Investigating the versatility of a primary fish gill cell culture system for environmental monitoring

Transcription

1 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. 7, 1, ; Toxicol. Appl. Pharmacol. 8, 3, 7 77

2 FIGCS is a functional transporting epithelium Transepithelial Resistance PEG - flux TER KΩ cm 1 TER Ω cm Walker et al. Environ. Sci. Technol., 7, 1, Registration, Evaluation, Authorisation and restriction of new CHemicals (REACH) legislation (7). Over 3, (19,!) registered chemicals.

3 Environmental Impact assessment if production exceeds 1 tonne. Toxicity tests on an alga, invertebrate and aquatic vertebrate. Derive values for LC5 and NOEC that can be used to determined Predicted no effect concentration (PNEC) Limit tests 1 animals or tox-test animals If a compound is produced in excess of 1 tonnes and/or log Kow >3 then need to undertake an OECD35 Bioconcentration Factor (BCF) study. This uses 18 fish per test and there is estimated to be 1 chemicals in this category.

4 USEPA Whole Effluent Toxicity (WET) EU Direct toxicity Assessment (DTA) Mandatroy requirement Integrated Pollution Prevention and control directive WET uses between 3- million fish per annum. Why? Water Framework Directive Biological monitoring Chemical monitoring Water body status Surveillance monitoring Physiochemical monitoring Fails Chemistry Fails Biology Additional Information Operational monitoring Investigative monitoring Investigative tasks e.g. Identification of contamination Understand the issue Remediation strategy selection

5 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., (8) Toxicol. Appl. Pharmacol. 3(1): 7-77

6 FIGCS gene expression profiles on exposure to Ag, Cd, and Cu Walker et al., (8) Toxicol. Appl. Pharmacol. 3(1): 7-77 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?

7 River Metal Concentrations [Cu] (μg/l) Copper Nickel 3 1 [Ni] (μg/l) St Ives Bay RED RIVER CAMBORNE HAYLE [Zn] (μg/l) Zinc Cadmium Drym Binner Bridge Godolphin Relubbus St. Erth [Cd] (μg/l) Km Lower Region Relubbus St. Erth RIVER HAYLE Godolphin Middle Region Drym Binnerton Upper Region 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 LC5 Drym Binner Godolphin Relubbus St Erth Sept 11 Cu Zn Cd Dec 11 Cu Zn Cd Jan 1 Cu Zn Cd % 5 9.9% % >1%

8 Effect of natural water on in vivo Na + influx rate 1. Na+ influx rate (μmol/ (g x h)).8... *. Aquarium Drym Binnerton Godolphin Relubbus St. Erth Drym Binner Godolphin Relubbus St Erth Jan 1 Cu Experiments 1. September - Water collected from site and cells exposed in the lab 5 Sites. December - Water collected from the site and cells exposed in the lab - sites 3. January Cells taken to the field and exposed to water at site as well as water brought back

9 Experiments In each experiment cells exposed directly to either natural water,.5μm or.μm (sterile) for hrs. N= or 5 for each condition. L-15 OECD MSW Test Water Water chemistry: ph, T o C, hardness, alkalinity, cations and anions, DOC and TOC. Total and dissolved metals (.5 and/or.μm); Cu, Zn, Cd, Ni, Ag, Fe, Co, Cr Pb, Sn. Ionic metal concentrations (MINTEQ) and prediction of toxicity (HydoQual Inc. BLM) Endpoints: TER, MTT assay Experiments: QPCR - Expression levels of: Metallothionein A and B, Glutathione-S-transferase, Glucose--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

10 Effect of OECD water on gene expression Assymetrical conditions (OECD Water) Expression levels normalised to the housekeeping genes Symetrical Conditions Expression levels normalised to the housekeeping genes MTA MTB ZnT DMT GPD GsT GR Na/KATPase ATP7A CYP1A Response of cell culture to River Hayle water

11 Effect of natural water on cell viability (MTT).5 Absorbance 57nM Control Drym Binnerton Godolphin Relubbus St Erth Gene expression levels Gene Sept Dec Jan D B G R E D G R E D G R METALS MT A MT B ATP7A DMT1 ZnT1 GsT GPD GR Na/K ATPase CYP1A Fold induction of expression levels < >.5

12 Gene expression levels Gene Sept Dec Jan D B G R E D G R E D G R METALS MT A MT B ATP7A DMT1 ZnT1 GsT GPD GR Na/K ATPase CYP1A Fold induction of expression levels < >.5 Natural water,.5 and.μm filtration on gene expression 7 Total.5μm.μm MTA fold induction Drym Godolphin Relubbus St Erth

13 Effect of in field exposure 1kms, 3hrs Effect of the field and.μm filtration on TER % of symetrical values after hrs exposure to water Symetrical Control Travel Laboratory Asymetrical Control Drym Total Drym Filtered Godolphin Total Godolphin Filtered

14 Field v Bench expression levels Bench expression levels normalised to housekeeping genes Field expression levels normalised to housekeeping genes MT-A MTA MTB ZnT DMT1 ATP7A GPD GsT GR Na/KATPase CYP1A Measured dissolved Cu and Zn v MTA expression MT-A fold induction R =.1 R = [Dissolved Zinc] (μg/l) [Dissolved Copper] (μg/l)

15 Predicted Biotic Ligand Zn and Cu v MTA expression MTA - Fold induction /11 1/11 1/1 R =.88 R = [Biotic ligand - Zn] (nmol/g) 8 [Biotic Ligand - Cu] (nmol/g) Conclusions on FIGCS for Environmental Monitoring? 1. Can the primary gill cell culture tolerate natural river water? - YES. 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

16 Thanks : Matteo Minghetti Lucy Stott Wolfgang Maret Christer Hogstrand Sabine Schell Cumulative BLM toxicity v MTA & B expression MTA Fold Induction /11 1/11 1/1 MTB Fold Induction 8 8 Cumulative BLM (Zn, Cu, Cd) toxicity 8 Cummulative BLM (Zn, Cu, Cd) toxicity

17 Calculated ionic Cu and Zn v MTA expression MTA Fold Induction R = [Zn + ] (μm) [Cu + ] (μm) Predicted Biotic Ligand Zn and Cu v MTB expression 8 9/11 8 R =.5 MTB Fold Induction 1/11 1/1 8 1 [Biotic Ligand - Zn] (nmol/g) 8 [Biotic Ligand - Cu] (nmol/g)

18 Calculated ionic Cu and Zn v MTB expression MTB - Fold Induction 1 8 R =.5 8 R = [Zn + ] (μm) [Cu + ] (μm)