Mechanism(s) of Toxicity of Oil Sands ProcessAffected 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 284,025,638Other SE 75bp 72,290,015DTW x3 PE 100bp 241,258,966OSPW x3 PE 100bp 240,554,734O3-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

OSPWAnnotated 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.

BiotransformationTranscript 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 3.3Aldehyde 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 - IITranscript Fold

ChangeNADH 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

ApoptosisTranscript 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

Conc

entra

tion

ROS

0.00.20.40.60.81.01.21.41.61.82.0

*

cyp1a cyp3a

Fold-

chan

ge in

abun

danc

e o

f tra

nscri

pt

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-

chan

ge in

abun

danc

e o

f tra

nscri

pt

*

**

Control OSPW

Phase I biotransformation

casp3 casp9 apopEn

Fold-

chan

ge in

abun

danc

e o

f tra

nscri

pt

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