Mechanism(s) of Toxicity of Oil Sands Process Affected Water Steve Wiseman Toxicology Centre...
-
Upload
aldous-benjamin-gallagher -
Category
Documents
-
view
217 -
download
3
Transcript of Mechanism(s) of Toxicity of Oil Sands Process Affected Water Steve Wiseman Toxicology Centre...
Mechanism(s) of Toxicity of Oil Sands
Process
Affected Water
Steve WisemanToxicology Centre
University of Saskatchewan
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
Deposits of Oil Sands
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
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
Effects of OSPW on Aquatic Organisms
Mechanism(s) of Toxicity of OSPW
Because NAs are surfactants, it has been proposed that OSPW mighthave toxicity via narcosis.
Cholesterol loadedControl Cholesterol stripped OSPW
Greater concentrations of cholesterol in membranes
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.
Control
OSPW
Annotated reference
NormalizedAbundance
NormalizedAbundance
Change in Abundance
Results 1 : Global Gene Expression
UP(109)
Down
(95)
Freshwater -vs- Untreated OSPW
Functional annotation using GO terms and KEGG mapping to identify process indicative of effects of OSPW.
Biotransformation
Transcript Fold ChangeCYP1A 2.1CYP2k19 11.3CYP2k6 10.1CYP2N 2.7CYP2AD2 2.2UGT 5B4 6.3UGT 5F1 -4.3Sulfotransferase 1,3 1.8GST (mitochondrial) 4.5GST (cytosolic) >23.3MDR-2 3.3
Aldehyde oxidase 1 3.1Aldehyde dehydrogenase
3.6
Monoamine oxidase 3.2Epoxide hydrolase 2.0
Phase I
Phase II
Phase III
Oxidative metabolism
AhR PXRCAR
OSPW-OC
CYP1AGSTMDRUGT
CYP2 GSTUGTMDRST
Transcript Fold Change
Glutathione synthase 3.1Glutathione reductase 3.2Glutathione peroxidase 1.7Transketolase 2.46-phosphogluconate dehydrogenase 10.1Glucose-6-phosphate dehydrogenase 2.7Nuclear factor like 2 1.8
Oxidative Stress - I
Glutathione metabolism
Pentose-phosphate pathway
Glutathione
Reductase
GSH
GSSG
GSH Synthase
Glutathione Peroxidase
NADP
NADPH
G-6-PDH6-PGDH
Transketolase
H202
H20 + 02
NRF2
AO MOA AlDH EH
GST UGT MDR
ROS
Transcription factor
Oxidative Stress - II
Transcript Fold Change
NADH dehydrogenase 1 beta subcomplex subunit 1
1.8
Acyl carrier (mitochondrial precursor)
1.5
Cytochrome b-c1 complex subunit 9
1.5
Cytochrome b561 domain 2 3.3Cytochrome b5a 8.8
Complex I
Complex III
Complex I and III are major sites of productionof ROS
http://en.wikipedia.org/wiki/File:Mitochondrial_electron_transport_chain%E2%80%94Etc4.svg
ROS
Apoptosis
Transcript Fold ChangeApoptosis-inducing factor 3 4.3Apoptosis-inducing factor mitochondrial associated-2
4.1
Poly [ADP-ribose] polymerase 4.8Programmed cell death 4a 1.5DNA damage-regulated autophagy modulator protein 2
> 23.3
Cathepsin b 1.5BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
-1.8
Forkhead box transcription factor O3A -3.3AIF
PARP
Cathepsin b
AIF AIF
ROS
AhR PXR
CAR
OSPW-OC
CYP1ACYP2KCYP2ADCYP2NCYP3A*
OSPW-OC /Endobiotics
nucleus
nrf2AOMOAAlDHEH ROS
Complex IComplex III
Mechanism of Toxicity
GSTUGTMDR
Apoptosis
GSTUGTMDR
mitochondria
Effects of OSPW on Early Life Stages of the Fathead Minnow
Hemorrhage Pericardial edema Malformation of spine
Reactive oxygen species (ROS)
Control OSPW
Con
cent
ratio
n RO
S
0.00.20.40.60.81.01.21.41.61.82.0
*
cyp1a cyp3a
Fold
-cha
nge
in a
bund
ance
of tr
ansc
ript
0.0
0.5
1.0
1.5
2.0
2.5
3.0Control OSPW *
Oxidative stress response genes
gst sod cat0
1
2
3
4
5
Fold
-cha
nge
in a
bund
ance
of tr
ansc
ript
*
**
Control OSPW
Phase I biotransformation
casp3 casp9 apopEn
Fold
-cha
nge
in a
bund
ance
of tr
ansc
ript
0
1
2
3
4
5
**
*
Control OSPW
Apoptosis
Molecular and Biochemical Effects
Conclusions
RNAseq - apoptosis induced by ROS that result from metabolism of organiccompounds 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.
Jon MartinMohamed Gamal El-Din
John GiesyYuhe HeRishi MandinkyMarkus HeckerPaul JonesSarah Peterson
Warren Zubot