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The Identification of Metallothionein in Rare Minnow (Gobiocypris Rarus

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The Identification of Metallothionein in Rare Minnow (Gobiocypris Rarus e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1283–1291 Available online at www.sciencedirect.com ScienceDirect jo urnal homepage: www.elsevier.com/locate/etap The identification of metallothionein in rare minnow (Gobiocypris rarus) and its expression following heavy metal exposure a,b,c a,b,c,∗ a,b,c Chunling Wang , Futie Zhang , Wenxuan Cao , a,b,c Jianwei Wang a Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, PR China b The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy Of Sciences, Wuhan, Hubei 430072, PR China c University of Chinese Academy of Sciences, Beijing 100039, PR China a r t a b i c s t l e i n f o r a c t Article history: Heavy metal, such as cadmium (Cd), lead (Pb) and copper (Cu) poses serious toxin to aquatic Received 7 November 2013 organisms. These exogenous materials affect biological processes including physiology, Received in revised form biochemistry and development. Metallothionein (MT), one of the metal-regulated genes, 15 April 2014 participates in regulating essential and detoxifying non-essential metals in living animals. Accepted 18 April 2014 In this study, MT EST in rare minnow (Gobiocypris rarus) (GrMT) was obtained from the cDNA Available online 28 April 2014 subtraction library and the GrMT cDNA was firstly cloned by RACE with a sequence of 379 bp, which can code 60 amino acids. After Cd exposure, the GrMT expression levels dramati- Keywords: cally changed in liver, spleen, gill, kidney, intestine, but moderately in muscle. Significantly Heavy metals positive relationships were found between Cd dosages and MT expression levels in liver. Rare minnow And there distinction existed at the GrMT transcript level in fish gender and developmen- Metallothionein tal stages during Cd exposure. While Pb exposure, the expression alteration happened in Gene expression spleen, gill, intestine, muscle, and moderately in liver. In the Cu treatment test, there were slight changes in intestine and kidney, but more significant changes occurred in spleen, gill, muscle, and liver. This study had investigated the effects of diverse heavy metals on GrMT expression patterns. The results suggest that GrMT would be a potential biomarker to metal contamination monitoring in aquatic environments and rare minnow could be one of the perfect experimental fishes for surveying the freshwater pollution in China. © 2014 Elsevier B.V. All rights reserved. The heavy metal contamination in aquatic ecosystems has 1. Introduction attracted worldwide attention. These exogenous materials affect biological processes including physiology, biochem- Heavy metals, such as cadmium (Cd), lead (Pb) and copper istry and development (Zhu et al., 2011). Cd is a ubiquitous (Cu), are in high concentrations in many aquatic environment environment contaminant that has been detected in vari- (Zenk, 1996). They pose serious toxin to aquatic organisms. ous aquatic organisms. Cd, even in low concentrations, could ∗ Corresponding author at: The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy Of Sciences, Wuhan, Hubei 430072, PR China. Tel.: +86 27 68780723; fax: +86 27 68780065. E-mail address: [email protected] (F. Zhang). http://dx.doi.org/10.1016/j.etap.2014.04.021 1382-6689/© 2014 Elsevier B.V. All rights reserved. 1284 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1283–1291 2+ induce oxidative stress response and DNA damage in the liv- (Wang et al., 2013). Mean lethal concentration (LC50) of Cd ers of Cyprinus carpio var. color (Jia et al., 2011). When in high was evaluated among different groups with 10 fish in each level, Cd would cause cytotoxicity (George et al., 1996). Cu is group at 48 h post-exposure. an essential micronutrient for fish function. However, redun- 2+ dant Cu would exert a variety of physiological effects in fish, 2.1.2. Cd dosage-dependent experiment such as increasing heart rates and retarding the development Rare minnow adult fish without gender difference, described of zebra fish embryos (Johnson et al., 2007). Short-term expo- in Section 2.1.1, were exposed to six differential concentra- sure to high concentration Pb can cause brain and kidney tions of Cd ion including: 0 ␮g/L, 100 ␮g/L, 200 ␮g/L, 400 ␮g/L, damage and gastrointestinal distress in humans (Jarup, 2003). 800 ␮g/L, and 1600 ␮g/L. There were two tanks in each concen- Gene expressions affected by heavy metals has arouse certain tration with 10 fish in each tank. Several fish were sacrificed notices recently, and metallothionein (MT) is one of the genes and livers were collected at 0 h and 12 h exposure, then quickly that are regulated by various heavy metal ions. removed and homogenized in RNAlater (Sigma) reagent and ◦ MTs are a family of cysteine-rich, low-molecular-weight, stored at −20 C until future analysis. metal-binding proteins that have been reported for a variety of 2+ living organisms from prokaryotes to eukaryotes. MT has high 2.1.3. Male and female fish exposed to Cd solution cysteine content (30%) and is highly conservative in the pro- separately cess of evolution. It participates in regulating essential metals Male and female fish, described in Section 2.1.1, were sep- 2+ (zinc and copper) and detoxifying non-essential metals (cad- arately exposed to Cd solution. The concentration was 2+ mium, lead, and mercury) in living animals. The role of MT selected based on Cd 48hLC50. Male fish were treated in four in detoxification of heavy metals and maintaining of essential tanks with 10 fish in each tank, 2 were metal treated groups metal homeostasis, which is due to its high affinity for these while the other were blank. Several fish were sacrificed and metals, is mostly investigated in recent years (Bervoets et al., kidney, liver, muscle, and spleen were collected at 0 h and 2013). 12 h exposure. Tissues were quickly removed and individually ◦ We employed rare minnow as a model fish in this study. homogenized in RNAlater (Sigma) reagent and stored at −20 C Rare minnow, a native endemic species in China, has many until future analysis. Female fish were treated just as the male. excellent biological characteristics for aquatic toxicity tests: 2+ small size (38–85 mm for adult), ease of culture in laboratory, 2.1.4. Fish zygote and larvae exposure to Cd solution frequent spawning of transparent eggs (every 4 days), short Fish zygote not later than 8 h post-fertilization and newly ◦ 2+ generation span, wide range of temperature (0–35 C). More- hatched larvae were collected and exposed to Cd solu- 2+ over, it has been approved to be sensitive to Cd and other tion. The concentration was selected based on Cd 48hLC50. environmental contaminant (Wei et al., 2008). After 0 h and 12 h exposure, zygote and larvae were collected, In the present work, our goal was to study MT gene expres- quickly removed and individually homogenized in RNAlater ◦ sion pattern in rare minnow including: MT expression after (Sigma) reagent and stored at −20 C until future analysis. 2+ 2+ exposure to Cd ; the relationship between Cd concentra- tion and MT expression level in liver; the influence of fish 2.1.5. Adult fish without gender difference exposed to 2+ 2+ development stage and adult gender on gene expression; and various concentrations of Pb and Cu solution 2+ 2+ 2+ 2+ 2+ the regulations of other metals such as Cu and Pb on MT Fish exposed to Pb and Cu were treated as the Cd exper- expression, to offer the baseline biological data of rare minnow iment described in Section 2.1.1. Different groups included 2+ exposure to heavy metal pollution in water. 0 mg/L, 1 mg/L, 2 mg/L, 4 mg/L and 8 mg/L Pb solution, while different groups included 0 ␮g/L, 3 ␮g/L, 6 ␮g/L, 12 ␮g/L and ␮ 2+ 2+ 24 g/L Cu solution. Mean lethal concentration (LC50) of Pb 2. Materials and methods 2+ and Cu was evaluated among different groups with 10 fish in each group at 48 h after exposure. 2.1. Fish and heavy metals exposure experiments 2.2. Total RNA isolation and cDNAs synthesis CdCl2, Pb(NO3)2, and CuSO4 were purchased from Geel (Belgium) for analysis. The ultra-pure grade water was from Total RNA extraction was performed as described in our pre- Millipore instrument (Hi-tech). vious study (Wang et al., 2013). 2.1.1. Adult fish without gender difference exposed to 2.3. Construction of cDNA subtraction library and 2+ various concentrations of Cd solution rapid amplification of cDNA ends (RACE) Rare minnow adult fish (2 months old) with an average length ± 2.1 0.2 mm were obtained from a laboratory hatchery and cDNA subtraction library was constructed as described in our maintained in 5 L glass tanks (2 bodies/L) for 2 days before previous study (Wang et al., 2013). metal exposure. During feeding, fish were maintained at To obtain full-length rare minnow MT cDNA sequence, ± ◦ 25 1 C and subjected to a photoperiod of 16 h:8 h (light:dark). 3 and 5 rapid amplification of cDNA ends (RACE) reac- TM The average pH and dissolved oxygen (DO) of tank water tion was performed using the SMART RACE cDNA were 7.4 ± 0.1 and 5.8± 0.2 mg/L, respectively.
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