Investigating the Versatility of a Primary Fish Gill Cell Culture System for Environmental Monitoring

Investigating the versatility of a primary fish gill cell culture system for environmental monitoring Matteo Minghetti, Sabine Schnell, Christer Hogstrand, Nic Bury Fish Gill In vitro Cell culture System (FIGCS) Walker et al. Environ. Sci. Technol. 2007, 41, 6505-6513; Toxicol. Appl. Pharmacol. 2008, 230, 67–77 FIGCS is a functional transporting epithelium Transepithelial Resistance PEG4000 - flux 1 ‐ cm Ω K TER TER Ω cm2 Walker et al. Environ. Sci. Technol., 2007, 41, 6505-6513 Registration, Evaluation, Authorisation and restriction of new CHemicals (REACH) legislation (2007). Over 30,000 (149,000!) registered chemicals. Environmental Impact assessment if production exceeds 1 tonne. Toxicity tests on an alga, invertebrate and aquatic vertebrate. Derive values for LC50 and NOEC that can be used to determined Predicted no effect concentration (PNEC) Limit tests – 14 animals or tox-test 42 animals If a compound is produced in excess of 100 tonnes and/or log Kow >3 then need to undertake an OECD305 Bioconcentration Factor (BCF) study. This uses 108 fish per test and there is estimated to be 1000 chemicals in this category. USEPA – Whole Effluent Toxicity (WET) EU – Direct toxicity Assessment (DTA) Mandatroy requirement – Integrated Pollution Prevention and control directive WET uses between 3- 6 million fish per annum. Why? Water Framework Directive Surveillance monitoring Biological Chemical Physiochemical monitoring monitoring monitoring Water body status Fails Chemistry Fails Biology Operational monitoring Additional Information Investigative monitoring Investigative tasks e.g. Identification of contamination Understand the issue Remediation strategy selection Can the FIGCS be used for Environmental Monitoring? Can it be used to identify “biologically active compounds in natural waters. ..... enables us to identify pollutants that definitely induce a biological response...” In vivo versus In vitro ZnT1 MTs Walker et al., (2008) Toxicol. Appl. Pharmacol. 230(1): 67-77 FIGCS gene expression profiles on exposure to Ag, Cd, and Cu Walker et al., (2008) Toxicol. Appl. Pharmacol. 230(1): 67-77 Can the FIGCS be used for Environmental Monitoring? Can the primary gill cell culture tolerate natural river water? Can the primary gill cell culture withstand transport to the field for site specific monitoring? Do the cells respond in a predictable way to pollutants - polymetal gradient? River Metal Concentrations 120 40 Copper 100 Nickel 30 80 20 St Ives Bay g/L) 60 g/L) μ μ RED RIVER 40 10 CAMBORNE [Ni] ( [Cu] ( [Cu] 20 0 0 HAYLE 1200 2.5 Zinc 1000 St. Erth Cadmium 2.0 Lower RIVER HAYLE 800 1.5 Region g/L) g/L) 600 Drym μ μ 1.0 400 [Zn] ( [Zn] [Cd] ( [Cd] Relubbus Binnerton Upper 0.5 200 Godolphin Region 0 0.0 Middle Km Region ge hin rth d p bus Drym l b o lu t. E od S G Re inner Bri B No change in Pb, Fe, As, Cr, Co Site Specific Metal Toxicity Predicted by the Biotic Ligand model Predicted BL‐metal (nmol/gw) as a % of the site specific BL‐metal at a LC50 Drym Binner Godolphin Relubbus St Erth Sept 2011 Cu 0.3 1.1 114 16.1 7.2 Zn 8.1 62.8 574 418 419 Cd 0.4 1.9 66.1 45.6 40.8 Dec 2011 Cu 1.69 149 31.5 31.2 Zn 29.7 431 375 304 Cd 6.9 37.6 28.7 45.5 Jan 2012 Cu 0.4 2.6 200 86.9 28.4 Zn 28.2 90.0 457 469 413 Cd 6.9 17.8 55.1 50.0 39.4 0 – 24.9% 25 – 49.9% 50 – 99.9% >100% Effect of natural water on in vivo Na+ influx rate 1.0 0.8 mol/ (g x h)) (g x mol/ 0.6 μ * 0.4 0.2 Na+ influx rate ( rate influx Na+ 0.0 n h i rt ium rton E ar Drym e olph . inn od St Aqu B G Relubbus Drym Binner Godolphin Relubbus St Erth Jan 2012 Cu 0.4 2.6 200 86.9 28.4 Experiments 1. September - Water collected from site and cells exposed in the lab – 5 Sites 2. December - Water collected from the site and cells exposed in the lab - 4 sites 3. January – Cells taken to the field and exposed to water at site as well as water brought back Experiments • In each experiment cells exposed directly to either natural water, 0.45μM or 0.2μM (sterile) for 24 hrs. N=4 or 5 for each condition. OECD Test L-15 MSW Water • Water chemistry: pH, T oC, hardness, alkalinity, cations and anions, DOC and TOC. Total and dissolved metals (0.45 and/or 0.2μM); Cu, Zn, Cd, Ni, Ag, Fe, Co, Cr Pb, Sn. Ionic metal concentrations (MINTEQ) and prediction of toxicity (HydoQual Inc.– BLM) Experiments: • Endpoints: TER, MTT assay • QPCR - Expression levels of: Metallothionein A and B, Glutathione-S-transferase, Glucose-6-phosphate, Glutathione reductase ATP7A, Zinc Transporter 1 (ZnT1), Divalent Metal Transporter 1 (DMT1), Na/K-ATPase, CYP1A, geNorm normalisation Elongation factor 1 alpha, Ubiquitin, 18S, ARP, ee1fb Effect of OECD water on gene expression 106 105 104 103 102 101 Assymetrical conditions (OECD Water) (OECD conditions Assymetrical 100 100 101 102 103 104 105 106 Expression levels normalised to the housekeeping genes housekeeping the to normalised levels Expression Symetrical Conditions Expression levels normalised to the housekeeping genes MTA MTB ZnT DMT ATP7A G6PD GsT GR Na/KATPase CYP1A Response of cell culture to River Hayle water Effect of natural water on cell viability (MTT) 0.05 0.04 0.03 0.02 Absorbance 570nM Absorbance 0.01 0.00 l m n rton Dry olphi St Erth Contro inne elubbus B God R Gene expression levels Gene Sept Dec Jan D B G R E D G R E D G R METALS Fold induction of expression levels MT‐A MT‐B < 1 1 –1.5 ATP7A 1.5 –2.5 DMT1 >2.5 ZnT1 GsT G6PD GR Na/K‐ATPase CYP1A Gene expression levels Gene Sept Dec Jan D B G R E D G R E D G R METALS Fold induction of expression levels MT‐A MT‐B < 1 1 –1.5 ATP7A 1.5 –2.5 DMT1 >2.5 ZnT1 GsT G6PD GR Na/K‐ATPase CYP1A Natural water, 0.45 and 0.2μm filtration on gene expression 7 Total 6 0.45μm 0.2μm 5 4 3 2 MTA fold induction 1 0 n hi th p Drym t Er dol S Go Relubbus Effect of in field exposure 1000kms, 30hrs Effect of the field and 0.2μm 200 180 filtration on TER 160 140 120 Travel 100 Laboratory 80 60 40 20 % of symetrical values after 24hrs exposure to water 0 Symetrical Control Asymetrical Control Drym Total Drym Filtered Godolphin Total Godolphin Filtered Field v Bench expression levels 106 105 MT-A 104 103 102 Bench expression levels levels expression Bench normalised to housekeeping genes to housekeeping normalised 101 101 102 103 104 105 106 Field expression levels normalised to housekeeping genes MTA MTB ZnT DMT1 ATP7A G6PD GsT GR Na/KATPase CYP1A Measured dissolved Cu and Zn v MTA expression 12 12 2 R2=0.61 R =0.53 10 10 8 8 6 6 4 4 MT-A induction fold 2 2 0 200 400 600 800 0 10203040506070 [Dissolved Zinc] (μg/L) [Dissolved Copper] (μg/L) Predicted Biotic Ligand Zn and Cu v MTA expression 12 12 09/11 12/11 01/12 10 10 R2=0.88 8 8 6 6 4 4 MTA - Foldinduction 2 2 R =0.87 2 0246810 02468 [Biotic ligand - Zn] (nmol/g) [Biotic Ligand - Cu] (nmol/g) Conclusions on FIGCS for Environmental Monitoring? 1. Can the primary gill cell culture tolerate natural river water? - YES 2. Can the primary gill cell culture withstand transport to the field for site specific monitoring? - YES 3. Do the cells respond in a predictable way to polymetal gradient? -YES Thanks : Matteo Minghetti Lucy Stott Wolfgang Maret Christer Hogstrand Sabine Schell Cumulative BLM toxicity v MTA & B expression 12 09/11 8 10 12/11 01/12 6 8 6 4 4 MTA Fold Induction MTA Fold MTB Fold Induction 2 2 0 0 200 400 600 800 0 200 400 600 800 Cumulative BLM (Zn, Cu, Cd) toxicity Cummulative BLM (Zn, Cu, Cd) toxicity Calculated ionic Cu and Zn v MTA expression 12 12 R2=0.46 10 10 8 8 6 6 4 4 MTA FoldMTA Induction 2 2 0246810121416 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [Zn2+] (μM) [Cu2+] (μM) Predicted Biotic Ligand Zn and Cu v MTB expression 2 8 09/11 8 R =0.5 12/11 6 01/12 6 4 4 MTB Fold Induction MTB Fold 2 2 0 0 0246810 02468 [Biotic Ligand - Zn] (nmol/g) [Biotic Ligand - Cu] (nmol/g) Calculated ionic Cu and Zn v MTB expression 10 R2=0.45 8 R2=0.61 8 6 6 4 4 2 MTB - Fold Induction 2 0 0 0246810121416 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [Zn2+] (μM) [Cu2+] (μM).

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