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