High Throughput Screening Of Nanotoxicity Based On Paradigm
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Nanotoxicology Research and Training Program
December 11 2008
High throughput screening of nanotoxicity based on oxidative stress paradigm
Saji George Dr Andre Nel’s lab Dept. of Medicine (Division of Nanomedicine) UCLA THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
Growing nanotech industry- need of alternate toxicity screening strategies
Traditional Toxicological Approach -Based on observable experimental animals
outcomes
in
-Time consuming -Labor and cost intensive
Nature- Vol 444|16 2006
Skincare and consumer products, healthcare, electronics, photonics, biotechnology, engineering products, pharmaceuticals, drug delivery, and agriculture.
US national academy of science calls for a paradigm shift in toxicity evaluation http://www.nap.edu/openbook.php?record_id=11970&page=1
I trillion $ worth business over 15-20 yrs
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
HTS for faster screening of toxicity of nanomaterials Cell growth and interaction with NPs Probes for assaying toxicity markers
Automated imaging/readin g
Advantages of HTS
Automatic image analysis
Assay Design
Data storage and management
Less labor and time consuming Data analysis
Wide range of NMs properties Large batches of NMs Comprehensive array of predictive in vitro tests
Cellular knowledge
Easy visualization and interpretation of data Cytotoxicity assessment
www.api.com/images/lifescience/well_samples.jpg
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
Major pathways of toxicity
Oxidative damage of proteins, DNA and membrane Inflammation:cytokines/chemokines Mitochondrial damage- Apoptosis Lysosomal damage Membrane damage
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
December 11 2008
Nanotoxicology Research and Training Program
December 11 2008
Mechanism of NP mediated toxicity- Hierarchical oxidative stress paradigm
High GSH/GSSG ratio
Low GSH/GSSG ratio
Tier 2
Tier 3
ss ert s e vit a di xo f o l e ve L
Tier 1 Cell response pathway
Normal
Anti-oxidant Defense
Inflammation
cytotoxicity
Signaling pathway:
Nrf-2
MAPK
Mitochondrial perturbation-PT pore
Genetic response:
ARE
AP-1
N/A
Outcome
PhaseII enzyme
Cytokines chemokines
Apoptosis
http://www.nap.edu/openbook.php?record_id=11970&page=7 Nel et al. Science 2006, 311: 622-7
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
Preliminary results
(Xia et al. ACS Nano 2008)
*
6 5
ZnO
4
*
3
CeO2
2 1
TiO2
0 0
B
5
2.5
10
15
ZnO
60 40
ROS
*
* *
20
20
1.5
CeO2
0
ZnO
0.5
5
10 15 Time (h)
D
ZnO
5
B
CeO2
*
ZnO
* *
20 10
CeO2
1 00
10
Time (h)
15
Dead cells
* *
1.5
Tier 3
*
ZnO
* CeO2
1
TiO2
0.5
20
5
10
Time (h)
15
ZnO
40
*
* *
20
TiO2
5
10
15
Time (h)
15
20
* *
25
CeO2 5
100
20
p-JNK JNK
10
Time (h)
15
Dead cells
ZnO
*
5
*
4
*
3 2
CeO2
1
TiO2
TiO2
0
20
*
[Ca2+]i
6
ZnO
50
0
5
10
Time (h)
15
20
*
75
ZnO
50 25
*
* 0
0 0
10
Time (h)
7
*
75
CeO2 TiO2
0
CeO2
5
TiO2
Lowered membrane potential
0 0
TiO2
TiO2
CeO2
0
*
80 60
CeO2
ZnO
100
% PI + Cells (M1)
5
ZnO
Φ
JNK
0
0
0
20
p54 p46 p54 p46
TiO2
0
% PI + Cells (M1)
p-JNK
2
Fold ↑ in Fluo-4
% JC-1 low Cells
50
30
15
Tier 2
[Ca2+]i
CeO2
Actin Φ
2.5
*
HO-1
TiO2
Lowered membrane potential
40
10
Tier 1
D
60
*
25
C
Tier 2 Φ
50
Time (h)
TiO2 p54 p46 p54 p46
* ZnO
0 0
20
Superoxide
75
0
Actin CeO2
TiO2
1
CeO2 TiO2
HO-1
ZnO
100
H2O2
2
80
Tier 1
Tier 3
A
*
0
Time (h)
Φ
C
*
Superoxide
100
% JC-1 low Cells
Fold ↑ in DCF
ROS
*
7
120
H2O2
Fold ↑in Fluo-4
*
8
Fold ↑ in DCF
9
% MitoSOX Red+ Cells
A
Nanoparticle stimulated oxidant injury in human epithelial cells.
% MitoSOX Red+ Cells
Nanoparticle stimulated oxidant injury in murine macrophage cells.
5
10
Time (h)
15
20
20
Legend: At each Tier of the Hierarchical Oxidative Stress Model, ZnO nanoparticles exhibited significantly more effects than either CeO2 or TiO2.
THE CALIFORNIA
Legend: At each Tier of the Hierarchical Oxidative Stress Model, ZnO exhibited significantly more effects than either CeO2 or TiO2.
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
In vitro toxicity assessment based on oxidative stress paradigm- Project outline
Rat Macrophage RAW 264.7
Human lung epithelial cells TiO2
CeO2
ZnO
PS-NH2
Dimensio n
Dose
Tier 1
Tier 2
HO-1 expression
TNF-α
NQO-1 expression
IL-8 Junk
PhaseII enzymes
Cox
Duration
Tier 3 Mitochondrial membrane potential Cytoplasmic Ca content PI uptake Apoptosis
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
Tier 3 response in BEAS-2B cells subjected to nanoparticles
Confocal laser scanning microscopic images of BEAS-2B cells stained with a ‘dye-mix’ containing Hoechst, fluo4 and propidium iodide, after subjecting them to different treatment conditions.
THE CALIFORNIA
Confocal laser scanning microscopic images of BEAS-2B cells stained with a ‘dye-mix’ containing Hoechst, JC1 and propidium iodide, after subjecting them to different treatment conditions.
NANOSYSTEMS INSTITUTE
December 11 2008
High throughput screening of nanotoxicity based on oxidative stress paradigm
Saji George Dr Andre Nel’s lab Dept. of Medicine (Division of Nanomedicine) UCLA THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
Growing nanotech industry- need of alternate toxicity screening strategies
Traditional Toxicological Approach -Based on observable experimental animals
outcomes
in
-Time consuming -Labor and cost intensive
Nature- Vol 444|16 2006
Skincare and consumer products, healthcare, electronics, photonics, biotechnology, engineering products, pharmaceuticals, drug delivery, and agriculture.
US national academy of science calls for a paradigm shift in toxicity evaluation http://www.nap.edu/openbook.php?record_id=11970&page=1
I trillion $ worth business over 15-20 yrs
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
HTS for faster screening of toxicity of nanomaterials Cell growth and interaction with NPs Probes for assaying toxicity markers
Automated imaging/readin g
Advantages of HTS
Automatic image analysis
Assay Design
Data storage and management
Less labor and time consuming Data analysis
Wide range of NMs properties Large batches of NMs Comprehensive array of predictive in vitro tests
Cellular knowledge
Easy visualization and interpretation of data Cytotoxicity assessment
www.api.com/images/lifescience/well_samples.jpg
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
Major pathways of toxicity
Oxidative damage of proteins, DNA and membrane Inflammation:cytokines/chemokines Mitochondrial damage- Apoptosis Lysosomal damage Membrane damage
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
December 11 2008
Nanotoxicology Research and Training Program
December 11 2008
Mechanism of NP mediated toxicity- Hierarchical oxidative stress paradigm
High GSH/GSSG ratio
Low GSH/GSSG ratio
Tier 2
Tier 3
ss ert s e vit a di xo f o l e ve L
Tier 1 Cell response pathway
Normal
Anti-oxidant Defense
Inflammation
cytotoxicity
Signaling pathway:
Nrf-2
MAPK
Mitochondrial perturbation-PT pore
Genetic response:
ARE
AP-1
N/A
Outcome
PhaseII enzyme
Cytokines chemokines
Apoptosis
http://www.nap.edu/openbook.php?record_id=11970&page=7 Nel et al. Science 2006, 311: 622-7
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
Preliminary results
(Xia et al. ACS Nano 2008)
*
6 5
ZnO
4
*
3
CeO2
2 1
TiO2
0 0
B
5
2.5
10
15
ZnO
60 40
ROS
*
* *
20
20
1.5
CeO2
0
ZnO
0.5
5
10 15 Time (h)
D
ZnO
5
B
CeO2
*
ZnO
* *
20 10
CeO2
1 00
10
Time (h)
15
Dead cells
* *
1.5
Tier 3
*
ZnO
* CeO2
1
TiO2
0.5
20
5
10
Time (h)
15
ZnO
40
*
* *
20
TiO2
5
10
15
Time (h)
15
20
* *
25
CeO2 5
100
20
p-JNK JNK
10
Time (h)
15
Dead cells
ZnO
*
5
*
4
*
3 2
CeO2
1
TiO2
TiO2
0
20
*
[Ca2+]i
6
ZnO
50
0
5
10
Time (h)
15
20
*
75
ZnO
50 25
*
* 0
0 0
10
Time (h)
7
*
75
CeO2 TiO2
0
CeO2
5
TiO2
Lowered membrane potential
0 0
TiO2
TiO2
CeO2
0
*
80 60
CeO2
ZnO
100
% PI + Cells (M1)
5
ZnO
Φ
JNK
0
0
0
20
p54 p46 p54 p46
TiO2
0
% PI + Cells (M1)
p-JNK
2
Fold ↑ in Fluo-4
% JC-1 low Cells
50
30
15
Tier 2
[Ca2+]i
CeO2
Actin Φ
2.5
*
HO-1
TiO2
Lowered membrane potential
40
10
Tier 1
D
60
*
25
C
Tier 2 Φ
50
Time (h)
TiO2 p54 p46 p54 p46
* ZnO
0 0
20
Superoxide
75
0
Actin CeO2
TiO2
1
CeO2 TiO2
HO-1
ZnO
100
H2O2
2
80
Tier 1
Tier 3
A
*
0
Time (h)
Φ
C
*
Superoxide
100
% JC-1 low Cells
Fold ↑ in DCF
ROS
*
7
120
H2O2
Fold ↑in Fluo-4
*
8
Fold ↑ in DCF
9
% MitoSOX Red+ Cells
A
Nanoparticle stimulated oxidant injury in human epithelial cells.
% MitoSOX Red+ Cells
Nanoparticle stimulated oxidant injury in murine macrophage cells.
5
10
Time (h)
15
20
20
Legend: At each Tier of the Hierarchical Oxidative Stress Model, ZnO nanoparticles exhibited significantly more effects than either CeO2 or TiO2.
THE CALIFORNIA
Legend: At each Tier of the Hierarchical Oxidative Stress Model, ZnO exhibited significantly more effects than either CeO2 or TiO2.
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
In vitro toxicity assessment based on oxidative stress paradigm- Project outline
Rat Macrophage RAW 264.7
Human lung epithelial cells TiO2
CeO2
ZnO
PS-NH2
Dimensio n
Dose
Tier 1
Tier 2
HO-1 expression
TNF-α
NQO-1 expression
IL-8 Junk
PhaseII enzymes
Cox
Duration
Tier 3 Mitochondrial membrane potential Cytoplasmic Ca content PI uptake Apoptosis
THE CALIFORNIA
NANOSYSTEMS INSTITUTE
Nanotoxicology Research and Training Program
December 11 2008
Tier 3 response in BEAS-2B cells subjected to nanoparticles
Confocal laser scanning microscopic images of BEAS-2B cells stained with a ‘dye-mix’ containing Hoechst, fluo4 and propidium iodide, after subjecting them to different treatment conditions.
THE CALIFORNIA
Confocal laser scanning microscopic images of BEAS-2B cells stained with a ‘dye-mix’ containing Hoechst, JC1 and propidium iodide, after subjecting them to different treatment conditions.
NANOSYSTEMS INSTITUTE
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