Mechanism(s) of Toxicity of Oil Sands Process
Affected Water
Steve Wiseman
Toxicology Centre
University of Saskatchewan
Deposits of Oil Sands
 Canada is home to the third largest oil reserves,
mostly in Alberta’s Athabasca site
 Over 200 billion m3 of oil in deposit; 178 billion
barrels recoverable with current technologies
 Economic Benefits
• Over the next 25 years, employment is
expected to grow from 75,000 jobs to
905,000, and create $444 billion in tax
revenue
Surface Mining of Oil Sands
Bitumen
Clarke Hot Water Extraction
Process Affected Water
Oil Sand Process-Affected Water (OSPW)
 Oil Sands Process-affected Water (OSPW)
• Sands, clay, metals, unrecoverable bitumen
• Polycyclic aromatic hydrocarbons (PAHs; particle bound)
• Dissolved organic fraction containing >250,000 chemicals,
including naphthenic acids (NAs)
• Held in on-site tailings ponds under a policy of no release
Effects of OSPW on Aquatic Organisms
 Endocrine disruption
• Changes in concentrations of T and E2
• Impaired reproduction of fathead minnows exposed
to OSPW
 Embryotoxicity
• Reduced growth
• Greater mortality, hemorrhages, malformations
• Greater EROD activity (sediment/tailings)
 Immunotoxicity
• Greater incidences of fin erosion and viral lesions
• Decreases leukocytes, thrombocytes, and
granulocytes
Mechanism(s) of Toxicity of OSPW
 Because NAs are surfactants, it has been proposed that OSPW might
have toxicity via narcosis.
Control
Cholesterol loaded
Cholesterol stripped
Greater concentrations of cholesterol in membranes
OSPW
Transcriptomics
Given the complexity of OSPW it is likely that there are multiple mechanisms of toxicity.
Goal: Quantify abundances of transcripts in livers of male fathead minnows
exposed to OSPW to gain insight into potential mechanisms of toxicity.
Step 1 : De novo Assembly and Annotation
the Reference Transcriptome
Sample
Type of Read # of Reads (filtered)
Other
PE 100bp
284,025,638
Other
SE 75bp
72,290,015
DTW x3
PE 100bp
241,258,966
OSPW x3
PE 100bp
240,554,734
O3-OSPW x3
PE 100bp
268,336,086
Total Reads
1,106,465,439
Reads assembled into 61,103 contigs of 200bp or greater (CLC genomics)
BLAST2GO - Annotation of the 62,103 contigs using BLASTX identified 25,342
contigs with an e-value of ≤ 10-5
Step 2: Mapping Reads and RNAseq
 Abundances of transcripts determined using the RPKM method
 Read mapping
• Minimum of 5 reads from each of the three samples in at least one of the two
treatments.
• If reads were present in each of the three samples from one condition it did not
matter if reads were present in any of the three samples from the other condition.
 Significant (p < 0.05) change of ±1.5-fold deemed biologically relevant.
Normalized
Abundance
Control
Change in
Abundance
OSPW
Annotated reference
Normalized
Abundance
Results 1 : Global Gene Expression
Freshwater -vs- Untreated OSPW
Down
(95)
UP
(109)
Functional annotation using GO terms and
KEGG mapping to identify process
indicative of effects of OSPW.
Biotransformation
OSPW-OC
Transcript
Fold Change
CYP1A
CYP2k19
CYP2k6
CYP2N
CYP2AD2
UGT 5B4
UGT 5F1
Sulfotransferase 1,3
GST (mitochondrial)
GST (cytosolic)
MDR-2
Aldehyde oxidase 1
Aldehyde dehydrogenase
Monoamine oxidase
Epoxide hydrolase
2.1
11.3
10.1
2.7
2.2
6.3
-4.3
1.8
4.5
>23.3
3.3
3.1
3.6
3.2
2.0
Phase I
Phase II
Phase III
Oxidative
metabolism
AhR
CAR
PXR
CYP1A
GST
MDR
UGT
CYP2
GST
UGT
MDR
ST
Oxidative Stress - I
Transcript
Fold Change
Glutathione synthase
Glutathione reductase
Glutathione peroxidase
Transketolase
6-phosphogluconate dehydrogenase
Glucose-6-phosphate dehydrogenase
Nuclear factor like 2
3.1
3.2
1.7
2.4
10.1
2.7
1.8
ROS
Transcription factor
GSH
NADP
NADPH
H202
Glutathione
Peroxidase
Glutathione
Reductase
G-6-PDH
6-PGDH
Transketolase
GST UGT MDR
Pentose-phosphate
pathway
GSH Synthase
NRF2
AO MOA AlDH EH
Glutathione metabolism
GSSG
H20 + 02
Oxidative Stress - II
Transcript
Fold Change
NADH dehydrogenase 1 beta
subcomplex subunit 1
Acyl carrier (mitochondrial precursor)
Cytochrome b-c1 complex subunit 9
Cytochrome b561 domain 2
Cytochrome b5a
1.8
Complex I
1.5
1.5
3.3
8.8
ROS
http://en.wikipedia.org/wiki/File:Mitochondrial_electron_transport_chain%E2%80%94Etc4.svg
Complex III
Complex I and III are
major sites of production
of ROS
Apoptosis
Transcript
Fold Change
Apoptosis-inducing factor 3
4.3
Apoptosis-inducing factor mitochondrial associated-2
4.1
Poly [ADP-ribose] polymerase
4.8
Programmed cell death 4a
1.5
DNA damage-regulated autophagy modulator protein 2
> 23.3
Cathepsin b
1.5
BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 -1.8
Forkhead box transcription factor O3A
-3.3
AIF
PARP
ROS
AIF
Cathepsin b
AIF
Mechanism of Toxicity
OSPW-OC
CAR
AhR
mitochondria
Complex I
Complex III
PXR
nucleus
GST
UGT
MDR
CYP1A
CYP2K
CYP2AD
CYP2N
CYP3A*
GST
UGT
MDR
AO
MOA
AlDH
EH
nrf2
ROS
OSPW-OC /
Endobiotics
Apoptosis
Effects of OSPW on Early Life Stages of the
Fathead Minnow
Hemorrhage
Pericardial edema
Malformation of
spine
Molecular and Biochemical Effects
Reactive oxygen species
(ROS)2.0
Phase I biotransformation
Control
OSPW
Concentration ROS
2.5
*
2.0
1.5
1.0
0.5
0.0
cyp1a
Control
cyp3a
Apoptosis
Fold-change in abundance
of transcript
5
*
OSPW
Oxidative stress response genes
Control
4
3
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
OSPW
5
*
*
*
2
1
0
casp3
casp9
apopEn
Fold-change in abundance
of transcript
Fold-change in abundance
of transcript
3.0
Control
4
*
3
2
OSPW
*
*
1
0
gst
sod
cat
Conclusions
 RNAseq - apoptosis induced by ROS that result from metabolism of organic
compounds in OSPW and from changes in mitochondrial respiration might cause
toxicity of OSPW.
 Results of the RNAseq are supported by results from embryotoxicity of OSPW.
 Abundances determined by RNAseq match changes determined by qPCR.
Where next ?
 What are the chemicals in OSPW that are causing these effects?
Targeted studies to further establish this mechanism of toxicity.
Development of a PCR array.
John Giesy
Yuhe He
Rishi Mandinky
Markus Hecker
Paul Jones
Sarah Peterson
Warren Zubot
Jon Martin
Mohamed Gamal El-Din