SETAC NORTH AMERICA 33rd ANNUAL MEETING, LONG BEACH,
CALIFORNIA, NOV 11-15, 2012
Session:
High Throughput Screening of
Environmental Pollutant on
Toxicity
Xiaowei Zhang, PhD., Nanjing University, China
Yong-Lai Feng, PhD., Health Canada, Canada
Motivation
• Increasing test tasks
– Larger chemical inventory
– Environmental Samples/Extracts
• Increasing demands on toxicological knowledge
– LC50, EC50, NOEC, etc.
– Enzymes/Receptor interactions (Multiple)
– Gene expression (Genome wise)
• Increasing test capability
– High throughput instruments
– Miniaturized Assays
• Better strategies to assess toxicity
– Screening, Classification, Prioritization, Toxicity/Risk assessment
– Toxicant identification, Effect-directed analysis
Presentations in Our Session
• Chemical tested
– Organophosphate Flame retardants (Crump et al), PCBs(Fritsch et al),
Endocrine disrupting chemicals(Paul et al.), nanomaterials(Cole et al.)
– Micropollutants (Allionson et al)
• Ecological species
– E coli (Zhang et al., Lan et al.), Yeast (Lan et al.), Fish (Fritsch et al.), Chicken
(Crump et al.), Phytoplankton (Cole et al.)
• Testing systems
– Cell line, primary cells, in vivo
– reporter gene system, mRNA expression
• Statistical issues
– Z score (Paul et al.)
– Multi variants issues (Lan et al.)
• Testing Strategies
– Tiered approach/stepwise approach(Paul et al.)
– In combination with Instrument analysis (Allionson et al.)
To: SETAC NORTH AMERICA 33rd ANNUAL MEETING, LONG BEACH, CALIFORNIA,
NOV 11-15, 2012
Toxicogenomic Screening of
Environmental Pollutants on
Toxicity
Xiaowei Zhang
School of the Environment
Nanjing University
Toxicogenomic Screening
• Genome wise study on gene expression profile by toxic
chemical exposure
•
•
Transcriptomics: Microarray/ RNA-seq
Proteomics, Metabolomics
• Higher throughput genomic assay/Functional Genome
assays
– Genome wide reporter gene assay
– Gain of function assay
• ORF screening assay
– Loss of function assay
• Mutant library screening assay
• RNAi screening assay
• Toxicogenomic Screening
– Higher throughput
– Lower cost
Outline
1. Background
2. Technology and principle
3. Case studies
 Case 1: naphthenic acids (NAs)
 Case 2：PBDEs and its Analogs
 Case 3：Toxicological Mechanisms of ZnO
nanoparticles
 Case 4：Chemical screening: OPFRs
 Case 5：Environmental Monitoring
4. Perspectives
Genome Wide Live Cell Reporter Array
Reporter Gene Assay---
A Successful Tool for Chemical Toxicity Assessment
damage
TF
Activation
Promoter
Luciferase/GFP
Gene Expression
(Transcript/Protein)
Cell pre-culture, over night
1800 strains/ 70% genome
one strain each well
Cell innoculation, 2-3 hour
Chemical dosing, 2-5 min
Incubation & Fluo/OD
measurement, 3-6 hour
Culture
Data Analysis
Measurement
Cytotoxicity Assay
(Alamar Blue)
Outline
1. Background
2. Technology and principle
3. Case studies
 Case 1: naphthenic acids (NAs)
 Case 2：PBDEs and its Analogs
 Case 3：Toxicological Mechanisms of ZnO
nanoparticles
 Case 4：Chemical screening: OPFRs
 Case 5：Environmental Monitoring
4. Perspectives
Case 1、Toxicogenomic mechanims of NAs
Naphthenic Acids (NAs): Pollutants
from Oil Sands industry
Mechanism of Toxicity
16
4
1
0.2
0.1
Fold Change
inaA
ilvC
yfcD
marR
uhpT
chbA
insA_2
yajO
ycaD
yjeB
yfbM
xthA
ypeA
rbsD
ileX
rluE
ppiD
gadW
ydcM
yahD
yaaW
yfbE
ybiH
alaS
insA_7
crl
rph
ybhK
ydgH
modF
ribA
feaB
serC
ppiB
greA
ptsG
yfdU
rpiA
gnd
araF
yfeN
sodA
ybjP
yjbJ
hemC
pitA
gadX
somA
ybgI
atpI
yjbQ
ymcC
ybeB
brnQ
ligA
cvrA
yjfI
trxA
b2641
yjgA
rob
ftsK
aceB
ihfB
htrL
rpsM
msrB
pmrD
yhiD
rrnA
accB
trmU
yejA
ycfD
bolA
dppA
ydcS
ybfE
ssb
ybaY
Transcriptional network database: Regulon DB
Active modules identification: jActiveModules
Network analysis platform: Cytoscape
Time: 0 -----------3-------------6h
Zhang et al., 2011. Environ Sci Technol. 45(5):1984-91
NOTEC: A promising toxicological endpoints for
industrial chemicals
Transcriptional Response
Cytoxicity
Concentration
NOTEC – no observed transcriptional effect concentration
Zhang et al EST 2011
Case 2、PBDEs & Analogs comparison
BDE-47,
HO-BDE-47,
MeO-BDE-47
Su et al., 2012. Environ Sci Technol. 46(2):1185-91
OH-BDEs caused similar gene expression profiles
AhR Activity of OH-BDEs
Su et al., 2012. Environ Sci Technol. 46(19):10781-8
Case 3、is the toxicity of Nanoparticles due to
release of metals?
Different mechanisms associated with the cytoxicity by ZnO NPs and Zn2+
Lg (Concentration of released
zinc ion)
3
2.5
y = 0.577x - 0.1636
R² = 0.983
2
1.5
1
0.5
0
1.5
3.5
Lg (Concentration of nano-ZnO)
5.5
Su et al EST (Submitted)
Case 4、Alternative Flame Retardants
Toxicity Screening
No
Chemicals
CAS number
T est Concentration
Range (ppm, mg/L)
Maxium Inhibition
Ratio (%)
IC10 after a 4hour exposure
IC50 after a 4hour exposure
1
Bis(2-ethychexyl)
hydrogenphosphate
298-07-7
0-5518
100
66.3
399.4
2
1,4,5,6,7,7-Hexachloronorbornene-2,3-dicarboxylic acid
115-28-6
0-3259
95
13.5
443.3
3296-90-0
0-5206
81
90.9
2403.7
4
2,2-Bis(bromomethyl)-1,3propanediol
T ris(2-butoxyethyl) phosphate
78-51-3
0-6181
33
215.9
4944.5
5
T riethyl phosphate
78-40-0
0-7145
33
561.4
NA
6
T ributyl Phosphate
126-78-3
0-9768
42
105
NA
7
Metamine
107-78-1
0-1289
NA
NA
NA
8
T CPP
13674-84-5
0-2509
NA
NA
NA
3
3194-55-6
0-5814
NA
NA
NA
10
1,2,5,6,9,10Hexabromocyclododecane
T DCPP
136374-87-8
0-1636
NA
NA
NA
11
T ricresyl phosphate
1330-78-5
NA
NA
NA
9
•
•
•
Six alternative flame retardants （CAS#：29807-7，115-28-6，3296-90-0，78-51-3，7840-0和126-78-3）demonstrated obvious
cytotoxicity .
The chemicals can be classified based on gene
expression profile
GO terms: protein binding, plasma membrane,
and stress responses
Case 5、Environmental Monitoring
Sediment extracts from Yangtz River（1－14）
900
800
TELIs
700
600
Low
500
Middle
400
High
300
200
100
0
Gene function
Discussion & Future application
• A novel genomic tools for toxicity assessment
–
–
–
–
–
–
High throughput technology coupled with genomic information
Affordable to most lab
Real time monitoring of gene expression
Screening: molecular pathways vs chemicals
Chemical classification
Field study: Effect-based Monitoring, Detection and Prioritization of chemical
pollution
• Tools for hypothesis testing in Predictive toxicology
– Mechanism based Chemical classification
– Aids in quantitative structure & activity relationship analysis
– Mixture effects: mode of action
• Application and future research
– Chemical (eco)toxicity test
– Contamination identification by the approach of effect-directed analysis
– Mammalian cell based reporter array system
Acknowledgement
• Students
– Mr. Guanyong Su (PhD candidate)
– Dr Jie Fu (Postdoc )
– Miss Miao Guan
• Funding Agents
– NFSC
– New Talents Funding (NJU)
– Major State Basic Research Development Program
(2008CB418102)
– Program for New Century Excellent Talents in University
(NCET)
– Environment Canada
Laboratory of Ecotoxicology and Environmental Health
Thank you!
• Xiaowei Zhang, PhD, Prof
•
•
•
•
•
Laboratory of Ecotoxicology & Environmental Health
School of the Environment
Nanjing University
Nanjing, China
Tel: (86)-25-8968 0623