D. Bello, 1,2,4 M. Khatri, 3 A. Pal, 1,3 J. Martin, S. Pirella, 2,4 - PowerPoint PPT Presentation

10 th US ‐ Korea Conference on XX Destination Nano: Creating the Next Industrial Revolution Integrated Approaches to Meet NanoEHS Challenges Along the Life Cycle of Nano- Enabled 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. Demokritou 2,4 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 1 100 nm at Harvard School of Public Health

Destination Nano: Creating the Next Industrial Revolution Disease – A Complex Process Exposure Tissue Dose Biological interaction Perturbation System Biological Inputs Function Impaired function Disease Morbidity & Mortality Adaptation / Repair 2 Andersen et al, Trends in Biotech, 2005,23,3, 122 ‐ 127

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 Episodic illness not limiting activity Degree of Adversity Subclinical pathophysiological changes Physiological responses potentially contributing to pathology Region of Uncertainty CELLULAR Homeostatic physiological responses Dose of pollutant or reactive product to target organs Exposure Proportion of Population Affected ACELLULAR Mauderly et al., Chapt. 4 in: Technical Aspects of Multipollutant air Quality Management , Springer. 2011 DHIMITER_BELLO@UML.EDU 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 dhimiter_bello@uml.edu 4

Destination Nano: Creating the Next Industrial Revolution Nanomanufacturing: Diverse, Dynamic & Challenging to Study Bekker et al, AnnHyg 2012 dhimiter_bello@uml.edu 5

Destination Nano: Creating the Next Industrial Revolution An Integrated Approach to Fast Track NanoEHS CHARACTERIZE REALISTIC EXPOSURES CHARACTERIZE REALISTIC EXPOSURES Levels; PCM Variability CALCULATE EQUIVALENT DOSE to HUMANS CALCULATE EQUIVALENT DOSE to HUMANS Regional Deposition Model Sensitivity Analysis IN ‐ VITRO Toxicity Assessment IN ‐ VITRO Toxicity Assessment Equivalent Dose Estimates Mechanistically Endpoints VALIDATE OBSERVATIONS in ANIMALS / VALIDATE OBSERVATIONS in ANIMALS / HUMANS HUMANS

Destination Nano: Creating the Next Industrial Revolution We started here…. VOC METER APS OZONE METER CPC FMPS X 2012 DHIMITER_BELLO@UML.EDU 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/(m 2 min) & Exposure time of 480 min= 8hr Estimated lung surface dose of 34.6 µg/m 2 Nasal Cavity : 150 cm 2 Deep Lungs: 140 m 2 SA Lungs/Nasal Ratio = ~1.2 x 10 4 Deposited Fraction ~5x Nose/Alveolar Dose (cm ‐ 2 ) ~ 2,500x 10 Khatri et al Part Fiber Tox 2013

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 C) Traps are connected with the small 2.7 mm in diameter, and trapped in portable evacuator mucous trap B) C) DHIMITER_BELLO@UML.EDU 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 ‐ 1 β I FN  , MCP ‐ 1, IL ‐ 1 α  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 Particle collection epithelial cells from copy centers and THP ‐ 1 cell line exposed PM 0.1 , PM 0.1 ‐ 2.5 30 ug/ml 100ug/ml 300ug/ml Cell Viability 6h Inflammatory cytokines DNA damage/ROS In vitro exposure 24 h Apoptosis Gene Expression 13

Destination Nano: Creating the Next Industrial Revolution Delivered Dose is Critical Printex 100 Administered 120 Depsoited 100 % Cell viability 80 60 y = -2.8027x + 97.008 y = -8.2431x + 97.008 40 20 0 0 1 2 3 4 5 6 7 8 9 10 Mass (ug) SWCHN-ox Administered 100 Depsoited 80 % Cell viability 60 y = -2.4552x + 79.105 40 y = -13.64x + 79.105 20 0 0 1 2 3 4 5 6 7 8 9 10 Mass (ug) TiO 2 P25 Administered 120 Depsoited 100 % Cell viability 80 y = -1.7322x + 89.866 60 y = -1.7604x + 89.866 40 Pal et al (manuscript in preparation) 20 0 0 1 2 3 4 5 6 7 8 9 10 Mass (ug)

Destination Nano: Creating the Next Industrial Revolution THP ‐ 1 Cell Line ‐ Inflammatory Cytokines IL-I  GM-CSF GCSF 6000 1500 20000 Concentration in pg/ml Concentration in pg/ml Concentration in pg/ml 15000 * 4000 1000 * * 10000 * * * * * 2000 * * 500 5000 * * 0 0 0 h h h h h h 6 4 6 4 2 6 4 2 2 IL-6 IL-1  TNF-  5000 60 Concentration in pg/ml 8000 Concentration in pg/ml Concentration in pg/ml 4000 6000 * * * 3000 40 * * 4000 * 2000 * * * * 20 * * 2000 1000 0 0 0 h h h h h h 6 4 6 4 2 2 6 4 2 MCP-1 VEGF IL-8 8000 * * 3000 20000 Concentration in pg/ml * Concentration in pg/ml * * Concentration in pg/ml * * * * * * 6000 * 15000 * * * 2000 4000 * 10000 * * 1000 2000 5000 0 0 h h 0 h h 6 4 6 4 2 2 h h 6 4 15 2

Destination Nano: Creating the Next Industrial Revolution Delivered dose makes a difference 6 hr time point Ratio of Slopes Copier NP/CuO ~ 7x 15000 y = 93.21x + 1010.5 Copier ‐ R² = 0.77 NP TNF ‐ a (pg/mL) 10000 CuO y = 12.929x + 371.43 5000 R² = 0.97 0 0 50 100 150 200 Delivered Dose (ug/mL)

Destination Nano: Creating the Next Industrial Revolution Apoptosis Results 2.5 (Fold change over control) * 30 * * * 100 2.0 * Annexin V + cells * 300 * 1.5 1.0 0.5 0.0 P s o P u N N C d e n t o c b e r l a l o C C 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 PM 0.1 ‐ 2.5 , Cytokines in THP ‐ 1 Khatri et al Inh Tox 2013

Destination Nano: Creating the Next Industrial Revolution PM 0.1 ‐ 2.5 , Apoptosis in THP ‐ 1

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 2.5 BALF Neutrophils (millions) * 2.0 1.5 1.0 # 0.5 0.0 S ) ) ) ) ) ) ) ) ) ) ) ) g g g g g g g g g g g g B k k k k k k k k k k k k F RPMI/10% FBS / / / / / / / / / / / / g g g g g g g g g g g g % m m m PM 0.1 m m m PM 0.1-2.5 Fe 2 O 3 m m Welding m m m m 0 2 6 2 2 6 2 2 6 2 2 6 2 (mg/kg bw) (mg/kg bw) (mg/kg bw) fumes 1 . . ( . . ( . . . . ( ( 0 0 0 0 0 0 0 0 / 1 5 I 3 F (mg/kg bw) ( ( ( ( M ( ( ( ( . . O W 1 1 0 5 5 2 3 3 F F P . . . . - 2 O O W W 0 0 < 2 2 1 R e - - . 2 2 < < M 1 1 0 F e e . . M M P 0 0 M F F P P M M P P P Pirella et al Inh Tox 2013

Summary of cytokine production Destination Nano: Creating the Next Industrial Revolution Khatri et al 2013, Inh Tox Test System In Vitro ( Nanoparticle dosing) In Vivo In Vitro (Fine particle dosing) Khatri et al 2013 Nasal Small Airway Nasal Small Airway Nasal lavage of THP-1 Epithelial Epithelial THP-1 Epithelial Epithelial Healthy Volunteers Cells Cells Cells Cells (Khatri et al 2012) Cytokine              IL-8    - - - - IL-6     IL-1  - - -   - - - - - GM-CSF     TNF-  - -   - - - - - MCP-1     - - - VEGF   IL-1  - - - - -  - - - - - - G-CSF   - - - - - Fractalkine    - - - - EGF  IFN-  - - - - -