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. Cholesterol loaded OSPW Control Cholesterol stripped 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 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 Normalized Abundance Annotated reference
Results 1 : Global Gene Expression Functional annotation using GO terms and KEGG mapping to identify process indicative of effects of OSPW.
Biotransformation OSPW-OC AhR CAR PXR Phase I GST UGT MDR ST CYP1A GST MDR UGT CYP2 Phase II Phase III Oxidative metabolism
Oxidative Stress - I Glutathione metabolism Pentose-phosphate pathway Transcription factor GSH Synthase ROS NRF2 GSH H202 NADP Glutathione Peroxidase Glutathione Reductase G-6-PDH 6-PGDH Transketolase AO MOA AlDH EH GST UGT MDR H20 + 02 GSSG NADPH
Oxidative Stress - II ROS Complex I Complex III Complex I and III are major sites of production of ROS http://en.wikipedia.org/wiki/File:Mitochondrial_electron_transport_chain%E2%80%94Etc4.svg
Apoptosis AIF AIF AIF PARP Cathepsin b ROS
Mechanism of Toxicity OSPW-OC CAR mitochondria AhR Complex I Complex III PXR Apoptosis nucleus GST UGT MDR nrf2 AO MOA AlDH EH GST UGT MDR CYP1A CYP2K CYP2AD CYP2N CYP3A* ROS OSPW-OC / Endobiotics
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) Phase I biotransformation * * Apoptosis Oxidative stress response genes * * * * * *
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.
Jon Martin Mohamed Gamal El-Din John Giesy Yuhe He RishiMandinky Markus Hecker Paul Jones Sarah Peterson Warren Zubot