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

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

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

• Availability: Predominantly Rutile and Anatase crystal forms

http://www.nanotechproject.org/inventories/consumer/analysis_dra

ft/

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50

100

150

200

250

300

350

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2011

• Good fatigue strength

• Corrosion resistance

• Machinability

• Whitening

• Thermal stability

• Good electrical

properties

• Photocatalysis

Anatase

Rutile

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

Respiratory Dermal

(1-10% of the

product content)

TiO2 NPs Exposure to Biological Systems

Oral(0.01 to 2 μg TiO2/mg

food)

Intravenous/

Subcutanous

Introduction Exp. Design Results Conclusion Future Studies

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).

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 Particle (size nm) Exposure Effect

Tang et al.,

2011Anatase TiO2 (5±1) 0.8-20 mg/kg TiO2

Histology: lung gaps expanded,

hyperemia, alveolar thickness.

Scuri et

al., 2010

P25 Degussa

TiO2 (21)

12 mg/m3 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.

Li et al.,

2010Anatase TiO2 (3)

3.3 mg/kg TiO2 once a wk for 4

wks.

Inflammatory action: ACP, ALP

increased in BAL.

Histology: destroyed alveolar walls.

Liu et al.,

2010

TiO2 (5)

TiO2 (200)0.5–50 mg/kg TiO2

AM phagocytic and chemotactic

ability: reduced by TiO2 NPs.

Introduction Exp. Design Results Conclusion Future Studies

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).

Liver and TiO2 NPs

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

Introduction Exp. Design Results Conclusion Future Studies

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

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

Isolation of primary hepatocytes by two step collagenase digestion

Seeding cells

Addition of TiO2 to the cells

Hepatocytes exposed to 100 ppm TiO2 everyday

Stirring

Introduction Exp. Design Results Conclusion Future Studies

1. Rutile 50nm

2. Anatase 50nm

3. Degussa P25

• 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

Experiments

Introduction Exp. Design Results Conclusion Future Studies

TiO2 NPs characterization- Dynamic Light Scattering

Introduction Exp. Design Results Conclusion Future Studies

Particle Type 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

Scale = 200 microns

Cell Morphology

Introduction Exp. Design Results Conclusion Future Studies

Day 1

Day 4

Untreated RutileAnataseP25

Scale = 200 microns Day 4 of culture with 100 ppm nanoparticle treatment

Cell Viability

Introduction Exp. Design Results Conclusion Future Studies

Control

RutileAnataseP25

Live cells: Calcein AM (Green) , Dead cells: Ethidium Bromide (Red)

Day 4 of culture with 100 ppm nanoparticle treatment

Cell Viability Study: MTT Cytotoxicity Assay

Introduction Exp. Design Results Conclusion Future Studies

0

0.2

0.4

0.6

0.8

1

1.2

Untreated P25 Anatase Rutile

Re

lati

ve O

pti

cal D

en

sity

at

57

0 n

m

** *

Day 4 of culture with 100 ppm nanoparticle treatment

Liver-Specific Functions: Urea Synthesis

Introduction Exp. Design Results Conclusion Future Studies

0

20

40

60

80

100

120

140

160

180

Ctrl P25 Anatase Rutile

Ure

a Sy

nth

esi

s (

μg/

ml/

mill

ion

ce

lls)

*

*

*

Day 4 of culture with 100 ppm nanoparticle treatment

Liver-Specific Functions: Albumin Synthesis

Introduction Exp. Design Results Conclusion Future Studies

0

0.5

1

1.5

2

2.5

3

3.5

Untreated P25 Anatase Rutile

Alb

um

in s

ecr

ete

d (

μg/

ml/

mill

ion

ce

lls)

** *

Day 4 of culture with 100 ppm nanoparticle treatment

Gene expression Study: Oxidative Stress

Introduction Exp. Design Results Conclusion Future Studies

0

0.5

1

1.5

2

2.5

Untreated P25 Rutile Anatase

Re

lati

ve L

DH

ge

ne

exp

ress

ion

*

**

• 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 Future Studies

• 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

Introduction Exp. Design Results Conclusion Future Studies

ACKNOWLEDGEMENT

Dr. Srivatsan Kidambi

Dr. Edward Harris, Department of Biochemistry, UNL

Focμs Lab, Department of Chemical and BiomolecularEngineering

University of Nebraska-Lincoln