Effect of Oil Sands Process Water on Activity of Drug Efflux
Transporters in Caco-2 Cells and Japanese Medaka
Hattan Al Harbi1, David Saunders1, Jonathan Martin2, Alberto Pereira2, John P. Giesy1,3,4, Steve Wiseman1
Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada, Division of Analytical & Environmental Toxicology, Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, AB, Canada
3 Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada, 4 Department of Biology & Chemistry, City University of Hong Kong, and State Key Laboratory for Marine Pollution, Kowloon,
Hong Kong, SAR, China
1
RESULTS 1 – CACO 2 CELLS
Oil sands process affected water (OSPW) is generated during
extraction of bitumen from oil sands during surface mining.
2.00

Fresh OSPW is acutely toxic to aquatic organisms. As OSPW
ages in tailings ponds potency for acute toxicity decreases.
1.75

Fold change
1.50
***
1.25
B
***
1.5
**
*
1.0
C
***
2.0
1.5
1.0
Ne
ut
ra
l
Ba
si
c
x
x
5
1
2.
5
x
x
0.
5
µM
Determine if organic chemicals in fresh and aged OSPW affect activity
of ABC proteins.

Dose-dependent increase in accumulation of calcein-AM in cells exposed to different concentrations of, verapamil, an inhibitor of MDR1/P-gp.
METHODS

Dose-dependent increase in accumulation of calcein-AM in cells exposed to different concentrations of the dissolved organic fraction of BMLOSPW.
RESULTS – JAPANESE MEDAKA FRY
2
Figure 2. Illustration of the assay
to determine function of MRP in
embryos of Japanese medaka.
Created by Peter Downing – Educational Media Access and Production © 2011
Wash 3×
in cold water
6
4
Aging of OSPW attenuates this effect as accumulation of
calcein AM was not greater in fry of Japanese medaka exposed
to the dissolved organic fraction of OSPW from the TPW
experimental reclamation pond.

Chemicals that cause the effect of the dissolved organic
fraction of BML-OSPW are found in neutral and basic fractions,
but the identities of these compounds are not known.
Future Work

Chemical structures of the major components of basic and
neutral fractions are being elucidated to understand structureactivity relationships.

Polycyclic aromatic hydrocarbons (PAHs), which are found in
tailings ponds, are excreted by ABC proteins. Studies are being
conducted to determine if dissolved organic compounds in BMLOSPW impair excretion of metabolites of PAHs from cells, and
whether this affect might have toxicological implications.
0.0
(5µ
M)
M
5x
0
5X
A
B
ACKNOWLEDGEMENTS
5.0
0
2.5X
*
7.5
2.5
1X
C
10.0
2
BML-OSPW
C
TPW-OSPW
Figure 4. Fold change accumulation of calcein-AM in fry of Japanese medaka
exposed to (A) MK571 (positive control), (B) different concentrations of the
dissolved organic fraction of BML-OSPW or TPW-OSPW, and (C) fractions of
BML-OSPW relative to control (* ρ < 0.5, ** ρ < 0.1, *** ρ < 0.01).
 JAPANESE MEDAKA EMBRYO ASSAY
Incubate at 26oC for 15 min
then add calcein AM
12.5
1
K5
71
Add new media
+ calcein-AM
and inhibitors, and
incubate (37oC
for 60 min)
**
8
0.1X
Incubate
(37oC for 15 min)
15.0
Figure 5. Representative images
showing accumulation of calcein-AM in
fry of J. medaka exposed to (A) solvent
control, (B) 5 µM of MK571 (C) 2.5x of
BML-OSPW, or (D) 5X of BML-OSPW.

Accumulation of calcein-AM was greater in fry exposed to 5 μM of MK571, an inhibitor of MRP activity.

Accumulation of calcein-AM was greater in fry exposed to the dissolved organic fraction of BML-OSPW but not TPW-OSPW.

Accumulation of calcein-AM was greater in fry exposed to 2.5× or 5 × of the dissolved organic fraction of BML-OSPW.

Accumulation of calcein-AM was greater in fry exposed to the neutral or basic fractions, but not the acid fraction, of BML-OSPW.

Collaborative Research and Development Grant from the
Natural Science and Engineering Council of Canada (NSERC-CRD)
and Syncrude Canada Ltd to J. Martin and J.P Giesy.

Discovery Grant from the Natural Science and Engineering
Research Council of Canada (NSERC) to J.P. Giesy.

Grant from Western Economic Diversification Canada to J.P.
Giesy.

J.P. Giesy is supported by the Canada Research Chair program.

OSPW was supplied by Warren Zubot of Syncrude Canada Inc.

Hattan was supported by KSU

Japanese medaka were raised and bred in the Aquatic Toxicology
Research Facility (ATRF) at the University of Saskatchewan
D
Ne
ut
ra
l
3
Figure 1. Illustration of the assay to determine function of
MDR1/p-GP in Caco-2 cells.
Fry (24 hph) placed
in 24-well plate
containing inhibitor
Fold change
Add new media
+ inhibitors
B
Ba
si
c
10
***
Ac
id
A
12
**
Fold change
5
4
Quantify
fluorescence

Accumulation of calcein-AM was greater in cells exposed to the acid, basic, or neutral fractions of the dissolved organic fraction of BML-OSPW.
Accumulation of calcein-AM was greatest in cells exposed to the basic or neutral fractions.

 CACO-2 ASSAY
• Intestinal epithelial cells that express several ABC proteins,
including MDR1/p-GP.
Was 3× in
ice cold media
Inhibition of MXR activity in fry of Japanese medaka occurred
only at concentrations of the dissolved organic fraction of BMLOSPW that were greater than found in the BML end pit lake.
Figure 3. Fold change accumulation of calcien-AM in Caco-2 cells exposed to different concentrations of the dissolved
organic fraction relative to control of ((A) Verapamil (positive control), B) BML-OSPW, and (C) fractions of BML-OSPW (* ρ
< 0.5, ** ρ < 0.1, *** ρ < 0.01).
OBJECTIVES
Incubate
(37oC for 24h)

***
0.5
0.5
It is not known if OSPW effects activity of ABC proteins.
Plate Cells
(103 cells/ml)
Dissolved organic chemicals in fresh OSPW collected from Base
Mine Lake, inhibited activity MXR proteins in vitro and in fry of
Japanese medaka.
***
Ac
id
Members of the ABC family of proteins include p-glycoproteins
(P-gp)/multiple drug resistance proteins (MDR), and multidrug
resistance-associated protein (MRP).
 OSPW was collected form two sources:
• Base Mine Lake (BML)– The first experimental end pit lake
constructed in the oil sands industry. Established in 2013.
• TPW - Experimental reclamation pond constructed in 1993.

1.00
1

***
µM
Crude oil can inhibit cellular detoxification by inhibition of the
activity of ABC transporters proteins that efflux parents or
metabolites of organic compounds from cells.
2.5
***
10
0

A
µM
Aged OSPW has chronic toxicity to aquatic organisms.
2.0
10

Fold change

Discussion
Fold change
BACKGROUND