Bioavailability of metals to fish: its molecular basis, toxicological consequences and how it can be assessed. Christer Hogstrand

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1 Bioavailability of metals to fish: its molecular basis, toxicological consequences and how it can be assessed Christer Hogstrand Metal Metabolism Group King s College London Global maps for the year 2000 of in-use (A) copper stock intensities (Gg/grid cell) compared with in-ground (B) copper resources Rauch J N PNAS 2009;106:

2 Relationship between the background concentration of zinc in water and toxicity expressed as the no observed effect concentration (NOEC) Bodar et al., (2005) Integr Environ Assess Manag 1 Metal concentrations in The River Hayle, Cornwall

3 Comparative acute toxicity of metals to rainbow trout McDonald (1982) The teleost gill is a target organ for metal toxicity Eckert et al. (1997) Animal Physiology

4 Na + Water Gill Plasma mosmol 300 mosmol Na + 3 Na + ATP H + Mg ATP 2 K + NH 3 H + NH 3 Na + H +, NH 4 Zn CA CO 2 HCO 3 - K + Cl - Ca 2+ HCO 3-, OH - GSH MT Cl - Ca2+ ATP? H 2 O O 2 Apical -70 mv Basolateral CO 2 Ca 2+ Cl -

5 Zinc inhibits calcium uptake leading to acute hypocalcaemia Am. J. Physiol. 270 Speciation dictates metal bioavailability and toxicity

6 Metal impact on biota in Big Bayou Creek, Kentucky Birge et al. (2000) Environ. Toxicol. Chem. 19 Impact correlates to bioavailable rather than total dissolved metal Calculated bioavailable Cu, Ag and Cd fractions correlated significantly with BA score (r > -0.95) and number of taxa ( r = -0.95). Birge et al. (2000) Environ. Toxicol. Chem. 19

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8 Fish gill physiology

9 Expression of zinc transporters in gill changes in response to changes in water zinc concentrations Zheng et al. (2008) Physiol Genomics 34: ,.

10 Physiology modifies metal affinity and accumulation Walker et al. Environ. Sci. Technol. 2007, 41, ; Toxicol. Appl. Pharmacol. 2008, 230, 67 77

11 Fish Gill In vitro Cell culture System (FIGCS) is a functional transporting epithelium TER KΩ cm -1 [Zn 2+ ] Walker et al. Environ. Sci. Technol., 2007, 41, Comparison of effects of waterborne Ag(I) on rainbow trout and FIGCS Walker et al 2008 Toxicol Appl Pharmacol 230: 67-77

12 Comparison of the effects of water chemistry on whole organism ion uptake and gene expression response in vitro ZnT1 MTs Walker et al., (2008) Toxicol. Appl. Pharmacol. 230(1): Chronic zinc toxicity Species Average NOEC (µg/l) Jornanella floridae 44 2 Eurasian minnow 50 1 Fathead minnow 78? Rainbow trout Brook trout 530? Zebrafish Number of studies Fish toxicity data used in the EU Environmental Risk Assessment for Zn End-points to set NOEC were: -Survival -Reproduction -Growth Species LOEC Response Hardness ph Reference µg/l mg/l CaCO3 Rainbow trout 5.6 Avoidance Sprague (1968) Lake whitefish 10 Avoidance Scherer and McNicol (1998) Rainbow trout 10 Avoidance Svecevicius (1999) Rainbow trout 47 Avoidance Black and Birge (1980) Atlantic salmon 53 Avoidance Sprague (1964) Rainbow trout 144 Ventilation rate 25 7 Cairns et al. (1982) Vimba bream 220 Avoidance* Svecevicius (1999) Brook charr 1,390 Cough rate Drummond and Carlson (1977) Bluegill 3,640 Movement pattern Waller and Cairns (1972)

13 Normal Water: 0.5 um Diet: 1.2 nmol g -1 day -1 Excess Water: 5 um Diet: 120 nmol g -1 day -1 n=9 Zheng et al. BMC Genomics 2010, 11:553 Annotation Enrichment on day Clusters Category Name Genes P-value Development BP developmental process E-06 BP anatomical structure development E-05 BP system development E-04 BP multicellular organismal process E-03 BP nervous system development E-03 BP cellular developmental process E-03 BP cell differentiation E-03 BP cell development E-03 BP multicellular organismal development E-03 BP brain development E-03 BP central nervous system development E-03 BP organ development E-03 BP cell fate commitment E-03 Metabolism BP primary metabolic process E-05 BP metabolic process E-05 BP cellular metabolic process E-05 BP lipid biosynthetic process E-03 BP biopolymer metabolic process E-03 BP macromolecule metabolic process E-03 BP multicellular organismal catabolic process E-03 Gene expression MF sequence-specific DNA binding E-06 BP transcription from RNA polymerase II promoter E-04 BP positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process E-03 MF transcription factor activity E-03 BP positive regulation of transcription E-03 BP positive regulation of transcription, DNA-dependent E-03 MF transcription factor binding E-03 Nuclear receptors BP response to steroid hormone stimulus E-04 MF steroid hormone receptor activity E-03 MF retinol binding E-03 MF ligand-dependent nuclear receptor activity E-03 Regulation BP positive regulation of biological process E-04 BP positive regulation of cellular process E-03 BP regulation of multicellular organismal process E-03 BP regulation of biological process E-03 BP biological regulation E-03 Others MF oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen E-03 BP microtubule cytoskeleton organization and biogenesis E-03 BP cell cycle process E-03 MF 5'-nucleotidase activity E-03 MF oxygen binding E-03 MF iron ion binding E-03 MF nucleotidase activity E-03 BP detection of abiotic stimulus E-03 >0.1 <0.1 <0.05 <0.01 <0.005 <0.001 Development Metabolism Gene expression Nuclear receptors Regulation Others (e.g. Cell cycle process, iron binding, microtuble cytoskeleton organisation) Zheng et al. BMC Genomics 2010, 11:553

14 Zn 2+ is a potent inhibitor of protein tyrosine phosphatases RPTP-β, K i = 21 ± 7 pm 2012 by American Society for Biochemistry and Molecular Biology Wilson et al. (2012) J Biol Chem 287, 9322 Zn 2+ inhibits enzymes at physiological [Zn 2+ ] i 1 0 Saturation of Zn binding sites in proteins ( ) [Zn 2+ ] i Saturation of Zn binding sites in MT ( ) pzn ( log[zn 2+ ]) Modified from Maret (2011) J Biol Inorg Chem 16:

15 Total cumulative number of eggs No. of eggs Total cumulative number of spawns No. of spawns Day Day Metal Concentration (μg g -1 dry weight) * * * * * Ag As Cd Cu Fe Zn Mean hatch rate (%) * 150 * Mean number eggs per spawning pair Day Day Hatch rate Gene express Vtg MT-II 180 Eggs/spawn Environ. Sci. Technol. 2008, 42, Metal storage strategies in Gammarus Khan et al., Aquat. Toxicol. 96 (2010) / J. exp. Biol., in revision

16 Effect of metal storage strategy on dietary metal uptake and toxicity in zebrafish Khan et al., 2009 Aquat. Toxicol. / 2010 Aquatic Toxicol Acknowledgments King s Dr Nic Bury Dr Wolfgang Maret Dr Dongling Zheng Mr Phil Cunningham Dr Andong Qiu Dr David Boyle Dr Paul Walker Dr Farhan Khan Dr Issa Muraina Mr Matt Wilson Dr Peter Kille Dr Graham Feeney Prof Richard Handy Dr Rod Wilson Dr Fernando Galvez Prof Chris Wood Prof Wesley Birge Mr Steve Munger Dr Rick Playle