SETAC NA 32nd Annual Meeting, Boston, USA, Nov 13-17,
2011
Application of a Genome Wide Live Cell
Array system in Ecotoxicity Assessment
Xiaowei Zhang
State Key Laboratory of Pollution Control and Resource Reuse
School of the Environment
Nanjing University, Nanjing
Laboratory of Ecotoxicology and Environmental Health
1) What kinds of ADVERSE EFFECTS
can chemicals cause?
2) What is the MECHANISMs of the
adverse effects caused by the
chemicals?
Chemicals/
Pollutants
4) Which BIOMARKERS can be
used to indicate the adverse
effects/Chemical exposure?
3) What is the THRESHOLD of
chemical to cause the adverse
effects?
Environment
(Living Organisms)
Chemical Safety Assessment
• Environmental Quality Standards
• Chemical Risk Assessment
Contaminants Identification
• Toxicity Identification & Evaluation
• Effect-directed Analysis
Mixture Toxicity Assessment
Conventional Toxicity Testing
•
•
•
•
Animal based tests
Time, Labor, Cost
Poor mechanistic information
Demands not met
Predictive Toxicology
•
•
•
•
In vitro based tests
High through-put
Genomic, system Biology,
bioinformatics
Mechanistic information
Laboratory of Ecotoxicology and Environmental Health, School of the Environment, Nanjing University, Nanjing
Application of Genomics
Challenges
Mechanism of Toxicity
Expensive
Chemical Prioritizing
Biomarker Screening
Toxicity Prediction
Complicated
One Shot analysis
Toxicity = Exposure(Concentration) X Time
Laboratory of Ecotoxicology and Environmental Health, School of the Environment, Nanjing University, Nanjing
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)
Naphthenic Acids
ZnO nano-particles
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
Time: 0 --------------3---------------6h
8
2
0.1
0.5
Fold Change
16
4
1
0.2
0.1
Fold Change
uspB
ghrA
ybiF
yegE
ugpB
yjgR
yncE
dppB
yafD
tktA
ftsK
dksA
pykF
araF
lgt
mdtO
yibK
yhcF
rpmE
msbB
fryB
yoaD
sieB
sodB
wrbA
yfbM
yneJ
ppiB
ybjG
yejH
soxS
rof
rpsT
yfbU
ecfK
yhbO
yhdY
selA
rhsE
b0671
yhbQ
yejK
ybgH
ynfA
glpA
gatY
tyrS
ydgD
ybfE
pck
dpbA
serA
ybjM
ybiV
gcvA
rluB
yccA
cdd
ydcJ
mdtN
yqaE
galR
mcrB
yhiI
yciK
nohB
pspE
hyaA
yhhZ
trpR
b0501
yfaY
ybiH
pinH
folE
folA
yeaZ
pmbA
yfeY
seqA
yiiR
ftsY
gpt
cysH
ymcE
dppA
elaB
yaiW
dps
tolB
glpE
yajR
ymfI
garD
rluC
yebS
codB
guaC
yicC
yoaB
selD
ytfM
rcsC
parC
lacY
trmA
yrbA
ybhK
ygaV
yebQ
slyX
yhfZ
sbmC
ung
yciA
thiI
mdtK
yohJ
yeaM
yqjC
yhiR
yecF
mhpR
ftsQ
ycgF
ydgA
ecfI
slyA
yhbT
yciC
lolB
hemH
ygcF
allS
yggH
ycjM
ygjO
hupA
b0582
yohL
Zhang et al EST 2009
A) Altered Gene Expression
Concentration-dependent relationship
Zhang et al EST 2011
B) Concentration-dependent response of transcription
Transcriptional Response
Cytoxicity
Concentration
NOTEC – no observed transcriptional effect concentration
Zhang et al EST 2011
C) Fold change vs # gene altered
100
80
Algae
Fish
Molluscs
Worms
Daphnid
Mysid Shrimp
60
Percent Rank (%)
80
*
*
20
20
40
40
60
Response (%)
Discrete variable
Continuous variable
0
0
20
40
60
80
Discrete variable
Continuous variable
0
Response (%)
100
B: 2 fold change cut-off
100
A: 1.5 fold change cut-off
D) Ecotoxicity data plot (TPTC)
10
100
1000
NAs concentration (mg/L)
10
100
1000
NAs concentration (mg/L)
-1
0
1
2
3
4
5
Log10(ug/L)
NOTEC – no observed transcriptional effect concentration
Zhang et al EST 2011
E. Coli Transcriptional Regulatory Network
http://regulondb.ccg.unam.mx/index.jsp
Mechanism of Toxicity
Naphthenic Acids (NAs)
Transcriptional network database: Regulon DB
Active modules identification: jActiveModules
Network analysis platform: Cytoscape
Examples: BDEs and derivatives
High Concentration
Natural Source
CH3
Br
Br
O
Br
Human Blood
Br
BDE-47
O
Br
O
O
Br
Br
Br
6-MeO-BDE-47
OH
Br
Br
Br
6-HO-BDE-47
Br
Cytotoxicity
Inhibition profile of E. coli growth by 6-HO-BDE-47 at different
concentrations
6-HO-BDE-47
Real-time gene expression profiles of differentially expressed gens in E. coli.
Su et al EST 2011 (Accepted)
Pathways altered by OH-BDEs
BDE-47, HO-BDE-47,MeO-BDE-47
Discussion & Future application
• A novel genomic tools for toxicity assessment
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–
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High throughput technology coupled with genomic information
Affordable to most lab
Real time monitoring of gene expression
Increased resolution of biological responses
• 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
Thank you!
• Xiaowei Zhang, PhD
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Professor in Ecotoxicology
School of the Environment
Nanjing University
Nanjing, China
Tel: (86)-25-8968 0623; Fax: (86)-25- 8968 0623
• Email: howard50003250@yahoo.com
• Web: http://hjxy.nju.edu.cn/files/faculty/zhangxw.htm
Ecotoxicology and Environmental Health Research Laboratory