Assessing the mechanisms of toxicity of oil sands process affected water
by use of a genome wide live cell array reporter system
1Garrett
Morandi, 2Guan Miao, 1Hattan Alharbi, 2Xiaowei Zhang, 3Alberto dos Santos Pereira, 3Jonathan W. Martin, 1,4,5,6John P. Giesy, 1Steve Wiseman
1Toxicology
Centre, University of Saskatchewan, SK, Canada,2 State Key Laboratory of Pollution Control & Resource Reuse, School of Environment, Nanjing University, Jiangsu province, Peoples Republic of China,
3Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Chemistry, University of Alberta, Edmonton, AB, Canada, 4Department of Veterinary and Biomedical Sciences, University of Saskatchewan, Saskatoon, SK,
Canada, 5State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, Peoples Republic of China, 6Department of Zoology and Centre for Integrative Toxicology, Michigan
State University, East Lansing, MI, USA.
INTRODUCTION
• OSPW is a by-product from the extraction and
separation of bitumen in the Alberta Oil Sands(1).
• OSPW is restricted to a zero-discharge policy and
requires remediation before release(2).
• OSPW has acute and chronic toxicity to a range of
species(3, 4, 5).
• Causative agents and mechanism of toxicity are
unknown but studies have shown that the dissolved
organic fraction of OSPW is acutely toxic(3, 6).
• Naphthenic acids, class of cyclic and acyclic
compounds with a -COOH functional group, have
been proposed as the main toxic constituents of the
organic fraction of OSPW.
• Oxidative stress might contribute to the toxicity of
OSPW (3, 6).
Escherichia coli K-12 MG-1655 gene reporter
system
• Open-format approaches for investigating the
adverse effects of chemicals have gained popularity
in recent years(7).
• The Live Cell Array (LCA) system facilitates
measurement of promoter activity by use of
transcriptionally fused fast-folding fluorescent
proteins(7).
• E. coli MG-1655 system is composed of 1820/2500
promoters in its genome.
• An acutely toxic fraction (NEF2) of OSPW was identified
using a suite of bioassays and contains the enriched
chemical classes:
1. OSPW Fractionation and acute toxicity assays
A) 15-minute
EC50 Microtox®
Primary fractionation
Primary fractions
AE
NE
2. LCA
Workflow
BE
Fractions identified
as toxic during
fractionation
B) 96-hr acute lethality
(Chironomus dilutus)
Secondary fractionation
NEF1
Secondary fractions
NEF2
Fractions
screened by
use of LCA
assay
C) 96-hr embryotoxicity
(Fathead minnow)
Figure 1. Fractionation of the OSPW organics. Round 1 fractionation was
sequential- solvent extraction at pH 7, 2 and 11. Round 2 fractionation was
liquid-liquid washing at pH 2 and 12. Acutely toxic fractions were identified
using A) The Microtox® system, B) 96-hr growth and lethality test Chironomus
dilutus and C) 96-hr embryotoxicity test Pimephales promelas. The OSPW was
collected from the Base mine lake and is the first end pit lake in the industry.
1. E. coli strains grown at 37°C in a
2X LB Lennox media with 25
ug/mL kanamycin.
1.50E+07
Fraction
Screened
on LCA
system
# altered
genes
(n=68 total)
# Downregulated
genes
# Upregulated
genes
NE
37
29
8
NEF1
55
42
13
NEF2
43
33
10
Peak area
3. Analyze gene expression profiles by use of DAVID(9)
to identify enriched pathways.
Figure 3. Clustering of the fraction- and time dependent
expression of the 68 genes altered at least 2-fold change
over background. The fold change of gene expression is
indicated by the colour gradient, and time course
expression changes from left to right.
• Pathway analysis revealed significant responsiveness in
the KEGG pathway (Kyoto Encyclopedia of Genes and
genomes) ubiquinone and terpenoid biosynthesis:
• Ionizable compounds are known to effect the electron
transport chain… a role for naphthenic acids
contributing to toxicity?
FUTURE WORK
• Continue EDA and fractionate NEF2 fraction.
• Screen fractions of NEF2 by use of LCA system.
• Investigate role of electron transport chain in toxicity of
BML-OSPW.
REFERENCES
(1) Government
of Alberta. (2008). Oil Sands discovery Centre. www.history.alberta.ca.
Province of Alberta. (2014). Environmental Protection and Enhancement Act.
www.qp.alberta.ca. Accessed January 26, 2014.
(3) He, Y. et al. (2012a). Water Res. 46, 6359-6368.
(4) Colavecchia et al. 2004. Environ. Toxicol. Chem. 23, 1709–1718.
(5)Anderson et al. (2011). Water Research. 46 (6), 1662-1672.
(6) Wiseman et al. (2013). Aquat. Toxicol. 142, 414-421.
(7) Ronen et al. (2002). Proc. Natl. Acad. Sci. U. S. A, 99, 10555–10560 [119].
(8 )R Core Team (2014). R Foundation for Statistical Computing, Vienna, Austria. URL
http://www.R-project.org/.
(9) Nature Protocols 2009; 4(1):44 & Genome Biology 2003; 4(5):P3
(2)
1.80E+07
B
A) ESI-
1.50E+07
1.20E+07
NE F1
9.00E+06
NE F2
1.20E+07
9.00E+06
6.00E+06
6.00E+06
3.00E+06
3.00E+06
0.00E+00
0.00E+00
Functioning of the electron transport chain (ydhD, trxC, ykgF)
Stress response (clpX, dnaK, iscR, ssb, mfd)
specifically affecting genes related to Oxidative
Phosphorylation.
• UbiG, UbiC > 2-fold down-regulation.
• Linear regression was used to assess the effect of time on the
response of the genes (p≤ 0.001, R 2.3.0(8)).
• 2-fold change in gene expression was used as a cut-off to
generate gene list.
• Gene enrichment and KEGG analysis completed by use of
DAVID(8).
1.80E+07
• Gene enrichment analysis identified two processes that
responded to the toxic fractions (NE, NEF2) of OSPW:
•
Table 1. Fraction induced response in the number of
differentially expressed genes from three fractions
of OSPW organics.
2. Assess the molecular mechanisms of toxicity of
fractions of organic compounds from OSPW by use
of the E-coli MG1655 gene reporter system.
Created by Peter Downing – Educational Media Access and Production © 2011
4. Add 3.75 uL exposure/
control solution per well.
5. Read GFP and OD every
6. Statistical and data analysis: 10-minutes for 4 hrs.
Figure 4. Fraction induced response
in the number of differentially
expressed genes from three fractions
of OSPW organics.
• ESI-: -O2, -OS, -O2S and ESI+: -O, -O2, -O3 , -OS, -ON, O2N
•
•
RESULTS
1. Perform an effects-directed analysis (EDA) of the
organic fraction of OSPW to identify compounds
responsible for acute toxicity.
5. Compare results with chemical profile of fractions
to identify chemicals in OSPW that cause toxicity.
3. Addition of 71.25 uL of LB
medium to 384-well optical
bottom plate.
2. Incubate for 3.5 hrs
at 37°C.
OBJECTIVES
4. Combine acute toxicity data and gene enrichment
profiles to compare molecular mechanisms of
toxicity between toxic and non-toxic fractions.
CONCLUSIONS
Peak area
Oil Sands Process Affected Waters (OSPW)
MATERIALS & METHODS
ACKNOWLEDGEMENTS
B) ESI+
NE F1
NE F2
Figure 5. Total abundance of species by heteroatom class based on the sum of the
peak areas in the chromatograms of fractions of OSPW, by use of Orbitrap Mass
spectrometry. A) NEF1 and NEF2 in ESI-, B) NEF1 and NEF2 in ESI+.
•
•
•
•
•
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Collaborative Research and Development Grant from the Natural Science and
Engineering Research Council of Canada and Syncrude Canada Ltd. (NSERC-CRD) to
J.P. Giesy and J.W. Martin.
Discovery Grant from the Natural Science and Engineering Research Council of
Canada (NSERC) to J.P. Giesy.
Grants from Western Economic Diversification Canada to J.P. Giesy.
Grants from the Helmoltz Alberta (HAI) Initiative to J.W. Martin.
J.P. Giesy was supported by the Canada Research Chair program, and an at large
Chair Professorship at the Department of Biology and Chemistry and State Key
Laboratory in Marine Pollution, City University of Hong Kong.
Syncrude Canada Ltd. for supplying the OSPW.