I. Pulmonary Toxicology The Nanotoxicology Research ...

1 1 The Nanotoxicology Research Program in NIOSHV incent Castranova, PhDNIOSH Nanotoxicology Research Toxicology (8 projects) Effects(3 projects)III. Dermal Effects(1 project)IV. Predictive Algorithms(2 projects) Toxicity - IssuesA. Deposition pattern, interstitialization, clearanceB. Pulmonary reactionsC. Relevance of intratracheal instillation or aspiration to inhalation exposureD. Genotoxicity, carcinogenicityE. Relevance of exposure doses in animal models to occupational Toxicity - Projects1. Pulmonary toxicity of carbon nanotubeparticles Dr. Shvedova, PIa) Raw SWCNT (30% iron by weight) generate ROS with bronchial epithelial cells in vitro(Shvedova et al, 2004).b) Purified SWCNT (acid treated) generate low ROS and are less cytotoxic (Kagan et al, 2006).c) Aspiration of purified SWCNT in a mouse model (Shvedova et al, 2005):1) Rapid but transient inflammation and damage2) Agglomerates deposit at the terminal bronchioles and proximal alveoli; cause ) More dispersed structures cause rapid and progressive interstitial Toxicity Projects1.

2 Pulmonary toxicity of carbon nanotube particles (continued)d) Aspiration of purified SWCNT in vitamin E-deficient mice (Shvedova et al, 2007):1) More prolonged oxidant stress2) Greater acute inflammation3) Greater interstitial fibrosise) Aspiration of purified SWCNT in NADPH oxidase-deficient mice (Shvedova et al, 2008):1) Less acute inflammation and decreased fibrosisf)Aspiration of purified SWCNT in a mouse model (Shvedova et al, 2008):1) Increased susceptibility to Pulmonary Toxicity Projects1. Pulmonary toxicity of carbon nanotubeparticles (continued)g) Inhalation of SWCNT (Shvedova et al, 2008):1) Designed a system to aerosolize SWCNT (Baron et al, 2008)2) Greater particle dispersion than with aspiration3) Qualitatively similar inflammation/damage as with aspiration4) Fewer granulomas; greater interstitial Toxicity Projects2.

3 Evaluation of the Pulmonary deposition and translocation of nanomaterials Dr. Mercer, PIa) Pretreatment of purified SWCNT with acetone and sonication results in greater dispersion in aqueous mediab) Label SWCNT with colloidal goldc) Pharyngeal aspiration of dispersed vs non-dispersed SWCNT:1) Dispersed SWCNT rapidly enter the alveolar septa2) Fewer granulomas3) 4-fold greater interstitial fibrotic Toxicity Projects2. Evaluation of the Pulmonary deposition and translocation of nanomaterials(continued)d) Dispersed SWCNT not avidly phagocytized by alveolar macrophages(Mercer et al, 2008) Toxicity Projects3. In vivoinvestigation of multi-walled carbon nanotube toxicity Dr. Porter, PIa) Suspending MWCNT in a DPPC/albumin solution improves dispersion (Porter et al, 2008).b) Aspiration of purified MWCNT in mice results in acute inflammation which slowly resolves (Sriram et al, 2007; Porter et al, 2009)c) Designed a system to aerosolize MWCNT (McKinney et al, 2009)d) Inhalation of MWCNT results in qualitatively similar results as aspiration (Porter et al, 2009).

4 Toxicity Projects4. Potential aneuploidy following exposure to carbon nanotubes Dr. Sargent, PIa) Expose bronchial epithelial cells in culture to SWCNTb) SWCNT appear to interfere with mitotic spindles during division (Sargent et al, 2009).5. Pulmonary toxicity of metal oxide nanospheres and nanowires Dr. Porter, PIa) Titanium dioxide nanowires generate OH radicals in ) Intratracheal instillation of TiO2nanowires causes greater acute inflammation than TiO2nanospheres (Porter et al, 2008; Hamilton et al, 2008). Toxicity Projects6. Potential effects of silica-based nanowireson lung toxicity Drs. Leonard and Roberts, PIsa) Silicon nanowires generate ROS in vitro (Chapman et al, 2009).b) Will measure the inflammatory response in vivoc) Will determine the effects of surface modification on Toxicity Projects7. Induction of lung fibrosis by cerium oxide in diesel exhaust Dr.

5 Ma, PIa) In vitroeffects of cerium oxide on AM1) No generation of intracellular oxidants2) Induces apoptosis, increases TNF- , reduces IL-123) Decreases LPS-induced NO production (Ma et al, 2008)b) IT exposure of rats to cerium oxide1) Induces Pulmonary inflammation and damage2) Elevates AM phagocytotoxic activity(Ma et al, 2009) Toxicity Projects8. WC-Co nanoparticles in initiating angiogenesis by reactive oxygen species Dr. Ding, PIa) WC-Co nanoparticles induce ROS in lung epithelial cellsb) WC-CO nanoparticles activate Akt and ERK signaling Effects - IssuesA. Translocation to systemic sitesB. Cardiovascular effects of Pulmonary exposureC. CNS effects of Pulmonary Effects - Projects1. Role of carbon nanotubes in cardiovascular inflammation and COPD-related disease Dr Simeonova, PIa) Aspiration of purified SWCNT in miceb) Oxidant damage to mitochondrial DNA in aortic and cardiac tissue 7 days post-exposurec) Increased heme oxygenase I activity in aortic and cardiac tissue 7 days post-exposured) Multiple exposures (every 2 weeks for 2 months) to purified SWCNT in ApoE-/-mice increased aortic plaque formation(Li et al, 2007).

6 E) Elevated gene expression for inflammatory mediators in blood and aortic tissue 4 hr post-exposure (Erdely et al, 2009) Effects - Projects2. Systemic microvascular dysfunction effects of ultrafine vs fine particles Dr. Castranova, ) IT exposure of rats to particles causes:1) Adherence of PMN to systemic microvessels2) Generation of ROS at systemic microvessels3) Decreased ability of systemic microvessels to dilate(Nurkiewicz et al, 2004; 2006)b) On a mass basis, inhalation of nano-TiO2is more effective than fine TiO2(Nurkiewicz et al, 2008)c) Inhalation of nano-TiO2decreases NO levels in systemic microvessel endothelial cels (Nurkiewicz et al, 2008)d) Inhalation of nano-TiO2 decreases dilation of coronary arterioles (LeBlanc et al, 2008). Effects - Projects3. Occupational exposures and potential neurological risks Dr.

7 Sriram, PIa) Aspiration of MWCNT in mice1) Increased inflammatory cytokines 24 hr post in olfactory bulb, hippocampus and frontal cortex2) Increased E-selectin (marker of endothelial cell damage)(Sriram et al, 2007; 2009)b) Aspiration of TiO2nanowires vs nanospheres in mice1) Inflammatory brain responses with nanowires being more potent than nanospheres. (Porter et al, 2008)III. Dermal Effects - IssuesA. Penetration of nanoparticles into skinB. Effects of topical exposure4 III. Dermal Effects - Projects1. Dermal effects of nanoparticles Drs. Shvedova and Ding, PIsa) Effects of SWCNT1) In vitroexposure of keratinocytes to raw OH radicalsii. Causes oxidant stress and cytotoxicity(Shvedova et al, 2003)2) In vitroexposure to purified radical productionii. Less cytotoxicity3) Topical application of raw SWCNT, but not purified SWCNT, to mice causes increased inflammation (Murray et al, 2009)III.

8 Dermal Effects - Projects1. Dermal effects of nanoparticles (continued)b) Effects of metal nanoparticles1) In vitroexposure of dermal cells to ultrafine OH radicalsii. Activates MAPK pathwaysiii. Activates AP-1(Ding et al, 2006)2) Similar responses after ultrafine WC-Co in vitroIV. Predictive Algorithms - IssuesA. Best dose metricB. Effect of physicochemical propertiesC. Effect of agglomeration on bioactivityD. Predictive in vitroscreening tests for in vivobioactivityIV. Predictive Algorithms surface area as a dose metric Dr. Castranova, PIa)Suspension of nanoparticles in diluted alveolar lining fluid improves dispersion without masking surface activity (Sager et al, 2007)b)Improved dispersion of ultrafine carbon black increases the in vivoinflammatory potential by 8 fold (Shvedova et al, 2007)c)On an equivalent mass basis, ultrafine particles are more inflammatory in rats than fine particles of the same composition.

9 This potency difference is decreased when a particle surface area dose metric is used (Sager et al, 2008)d)In vitroROS generation of metal oxide nanoparticlescorrelates with in vivoinflammatory Predictive Algorithms -Projects2. Specific biomarkers for unusual toxicity of nanoparticles Dr. Rojanasakul, PIa) Fibroblasts exposed in vitroto purified SWCNT1) SWCNT enhance cell proliferation2) SWCNT increase collagen production(Wang et al, 2008; 2009).Exposure Assessment(particle characterization,measurement methods,and field evaluations) Nanotoxicology (exposure route,deposition and translocation, dose-response, and time course) Modeling and Risk Assessment Recommended Controls and Good Handling PracticesNIOSH Nanotechnology Program