Carla Labarrère1, John. P. Giesy2,5,6,7,8,9,Christy Morrissey2,3,4
1Toxicology
Graduate Program, University of Saskatchewan, Saskatoon, Canada; 2Toxicology Centre, University of Saskatchewan, Saskatoon, Canada; 3School of Environment and Sustainability, University of
Saskatchewan, Saskatoon, Canada; 4Department of Biology, University of Saskatchewan, Saskatoon, Canada; 5Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada; 6Zoology
Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA; 7Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China; 8School of Biological
Sciences, University of Hong Kong, Hong Kong, SAR, China; 9State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China.
Correspondence: carla.labarrere@usask.ca
Sediment
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*
7
A
6
5
4
3
2
Accelerated Solvent Extraction (ASE)
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*
*
PAHs, PCBs and Dioxins
relevant to environmental
contamination
Luciferase
bioassay
Potency of the sediment
extracts to activate the aryl
hydrocarbon receptor (AhR) in
the rat hepatoma (H4IIE-Luc)
cell line
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*
relative toxicity of extraction
Figure 1. Sediment sampling
represented by yellow stars.
1) Identify to what extent shorebirds are
exposed to polycyclic aromatic
Novel application of CYP1A detection technique
hydrocarbons (PAHs), polychlorinated
A
biphenyls (PCBs) and dioxins, across
EROD
The existing approaches to detect CYP1A in birds are developed
their range in North and South
liver slices
only for embryos. This new application has been developed to be
America.
used in ADULT birds, increasing the utility for applications of:
CYP1A
EROD
2) Assess in vitro sensitivity to a range
detection
monolayer
of PAHs and other dioxin like
- Species at risk or rare species
compounds, as well as he mixture
- Species without access to breeding areas
mRNA
compounds found in the winter and
liver slices
- Species that cannot be held in captivity
stop over areas.
B
Dosing
Pure compounds
Contaminant extracts
from sediment
3
2
0
TCDD 0.01
TCCD 0.1
TCCD 1
Dosing Solution (nM)
TCCD 10
DMSO
PAH 10
PAH 100
PAH 1000
Dosing Solution (nM)
PAH 10000
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Testing on Sanderling liver
slices
CYP1A induction potency
4
Figure 3. Sanderling CYP 1A4 gene expression.
Liver slices from three control Sanderlings were dosed with
different concentrations of TCDD (a) and Benzo-kfluoranthene (b), a PAH for 24 hours. Bar graphs represent
mean CYP 1A4 expression.
Contaminant extracts
Residue analyses
5
1
0
*
B
1
DMSO
Polycyclic aromatic hydrocarbons
(PAHs), founded in petroleum based
products,
and
other
dioxin-like
compounds
have
been
largely
overlooked as a cause of the decline
shorebirds population despite their
global distribution, toxicity, cumulative
effects, and the large annual quantities
introduced into the marine environment.
6
qPCR Fold Change - CYP 1A4
Sediments from shorebird migratory stopover sites in Canada, United States (pre and post
oil spill), Colombia, Ecuador, Uruguay and Brazil were collected during 2013 and 2014.
qPCR Fold Change - CYP 1A4
Evidence suggests that widespread
marine oil pollution is leading to the
exposure of shorebird populations to
petroleum
derivatives
and
other
industrial contaminants, across their
migratory range.
7
8
PCR
analysis
CYP1A genes
amplification
Figure 2. (a) Multiple approaches were tested to detect CYP1A in adult birds. Ethoxyresorufin-O-deethylase (EROD) activity could not be validated for either
monolayer and liver slices. (b) Methodology used to detect CYP1A induction potency using mRNA: Sanderlings were euthanized to obtains liver slices, which
have been dosed with pure compounds and sediment extracts to assess their relative toxicity. Pure compounds used are TCDD (0.0003, 0.001, 0.003, 0.01, 0.03,
0.1, 0.3, 1, 3, 10 nM), Benzo-k-fluoranthene, Benzo-a-pyrene, Aroclor 1254 (0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 nM).
Sediment sampling
1- Sediments were collected in Brazil (north and
south), Colombia, Ecuador, Uruguay, United States
(Texas before and after oil spill, and Delaware) and
Canada (James Bay and Chaplin Lake).
2- Samples from Colombia, Ecuador, Uruguay, Texas
(after oil spill), Delaware, and James bay have been
collected and are being shipped to the University of
Saskatchewan for future analysis.
3- Luciferese reporter gene bioassay is being
adapted to identify toxic potency of sediment extracts.
In vitro Assay
1- Pure compounds are being used on Sanderling
liver slices to identify toxic potency.
2- Liver slices are being dosed with 10 doses of each
pure compound.
3- Sediment extracts will be used to dose liver slices
during summer 2014.
We would like to acknowledge Dr. Sean Kennedy for the
information and in vitro protocol; Stephanie Jones for the assistance
on the method development and mRNA analysis, and all partners in
United States and South America for collection of sediment
samples. Stipend support from NSERC CREATE HERA Program.
Thank you to the Toxicology Graduate Program.