Hostname: page-component-cd9895bd7-hc48f Total loading time: 0 Render date: 2024-12-29T10:19:33.342Z Has data issue: false hasContentIssue false
  • English
  • Français

Evaluation of reference genes for reverse transcription-quantitative PCR assays in organs of zebrafish exposed to glyphosate-based herbicide, Roundup

Published online by Cambridge University Press:  27 November 2017

M. L. Jaramillo ,
A. G. Pereira ,
C. E. Davico ,
L. Nezzi ,
D. Ammar ,
Y. M. R. Müller  and
E. M. Nazari
M. L. Jaramillo
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
A. G. Pereira
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
C. E. Davico
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
L. Nezzi
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
D. Ammar
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil Centro Universitário – Católica de Santa Catarina, 89203-005 Joinville, Santa Catarina, Brazil
Y. M. R. Müller
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
E. M. Nazari*
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
*
†E-mail: evelise.nazari@ufsc.br
Get access

Abstract

Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, β-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0.065, 0.65 and 6.5 mg N-(phosphonomethyl) glycine/l) for 7 days. Genes were ranked by geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder, and their best combinations were determined by geNorm and NormFinder programs. The two most stable ranked genes were specific to each organ: gill (β-act; rpl8); brain (rpl8; β-act); testis (ef1α; gapdh); and ovary (rpl8; hprt1). The cat transcript level was used to evaluate the effect of normalization with these reference genes. These are the first suitable reference genes described for the analysis of gene expression in organs of Roundup-exposed zebrafish, and will allow investigations of the molecular mechanisms of Roundup toxicity.

Keywords

Roundup WGnormalizationexpression stabilityreverse transcription-quantitative PCRDanio rerio
Type
Research Article
Information
animal , Volume 12 , Issue 7 , July 2018 , pp. 1424 - 1434
Copyright
© The Animal Consortium 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Acquavella, JF, Alexander, BH, Mandel, JS, Gustin, C, Baker, B and Chapman, P 2004. Glyphosate biomonitoring for farmers and their families: results from the farm family exposure study. Environmental Health Perspectives 112, 321–326.Google Scholar
Andersen, CL, Jensen, JL and Ørntoft, TF 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64, 5245–5250.Google Scholar
Armiliato, N, Ammar, D, Nezzi, L, Straliotto, M, Muller, YMR and Nazari, EM 2014. Changes in ultrastructure and expression of steroidogenic factor-1 in ovaries of zebrafish Danio rerio exposed to glyphosate. Journal of Toxicology and Environmental Health, Part A 77, 405–414.Google Scholar
Arregui, MC, Lenardón, A, Sanchez, D, Maitre, MI, Scotta, R and Enrique, S 2004. Monitoring glyphosate residues in transgenic glyphosate-resistant soybean. Pest Management Science 60, 163–166.Google Scholar
Bower, NI and Johnston, IA 2009. Selection of reference genes for expression studies with fish myogenic cell cultures. BMC Molecular Biology 10, 1–11.Google Scholar
Chapman, JR and Waldenström, J 2015. With reference to reference genes: a systematic review of endogenous controls in gene expression studies. PLoS One 10, 1–18.Google Scholar
Chen, D, Pan, X, Xiao, P, Farwell, MA and Zhang, B 2011. Evaluation and identification of reliable reference genes for pharmacogenomics, toxicogenomics, and small RNA expression analysis. Journal of Cellular Physiology 226, 2469–2477.Google Scholar
Conselho Nacional do Meio Ambiente (CONAMA) 2005. Classification of water bodies and environmental guidelines for its framework and establishes the conditions and standards for effluent discharge, and other measures. Resolução no. 357, Conselho Nacional do Meio Ambiente, Brasilia/DF, Brazil, 17 March.Google Scholar
Conselho Nacional de Controle de Experimentação Animal (CONCEA) 2013. Diretrizes da pratica de eutanasia do CONCEA. Ministério da Ciência, Tecnologia e Inovação, Brasilia/DF, Brazil, pp. 1–54.Google Scholar
Dai, Y-J, Jia, Y-F, Chen, N, Bian, W-P, Li, Q-K, Ma, Y-B, Chen, Y-L and Pei, D-S 2014. Zebrafish as a model system to study toxicology. Environmental Toxicology and Chemistry 33, 11–17.Google Scholar
Environmental Protection Agency (EPA) 2002. Occurrence summary and use support document for the six-year review of national primary drinking water regulations, EPA-815-D-02-006 453, USA.Google Scholar
Filby, AL and Tyler, CR 2007. Appropriate ‘housekeeping’ genes for use in expression profiling the effects of environmental estrogens in fish. BMC Molecular Biology 8, 10.Google Scholar
Folmar, LC, Sanders, HO and Julin, AM 1979. Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates. Archives of Environmental Contamination and Toxicology 8, 269–278.Google Scholar
Giesy, J, Dobson, S and Solomon, K 2000. Ecotoxicological risk assessment for roundup herbicide. In Reviews of environmental contamination and toxicology (ed. G Ware), pp. 35–120. Springer-Verlag, New York, USA.Google Scholar
Giroux, I, Roy, N and Lamontagne, C 2010. Présence de Pesticides dans l’Eau Souterraine en Milieu Agricole: Étude Pilote du Bassin Versant de la Rivière Châteauguay. Canadian Water Resources Journal 35, 527–542.Google Scholar
Gong, H, Sun, L, Chen, B, Han, Y, Pang, J, Wu, W, Qi, R and Zhang, TM 2016. Evaluation of candidate reference genes for RT-qPCR studies in three metabolism related tissues of mice after caloric restriction. Scientific Reports – Nature 6, 38513.Google Scholar
Gubern, C, Hurtado, O, Rodríguez, R, Morales, JR, Romera, VG, Moro, MA, Lizasoain, I, Serena, J and Mallolas, J 2009. Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia. BMC Molecular Biology 10, 57.CrossRefGoogle ScholarPubMed
Guyton, KZ, Loomis, D, Grosse, Y, El Ghissassi, F, Benbrahim-Tallaa, L, Guha, N, Scoccianti, C, Mattock, H, Straif, K, Blair, A, Fritschi, L, McLaughlin, J, Sergi, CM, Calaf, GM, Le Curieux, F, Baldi, I, Forastiere, F, Kromhout, H, Mannetje, A, Rodriguez, T, Egeghy, P, Jahnke, GD, Jameson, CW, Martin, MT, Ross, MK, Rusyn, I and Zeise, L 2015. Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate. Lancet Oncology 16, 490–491.Google Scholar
Hu, Y, Xie, S and Yao, J 2016. Identification of novel reference genes suitable for qRT-PCR normalization with respect to the zebrafish developmental stage. PLoS One 11, e0149277.Google Scholar
Ingerslev, HC, Pettersen, EF, Jakobsen, RA, Petersen, CB and Wergeland, HI 2006. Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Molecular Immunology 43, 1194–1201.Google Scholar
Jofré, DM, Alvarez, M, Perez, E, Mohamed, F, Jerez, MB, Juri Ayub, M, Enriz, RD and Giannini, GF 2016. Studies of acute and chronic toxicity of commercial herbicides with glyphosate against Danio rerio . Journal of Environmental and Analytical Toxicology 6, 1–5.Google Scholar
Lang, X, Wang, L and Zhang, Z 2015. Stability evaluation of reference genes for real-time PCR in zebrafish (Danio rerio) exposed to cadmium chloride and subsequently infected by bacteria Aeromonas hydrophila . Aquatic Toxicology 170, 240–250.Google Scholar
Livak, KJ and Schmittgen, TD 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-∆∆C(T)) Method. Methods 25, 402–408.CrossRefGoogle ScholarPubMed
Lopes, FM, Caldas, SS, Primel, EG and da Rosa, CE 2017. Glyphosate adversely affects Danio rerio males: acetylcholinesterase modulation and oxidative stress. Zebrafish 14, 97–105.Google Scholar
Mann, RM and Bidwell, JR 1999. The toxicity of glyphosate and several glyphosate formulations to four species of southwestern Australian frogs. Archives of Environmental Contamination and Toxicology 26, 193–199.Google Scholar
McCurley, AT and Callard, GV 2008. Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Molecular Biology 9, 1–12.Google Scholar
Menéndez-Helman, RJ, Ferreyroa, GV, Dos Santos Afonso, M and Salibián, A 2012. Glyphosate as an acetylcholinesterase inhibitor in Cnesterodon decemmaculatus . Bulletin of Environmental Contamination and Toxicology 88, 6–9.CrossRefGoogle ScholarPubMed
Mo, F, Zhao, J, Liu, N, Cao, LH and Jiang, SX 2014. Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp. Genetics and Molecular Biology 37, 500–507.CrossRefGoogle ScholarPubMed
Murussi, CR, Costa, MD, Leitemperger, JW, Guerra, L, Rodrigues, CCR, Menezes, CC, Severo, ES, Flores-Lopes, F, Salbego, J and Loro, VL 2016. Exposure to different glyphosate formulations on the oxidative and histological status of Rhamdia quelen . Fish Physiology and Biochemistry 42, 445–455.Google Scholar
OECD (Organisation for Economic Co-operation and Development) 1992. Guideline for the testing of chemicals: fish, acute toxicity test, no. 203, 8. OECD, Paris, France, pp. 1–8.Google Scholar
Peixoto, F 2005. Comparative effects of the Roundup and glyphosate on mitochondrial oxidative phosphorylation. Chemosphere 61, 1115–1122.Google Scholar
Pfaffl, MW, Tichopad, A, Prgomet, C and Neuvians, TP 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnology Letters 26, 509–515.CrossRefGoogle ScholarPubMed
Portier, CJ, Armstrong, BK, Baguley, BC, Baur, X, Belyaev, I, Bellé, R, Belpoggi, F, Biggeri, A, Bosland, MC, Bruzzi, P, Budnik, LT, Bugge, MD, Burns, K, Calaf, GM, Carpenter, DO, Carpenter, HM, López-Carrillo, L, Clapp, R, Cocco, P, Consonni, D, Comba, P, Craft, E, Dalvie, MA, Davis, D, Demers, PA, AJDe, Roos, Dewitt, J, Forastiere, F, Freedman, JH, Fritschi, L, Gaus, C, Gohlke, JM, Goldberg, M, Greiser, E, Hansen, J, Hardell, L, Hauptmann, M, Huang, W, Huff, J, James, MO, Jameson, CW, Kortenkamp, A, Kopp-Schneider, A, Kromhout, H, Larramendy, ML, Landrigan, PJ, Lash, LH, Leszczynski, D, Lynch, CF, Magnani, C, Mandrioli, D, Martin, FL, Merler, E, Michelozzi, P, Miligi, L, Miller, AB, Mirabelli, D, Mirer, FE, Naidoo, S, Perry, MJ, Petronio, MG, Pirastu, R, Portier, RJ, Ramos, KS, Robertson, LW, Rodriguez, T, Röösli, M, Ross, MK, Roy, D, Rusyn, I, Saldiva, P, Sass, J, Savolainen, K, PTJ, Scheepers, Sergi, C, Silbergeld, EK, Smith, MT, Stewart, BW, Sutton, P, Tateo, F, Terracini, B, Thielmann, HW, Thomas, DB, Vainio, H, Vena, JE, Vineis, P, Weiderpass, E, Weisenburger, DD, Woodruff, TJ, Yorifuji, T, Yu, IJ and Zambon, P 2016. Differences in the carcinogenic evaluation of glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA). Journal of Epidemiology and Community Health 70, 741–745.Google Scholar
Potashnikova, D, Gladkikh, A and Vorobjev, IA 2015. Selection of superior reference genes’ combination for quantitative real-time PCR in B-cell lymphomas. Annals of Clinical Laboratory Science 45, 64–72.Google Scholar
Radonić, A, Thulke, S, Mackay, IM, Landt, O, Siegert, W and Nitsche, A 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 313, 856–862.Google Scholar
Richard, S, Moslemi, S, Sipahutar, H, Benachour, N and Seralini, GE 2005. Differential effects of glyphosate and Roundup on human placental cells and aromatase. Environmental health perspectives 113, 716–720.Google Scholar
Ronco, AE, Marino, DJG, Abelando, M, Almada, P and Apartin, CD 2016. Water quality of the main tributaries of the Paraná Basin: glyphosate and AMPA in surface water and bottom sediments. Environmental Monitoring and Assessment 188, 458.Google Scholar
Roy, NM, Carneiro, B and Ochs, J 2016. Glyphosate induces neurotoxicity in zebrafish. Environmental Toxicology and Pharmacology 42, 45–54.Google Scholar
Sarmiento, J, Leal, S, Quezada, C, Kausel, G, Figueroa, J, Vera, MI and Krauskopf, M 2001. Environmental acclimatization of the carp modulates the transcription of β-actin. Journal of Cellular Biochemistry 80, 223–228.Google Scholar
Scribner, EA, Battaglin, WA, Gilliom, RJ and Meyer, MT 2007. Concentrations of glyphosate, its degradation product, aminomethylphosphonic acid, and glufosinate in ground- and surface-water, rainfall, and soil samples collected in the United States, 2001-06. US Geological Survey Scientific Investigations Report, Virginia, No. 5122, 111pp.Google Scholar
Silberberg, G, Baruch, K and Navon, R 2009. Detection of stable reference genes for real-time PCR analysis in schizophrenia and bipolar disorder. Analytical Biochemistry 391, 91–97.Google Scholar
Silver, N, Best, S, Jiang, J and Thein, SL 2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Molecular Biology 7, 1–9.CrossRefGoogle ScholarPubMed
Small, BC, Murdock, CA, Bilodeau-Bourgeois, AL, Peterson, BC and Waldbieser, GC 2008. Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions. Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology 151, 296–304.Google Scholar
Spiegelaere, W, Dern-Wieloch, J, Weigel, R, Schumacher, V, Schorle, H, Nettersheim, D, Bergmann, M, Brehm, R, Kliesch, S, Vandekerckhove, L and Fink, C 2015. Reference gene validation for RT-qPCR, a note on different available software packages. PLoS One 10, e0122515.Google Scholar
Struger, J, Thompson, D, Staznik, B, Martin, P, McDaniel, T and Marvin, C 2008. Occurrence of glyphosate in surface waters of southern Ontario. Bulletin of Environmental Contamination and Toxicology 80, 378–384.Google Scholar
Tsui, MTK and Chu, LM 2003. Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52, 1189–1197.Google Scholar
Uren Webster, TM, Laing, LV, Florance, H and Santos, EM 2014. Effects of glyphosate and its formulation, roundup, on reproduction in zebrafish (Danio rerio). Environmental Science and Technology 48, 1271–1279.CrossRefGoogle ScholarPubMed
Uren Webster, TM and Santos, EM 2015. Global transcriptomic profiling demonstrates induction of oxidative stress and of compensatory cellular stress responses in brown trout exposed to glyphosate and Roundup. BMC Genomics 16, 32.Google Scholar
Vandesompele, J, De Preter, K, Pattyn, F, Poppe, B, Van Roy, N, De Paepe, A and Speleman, F 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, 1–12.CrossRefGoogle ScholarPubMed
Vanhauwaert, S, Van Peer, G, Rihani, A, Janssens, E, Rondou, P, Lefever, S, De Paepe, A, Coucke, PJ, Speleman, F, Vandesompele, J and Willaert, A 2014. Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies. PLoS One 9, 1–10.Google Scholar
Vanhauwaert, S, Lefever, S, Coucke, P, Speleman, F, De Paepe, A, Vandesompele, J and Willaert, A 2016. Chapter 17 – RT-qPCR gene expression analysis in zebrafish: Preanalytical precautions and use of expressed repetitive elements for normalization. In the zebrafish genetics, genomics, and transcriptomics. Vol 135. Methods in cell biology (eds. HW Detrich III , L Zon and M Westerfield), pp. 329–342. Academic Press, San Diego, USA.Google Scholar
Velasques, RR, Sandrini, JZ and da Rosa, CE 2016. Roundup in zebrafish: effects on oxidative status and gene expression. Zebrafish 13, 432–441.Google Scholar
Xie, F, Xiao, P, Chen, D, Xu, L and Zhang, B 2012. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Molecular Biology 80, 75–84.Google Scholar
Xu, H, Li, C, Zeng, Q, Agrawal, I, Zhu, X and Gong, Z 2016. Genome-wide identification of suitable zebrafish Danio rerio reference genes for normalization of gene expression data by RT-qPCR. Journal of Fish Biology 88, 2095–2110.Google Scholar