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 TER KΩ cm‐1 Over 30,000(149,000!)registered chemicals. FIGCS is a functional transporting epithelium transporting FIGCS isafunctional Transepithelial Resistance Transepithelial (REACH) legislation(2007). and restrictionofnewCHemicals Registration, Evaluation, Authorisation Walker Walker et al. et Environ. Sci. Technol., 2007, 41, 6505-6513 Sci. Technol., Environ. E40 flux PEG4000 - TER
Ω cm 2 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 filtration on TER
200
180 Travel
160 Laboratory
140
120
100
80
60
40
20
0
% of symetrical values after 24hrs exposure to after water 24hrs exposure % ofvalues symetrical l a d trol e tal ntrol ot r o T te Co Drym olphin T d lphin Filtered Drym Fil o ymetrical Go S Asymetrical Con God 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)