Sustainable Nanotechnology Organization (SNO) 2013 Conference

Investigating the Toxicological Effect of Titanium Dioxide Nanoparticles on Liver Vaishaali Natarajan, Christina Davis and Srivatsan Kidambi, Ph.D Department of Chemical and Biomolecular Engineering University of Nebraska-Lincoln www.focuslab.unl.edu

OUTLINE 1. Introduction 2. Experimental Design

3. Results/Discussion 4. Conclusions 5. Future Studies

Introduction

Exp. Design

Results

Conclusion

Future Studies

Titanium Dioxide Nanoparticles (TiO2 NPs) • One of the highest manufactured nanoparticles according to National Nanotechnology Initiative of America (Liang, G. et al, 2009)

• Nanoparticle form: Excellent physicochemical properties • Versatile applications: Paints, cosmetics, water/air purification, pharmaceuticals and food products Anatase

• Availability: Predominantly Rutile and Anatase crystal forms •

Good fatigue strength

•

Corrosion resistance

•

Machinability

•

Whitening

50

•

Thermal stability

0

•

Good electrical

Number of Products

350 300 250 200 150

2006

100

2011

Rutile

properties • http://www.nanotechproject.org/inventories/consumer/analysis_dra ft/

Photocatalysis

Liang, G. et al. Influence of Different Sizes of Titanium Dioxide Nanoparticles on Hepatic and Renal Functions in Rats with Correlation to Oxidative Stress. J. Toxicol. Environ. Health, Part A 72, 740-745, (2009) Hussain, S.B et al. Crystal structure mediates mode of cell death in TiO2 nanotoxicity, Journal of Nanoparticle Research 11, 1361-1374 (2009)

Introduction

Exp. Design

Results

Conclusion

Future Studies

TiO2 NPs Exposure to Biological Systems Respiratory

Dermal (1-10% of the product content)

Oral (0.01 to 2 μg TiO2/mg food)

Intravenous/ Subcutanous

Shi, H., Magaye, R., Castranova, V. & Zhao, J. Titanium dioxide nanoparticles: a review of current toxicological data. Part. Fibre Toxicol. 10-15, (2013) http://www.nanotechproject.org/inventories/consumer/browse/products

Introduction

Exp. Design

Results

Conclusion

Future Studies

TiO2 NPs Toxicological Profile • Toxicological properties dependent on physicochemical properties; varies drastically from the bulk form • International Agency for Research on Cancer: “possibly carcinogenic to humans” • Respiratory system extensively studied; High exposure rate causes serious health concerns in animal models Reference Tang et al., 2011

Scuri et al., 2010

Li et al., 2010 Liu et al., 2010

Particle (size nm) Anatase TiO2 (5±1)

P25 Degussa TiO2 (21)

Anatase TiO2 (3)

TiO2 (5) TiO2 (200)

Exposure 0.8-20 mg/kg TiO2

Effect Histology: lung gaps expanded, hyperemia, alveolar thickness.

TiO2 for 5.6 hr a day, for 3 consecutive days.

Neurotrophin expression: NGF, BDNF and their receptors increased in rats. Airway resistance: increased in mice.

3.3 mg/kg TiO2 once a wk for 4 wks.

Inflammatory action: ACP, ALP increased in BAL. Histology: destroyed alveolar walls.

0.5–50 mg/kg TiO2

AM phagocytic and chemotactic ability: reduced by TiO2 NPs.

12 mg/m3

Iavicoli, I. et al Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies. Eur Rev Med Pharmacol Sci 15, 481-508 (2011).

Introduction

Exp. Design

Results

Conclusion

Future Studies

Liver and TiO2 NPs Bio-distribution studies on TiO2 NPs show retention in the liver

Meena, R. & Paulraj, R. Oxidative stress mediated cytotoxicity of TiO2 nano anatase in liver and kidney of Wistar rat. Toxicol. Environ. Chem. 94, 2012 Yamashita, K. et al. Silica and titanium dioxide nanoparticles cause pregnancy complications in mice. Nat. Nanotechnol. 6, 321-328, (2011) Fabian, E. et al. Tissue distribution and toxicity of intravenously administered titanium dioxide nanoparticles in rats. Archives of toxicology 82, 151-157 (2008).

Introduction

Exp. Design

Results

Conclusion

Future Studies

Liver and TiO2 NPs • Liver functions: Center for xenobiotic detoxification and clearance • Most functions performed by hepatocytes • Not many studies carried out to establish the toxicity of TiO2 NPs on liver cells

OBJECTIVE: To investigate the effect of different forms of Titanium Dioxide Nanoparticles on Primary Rat Hepatocyte viability, morphology and liver-specific functions

Meena, R. & Paulraj, R. Oxidative stress mediated cytotoxicity of TiO2 nano anatase in liver and kidney of Wistar rat. Toxicol. Environ. Chem. 94, 2012 http://www.akaike-lab.bio.titech.ac.jp/akaike/english/resarch/

Introduction

Exp. Design

Results

Conclusion

Future Studies

1. Rutile 50nm 2. Anatase 50nm 3. Degussa P25 Isolation of primary hepatocytes by two step collagenase digestion

Addition of TiO2 to the cells

Stirring

Seeding cells

Hepatocytes exposed to 100 ppm TiO2 everyday

Introduction

Exp. Design

Results

Experiments • Morphology study: Phase contrast • Viability study: Live/Dead fluorescent imaging MTT assay • Cell-specific functions study: Urea secretion Albumin secretion • Gene expression study: RT-PCR

Conclusion

Future Studies

Introduction

Exp. Design

Results

Conclusion

Future Studies

TiO2 NPs characterization- Dynamic Light Scattering

Particle Diameter

Effective Diameter in Hepatocyte Medium (nm)

Zeta Potential (mV)

Degussa P25

21nm

2178.2 ± 41

-6.76

Anatase

50nm

580.9 ± 6.3

-3.17

Rutile

50nm

459.5 ± 4.8

-8.13

Particle Type

Introduction

Exp. Design

Results

Conclusion

Future Studies

Cell Morphology Day 1

Untreated Day 4

Scale = 200 microns

P25

Anatase

Rutile

Introduction

Exp. Design

Results

Conclusion

Future Studies

Cell Viability

Control

P25

Anatase

Rutile

Live cells: Calcein AM (Green) , Dead cells: Ethidium Bromide (Red) Scale = 200 microns

Day 4 of culture with 100 ppm nanoparticle treatment

Introduction

Exp. Design

Results

Conclusion

Future Studies

Cell Viability Study: MTT Cytotoxicity Assay

Relative Optical Density at 570 nm

1.2 1 * 0.8

*

*

Anatase

Rutile

0.6 0.4 0.2 0 Untreated

P25

Day 4 of culture with 100 ppm nanoparticle treatment

Introduction

Exp. Design

Results

Conclusion

Future Studies

Liver-Specific Functions: Urea Synthesis

Urea Synthesis (μg/ml/million cells)

180 160 140 *

120 *

100 *

80 60 40 20

0 Ctrl

P25

Anatase

Rutile

Day 4 of culture with 100 ppm nanoparticle treatment

Introduction

Exp. Design

Results

Conclusion

Future Studies

Liver-Specific Functions: Albumin Synthesis Albumin secreted (μg/ml/million cells)

3.5 3 2.5

*

*

*

P25

Anatase

Rutile

2 1.5

1 0.5 0 Untreated

Day 4 of culture with 100 ppm nanoparticle treatment

Introduction

Exp. Design

Results

Conclusion

Gene expression Study: Oxidative Stress

Relative LDH gene expression

2.5

*

*

Rutile

Anatase

2 1.5

*

1 0.5 0 Untreated

P25

Day 4 of culture with 100 ppm nanoparticle treatment

Future Studies

Introduction

Exp. Design

Results

Conclusion

Future Studies

• The nanoparticles compromise the normal viability and morphology of primary rat hepatocytes at a concentration of 100 ppm • Hepatocytes specific functions are compromised when treated with nanoparticles • Oxidative stress is induced in the cells by the nanoparticles

Introduction

Exp. Design

Results

Conclusion

• Investigating the mechanistic aspects of the toxicological effects of TiO2 on primary hepatocytes – Oxidative Stress • Investigating the effect of the nanoparticles on in vitro liver model

Future Studies

ACKNOWLEDGEMENT Dr. Srivatsan Kidambi Dr. Edward Harris, Department of Biochemistry, UNL Focμs Lab, Department of Chemical and Biomolecular Engineering University of Nebraska-Lincoln