Athabasca oil sands petcoke extract elicits biochemical and

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Athabasca oil sands petcoke extract elicits biochemical and transcriptomic effects in avian hepatocytes Doug Crump, Kim Williams, Suzanne Chiu, Yifeng Zhang, and Jonathan W. Martin Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on May 3, 2017

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Environmental Science & Technology

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Athabasca oil sands petcoke extract elicits biochemical and transcriptomic effects in avian

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hepatocytes

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Doug Crumpa*, Kim L. Williamsa, Suzanne Chiua, Yifeng Zhangb, Jonathan W. Martinb

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a

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National Wildlife Research Centre, Carleton University, Ottawa, ON, Canada K1A 0H3

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b

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Canada, T6G 2G3

Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada,

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta,

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*To whom correspondence should be addressed:

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Doug Crump, Environment and Climate Change Canada, National Wildlife Research Centre,

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1125 Colonel By Drive, Raven Road, Ottawa, Ontario, Canada K1A 0H3 or K1S 5B6 (courier).

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Fax: +1 (613) 998-0458. E-mail: [email protected]

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Keywords: petroleum coke; polycyclic aromatic compounds; in vitro screening; EROD; avian;

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PCR array; mRNA expression

1 ACS Paragon Plus Environment


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Abstract Petroleum coke or “petcoke” is a granular carbonaceous material produced during the

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upgrading of heavy crude oils, including bitumen. Petcoke dust was recently reported as an

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environmental contaminant in the Athabasca oil sands region, but the ecotoxicological hazards

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posed by this complex bitumen-derived material – including those to avian species – have not

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been characterized. In this study, solvent extracts (x) of delayed and fluid petcoke (xDP and xFP)

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were prepared and dissolved in dimethylsulfoxide. A water accommodated fraction of delayed

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petcoke (waDP) was also prepared. Graded concentrations of xDP, xFP, and waDP were

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administered to chicken and double-crested cormorant hepatocytes to determine effects on 7-

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ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression.

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Polycyclic aromatic compounds (PACs) were characterized and xDP, xFP, and waDP had total

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PAC concentrations of 93000, 270, and 5.3 ng/ml. The rank order of biochemical and

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transcriptomic responses was xDP>xFP>waDP (e.g. EROD EC50s were lower for xDP compared

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to xFP and waDP). A total of 22, 18, and 4 genes were altered following exposure to the highest

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concentrations of xDP, xFP, and waDP, respectively, using a chicken PCR array comprising 27

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AhR-related genes. To provide more exhaustive coverage of potential toxicity pathways being

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impacted, two avian ToxChip PCR arrays – chicken and double-crested cormorant – were

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utilized and xDP altered the expression of more genes than xFP. Traditional PAC-related toxicity

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pathways and novel mechanisms of action were identified in two avian species following

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petcoke extract exposure. Extrapolation to real world exposure scenarios must consider the

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bioavailability of the extracted PACs compared to those in exposed organisms.

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Introduction

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Petroleum coke or “petcoke” is a by-product of petroleum upgrading. The process

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involves thermally cracking the complex hydrocarbons and separating the non-volatile and heavy

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hydrocarbons (petcoke) from the lighter and valuable hydrocarbons used for synthetic crude oil.

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In the Athabasca oil sands region, two such coking methods are used to produce marketable

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synthetic crude oil – delayed coking and continuous fluid coking.1,2 In Alberta, approximately

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15% of extracted bitumen is converted to petcoke, and this material cannot be used for fuel in

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most markets due to its high sulfur content.3 This granular, carbonaceous, hydrophobic coal-like

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industrial by-product can be stored in large piles near processing facilities where it is susceptible

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to wind erosion; a potential vector for widespread environmental and human exposure to petcoke

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dust. Zhang et al.1 recently reported that delayed petcoke was a major source of polycyclic

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aromatic hydrocarbon (PAH) deposition to the environment around oil sands development. PAH

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concentrations of 25-28 µg/g dry weight were reported in authentic delayed petcoke from the

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Athabasca oil sands region.1 Moreover, metals, including nickel and vanadium, have been

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measured in petcoke at concentrations greater than 100 ppm.1,4 PAHs are known mutagens and

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carcinogens that are included on the Toxic Substances List (Schedule 1) of the Canadian

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Environmental Protection Act and can elicit biochemical and molecular-level effects via aryl

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hydrocarbon receptor (AhR) activation.5,6

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Although PAHs are classified as toxic substances, the screening-level hazard

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characterization of petcoke (both green [CASRN 64741-79-3] and calcined [CASRN 64743-05-

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1]) conducted as part of the US Environmental Protection Agency’s (EPA) High Production

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Volume Challenge Program did not classify petcoke as a hazardous waste material based on the

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analysis of potential human health and environmental impacts.7 In fact, most toxicity analyses

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reported a low potential of petcoke to cause adverse effects on aquatic or terrestrial environments 3 ACS Paragon Plus Environment


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as well as a low health hazard potential in humans, with no observed carcinogenic, reproductive,

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or developmental effects. In rats and monkeys exposed via inhalation to dust aerosol

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concentrations of delayed process petcoke (10.2 and 30.7 mg/m3) for 5 days/week over 2 years,

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Klonne et al. reported no significant adverse effects with the exception of discolouration of lungs

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(due to deposition of petcoke) and increased lung weight; no mortality was observed up to the

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highest concentration tested.8 Klonne et al. also conducted a nose-only inhalation study with

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micronized petcoke for 52 days to assess reproductive/developmental toxicity and reported no

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effects up to the highest concentration tested; ~0.3 mg/L.8 No mortality occurred in fathead

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minnows (Pimephales promelas) exposed to water accommodated petcoke fractions under semi-

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static conditions for 96 h up to a maximum loading rate of 1000 mg/L; however, petcoke has

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negligible water solubility and attempts to measure unalkylated PAHs, metals and sulphur

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revealed that concentrations were below detection limits.7 However, it is not clear if these former

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studies are relevant to petcoke produced in the Canadian oil sands region. Furthermore, to the

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best of our knowledge, no studies have assessed the potential toxic effects of petcoke in birds,

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which could be exposed, for example, via the diet or by inhalation of airborne petcoke dust

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particles. In general, more comprehensive toxicity tests such as in vivo and in vitro assays using

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birds, fish, invertebrates, and mammals are warranted to assess the toxicity of petcoke in

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complex organisms, cell lines, and/or primary cell cultures.

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The objectives of the present study were to: 1) prepare solvent extracts from delayed and

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fluid petcoke (xDP and xFP) and a water accommodated fraction of delayed petcoke (waDP); 2)

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characterize the concentration of a suite of polycyclic aromatic compounds (PACs) in xDP, xFP

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and waDP; and 3) utilize a well-established, high-throughput avian in vitro assay (i.e. primary

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embryonic hepatocyte assay), two well-characterized biochemical assays, and custom-designed


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avian PCR arrays to elucidate biochemical and transcriptomic effects of petcoke extract exposure

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in both a domestic and wild avian species (i.e. chicken and double-crested cormorant). The

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inclusion of two avian species permits the comparison of end points in a lab model species that is

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typically considered highly sensitive to a wide range of environmental pollutants, with a wild

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species that could realistically be exposed to petcoke in the environment. Ultimately, the goal

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was to determine if subtle biochemical and molecular biological end points could be used to rank

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the petcoke extracts in a manner that corresponded to the actual PAC concentrations and

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determine predicted (i.e. AhR-mediated end points) and novel mechanisms of petcoke toxicity in

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avian cells.

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Experimental section

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Sample preparation for contaminant analysis and administration to hepatocytes

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For the DP and FP extract, 10 g of authentic DP and FP solids from the Athabasca oil

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sands region in Canada were extracted twice with 250 mL Optima-grade dichloromethane

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(DCM) and then with 250 mL Optima-grade methanol (MeOH). The combined solvent was

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filtered through a GB-140 glass fiber membrane filter (90 mm diameter, pore size = 0.4 µm).

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Rotary evaporation was used to concentrate the extract, which was subsequently transferred to a

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glass vial and blown to dryness under nitrogen gas. The final dry weights of pure solvent-

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extracted material from 10 g DP and FP were 79.8 mg and 1.8 mg, respectively.

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Given that the mass of total material extracted was so different between DP and FP,

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concentrations of test materials for toxicity testing are reported here in two ways. First, to best

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compare the toxic potency of the pure solvent-extracted material, concentrations are reported as

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mg extract/volume DMSO. Stock solutions were prepared for both xDP and xFP at nominal



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concentrations of 0.01 mg extract/µL dimethyl sulfoxide (DMSO). Specifically, for delayed

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petcoke, the 0.01 mg/µL stock was prepared by dissolving 3.9 mg of pure DP organic extract in

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378 µL DMSO yielding a final concentration of 0.0103 mg/µL (Table 1). For fluid petcoke, the

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entire extract (1.8 mg) was dissolved in 250 µL of DMSO, for a final concentration of 0.007

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mg/µL (Table 1). The second method of reporting the concentrations is based on the equivalent

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mass of original petcoke from which the solubilized test material originated. Thus, based on the

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above stock solutions, the highest corresponding xDP equivalent concentration was 0.00126 g

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DP/µL DMSO, and the highest corresponding xFP equivalent concentration was 0.04 g FP/µL

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DMSO (Table 1).

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The water accommodated fraction (waDP) was prepared from solid DP (1 gram) that was

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stirred in 10 mL Optima LC/MS water for 10 days at 23°C and then filtered as above. To remove

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suspended particulate, this mixture was centrifuged at 15800 x g, transferred into a glass vial,

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and serial dilutions were made from this stock for hepatocyte administration in Optima-grade

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LC/MS water. Concentrations for toxicity testing are based on the total loading of DP in water,

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thus, the highest equivalent concentration for waDP was 0.0001 g DP/µL water (1 g/10 000 µL;

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Table 1).

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Determination of PAC concentrations The polycyclic aromatic compounds that were analyzed are listed in the Supplemental

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Information along with the GC-MS parameters. Briefly, 16 parent PAH standards (unlabelled)

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and 16 internal standards (deuterium-labelled) were purchased from Wellington Laboratories

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(Guelph, Canada) and the 4 alkyl-PAH and dibenzothiopene (DBT) standards were purchased

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from Chiron (Norway). Accelerated solvent extraction was used for targeted analysis of xDP


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and xFP in DMSO, while a liquid-liquid extraction method was used for analysis of waDP,

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described in greater detail in the Supplemental Information. Solid phase extraction (SPE) was

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used to clean up all three sample extracts prior to concentrating them in 200 µL of hexane for

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quantitative GC-MS analysis. GC-MS analysis involved splitless injection of 1 µL from all

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extracts. Chromatographic separation was achieved on a DB-5MS column (Agilent; 20 m x 0.18

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mm x 0.18 µm). Additional details regarding the method and method detection limits (MDLs) for

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all individual analytes are available in the Supplemental Information.

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Preparation and dosing of chicken and double-crested cormorant hepatocytes Twenty-five fertilized, unincubated white leghorn chicken (Gallus domesticus) eggs were

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obtained from the Canadian Food Inspection Agency and 15 double-crested cormorant (DCCO)

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eggs were collected from nests containing one egg on May 20, 2016, at Doucet Rock, Lake

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Huron (46° 8'22.09"N, 82°51'7.35"W). Eggs of both species were artificially incubated until ~2

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days pre-hatch8,10,11 at which point, embryos were euthanized by decapitation and hepatocytes

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were prepared by collagenase digestion and filtration and distributed into 48-well plates at ~40

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µg/well, as described previously.9,10

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Following incubation for 24 hours at 37.5°C and 5% CO2, hepatocytes were treated with

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either the DMSO or water vehicle control (0.5% v/v) and serial dilutions of xDP, xFP or waDP

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(n=3 wells/treatment for 7-ethoxyresorufin-O-deethylase [EROD], and n=6 wells/treatment for

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PCR array). At the highest concentrations, final administered amounts (in g petcoke equivalents)

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were 0.00315 g xDP, 0.1 g xFP and 0.00025 g waDP (Table 1). Serial dilutions covering 7 orders

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of magnitude (1x10-3- 10-9 g petcoke equivalent/µL for xDP, 4x10-2-10-8 g petcoke equivalent/µL

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for xFP, and 1x10-4-10-10 g petcoke equivalent/µL for waDP) and a 2,3,7,8-tetrachloro-dibenzo-



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p-dioxin (TCDD) positive control (100 nM)10 were administered to chicken embryonic

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hepatocytes (CEH) for EROD activity determination. For PCR array analysis, serial dilutions

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covering 6 orders of magnitude were used for CEH, and two orders of magnitude for double-

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crested cormorant embryonic hepatocytes (DCEH). The number of concentrations and petcoke

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extracts tested in DCEH (i.e. waDP was not screened in DCEH due to the minimal response

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observed for EROD activity and gene expression in CEH) was lower due to the limited pool of

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cormorant hepatocytes available. The limited pool of DCEH also meant that the EROD and

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porphyrin assays were only conducted in chicken cells. Hepatocytes of both species were

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incubated for 24 h following extract administration until processing for subsequent RNA

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isolation, EROD/porphyrin determination or cell viability.10,11

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Cell viability determination

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Viability of CEH following exposure to concentrations of xDP, xFP, and waDP covering

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4 orders of magnitude from the most concentrated solution was estimated with the ViaLight Plus

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kit (Lonza), according to the manufacturer’s instructions. DCEH viability was not determined

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due to the limited pool of cells. Untreated cells were used as a negative control and cells dosed

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with a final, in-well concentration of 300 µM tris(1,3-dichloro-2-propyl) phosphate were used as

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a positive control.11 Luminescence data were analyzed and interpreted as described previously.10

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EROD and porphyrin assays EROD and total porphyrin assays were conducted as described previously9,12 and reagents were obtained from Sigma-Aldrich (St. Louis, MO). EROD activity, total protein


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concentration and total porphyrin concentration were measured using a fluorescence plate-reader

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(Beckman Coulter DTX880 multimode detector, Brea, CA, USA), as previously described.9,12

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PCR array Total RNA was extracted from CEH and DCEH (n=3 technical replicates/treatment

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group) using the Qiagen RNeasy 96-kit (Qiagen, Mississauga, ON), reverse transcribed (~200

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ng) to cDNA and then added directly to the RT2 SYBR Green Mastermix (Qiagen).10,13

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Mastermix (25 µL) was aliquoted to each well of the PCR array containing primers at

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preoptimized concentrations.

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Two custom chicken RT2 Profiler PCR Arrays were built by Qiagen/SABiosciences

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(Frederick, MD) according to our specifications: 1) a PCR array containing 27 genes14

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(Catalog#: CAPG12083) to determine the effects of petcoke extract exposure on the AhR

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pathway; and 2) a chicken ToxChip PCR array comprising 43 target genes, which permits a more

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exhaustive coverage in terms of potential toxicity pathways being impacted (see Table S2 for a

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list of genes and pathways; Catalog#: CAPG13553). The double-crested cormorant ToxChip

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PCR array was custom-designed and built in our lab and is described in detail elsewhere.10 Each

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96-well PCR array contained either three identical sets of 27 target genes or two identical sets of

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43 target genes plus 5 control genes. The 5 control genes included 2 internal control genes, a

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positive PCR control (tests the efficiency of the PCR among/within plates using a pre-dispensed

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artificial DNA sequence and the primer set that detects it), a reverse transcription control, and a

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genomic DNA contamination control. Arrays were run using the Stratagene MX3005P PCR

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system (Agilent Technologies, Santa Clara, USA) as described previously.10,13,14

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Data analysis A one-way analysis of variance (ANOVA) with Tukey’s honestly significant differences

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(HSD) post hoc test was used to determine significant differences in CEH viability between

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various concentrations of the 3 petcoke extract preparations and the vehicle control in GraphPad

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Prism v5.02 (San Diego, CA, USA).

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EROD activity data were fit to a modified Gaussian curve as described previously9,10 and

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EC50 values were calculated if possible (i.e. if a maximal EROD induction was reached).

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ECthreshold (ECthr) values were determined if a maximal EROD induction was not reached and for

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porphyrin concentration data, as described previously.10

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PCR array data were analyzed using MxPro v4.10 software (Agilent Technologies) and

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an example of raw cycle threshold data is provided in Table S3. Cycle threshold (Ct) data were

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normalized to 2 internal control genes (elongation factor 1-alpha [Eef1a1] and ribosomal protein

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L4 [Rpl4]) and the fold change of target gene mRNA abundance relative to the vehicle control

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was calculated using the 2-∆∆Ct method.15 The PCR Arrays demonstrate strong correlations across

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technical replicates with average correlation coefficients > 0.99 ensuring reliable detection of

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differences in expression between biological samples (Qiagen/SABiosciences). Significant fold

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change differences (pBaP>Chr~BaA~benz[ghi]perylene (BghiP).6 The most potent PAH, DahA, comprised

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3.5% of the ∑14PAHs in the delayed petcoke extract, while Chr and BaP (4th and 5th most potent

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in Head et al.6) comprised 40 and 11% of the ∑14PAHs, respectively. Therefore, it can be

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hypothesized that specific PAHs are disproportionally responsible for the observed EROD and

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porphyrin induction, but the potential for agonism, synergism, and/or antagonism among the

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mixture of PACs cannot be discounted.


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PCR array

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i)

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A custom-designed chicken PCR array was utilized to confirm the AhR pathway effects

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described above with those at the transcriptome level. The expression levels of the two internal

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control genes, Eef1a1 and Rpl4, were not affected by treatment with any of the petcoke extracts.

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There was no amplification observed in the genomic DNA contamination control and the reverse

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transcription control and positive PCR control met the appropriate quality control/assurance

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criteria. Unprocessed mRNA expression fold change data for the various petcoke extract

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concentrations are available in Table S4.

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AhR pathway PCR array

The hierarchical clustering of xDP, xFP, and waDP extracts based on their transcriptional

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alterations in CEH resulted in three main branches: 1) extracts that significantly altered ≥ 18/27

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genes; 2) extracts that significantly altered 12-13/27 genes; and 3) extracts that significantly

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altered ≤ 4 genes (Figure 2). To compare gene profiles of the petcoke extracts to a known AhR

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agonist (TCDD), data from a previous study from our laboratory14 were included in the

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hierarchical cluster analysis. The 3 most concentrated xDP extracts (1x10-3-10-5 g petcoke

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equivalent/µL) elicited the most gene expression changes (22-25/27 genes) and clustered with

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the most concentrated xFP extract (4x10-2 g petcoke equivalent/µL), which altered 18/27 genes,

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and TCDD (1 nM), which altered 19/27 genes (Figure 2). Exposure of CEH to xDP (1x10-7) and

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xFP (4x10-3) led to the modulation of 13 and 12 genes, respectively, and these extracts formed a

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distinct branch. Finally, 4 extract preparations led to the alteration of ≤ 4 genes and included an

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extremely diluted xDP extract (1x10-8, which only altered one gene), xFP (4x10-4), and waDP

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(1x10-4 and 10-6 g petcoke equivalent/µL) (Figure 2). These transcriptomic responses for AhR



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pathway-specific gene targets are in agreement with the EROD and porphyrin assays

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demonstrating concordance at multiple levels of biological organization.

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The AhR pathway PCR array has been used previously to determine gene expression

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profiles of TCDD, natural AhR agonists and photodegraded flame retardants.14,22 We observed

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82% directional concordance between CEH exposed to the petcoke extracts in the highly-

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responsive cluster and TCDD (Figure 2, Table S4). Cyp1a4 was the most responsive transcript

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on the PCR array and was up-regulated hundreds of fold in the highest 3 xDP treatment groups.

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In CEH exposed to 1 nM TCDD, Cyp1a4 was induced 1976-fold.14 In vitro measures of avian

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AhR activation (e.g. Cyp1a4 induction) have been shown to be good predictors of overt DLC

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toxicity in avian embryos.23 Due to the large Cyp1a4 induction observed following exposure of

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CEH to xDP and to a lesser extent xFP, future whole animal egg injection studies are warranted

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to explore the toxicological implications of these transcriptomic results.

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Regucalcin (Rgn) was significantly up-regulated (≥1.5-fold, p

Athabasca oil sands petcoke extract elicits biochemical and

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