10th US‐Korea Conference on XX Destination Nano: Creating the Next Industrial Revolution Integrated Approaches to Meet NanoEHS
Challenges Along the Life Cycle of NanoEnabled Products :
Nanoparticles from Photocopiers as an Example
D. Bello,1,2,4 M. Khatri,3 A. Pal,1,3 J. Martin, S. Pirella,2,4 P. Gaines 3 P. Demokritou2,4 1. University of Massachusetts Lowell; Department of Work Environment; 2. Harvard School of Public Health; Molecular and Integrative Physiological Science 3. Biomedical Engineering and Biotechnology Program 4. Harvard Center for Nanotechnology & Nanotoxicology Center for Nanotechnology and Nanotoxicology
at Harvard School of Public Health
1 100 nm Destination Nano: Creating the Next Industrial Revolution Disease – A Complex Process Exposure Tissue Dose Biological interaction Perturbation System Inputs Biological Function Impaired function Adaptation / Repair Andersen et al, Trends in Biotech, 2005,23,3, 122‐127 Disease Morbidity & Mortality 2 What Do We Want to Know? What Test Systems Should Be Used? Destination Nano: Creating the Next Industrial Revolution HUMAN Death
Permanent impairment
Progressive disease
Episodic incapacitating illness
Episodic illness limiting activity
ANIMAL CELLULAR De
gr
ee
of
Ad
ve
r
sit
y
Episodic illness not limiting activity
Subclinical pathophysiological changes
Physiological responses potentially contributing
to pathology
Region of
Homeostatic physiological responses
Uncertainty
Dose of pollutant or reactive product to target organs
Exposure
ACELLULAR Proportion of Population Affected
Mauderly et al., Chapt. 4 in: Technical Aspects of Multipollutant air Quality Management, Springer. 2011
[email protected] 3 Destination Nano: Creating the Next Industrial Revolution • ~60 studies in all, 25 studies in workplaces • Area Samplers • Number concentration & size distribution + STEM/EDS • Need for standardization & harmonization of measurements & approaches • Collection of systematic contextual information • Tiered Approach to understanding nature of exposures [email protected] 4 Destination Nano: Creating the Next Industrial Revolution Nanomanufacturing: Diverse, Dynamic & Challenging to Study Bekker et al, AnnHyg 2012 [email protected] 5 Destination Nano: Creating the Next Industrial Revolution An Integrated Approach to Fast Track NanoEHS CHARACTERIZE REALISTIC EXPOSURES Levels; PCM Variability CALCULATE EQUIVALENT DOSE to HUMANS Regional Deposition Model Sensitivity Analysis IN‐VITRO Toxicity Assessment Equivalent Dose Estimates Mechanistically Endpoints VALIDATE OBSERVATIONS in ANIMALS / HUMANS Destination Nano: Creating the Next Industrial Revolution We started here…. VOC METER APS OZONE METER CPC FMPS X 2012 [email protected] 7 Destination Nano: Creating the Next Industrial Revolution Nanoparticle Emissions from Commercial Photocopiers Bello et al Nanotoxicology 2012 8 Morphology & Chemistry
Destination Nano: Creating the Next Industrial Revolution Bello et al Nanotoxicology 2012 9 Destination Nano: Creating the Next Industrial Revolution Deposition Models & Dose: Linking in Vivo with In Vitro Dosimetry Mass Flux 0.072 µg/(m2min) & Exposure time of 480 min= 8hr Estimated lung surface dose of 34.6 µg/m2 Khatri et al Part Fiber Tox 2013 Nasal Cavity: 150 cm2 Deep Lungs: 140 m2 SA Lungs/Nasal Ratio = ~1.2 x 104 Deposited Fraction ~5x Nose/Alveolar Dose (cm‐2) 10 ~ 2,500x Destination Nano: Creating the Next Industrial Revolution Sample collection A) The nasal cavity is misted over with saline using a disposable plastic sprayer A) B) Nasal lavage fluid is collected by aspiration from both nostrils for 1 min each through a silicon tube 2.7 mm in diameter, and trapped in mucous trap C) Traps are connected with the small portable evacuator B) [email protected] C) 11 Destination Nano: Creating the Next Industrial Revolution Biomarkers in Nasal lavage & Urine 10 cytokines overexpressed (10‐2X): G‐CSF, IL‐8, VEGF, IL‐6, IL‐10, MCP1, Fractalkine, TNF‐α, EGF, IL‐1β IFN, MCP‐1, IL‐1α Total Protein Inflammatory cells 8‐OH‐dG in urine UP Khatri, Bello et al 2012 Nanotoxicology 12 Destination Nano: Creating the Next Industrial Revolution In vitro cytotoxicity and cytokine release 10ug/ml polymyxine B treatment for 60 min Human primary respiratory epithelial cells and THP‐1 cell line exposed PM0.1, PM0.1‐2.5 Particle collection from copy centers 30 ug/ml 100ug/ml 300ug/ml Cell Viability Inflammatory cytokines DNA damage/ROS Apoptosis Gene Expression 6h 24 h In vitro exposure 13 Destination Nano: Creating the Next Industrial Revolution Delivered Dose is Critical Printex 100
120
Administered
Depsoited
% Cell viability
100
80
60
y = -2.8027x + 97.008
y = -8.2431x + 97.008
40
20
0
0
1
2
3
4
5
6
Mass (ug)
7
SWCHN-ox
100
8
9
10
Administered
Depsoited
% Cell viability
80
60
y = -2.4552x + 79.105
40
y = -13.64x + 79.105
20
0
0
1
2
3
4
5
6
Mass (ug)
7
TiO2 P25
120
8
9
10
Administered
Depsoited
% Cell viability
100
Pal et al (manuscript in preparation) 80
60
y = -1.7322x + 89.866
y = -1.7604x + 89.866
40
20
0
0
1
2
3
4
5
6
Mass (ug)
7
8
9
10
Destination Nano: Creating the Next Industrial Revolution THP‐1 Cell Line‐ Inflammatory Cytokines IL-I
GCSF
h
h
0
24
24
6
h
IL-6
TNF-
3000
* * * 0
1000
h
6
h
24
h
0
h
15000
10000
5000
* * * * * * 0
h
* * * 20000
6
0
2000
IL-8
* * * Concentration in pg/ml
2000
Concentration in pg/ml
* * * 4000
6
Concentration in pg/ml
24
VEGF
3000
* * * 24
6
24
6
MCP-1
8000
6000
h
h
0
h
h
0
20
h
1000
40
6
2000
* * * Concentration in pg/ml
* 4000
h
2000
* * * * * 60
24
4000
Concentration in pg/ml
Concentration in pg/ml
8000
6000
IL-1
5000
h
h
6
500
h
0
0
* * * * * * 24
2000
* * * 24
5000
* * * 1000
h
10000
4000
6
15000
1500
Concentration in pg/ml
Concentration in pg/ml
20000
Concentration in pg/ml
GM-CSF
6000
15 Destination Nano: Creating the Next Industrial Revolution Delivered dose makes a difference 6 hr time point Ratio of Slopes Copier NP/CuO ~ 7x TNF‐a (pg/mL) 15000
y = 93.21x + 1010.5 R² = 0.77 Copier‐
NP
10000
CuO
y = 12.929x + 371.43 R² = 0.97 5000
0
0
50
100
150
Delivered Dose (ug/mL) 200
Destination Nano: Creating the Next Industrial Revolution 2.5
* 2.0
* * * 1.5
* * * 30
100
300
1.0
0.5
o
Cu
on
rb
Ca
l le
ct
ed
NP
s
NP
0.0
Co
Annexin V + cells
(Fold change over control)
Apoptosis Results 17 Destination Nano: Creating the Next Industrial Revolution DNA damage‐ Comet assay 18 Destination Nano: Creating the Next Industrial Revolution Gene Expression (RT‐qPCR), 5ug/mL April Gu’s group Destination Nano: Creating the Next Industrial Revolution PM0.1‐2.5, Cytokines in THP‐1 Khatri et al Inh Tox 2013 Destination Nano: Creating the Next Industrial Revolution PM0.1‐2.5, Apoptosis in THP‐1 2.5
2.0
1.5
1.0
#
0.5
0.0
PM0.1 (mg/kg bw) Pirella et al Inh Tox 2013 PM R
PM
BALF Neutrophils (millions)
<
PM 0 I/
. 1
< 1 ( 0%
0 0
PM PM .1 .2m FB
S
< (0. FBS g
RPMI/10%
0
PM .1 0. 6m /k -2 1 g g) 0. .5 (2 /k PM 1-2 (0 mg g) .
0. .5 ( 2m /kg Fe 1-2 0.6 g/k ) 2O .5 mg g) Fe 3 (2 /k 2O (0 mg g) .
Fe 3 ( 2m /kg 2O 0.6 g/k ) W 3 ( mg g) F 2m /k W (0. g g) F 2m /k (0 g g) W .6m /k F g g) (2 /k m g
g/ ) kg ) Destination Nano: Creating the Next Industrial Revolution In vivo instillation study in mice done at Harvard, Prof. Demokritou’s group Lavaged neutrophils 24 hours post-particle exposure
*
PM0.1-2.5 (mg/kg bw) Fe2O3 (mg/kg bw) Welding fumes (mg/kg bw) Summary of cytokine production
Destination Nano: Creating the Next Industrial Revolution Khatri et al 2013, Inh Tox Test System
In Vitro (Fine particle dosing)
THP-1
Nasal
Epithelial
Cells
Small Airway
Epithelial
Cells






-

-
Cytokine
IL-8
IL-6
IL-1
GM-CSF
TNF-
MCP-1
VEGF
IL-1
G-CSF
Fractalkine
EGF
IFN-
In Vitro (Nanoparticle dosing)
Khatri et al 2013
THP-1
Nasal
Epithelial
Cells
Small Airway
Epithelial
Cells





-








-












-
-
In Vivo
Nasal lavage of
Healthy Volunteers
(Khatri et al 2012)









-
Destination Nano: Creating the Next Industrial Revolution Chronic Exposures, NL 5 exposed in 3 centers; 2‐3 weeks & 8 controls Same picture for: 5
Ln(IL‐6) Total Protein 4
IL‐8 TNF‐α MCP‐1 8‐OH‐dG 3
lnIl_6
Acute Exposure, NL 6 2
 G‐CSF Under‐expressed 1
0
W1 C W2 W3 -1
Control
Exposed week 1
Exposed week 2
week_type
Exposed week 3
No change: VEGF, IL‐10, Fractalkine, EGF, IL‐1β 24 Destination Nano 2013: Creating the Next Industrial Revolution Destination Nano: Creating the Next Industrial Revolution Conclusions • Approach is generalizable • Suitable for a large number of life cycle scenarios of nano‐enabled products • It provides important context for risk and hazard assessment to Nanotech companies • Important methodological insights for in vitro testing and comparative assessment • Currently used to validate FRAS BOD as a screening assay Destination Nano: Creating the Next Industrial Revolution Questions? X 2012 [email protected] 26 Destination Nano: Creating the Next Industrial Revolution Laser Printer Emissions & Human Health “International foundation nano Control” database in Germany: • 2,500 persons who claim to be sick from laser printer emissions • 90% report respiratory tract complaints, glossalgia, chronic cough, rhinitis, and inflammation of the throat, tongue and paranasal sinuses • ~ 30% developed asthma • Inflammation of eyes, skin, diffuse pain and loss of hair [email protected] 27 Destination Nano: Creating the Next Industrial Revolution The hierarchical oxidative stress model Nel et al. Science 311, 622 (2006)