L, Huang J, Huang W, et al. Responses of antioxidant system in Arabidopsis thaliana suspension cells to the toxicity of microcystin-RR[J]. Toxicon, 2005, 46(8):859-864.
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