Expression of Metallothionein in White Sturgeon Following in vitro Exposure to Cd, Zn, or Cu
Bryanna Eisner1,3, Jon Doering2,3, Steve Wiseman3, Shawn Beitel2,3, David Vardy2,3, Shelby Reid3, John Giesy3,4,5, Markus Hecker3,6
1) Toxicology Undergraduate Program, University of Saskatchewan, Saskatoon, SK, CA. 2) Toxicology Graduate Program, University of Saskatchewan, Saskatoon, SK, CA. 3) Toxicology Centre, University of Saskatchewan, Saskatoon, SK, CA. 4) Dept. Veterinary
Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, CA. 5) Dept. Biology and Chemistry, City University of Hong Kong, Hong Kong, China. 6) School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, CA
Results
Introduction
• White sturgeon (WS; Acipenser transmontanus) are
endangered in some North American rivers and metal
contamination from metal mining and smelting activities is
one hypothesized contributors of these declines.
• Acute and chronic exposure studies indicate that WS might be
among the most sensitive species of fishes to certain metals,
especially during early life-stages (ELS) (Table 1; Figure 1)1,2.
• Metallothioneins (MTs) play an import role in metal detoxification in vertebrates, and thus, their regulation might play an
important role in the sensitivity of a species to certain elements.3
Fold-change from Solvent Control
6.0
Study Goal
*
A) Cd
5.0
*
4.0
3.0
*
*
2.0
*
1.0
0.0
Cadmium
Zinc
Copper
CWQG*
0.02 µg/L
30 µg/L
2 µg/L
Chronic ELS LC20
8.7 µg/L
102 µg/L
3.4 µg/L
Chronic ELS LC50
21.4 µg/L
340 µg/L
9.9 µg/L
*Chronic Water Quality Guideline (CWQG) acquired from Canadian Ministers of the
Environment-Canadian Environmental Quality Guidelines for the Protection of Aquatic
Life. Tompsett et al. (unpublished), Vardy et al (2011).
Figure 1: Juvenile White Sturgeon
Methods
In vitro Exposure: Livers from juvenile WS were minced, and
1mm X 1mm slices were added to 24 well plates with culture
medium and exposed to Cd, Zn, or Cu for 24 hrs at 15ºC.
Cloning and Sequencing of MT: Full-length cDNA for WS MT
was acquired by use of RACE-PCR.
Real-time PCR: Transcript abundance was quantified by
normalizing to Beta-actin according to the methods of Simon4.
Real-time PCR was conducted for n=4 individuals from in vitro
studies.
Statistics: Significance was determined by use of 1 Way ANOVA
followed by Tukey’s post-hoc test, by use of SPSS 19 software
(SPSS Inc.).
Figure 1: Example of the species sensitivity distribution (SSDs) for copper.
Values for species with life stages, expressed as days post-hatch (dph), are
from experiments conducted at the University of Saskatchewan. Atlantic,
shortnose, and shovelnose sturgeon values are from Dwyer et al.5, and all
other species values are from the ECOTOX database.6 The Species Mean
Acute Value (SMAV) is the geometrical mean LC50 for a given species.2
• WS MT encodes a protein of 64 amino acids in length
3.5
Fold-change from Solvent Control
Table 1: Chronic Lethal Concentrations for ELS of White Sturgeon
Solvent
1.2
73%
Dog MT-4
73%
Pacific Walrus MT-4
73%
Golden Hamster MT-4
72%
Opossum MT-4
71%
*Similarity of WS MT identified by blasting the full-length amino acid sequence through
the National Centre for Biotechnology Information (NCBI) Database.
30 ug/L
B) Zn
100 ug/L
*
Future Directions
• Studies to determine the mechanistic basis of differential
sensitivity of WS to Cu, Cd, and Zn focusing on the role of
the metal transcription factor (MTF) are ongoing.
• Characterization of other metal-related toxicities including, but
not limited to, disruption of ion-regulation and oxidative stress.
• Future in vivo studies with ELS of WS are planned to
determine life-stage specific sensitivity to Cu, Cd, and Zn
1.0
• It is suspected that WS express multiple isoforms of MT,
however attempts to clone additional MT genes in WS
has been unsuccessful to date.
Platypus MT-4
3 ug/L
1.5
0.0
% Similarity
0.3 ug/L
2.0
• Mammalian MT-4 is not known to be responsive to
metals
Most Similar MT Genes
0.03 ug/L
2.5
0.5
Table 2: Amino Acid Similarity between WS MT and MT of other Species
0.01 ug/L
• WS are among the most sensitive species of fishes to
metals, in particular, Cu (Table 1; Figure 1).
• Greater sensitivity of WS to Cd and Zn compared to other
fishes might be explained by the lesser up-regulation (2- to
4-fold) of MT gene expression in WS compared to other
species of fishes.
• Greater sensitivity of WS to Cu compared to other fishes
might be explained by the down-regulation of MT gene
expression.
• Lesser responsiveness of WS MT to exposure to Cd or Zn
and down-regulation following exposure to Cu might
impact the ability of WS to detoxify these metals.
• The expression of MT genes in sturgeon that have greater
homology to the MT of mammals raises interesting
evolutionary questions regarding sturgeon biology.
3.0
• WS MT had greatest homology to the MT-4 of mammals
Solvent
Fold-change from Solvent Control
Address uncertainties associated with the mechanism of
sensitivity of WS to metals such as Cd, Zn, and Cu through in
vitro investigations of MT gene expression in WS ELS.
Created by Peter Downing – Educational Media Access and Production © 2011
Discussion and Conclusions
0.3 ug/L
3 ug/L
30 ug/L
300 ug/L
1000 ug/L
1)
2)
3)
4)
5)
6)
C) Cu
1.0
*
0.8
*
*
*
0.6
*
0.4
0.2
0.0
Solvent
0.01 ug/L
0.03 ug/L
0.3 ug/L
3 ug/L
30 ug/L
References
100 ug/L
Figure 3: MT transcript abundance in tissue slices after 24h of exposure to
A) Cd., B) Zn, or C) Cu.. * Significant difference from solvent control (p<
0.05).
• MT gene expression was up-regulated in liver slices
exposed to Cd or Zn.
• MT gene expression was down-regulated in liver slices
exposed to Cu.
Vardy et al (2011) Enviro Toxicol Chem 11, 2497-2505.
Vardy et al. (2013) Ecotoxicol 22, 139-147.
Roesijadi (2000) Cell Mol Biol 46, 393-405.
Simon (2003) Bioinfo. 19, 1439-1440.
Dwyer et al. (2007) Arch Environ Contam Toxicol 48:143–154
EPA (2007a) http:/www.epa.gov/ecotox/. Accessed 20 Feb 2011
Acknowledgements
All experimental procedures were approved by the University Committee
on Animal Care and Supply (UCACS) at the University of
Saskatchewan.
Thanks to the Kootenay Trout Hatchery for their donation of white
sturgeon embryos.