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研究生:劉芝岑
研究生(外文):Chih-Tsen Liu
論文名稱:重金屬鎘對斑馬魚仔魚鈣運輸蛋白基因表現的影響
論文名稱(外文):Effects of Cadmium on Gene Expression of Calcium Transporters of Zebrafish Larvae (Danio rerio)
指導教授:吳淑美吳淑美引用關係
指導教授(外文):Su-Mei Wu
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:水生生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:68
中文關鍵詞:鈣離子運輸蛋白基因表現
外文關鍵詞:CadmiumCalciumCalcium transportergene expression
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鎘是環境中非生物必須且會累積的毒性重金屬,對水中生物造成毒害甚多。過去的許多研究,已知在鎘環境下魚類的鈣離子通常低下,其原因多數認為是因為鎘會與鈣競爭相同的離子通道的緣故,但是否也影響到與鈣吸收或排出的離子運輸蛋白有關呢?目前尚無報告提出,因此本研究首次以斑馬魚仔魚為實驗動物,探討其暴露在含有0.09μM 鎘的人工正常淡水(0.2 mM Ca2+)及高鈣水(2 mM Ca2+)連續96小時的情況下,對鈣離子吸收相關的離子通道之基因表現的影響,這些基因包括:上皮細胞鈣離子通道(Epithelial Calcium Channel; ECaC)、鈉鈣交換蛋白(Sodium/Calcium Exchanger 1b; NCX1b)和細胞膜鈣離子幫浦(Plasma Membrane Calcium ATPase 2;PMCA2)。利用即時定量PCR直接偵測此三個基因的表現量。結果發現仔魚在0.09μM 鎘的人工正常淡水中的總鈣量,從24-96小時皆顯著下降,而ECaC 和NCX1b表現量則在暴露後48小時受到抑制,至第96小時回復,另一方面PMCA2表現量直到第96小時才降低。仔魚在0.09μM 鎘的高鈣水中暴露96小時,ECaC無變化; PMCA2在暴露72小時後,表現量顯著提高;NCX1b則是在48與96小時顯著下降。綜合這些結果我們推論在低鎘濃度下(0.09μM Cd)就會對鈣的離子運輸蛋白造成傷害,但在高鈣補償下,ECaC和PMCA2可恢復其功能。由於ECaC是鰓上表皮頂端膜上最主要的鈣離子通道,因此將斑馬魚胚胎經由顯微注射 zECaC MO(Morpholino Oligonucleotides)使其表現量降低(Gene knockdown),並將其置於8.9μM 鎘(48h-LC50 of 3dpf larvae )的人工正常淡水,有將近97%以上的死亡率,反之當斑馬魚胚胎經微量注射ECaC cRNA使其過度表現(overexpression),則在相同鎘條件處理下其死亡率並無顯著下降,但體內的總鈣量增加,鈣的influex增加,顯示過度表現ECaC對斑馬魚胚胎對抗重金屬鎘並無絕對影響。

關鍵字:鎘,鈣,鈣離子運輸蛋白,基因表現
Abstract
Cadmium (Cd2+) is a non-essential biological element that is accumulated and known to have adverse effects in aquatic organism. In previous study, ambient Cd fish always appear hypo-calcium due to Cd2+ competitive inhibition calcium channel with Ca2+. However, the past studies almost focus on the effects of Cd2+ on the total Ca2+ content or Ca2+ uptake. This study was the first time to investigate the effect of Cd2+ explore on Ca- transporters in zebrafish (Danio rerio) larvae. The stage of 24hpf larvae were exposed with an artificial water (contained 0.2mM Ca2+) or exposed to Cd with high dose of calcium (2mM Ca2+) water. Under these conditions, the mRNA gene expression of different calcium transporter in gill including epithelial calcium channel (ECaC), sodium/calcium exchanger 1b (NCX1b) ,and plasma membrane calcium ATPase 2 (PMCA2) can be directly studied. Results showed that larvae treated with 0.09μM Cd2+ and 0.2mM Ca2+ water that was appeared significantly decrease on calcium content of whole body from 24 to 96 hours. Also, Quantitative-PCR analysis showed suppressed ECaC and NCX1b gene expression at 48 and 72 hours post Cd2+ treatment. On the other hand, decreased PMCA2 was only observed at 96 hours post Cd2+ exposure. ECaC expression will be changed upon larvae exposed to 0.09μM cadmium and maintained at 2mM Ca for 96 hours, and significantly improved PMCA2 expression after 72 hours post Cd exposure. On the contrary, NCX1b expression significantly decreased at 48 and 96 hours post Cd exposure. These results suggest that even low Cd2+ concentration impacts the calcium transporters but the functions of. ECaC and PMCA2 could be rescued after treatment with high dose of Ca. Moreover, the NCX1b seems more sensitive to cadmium than PMCA2. Besides, ECaC gene was treated with knowdown as morphants, which were appeared a higher mortality than wild-type zebrafish after exposed to 8.9μM Cd2+ (48h-LC50 of 3dpf larvae). But, ECaC cRNA was overexpressed, morphants appeared to similar mortality at the same treatment condition. Moreover, it was showed a significantly increase the Ca2+ influx.

Key words: Cadmium, Calcium, Calcium transporter, gene expression
目錄 I
Abstract V
前言 7
一、鎘與鈣的關係 7
二、哺乳動物與淡水魚調控鈣離子機制的比較 8
三、鎘對鈣離子調控的影響 11
材料與方法 13
一、預備實驗 13
(1)鎘對斑馬魚仔魚成長的無影響濃度(No Observed Effect Concentration, NOEC)之觀察 13
(2)鎘對斑馬魚仔魚的半致死濃度(Lethal concentration 50, LC50) 14
二、實驗動物 14
三、重金屬鎘(Cadmium,Cd)水之配置 15
四、魚體內總鈣量的測定 15
五、仔魚體長的量測 16
六、基因表現量測定 16
七、弱化ECaC基因(zECaC-Morpholino knockdown) 19
八、構築ECaC 基因 19
九、過度表現 ECaC(ECaC cRNA Overexpression) 24
十、鈣離子流進速率的測定(Ca2+ influx) 26
十一、實驗設計 27
十二、統計分析 29
結果 30
一、鎘對仔魚的總鈣量、體長和鈣運輸蛋白基因表現之影響 30
二、高鈣環境下,鎘暴露之仔魚其個體之總鈣量、體長和鈣運輸蛋白基因表現量之影響 30
三、顯微注射zECaC MO使ECaC表現量降低,對仔魚鈣離子吸收與對鎘耐受力之影響 31
四、 顯微注射cRNA使其過度表現ECaC,對仔魚鈣離子吸收與對鎘耐受力之影響 32
討論 33
總結 40
參考文獻 41
表 47
圖 52
附錄 66
Baldisserotto B, Chowdhury MJ, C.M. W (2005) Effects of dietary calcium and cadmium on cadmium accumulation, calcium and cadmium uptake from the water, and their interactions in juvenile rainbow trout. Aquat Toxicol, 72, 99-117.
Baldisserotto B, Kamunde C, Matsuo A, Wood CM (2004) A protective effect of dietary calcium against acute waterborne cadmium uptake in rainbow trout. Aquat Toxicol, 67, 57-73.
Bentley PJ (1992) Influx of zinc by channel catfish (Ictalurus punctatus): uptake from external environmental solutions. Comp Biochem Physiol C, 101, 215-217.
Blaustein MP, Lederer WJ (1999) Sodium/calcium exchange: its physiological implications. Physiol Rev, 79, 763 - 854.
Blechinger SR, Kusch RC, Haugo K, Matz C, Chivers DP, Krone PH (2007) Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish. Toxicology and Applied Pharmacology, 224, 72-80.
Blechinger SR, Warren JT, Kuwada JY, Krone PH (2002) Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line. Environmental Health Perspectives, 110, 1041-1046.
Chang M, Lin H, Hwang P (1997) Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus. Fish Physiology and Biochemistry, 16, 459-470.
Chen YY, Lu FI, Hwang PP (2003) Comparisons of calcium regulation in fish larvae. J Exp Zool, 295, 127 - 135.
Cheng SH, Wai AWK, So CH, Wu RSS (2000) Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos. Environmental Toxicology and Chemistry, 19, 3024-3031.
Chou M, Yang C, Lu F, Lin H, Hwang P (2002) Modulation of calcium balance in tilapia larvae (Oreochromis mossambicus) acclimated to low-calcium environments. Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 172, 109-114.
Chow ESH, Cheng SH (2003) Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis. Toxicological Sciences, 73, 149-159.
Clapham D (2003) TRP channels as cellular sensors. Nature, 426, 517-524.
Fraysse B, Mons R, Garric J (2006) Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicology and Environmental safety, 63, 253-267.
Galvez F, Franklin NM, Tuttle RB, Wood CM (2007) Interactions of waterborne and dietary cadmium on the expression of calcium transporters in the gills of rainbow trout: Influence of dietary calcium supplementation. Aquatic Toxicology, 84, 208-214.
Galvez F, Wong D, Wood CM (2006) Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout. Am J Physiol Regul Integr Comp Physiol, 291, R170 - 176.
Giles M (1984) Electrolyte and Water Balance in Plasma and Urine of Rainbow Trout(Salmo gairdneri) during Chronic Exposure to Cadmium. Canadian Journal of Fisheries and Aquatic Sciences, 41.
Glynn AW (2001) The influence of zinc on apical uptake of cadmium in the gills and cadmium influx to the circulatory system in zebrafish (Danio rerio). Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 128, 165-172.
Hoenderop JG, Dardenne O, Van Abel M, Van Der Kemp AW, Van Os CH, St -Arnaud R, Bindels RJ (2002) Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice. Faseb J, 16, 1398-1406.
Hoenderop JG, Nilius B, Bindels RJ (2005) Calcium absorption across epithelia. Physioll Rev, 85, 373 - 422.
Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH, Willems PH, Bindels RJ (1999) Molecular identification of the apical Ca2+ channel in 1, 25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem, 274, 8375-8378.
Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E (2003) Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest, 112, 1906 - 1914.
Hollis L, McGeer J, McDonald D, Wood C (2000) Effects of long term sublethal Cd exposure in rainbow trout during soft water exposure: implications for biotic ligand modelling. Aquatic Toxicology, 51, 93-105.
Hollis L, McGeer JC, McDonald DG, Wood CM (1999) Cadmium accumulation, gill Cd binding, acclimation, and physiological effects during long term sublethal Cd exposure in rainbow trout. Aquatic Toxicology, 46, 101-119.
Hwang P, Lin S, Lin H (1995) Different sensitivities to cadmium in tilapia larvae (Oreochromis mossambicus; teleostei). Archives of Environmental Contamination and Toxicology, 29, 1-7.
Hwang PP, Lee T (2007) New insights into fish ion regulation and mitochondria-rich cells. Comp Biochem Physiol A Mol Integr Physiol, 148, 479 - 497.
Hwang PP, Tsai YN, Tung YC (1994) Calcium Balance in Embryos and Larvae of the Fresh-Water-Adapted Teleost, Oreochromis Mossambicus. Fish Physio Biochem, 13, 325 - 333.
Hwang PP, Tung YC, Chang MH (1996) Effect of environmental calcium levels on calcium uptake in tilapia larvae (Oreochromis mossambicus). Fish Physiol Biochem, 15, 363 - 370.
Konishi T, Matsumoto S, Tsuruwaka Y, Shiraki K, Hirata K, Tamaru Y, Takagi M (2006) Enhancing the tolerance of zebrafish (Danio rerio) to heavy metal toxicity by the expression of plant phytochelatin synthase. Journal of Biotechnology, 122, 316-325.
Lee T, Hwang P, Lin H (1996) Morphological changes of integumental chloride cells to ambient cadmium during the early development of the teleost, Oreochromis mossambicus. Environmental Biology of Fishes, 45, 95-102.
Liao B-K, Deng A-N, Chen S-C, Chou M-Y, Hwang P-P (2007) Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells. BMC Genomics, 8, 354.
Liedtke W, Kim C (2005) Functionality of the TRPV subfamily of TRP ion channels: add mechano-TRP and osmo-TRP to the lexicon! Cellular and Molecular Life Sciences (CMLS), 62, 2985-3001.
Locknar SA, Barstow KL, Tompkins JD, Merriam LA, Parsons RL (2004) Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones. J Physiol, 555, 627-635.
McGeer JC, Szebedinszky C, Gordon McDonald D, Wood CM (2000) Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout 2: tissue specific metal accumulation. Aquatic Toxicology, 50, 245-256.
Meinelt T, Playle RC, Pietrock M, Burnison BK, Wienke A, Steinberg CE (2001) Interaction of cadmium toxicity in embryos and larvae of zebrafish (Danio rerio) with calcium and humic substances. Aquat Toxicol, 54, 205-215.
Montell C, Birnbaumer L, Flockerzi V, Bindels RJ, Bruford EA, Caterina MJ, Clapham DE, Harteneck C, Heller S, Julius D, Kojima I, Mori Y, Penner R, Prawitt D, Scharenberg AM, Schultz G, Shimizu N, Zhu MX (2002) A unified nomenclature for the superfamily of TRP cation channels. Mol Cell, 9, 229-231.
Nguyen LT, Janssen CR (2001) Comparative sensitivity of embryo-larval toxicity assays with African catfish (Clarias gariepinus) and zebra fish (Danio rerio). Environ Toxicol, 16, 566-571.
Nijenhuis T, Vallon V, van der Kemp AW, Loffing J, Hoenderop JG, Bindels RJ (2005) Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia. J Clin Invest, 115, 1651-1658.
Pan T-C, Liao B-K, Huang C-J, Lin L-Y, Hwang P-P (2005) Epithelial Ca2+ channel expression and Ca2+ uptake in developing zebrafish. Am J Physiol Regul Integr Comp Physiol, 289, R1202-1211.
Peng J-B, Chen X-Z, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, Hediger MA (1999) Molecular Cloning and Characterization of a Channel-like Transporter Mediating Intestinal Calcium Absorption. J. Biol. Chem., 274, 22739-22746.
Peng J, Chen X, Berger U, Vassilev P, Brown E, Hediger M (2000) A Rat Kidney-specific Calcium Transporter in the Distal Nephron. Journal of Biological Chemistry, 275, 28186-28194.
Perceval O, Couillard Y, Pinel-Alloul B, Giguere A, Campbell PG (2004) Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses. Aquat Toxicol, 69, 327-345.
Perry SF, Goss GG, Fenwick JC (1992) Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts. Fish Physiology and Biochemistry, 10, 327-337.
Pratap HB, Fu H, Lock RAC, Wendelaar Bonga SE (1989) Effect of waterborne and dietary cadmium on plasma ions of the teleost Oreochromis mossambicus in relation to water calcium levels. Archives of Environmental Contamination and Toxicology, 18, 568-575.
Pratap HB, Wendelaar Bonga SE (2007) Calcium homeostasis in low and high calcium water acclimatized Oreochromis mossambicus exposed to ambient and dietary cadmium. J Environ Biol, 28, 385-393.
Qiu A, Hogstrand C (2004) Functional characterisation and genomic analysis of an epithelial calcium channel (ECaC) from pufferfish, Fugu rubripes. Gene, 342, 113 - 123.
Reddy GN, Prasad MN (1992) Cadmium induced potassium efflux from Scenedesmus quadricauda. Bull Environ Contam Toxicol, 49, 600-605.
Rogers JT, Wood CM (2004) Characterization of branchial lead-calcium interaction in the freshwater rainbow trout Oncorhynchus mykiss. J Exp Biol, 207, 813-825.
Scott GR, Sloman KA (2004) The effects of environmental pollutants on complex fish behaviour: integrating behavioural and physiological indicators of toxicity. Aquat Toxicol, 68, 369-392.
Scott GR, Sloman KA, Rouleau C, Wood CM (2003) Cadmium disrupts behavioural and physiological responses to alarm substance in juvenile rainbow trout (Oncorhynchus mykiss). J Exp Biol, 206, 1779-1790.
Shahsavarani A, McNeill B, Galvez F, Wood CM, Goss GG, Hwang PP, Perry SF (2006) Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss). J Exp Biol, 209, 1928-1943.
Shahsavarani A, Perry SF (2006) Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss). Am J Physiol Regul Integr Comp Physiol, 291, R1490 - 1498.
Szebedinszky C, McGeer JC, McDonald DG, Wood CM (2001) Effects of chronic Cd exposure via the diet or water on internal organ-specific distribution and subsequent gill Cd uptake kinetics in juvenile rainbow trout (Oncorhynchus mykiss). Environmental Toxicology and Chemistry, 20, 597-607.
Tseng D-Y, Chou M-Y, Tseng Y-C, Hsiao C-D, Huang C-J, Kaneko T, Hwang P-P (2009) Effects of stanniocalcin 1 on calcium uptake in zebrafish (Danio rerio) embryo. Am J Physiol Regul Integr Comp Physiol, 296, R549-557.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 3, 0034.1-0034.11.
Verbost PM, Flik G, Pang PK, Lock RA, Wendelaar Bonga SE (1989) Cadmium inhibition of the erythrocyte Ca2+ pump. J Biol Chem, 264, 5613-5615.
Verbost PM, Senden MH, van Os CH (1987) Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium. Biochim Biophys Acta, 902, 247-252.
Voelker D, Vess C, Tillmann M, Nagel R, Otto G, Geisler R, Schirmer K, Scholz S (2007) Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae. Aquatic Toxicology, 81, 355-364.
Wang S, Shih Y, Ko W, Wei Y, Shih C (2008) Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway. Cellular and Molecular Life Sciences (CMLS), 65, 3640-3652.
Webster W (1990) The teratology and developmental toxicity of cadmium. Issue Rev. Teratol, 5, 255–282.
Wu SM, Ding HR, Lin LY, Lin YS (2008) Juvenile Tilapia ( Oreochromis mossambicus ) Strive to Maintain Physiological Functions After Waterborne Copper Exposure. Archives of Environmental Contamination and Toxicology, 54, 482-492.
Wu SM, Ho YC, Shih MJ (2007) Effects of Ca2+ or Na+ on metallothionein expression in tilapia larvae (Oreochromis mossambicus) exposed to cadmium or copper. Arch Environ Contam Toxicol, 52, 229-234.
Wu SM, Jong KJ, Kuo SY (2003) Effects of copper sulfate on ion balance and growth in tilapia larvae (Oreochromis mossambicus). Arch Environ Contam Toxicol, 45, 357-363.
Wu SM, Weng CF, Hwang JC, Huang CI, Hwang PP (2000) Metallothionein Induction in Early Larval Stages of Tilapia (Oreochromis mossambicus). Physiological and Biochemical Zoology, 73, 531-537.
Zalups RK, Ahmad S (2003) Molecular handling of cadmium in transporting epithelia. Toxicol Appl Pharmacol, 186, 163-188.
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