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Identification of copper-responsive genes in an early life stage of the fathead minnow Pimephales promelas

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Abstract

While physiological changes associated with copper toxicity have been studied in adult fathead minnow, Pimephales promelas, little is known about the effect of copper on newly hatched larvae. As a result we initiated an investigation on the mechanism of copper toxicity in 24 h post-hatch larvae using gene expression changes to identify responsive genes. Fish were exposed to copper concentrations of 0, 50, 125 and 200 μg/L in a 48 h toxicity test. Total RNA from survivors was used in a differential display assay to screen for differentially expressed gene products. Altogether, 654 copper-responsive differentially expressed bands were collected. Database searches found homology for 261 sequences. One hundred and sixty-one bands were homologous to NCBI genes of known function, of which 69 were individual genes. The most abundant categories of functional genes responding to copper were involved in protein synthesis/translational machinery and contractile proteins. Twenty-one dose-responsive genes were measured for expression changes using real-time quantitative PCR. Differential gene expression was validated for 11 of 13 genes, when a 1.2 times qPCR difference between the copper and control samples was observed. Transcripts identified as titin, cytochrome b, fast muscle specific heavy myosin chain 4, fast muscle troponin I, proteasome 26S subunit and troponin T3a were induced over twofold. Differential display bands identified as 60S ribosomal proteins L27 and L12 were repressed approximately threefold. We conclude that copper exposure affects several cellular pathways in larval fathead minnows with protein synthesis, ribosome structure, and muscle contractile proteins being the most sensitive to this stress.

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References

  • Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. doi:10.1093/nar/25.17.3389

    Article  CAS  Google Scholar 

  • Aruga J, Yokota N, Mikoshiba K (2003) Human SLITRK family genes: genomic organization and expression profiling in normal brain and brain tumor tissue. Gene 315:87–94. doi:10.1016/S0378-1119(03)00715-7

    Article  CAS  Google Scholar 

  • Baker JTP (1969) Histological and microscopical observations on copper poisoning in the winter flounder (Pseudopleuronectes americanus). J Fish Res Board Can 26:2785–2793

    CAS  Google Scholar 

  • Bartosiewicz M, Penn S, Buckpitt A (2001) Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo (a) pyrene, and trichloroethylene. Environ Health Perspect 109:71–74. doi:10.2307/3434924

    Article  CAS  Google Scholar 

  • Beretta L, Dubois MF, Sobel A et al (1995) Stathmin is a major substrate for mitogen-activated protein kinase during heat shock and chemical stress in HeLa cells. Eur J Biochem 227:388–395. doi:10.1111/j.1432-1033.1995.tb20401.x

    Article  CAS  Google Scholar 

  • Blaxter JHS (1988) Pattern and variety in development. In: Hoar WS, Randall DJ (eds) Fish physiology, vol XI, Part A. Academic Press, San Diego

  • Brooks ML, Boese CJ, Meyer JS (2006) Complexation and time-dependent accumulation of copper by larval fathead minnows (Pimephales promelas): implications for modeling toxicity. Aquat Toxicol 78:42–49. doi:10.1016/j.aquatox.2006.02.002

    Article  CAS  Google Scholar 

  • Carpenter KE (1927) The lethal action of soluble metallic salts on fishes. J Exp Biol 4:378–390

    CAS  Google Scholar 

  • Chan KM, Ku LL, Chan C-Y et al (2006) Metallothionein gene expression in zebrafish embryo-larvae and ZFL cell-line exposed to heavy metal ions. Mar Environ Res 62:S83–S87. doi:10.1016/j.marenvres.2006.04.012

    Article  CAS  Google Scholar 

  • Chen WY, John JA, Lin CH et al (2004) Expression of metallothionein gene during embryonic and early larval development in zebrafish. Aquat Toxicol 69:215–227. doi:10.1016/j.aquatox.2004.05.004

    Article  CAS  Google Scholar 

  • Cheung AP, Lam TH, Chan KM (2004) Regulation of Tilapia metallothionein gene expression by heavy metal ions. Mar Environ Res 58:389–394. doi:10.1016/j.marenvres.2004.03.084

    Article  CAS  Google Scholar 

  • David CPC (2003) Establishing the impact of acid mine drainage through metal bioaccumulation and taxa richness of benthic insects in a tropical Asian stream (The Philippines). Environ Toxicol Chem 22:2952–2959. doi:10.1897/02-529

    Article  CAS  Google Scholar 

  • Denslow ND, Bowman CJ, Ferguson RJ et al (2001) Induction of gene expression in sheepshead minnows (Cyprinodon variegatus) treated with 17β-estradiol, diethylstilbestrol, or ethinylestradiol: the use of mRNA fingerprints as an indicator of gene regulation. Gen Comp Endocrinol 121:250–260. doi:10.1006/gcen.2001.7605

    Article  CAS  Google Scholar 

  • Devlin EW, Brammer JD, Puyear RL et al (1996) Prehatching development of the fathead minnow Pimephales promelas Rafinesque. USEPA publication EPA/600/R-96/079, Cincinnati

  • Donaldson EM, Dye HM (1975) Corticosteroid concentrations in sockeye salmon (Oncorhynchus nerka) exposed to low concentrations of copper. J Fish Res Board Can 32:533–539

    CAS  Google Scholar 

  • Fei G, Guo C, Sun HS et al (2007) Chronic hypoxia stress-induced differential modulation of heat-shock protein 70 and presynaptic proteins. J Neurochem 100:50–61. doi:10.1111/j.1471-4159.2006.04194.x

    Article  CAS  Google Scholar 

  • Galkin O, Bentley AA, Gupta S et al (2007) Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5. RNA 13:2116–2128. doi:10.1261/rna.688207

    Article  CAS  Google Scholar 

  • Gardner GR, LaRoche G (1973) Copper induced lesions in estuarine teleosts. J Fish Res Board Can 30:363–368

    CAS  Google Scholar 

  • Hannan KM, Brandenburger Y, Jenkins A et al (2003) mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF. Mol Cell Biol 23:8862–8877. doi:10.1128/MCB.23.23.8862-8877.2003

    Article  CAS  Google Scholar 

  • Hernandez PP, Moreno V, Olivari FA et al (2006) Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio). Hear Res 213:1–10. doi:10.1016/j.heares.2005.10.015

    Article  CAS  Google Scholar 

  • Hilfiker S, Pieribone VA, Nordstedt C et al (1999) Regulation of synaptotagmin I phosphorylation by multiple protein kinases. J Neurochem 73:921–932. doi:10.1046/j.1471-4159.1999.0730921.x

    Article  CAS  Google Scholar 

  • Hua Y, Zhou J (2004) Survival motor neuron protein facilitates assembly of stress granules. FEBS Lett 572:69–74. doi:10.1016/j.febslet.2004.07.010

    Article  CAS  Google Scholar 

  • Infante C, Asensio E, Canavate JP et al (2008) Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole (Solea senegalensis Kaup): differential gene expression and thyroid hormones dependence during metamorphosis. BMC Mol Biol 9:19–36. doi:10.1186/1471-2199-9-19

    Article  Google Scholar 

  • Karan V, Vitorović S, Tutundžić V et al (1998) Functional enzymes activity and gill histology of carp after copper sulfate exposure and recovery. Ecotoxicol Environ Saf 40:49–55. doi:10.1006/eesa.1998.1641

    Article  CAS  Google Scholar 

  • Kyle HM (1926) The biology of fishes. The Macmillan Company, New York

    Google Scholar 

  • Landriscina M, Bagalá C, Mandinova A et al (2001) Copper induces the assembly of a multiprotein aggregate implicated in the release of fibroblast growth factor 1 in response to stress. J Biol Chem 276:25549–25557. doi:10.1074/jbc.M102925200

    Article  CAS  Google Scholar 

  • Lange S, Xiang F, Yakovenko A et al (2005) The kinase domain of titin controls muscle gene expression and protein turnover. Science 308:1599–1603. doi:10.1126/science.1110463

    Article  CAS  Google Scholar 

  • Larkin P, Villeneuve DL, Knoebl I et al (2007) Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26:1497–1506. doi:10.1897/06-501R.1

    Article  CAS  Google Scholar 

  • Lewis SS (2005) Identification of stress-responsive genes in the early larval stage of the fathead minnow Pimephales promelas. Ph.D. dissertation. University of Cincinnati, Cincinnati

  • Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971. doi:10.1126/science.1354393

    Article  CAS  Google Scholar 

  • Linskens MH, Feng J, Andrews WH et al (1995) Cataloging altered gene expression in young and senescent cells using enhanced differential display. Nucleic Acids Res 23:3244–3251. doi:10.1093/nar/23.16.3244

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25:402–408. doi:10.1006/meth.2001.1262

    Article  CAS  Google Scholar 

  • McKim JM (1977) Evaluation of tests with early life stages of fish for predicting long-term toxicity. J Fish Res Board Can 34:1148–1154

    CAS  Google Scholar 

  • McKim JM, Eaton JG, Holcombe GW (1978) Metal toxicity to embryos and larvae of eight species of freshwater fish-II: copper. Bull Environ Contam Toxicol 19:608–616. doi:10.1007/BF01685847

    Article  CAS  Google Scholar 

  • Moore JW, Ramamoorthy S (1984) Heavy metals in natural waters, applied monitoring and impact assessment. Springer, New York

    Google Scholar 

  • Mount DI (1968) Chronic toxicity of copper to fathead minnows (Pimephales promelas, Rafinesque). Water Res 2:215–223. doi:10.1016/0043-1354(68)90027-4

    Article  CAS  Google Scholar 

  • Newman MC, Unger MA (2003) Fundamentals of ecotoxicology, 2nd edn. Lewis, Boca Raton

    Google Scholar 

  • Patel PH, Tamanoi F (2006) Increased Rheb-TOR signaling enhances sensitivity of the whole organism to oxidative stress. J Cell Sci 119:4285–4292. doi:10.1242/jcs.03199

    Article  CAS  Google Scholar 

  • Rubin CI, Atweh GF (2004) The role of stathmin in the regulation of the cell cycle. J Cell Biochem 93:242–250. doi:10.1002/jcb.20187

    Article  CAS  Google Scholar 

  • Sanders BM, Nguyen J, Martin LS et al (1995) Induction and subcellular localization of two major stress proteins in response to copper in the fathead minnow Pimephales promelas. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 112:335–343

    CAS  Google Scholar 

  • Scudder BC, Carter JL, Leland HV (1988) Effects of copper on development of the fathead minnow, Pimephales promelas Rafinesque. Aquat Toxicol 12:107–124. doi:10.1016/0166-445X(88)90029-X

    Article  CAS  Google Scholar 

  • Shumyatsky GP, Malleret G, Shin RM et al (2005) Stathmin, a gene enriched in the amygdala, controls both learned and innate fear. Cell 123:697–709. doi:10.1016/j.cell.2005.08.038

    Article  CAS  Google Scholar 

  • Sompayrac L, Jane S, Burn TC et al (1995) Overcoming limitations of the mRNA differential display technique. Nucleic Acids Res 23:4738–4739. doi:10.1093/nar/23.22.4738

    Article  CAS  Google Scholar 

  • Stokes PM (1979) Copper accumulations in freshwater biota. In: Nriagu JO (ed) Copper in the environment. Part I. Ecological cycling. Wiley, New York, pp 358–381

    Google Scholar 

  • Takagi M, Absalon MJ, McLure KG et al (2005) Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell 123:49–63. doi:10.1016/j.cell.2005.07.034

    Article  CAS  Google Scholar 

  • Tan FL, Moravec CS, Li J et al (2002) The gene expression fingerprint of human heart failure. Proc Natl Acad Sci USA 99:11387–11392. doi:10.1073/pnas.162370099

    Article  CAS  Google Scholar 

  • Taylor EW, Beaumont MW, Butler PJ (1996) Lethal and sub-lethal effects of copper upon fish: a role for ammonia toxicity? In: Taylor EW et al (eds) Toxicology of aquatic pollution: physiological, cellular and molecular approaches. Cambridge University Press, New York, pp 85–114

    Google Scholar 

  • Thome J, Pesold B, Baader M et al (2001) Stress differentially regulated synaptophysin and synaptotagmin expression in hippocampus. Biol Psychiatry 50:809–812. doi:10.1016/S0006-3223(01)01229-X

    Article  CAS  Google Scholar 

  • Ton C, Stamatiou D, Dzau VJ et al (2002) Construction of a zebrafish cDNA microarray: gene expression profiling of the zebrafish during development. Biochem Biophys Res Commun 296:1134–1142. doi:10.1016/S0006-291X(02)02010-7

    Article  CAS  Google Scholar 

  • Ton C, Stamatiou D, Liew CC (2003) Gene expression profile of zebrafish exposed to hypoxia during development. Physiol Genomics 13:97–106

    CAS  Google Scholar 

  • Valko M, Morris H, Cronin MTD (2005) Metals, toxicity and oxidative stress. Curr Med Chem 12:1161–1208. doi:10.2174/0929867053764635

    Article  CAS  Google Scholar 

  • Weber CI (1993) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, 4th edn. EPA/600/4–90/027F, Cincinnati

  • Wintz H, Yoo LJ, Loguinov A et al (2006) Gene expression profiles in fathead minnow exposed to 2,4-DNT: correlation with toxicity in mammals. Toxicol Sci 94:71–82. doi:10.1093/toxsci/kfl080

    Article  CAS  Google Scholar 

  • Wright P, Felskie A, Anderson P (1995) Induction of ornithine-urea cycle enzymes and nitrogen metabolism and excretion in rainbow trout (Oncorhynchus mykiss) during early life stages. J Exp Biol 198:127–135

    CAS  Google Scholar 

  • Zhang F, Hamanaka RB, Bobrovnikova-Marjon E et al (2006) Ribosomal stress couples the unfolded protein response to p53-dependent cell cycle arrest. J Biol Chem 281:30036–30045. doi:10.1074/jbc.M604674200

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We acknowledge the contributions of J. Lazorchak, M. Smith, D. Lattier, M. Bagley, C. Tomlinson, R. Flick, D. Gordon, S. Jackson, M. Brown-Augustine, R. Haugland, J. Deddens, S. Keely and J. Stringer to this project. Funds for this project have been provided by the University of Cincinnati Research Foundation through the generosity of the Schlemm Family.

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  1. Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, 45221, USA

    Solange S. Lewis & Stephen J. Keller

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Correspondence to Stephen J. Keller.

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Lewis, S.S., Keller, S.J. Identification of copper-responsive genes in an early life stage of the fathead minnow Pimephales promelas . Ecotoxicology 18, 281–292 (2009). https://doi.org/10.1007/s10646-008-0280-3

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