Abstract
Chloride (Cl−) influences the bioavailability and toxicity of metals in fish, but the mechanisms by which it influences these processes is poorly understood. Here, we investigated the effect of chloride on the cytotoxicity, bioavailability (i.e., accumulation) and bioreactivity (i.e., induction of mRNA levels of metal responsive genes) of copper (Cu) and silver (Ag) in the rainbow trout gut cell line (RTgutGC). Cells were exposed to metals in media with varying Cl− concentrations (0, 1, 5 and 146 mM). Metal speciation in exposure medium was analyzed using Visual MINTEQ software. Cytotoxicity of AgNO3 and CuSO4 was measured based on two endpoints: metabolic activity and membrane integrity. Cells were exposed to 500 nM of AgNO3 and CuSO4 for 24 h in respective media to determine metal bioavailability and bioreactivity. Ag speciation changes from free ionic (Ag+) to neutral (AgCl), to negatively charged chloride complexes (AgCl2−, AgCl3−) with increasing Cl− concentration in exposure media whereas Cu speciation remains in two forms (Cu2+ and CuHPO4) across all media. Chloride does not affect Ag bioavailability but decreases metal toxicity and bioreactivity. Cells exposed to Ag expressed significantly higher metallothionein mRNA levels in low Cl− media (0, 1, and 5 mM) than in high Cl− medium (146 mM). This suggests that chloride complexation reduces silver bioreactivity and toxicity. Conversely, Cu bioavailability and toxicity were higher in the high chloride medium (146 mM) than in the low Cl− (0, 1, and 5 mM) media, supporting the hypothesis that Cu uptake may occur via a chloride dependent mechanism.
Clinical trials registration
This study did not require clinical trial registration.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alda JO, Garay R (1990) Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells. Am J Physiol Cell Physiol 259:C570–C576. https://doi.org/10.1152/ajpcell.1990.259.4.C570
Arredondo M, Muñoz P, Mura CV, Núñez MT (2003) DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells. Am J Physiol Cell Physiol 284:C1525–C1530. https://doi.org/10.1152/ajpcell.00480.2002
Bertinato J, Cheung L, Hoque R, Plouffe LJ (2010) Ctr1 transports silver into mammalian cells. J Trace Elem Med Biol 24:178–184. https://doi.org/10.1016/j.jtemb.2010.01.009
Bielmyer GK, Brix KV, Grosell M (2008) Is Cl- protection against silver toxicity due to chemical speciation? Aquat Toxicol 87:81–87. https://doi.org/10.1016/j.aquatox.2008.01.004
Blanchard J, Grosell M (2006) Copper toxicity across salinities from freshwater to seawater in the euryhaline fish Fundulus heteroclitus: is copper an ionoregulatory toxicant in high salinities? Aquat Toxicol 80:131–139. https://doi.org/10.1016/j.aquatox.2006.08.001
Burke J, Handy RD (2005) Sodium-sensitive and -insensitive copper accumulation by isolated intestinal cells of rainbow trout Oncorhynchus mykiss. J Exp Biol 208:391–407. https://doi.org/10.1242/jeb.01379
Bury NR, Walker PA, Glover CN (2003) Nutritive metal uptake in teleost fish. J Exp Biol 206:11–23. https://doi.org/10.1242/jeb.00068
Bury NR, Hogstrand C (2002) Influence of chloride and metals on silver bioavailability to Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) yolk-sac fry. Environ Sci Technol 36:2884–2888. https://doi.org/10.1021/es010302g
Bury NR, Wood CM (1999) Mechanism of branchial apical silver uptake by rainbow trout is via the proton-coupled Na+ channel. Am J Physiol Regul Integr Comp Physiol 277:R1385–R1391. https://doi.org/10.1152/ajpregu.1999.277.5.r1385
Bury NR, Grosell M, Grover AK, Wood CM (1999) ATP-dependent silver transport across the basolateral membrane of rainbow trout gills. Toxicol Appl Pharmacol 159:1–8. https://doi.org/10.1006/taap.1999.8706
Clifford RJ, Maryon EB, Kaplan JH (2016) Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu+ uptake system. J Cell Sci 129:1711–1721. https://doi.org/10.1242/jcs.173351
Coyle P, Philcox JC, Carey LC, Rofe AM (2002) Metallothionein: The multipurpose protein. Cell Mol Life Sci 59:627–647. https://doi.org/10.1007/s00018-002-8454-2
Di Toro DM, Allen HE, Bergman HL, Meyer JS, Paquin PR, Santore RC (2001) Biotic ligand model of the acute toxicity of metals. 1. Technical basis. Environ Toxicol Chem 20:2383–2396. https://doi.org/10.1897/1551-5028(2001)020<2383:blmota>2.0.co;2
Erickson RJ, Brooke LT, Kahl MD, Venter FV, Harting SL, Markee TP, Spehar RL (1998) Effects of laboratory test conditions on the toxicity of silver to aquatic organisms. Environ Toxicol Chem 17:572–578. https://doi.org/10.1002/etc.5620170407
Ferguson EA, Hogstrand C (1998) Acute silver toxicity to seawater‐acclimated rainbow trout: Influence of salinity on toxicity and silver speciation. Environ. Toxicol. Chem. 17:589–593. https://doi.org/10.1002/etc.5620170409
Fortin C, Campbell PG (2000) Silver uptake by the green alga Chlamydomonas reinhardtii in relation to chemical speciation: influence of chloride. Environ Toxicol Chem 19:2769–2778. https://doi.org/10.1002/etc.5620191123
Grosell M, Blanchard J, Brix KV, Gerdes R (2007) Physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates. Aquat Toxicol 84:162–172. https://doi.org/10.1016/j.aquatox.2007.03.026.
Grosell M, Wood CM (2002) Copper uptake across rainbow trout gills: mechanisms of apical entry. J Exp Biol 205:1179–1188. https://doi.org/10.1242/jeb.205.8.1179
Gustafsson JP (2013) Visual MINTEQ 3.1, https://vminteq.lwr.kth.se/ Accessed 4 Oct. 2021.
Handy RD, Musonda MM, Phillips C, Falla SJ (2000) Mechanisms of gastrointestinal copper absorption in the African walking catfish: copper dose-effects and a novel anion-dependent pathway in the intestine. J Exp Biol 203:2365–2377. https://doi.org/10.1242/jeb.203.15.2365
Ibrahim M, Oldham D, Minghetti M (2020) Role of metal speciation in the exposure medium on the toxicity, bioavailability and bioreactivity of copper, silver, cadmium and zinc in the rainbow trout gut cell line (RTgutGC). Comp Biochem Physiol C Toxicol Pharmacol 236:108816. https://doi.org/10.1016/j.cbpc.2020.108816
Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82:503–568
Kawano A, Haiduk C, Schirmer K, Hanner R, Lee LEJ, Dixon B, Bols NC (2011) Development of a rainbow trout intestinal epithelial cell line and its response to lipopolysaccharide. Aquac Nutr 17:241–252. https://doi.org/10.1111/j.1365-2095.2010.00757.x
Langan LM, Harper GM, Owen SF, Purcell WM, Jackson SK, Jha AN (2017) Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper. Ecotoxicol 26:1117–1133. https://doi.org/10.1007/s10646-017-1838-8
Matson CWW, Bone AJJ, Auffan M, Lindberg TTT, Arnold MCC, Hsu-Kim H, Wiesner MRR, Di Giulio RTT (2016) Silver toxicity across salinity gradients: the role of dissolved silver chloride species (AgClx) in Atlantic killifish (Fundulus heteroclitus) and medaka (Oryzias latipes) early life-stage toxicity. Ecotoxicol 25:1105–1118. https://doi.org/10.1007/s10646-016-1665-3
Minghetti M, Dudefoi W, Ma Q, Catalano JG (2019) Emerging investigator series: linking chemical transformations of silver and silver nanoparticles in the extracellular and intracellular environments to their bio-reactivity. Environ Sci Nano 6:2948–2957. https://doi.org/10.1039/C9EN00710E
Minghetti M, Schirmer K (2019) Interference of silver nanoparticles with essential metal homeostasis in a novel enterohepatic fish in vitro system. Environ Sci Nano 6:1777–1790. https://doi.org/10.1039/C9EN00310J
Minghetti M, Drieschner C, Bramaz N, Schug H, Schirmer K (2017) A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC. Cell Biol Toxicol 33:539–555. https://doi.org/10.1007/s10565-017-9385-x
Minghetti M, Schirmer K (2016) Effect of media composition on bioavailability and toxicity of silver and silver nanoparticles in fish intestinal cells (RTgutGC). Nanotoxicology 10:1526–1534. https://doi.org/10.1080/17435390.2016.1241908
Minghetti M, Leaver MJ, George SG (2010) Multiple Cu-ATPase genes are differentially expressed and transcriptionally regulated by Cu exposure in sea bream, Sparus aurata. Aquat Toxicol 97:23–33. https://doi.org/10.1016/j.aquatox.2009.11.017
Minghetti M, Leaver MJ, Carpenè E, George SG (2008) Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper. Comp Biochem Physiol C Toxicol Pharmacol 147:450–459. https://doi.org/10.1016/j.cbpc.2008.01.014
Ohgami RS, Campagna DR, McDonald A, Fleming MD (2006) The Steap proteins are metalloreductases. Blood 108:1388–1394. https://doi.org/10.1182/blood-2006-02-003681
Ohrvik H, Thiele DJ (2015) The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy. J Trace Elem Med Biol 31:178–182. https://doi.org/10.1016/j.jtemb.2014.03.006
Paquin PR, Gorsuch JW, Apte S, Batley GE, Bowles KC, Campbell PGC, Delos CG, Di Toro DM, Dwyer RL, Galvez F, Gensemer RW, Goss GG, Hogstrand C, Janssen CR, McGeer JC, Naddy RB, Playle RC, Santore RC, Schneider U, Stubblefield WA, Wood CM, Wu KB (2002) The biotic ligand model: A historical overview. Comp Biochem Physiol - C Toxicol Pharmacol 133:3–35. https://doi.org/10.1016/S1532-0456(02)00112-6
Połeć-Pawlak K, Ruzik R, Lipiec E (2007) Investigation of Cd (II), Pb (II) and Cu (I) complexation by glutathione and its component amino acids by ESI-MS and size exclusion chromatography coupled to ICP-MS and ESI-MS. Talanta 72:1564–1572. https://doi.org/10.1016/j.talanta.2007.02.008
Schecher WD, McAvoy DC (1992) MINEQL+: A software environment for chemical equilibrium modeling. Comput Environ Urban Syst 16:65–76. https://doi.org/10.1016/0198-9715(92)90053-T
Schirmer K, Chan AGJ, Greenberg BM, Dixon DG, Bols NC (1997) Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture. Toxicol In Vitr 11:107–119. https://doi.org/10.1016/S0887-2333(97)00002-7
Scott GR, Schulte PM, Wood CM (2006) Plasticity of osmoregulatory function in the killifish intestine: drinking rates, salt and water transport, and gene expression after freshwater transfer. J Exp Biol 209:4040–4050. https://doi.org/10.1242/jeb.02462
Shenberger Y, Marciano O, Gottlieb HE, Ruthstein S (2018) Insights into the N-terminal Cu (II) and Cu (I) binding sites of the human copper transporter CTR1. J Coord Chem 71:1985–2002. https://doi.org/10.1080/00958972.2018.1492717
Srikanth K, Pereira E, Duarte AC, Ahmad I (2013) Glutathione and its dependent enzymes’ modulatory responses to toxic metals and metalloids in fish—a review. Environ Sci Pollut Res 20:2133–2149. https://doi.org/10.1007/s11356-012-1459-y
Wood CM, McDonald MD, Walker P, Grosell M, Barimo JF, Playle RC, Walsh PJ (2004) Bioavailability of silver and its relationship to ionoregulation and silver speciation across a range of salinities in the gulf toadfish (Opsanus beta). Aquat Toxicol 70:137–157. https://doi.org/10.1016/j.aquatox.2004.08.002
Zhao CM, Campbell PGC, Wilkinson KJ (2016) When are metal complexes bioavailable? Environ Chem 13:425–433. https://doi.org/10.1071/EN15205
Zimnicka AM, Ivy K, Kaplan JH (2011) Acquisition of dietary copper: A role for anion transporters in intestinal apical copper uptake. Am J Physiol Cell Physiol 300:C588–C599. https://doi.org/10.1152/ajpcell.00054.2010
Acknowledgements
The authors thank the Interdisciplinary Toxicology Program at Oklahoma State University and the U.S. National Science Foundation (NSF; award no. CBET-1706093 to MM) for funding this research. We cordially thank Professor Kristin Schirmer (Eawag, CH) for providing the cell line RTgutGC used in this study.
Author information
Authors and Affiliations
Contributions
Both authors contributed to conceptualization, data curation, formal analyses, investigation, methodology, software, validation, and visualization. MM supervised this work and provided necessary resources to MI to conduct this research. MI wrote the first draft which was further edited and reviewed by MM. Both authors approved the final paper.
Funding
This research was funded by the Interdisciplinary Toxicology Program at Oklahoma State University through a fellowship awarded to MI and by the U.S. National Science Foundation (NSF) through award no. CBET-1706093 to MM.
Corresponding author
Ethics declarations
Conflict of Interest
The authors declare no competing interests.
Ethics Statement
This study did not use any vertebrate animal. All data was acquired using the cell line RTgutGC. RTgutGC cells were kindly gifted to MM by Professor Kristin Schirmer (Eawag, CH).
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
About this article
Cite this article
Ibrahim, M., Minghetti, M. Effect of chloride concentration on the cytotoxicity, bioavailability, and bioreactivity of copper and silver in the rainbow trout gut cell line, RTgutGC. Ecotoxicology 31, 626–636 (2022). https://doi.org/10.1007/s10646-022-02543-5
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10646-022-02543-5