Toxicology Centre
Untargeted strategy for identification of Toxic
Chemicals in OSPW
---- oxidative stress and PPARG agonistic activity in OSPW
Jianxian Sun, Hui Peng, Hattan Al Harbi, John P. Giesy,
Steve Wiseman
Oct 6, 2015
Toxicology Centre, University of Saskatchewan
Toxicology Centre
What is OSPW
 Oil Sands Process-Affected Water (OSPW) is produced during the surface
mining of oil sands in Alberta, The volume of OSPW is currently stored in
tailings is greater than 1 billion m3
PNAS, 2010, 951-952
Alberta Energy. Alberta Government. 2008.
http://www.energy .gov.ab.ca/OurBusiness/oilsands.asp
Toxicology Centre
OSPW: A Complex Mixture of Dissolved Organics
• Complex mixture of chemicals
• Uncharacterized compound
• Unknown mechanism of toxicity
 How to Identify causative chemicals in OPSW?
Chemicals
• Ox +/• SOx +/• NOx +/-
Thousands of dissolved organic compounds
Toxicity
?






Acute toxicity;
Deformity;
Nuclear receptors;
Endocrine disruption;
Immune toxicity;
Oxidative stress
Toxicology Centre
Methods to Identify Causative Chemicals
Unknown protein target
Effect-Directed Analysis (EDA)
Fractionation of
Active Fractions
Chemical
Analysis
Compare
Effects in
Bioassay
Identification of
Active Chemicals
Known protein Target
Pull down system
Toxicology Centre
Case 1: Oxidative Stress (no protein target)
Water Research, 2012, 6359-6368
Comparative Biochemistry and Physiology, 2013, 227-235
 Oxidative stress is the production and
accumulation of free radicals.
 Induction of oxidative stress in fathead minnow
by OSPW have been reported
 There are multiple pathways to induce ROS
with no certain protein targets.
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EDA: Chemical Analysis and Bioassay
Chemical Analysis:
High-Resolution Mass Spectrometry
Bioassay:
NRF2 Luciferase Reporter System
(High Throughput)
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1st Dimension Fractionation
OSPW
HLB
HLB
100% Hex
Hex/DCM
(5:1)
Hex/DCM
(1:1)
100% DCM
100% MeOH
F1
F2
F3
F4
F5
Polarity
 Dissolved organic phase of was separated into 5 fractions based on
polarity by use of HLB columns.
 Total extract created by pooling of samples.
Toxicology Centre
NRF2 Activity
4
F2
F1
***
3
F3
F4
***
2
***
**
*
TE
F5
***
***
**
**
1
0.
2
0.
04
1
0.
2
0.
04
1
0.
2
0.
04
1
0.
2
0.
04
1
0.
2
0.
04
0
1
0.
2
0.
04
1
Ctrl1
0.
2
tBHQ
Fold Change
HLB
 Oxidative stress responses of these five fractions were validated in
vivo (medaka)
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Reduced Responses by Co-exposure with GSH
***
***
2
**
tBHQ
0
Ctrl
1
***
3
***
2
1
0
1
0.2
0.04
tBHQ
tBHQ+GSH
TE
TE+GSH
***
Ctrl
3
4
tBHQ
tBHQ
tBHQ+GSH
F2
F2+GSH
Fold-change
Fold-change
4
 GSH is an anti-oxidant;
 Organic chemicals in OSPW cause ROS production
1
0.2
0.04
Toxicology Centre
Untargeted Mass Spec Analysis
positive
negative
Peaks
20000
Total peaks:
155892 in positive,
80830 in negative
15000
10000
5000
0
F1
ratio to max
1.5
F2
F3
F4
F5
 Greater than 10,000 peaks detected in 5
fractions
 Many peaks were specifically eluted in
F2, which may cause its greater potency.
 Potential causative chemicals were
narrowed in 1,000 chemicals from
10,000 compounds
F1
F2
F3
F4
F5
positive
1.0
negative
1.5
ratio to max
25000
0.5
F4
F1
F5
F2
F3
1.0
0.5
0.0
0.0
0
2000
4000
peak ID
6000
0
500
1000
1500
peak ID
2000
2500
Toxicology Centre
2nd Dimension HPLC Fractionation
OSPW
HLB
HLB
100% Hex
Hex/DCM
(5:1)
Hex/DCM
(1:1)
100% DCM
100% MeOH
F1
F2
F3
F4
F5
HPLC
F2-1, F2-2, F2-3……F2-60
Orbitrap
NRF2 System
Effective
Chemicals
1.4
0.8
Ctrl
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fold Change
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NRF2 Activity of 60 HPLC fractions
1.6
*
**
*
**
1.2
*
* *
1.0
Working on chemical analysis…
Toxicology Centre
Summary of EDA method
Effect-Directed Analysis (EDA)
Fractionation of
Active Fractions
Chemical
Analysis
Compare
 EDA is useful to narrow down
causative chemicals and to identify
active chemicals;
Effects in
Bioassay
Identification of
Active Chemicals
 Needs a lot of fractionation and
chemical analysis work. It is tricky to
identify the causative chemicals from
complex samples
Toxicology Centre
Case 2: PPARɣ Activation (known protein target)
 PPARs regulate intracellular lipid flux and adipocyte proliferation and
differentiation.
 PPARγ ligands may promote development of obesity.
 Activated by structurally diverse ligands
Toxicology Centre
PPARɣ Activation - Luciferase Reporter Gene Assay
Human PPAR-G
cell reporter system
PPAR response
80
60
40
20
0
0.1
1
10
100
Rosiglitazone (nM)
TE
Fractions
1000
10000
Toxicology Centre
PPARɣ mediated adipogenesis in 3T3-L1
 At low concentrations of OSPW (0.1×, 0.3×), accumulation of lipids was observed.
 At higher concentrations (1×, 3×), lipid droplet size was reduced but level was
increased.
Toxicology Centre
Ligand Identification: PPARɣ Pull-Down Assay
OSPW
Pull-down
Negative
control
Competitive
pull-down
intensity
His-PPARɣ
Putative ligands
Nonspecific binding
Rosiglitazone
Interfering compounds
Magnetic beads
mz
Toxicology Centre
Positive control: MTX-DHFR
30
Ratio
20
MTX
10
0
0
500
1000
Peak ID
1500
2000
H2N N NH2
N N
N
N
H O
N OH
O
O OH
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Profile of Compounds from Different Groups
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Compounds Detected as PPARɣ Ligands
Negative ion mode
Positive ion mode
 20 compounds in negative ion mode and 44 compounds in positive ion mode
were detected as potential PPARɣ ligands
Toxicology Centre
Conclusions and Perspectives
 Pull down system is a useful method for identifying causative
chemicals from complex environmental matrix
 OSPW can cause oxidative stress, we are still working on
identification of causative chemicals
 PPARɣ is sensitive target of OSPW organic chemicals, and
multiple ligands were identified
Toxicology Centre
Acknowledgements
Toxicology Centre
Thank you!
Toxicology Centre
Validation in 3T3-L1 Cell Line
The mouse embryonic fibroblast cell line 3T3-L1 is a favored model for
metabolism and obesity research, because the cells can be chemically
induced to differentiate into adipocytes.
Induce for 2 days
Exposure for 8 days
10 ug/ml insulin
1 uM DEX
0.5 mM MIX
10 ug/ml insulin
OSPW
Red Oil O staining of lipids
Toxicology Centre
Keap 1-NRF2
Under normal conditions, Nrf2 is constantly ubiquitinated through Keap1 and degraded in
the proteasome. Following Exposure to electrophiles or oxidative stress, Keap1 is inactivated.
Stabilized Nrf2 accumulates in the nucleus and activates many cytoprotective genes.
Yoichiro et al., 2012. Frontiers in Oncology
Toxicology Centre
in vivo:
Medaka larvae
Lipid Hydroperoxide (LPO)
3
**
Fold-change
Gene Expression
2
**
1
0
Ctrl
tBHQ
0.250.5
0.250.5
0.250.5
F1
F2
F5
 Oxidative stress related
genes were induced in
F2 group;
 F2 exposure significant
increased concentrations
of LPO
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Wnt-pathway
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PPARG-Wnt pathway
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Peak Detection and Match
NL: 1.16E6
Computation:
rt: 22.51 min
Intensity: 1.16E6
80
60
100
22.53
Relative Abundance
Relative Abundance
100
40
20
18.76
0 0
2
4
6
8 10 12 14 16 18 20 22 24
Time (min)
NL: 4.28E4
2.03
Computation:
rt: 2.42 min
Intensity: 4.28E4
80
60
40
20
0 0
2
4
6
8
10 12 14 16 18 20 22 24
Time (min)
Intensity cutoff: 1E4
positive
25000
negative
Peaks
20000
Total peaks:
155892 in positive,
80830 in negative
15000
10000
5000
0
F1
F2
F3
F4
F5
 Greater than 10,000 peaks detected in 5
fractions
 Rank order of peak abundance
 F1 > F3 > F4 > F2 > F5