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Titanium dioxide nanoparticles (TiO2 NPs) have found many practical applications in industry and daily life. A widespread application of TiO2 NPs rises the question about safety of their use in the context of potential occupational, environmental and intentional exposure of humans and biota. TiO2 NPs easily enter the body through inhalation, cross blood-brain barrier and accumulate in the brain, especially in the cortex and hippocampus. Toxicity of these NPs and the molecular mechanisms of their action have been studied extensively in recent years. Studies showed that TiO2 NPs exposure resulted in microglia activation, reactive oxygen species production, activation of signaling pathways involved in inflammation and cell death, both in vitro and in vivo. Consequently, such action led to neuroinflammation, further brain injury. A, spatial recognition memory and locomotor activity impairment has been also observed. Copyright © 2015. Published by Elsevier Ltd.
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Review
Toxicity of titanium dioxide nanoparticles in central nervous system
Magdalena Czajka
a,
, Krzysztof Sawicki
a
, Katarzyna Sikorska
b
, Sylwia Popek
a
, Marcin Kruszewski
a,b,c
,
Lucyna Kapka-Skrzypczak
a,c
a
Department of Molecular Biology and Translational Research, Institute of Rural Health, Lublin, Poland
b
Institute of Nuclear Chemistry and Technology, Centre for Radiobiology and Biological Dosimetry, Warsaw, Poland
c
Department of Medical Biology and Translational Research, Faculty of Medicine, University of Information Technology and Management, Rzeszów, Poland
article info
Article history:
Received 20 October 2014
Accepted 2 April 2015
Available online 18 April 2015
Keywords:
Neuroinflammation
Brain
Toxicity
Reactive oxygen species
Apoptosis
abstract
Titanium dioxide nanoparticles (TiO
2
NPs) have found many practical applications in industry and daily
life. A widespread application of TiO
2
NPs rises the question about safety of their use in the context of
potential occupational, environmental and intentional exposure of humans and biota. TiO
2
NPs easily
enter the body through inhalation, cross blood–brain barrier and accumulate in the brain, especially in
the cortex and hippocampus. Toxicity of these NPs and the molecular mechanisms of their action have
been studied extensively in recent years. Studies showed that TiO
2
NPs exposure resulted in microglia
activation, reactive oxygen species production, activation of signaling pathways involved in inflammation
and cell death, both in vitro and in vivo. Consequently, such action led to neuroinflammation, further brain
injury. A spatial recognition memory and locomotor activity impairment has been also observed.
Ó2015 Elsevier Ltd. All rights reserved.
Contents
1. Introduction ........................................................................................................ 1043
2. Exposure routes and biodistribution of TiO
2
NPs........................................................................... 1043
3. Toxicity in vitro: Inhibition of proliferation and cell death . . . . . . . . . . . . . . . . . .................................................. 1044
4. In vivo toxicity in nonmammalian vertebrates. . . . . . . . ..................................................................... 1044
5. In vivo toxicity in mammals . . . ........................................................................................ 1045
6. Inflammatory response . . . . . . . ........................................................................................ 1046
7. Other responses . . . . . . . . . . . . . ........................................................................................ 1046
8. The role of oxidative stress – induced toxicity and DNA damage. . . . . . . . . . . . .................................................. 1049
9. Offspring effect. . . . . . . . . . . . . . ........................................................................................ 1049
10. Implications of TiO
2
NPs physicochemical properties in their toxicity. . . . . . . .................................................. 1050
11. Summary . ........................................................................................................ 1051
Conflict of Interest . . . . . . . . . . . . ........................................................................................ 1051
Transparency Document . . . . . . . . ........................................................................................ 1051
Acknowledgement . . . . . . . . . . . ........................................................................................ 1051
References . ........................................................................................................ 1051
http://dx.doi.org/10.1016/j.tiv.2015.04.004
0887-2333/Ó2015 Elsevier Ltd. All rights reserved.
Abbreviations: 5-HT, 5-hydroxytryptamine; AChE, acetylcholinesterase (EC 3.1.1.7); APx, L-ascorbate peroxidase (EC 1.11.1.11); ATPase, adenosinetriphosphatase (EC
3.6.1.3); CAT, catalase (EC 1.11.1.6); DA, dopamine; Glu, glutamate; GSH, glutathione; GSH-Px, glutathione peroxidase (EC 1.11.1.9); GST, glutathione transferase (EC
2.5.1.18); HDG, hippocampal denate gyrus; IL-1b, interleukin 1b; LPS, lipopolysaccharide; LTP, long-term synaptic plasticity; NE, norepinephrine; NOS, nitric-oxide synthase
(NADPH dependent) (EC 1.14.13.39); NF-
j
B, nuclear factor kappa B; NPs, nanoparticles; Nrf-2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SOD,
superoxide dismutase (EC 1.15.1.1); SRXRF, synchrotron radiation X-ray fluorescence; TNF-
a
, tumor necrosis factor alpha.
Corresponding author at: Department of Molecular Biology and Translational Research, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland. Tel.: +48 81
7184539.
E-mail address: czajka.magdalenaa@gmail.com (M. Czajka).
Toxicology in Vitro 29 (2015) 1042–1052
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
1. Introduction
Fast development of nanoscience caused an increase in produc-
tion and use of nanomaterials (NMs), including nanoparticles
(NPs), nanomaterials that have all dimensions less than 100 nm.
Physical, chemical and biological properties of NPs are different
from those of individual atoms and molecules or bulky material,
thus development of nanotechnology is foreseen to bring a signif-
icant benefit to the society. NPs are applied in many sector mar-
kets, such as health, defence, energy, agriculture or environment
protection. With an increase of application of nanotechnology
products, there is increasing need to evaluate risk associated with
their use, due to the potential occupational and environmental
exposure of humans and biota to NPs and their intentional use in
diagnosis or therapy.
Titanium dioxide nanoparticles (TiO
2
NPs) are widely used in
many applications, e.g. paints, lacquers, laminates, food wraps,
foils, transparent plastics, papers, textiles, cosmetics, food prod-
ucts, medicines, pharmaceuticals and toothpaste. Due to their cat-
alytic properties they are also used in self-cleaning tiles, windows,
anti-fogging car mirrors, in the purification of water, sewage, com-
bustion gases, as a antimicrobial material in decontamination, as
catalyst in organic synthesis and as a photoactive material in solar
cells (Shi et al., 2013).
Owing to the common use of TiO
2
NPs in everyday life, it is very
important to gain the knowledge about their toxicity and their
potential harmful influence on human health and environment.
There are numerous experimental evidence, which suggest that
TiO
2
NPs exposure could be harmful and cause negative health
effects. Many in vitro studies showed that TiO
2
NPs was cyto-
and genotoxic, led to apoptosis, inflammation, induced ROS, chan-
ged enzyme activities and gene expression. In concordance with
in vitro experiments, many in vivo studies also showed that TiO
2
NPs, once entered the blood stream, could reach and accumulate
in important organs and cause their injury. TiO
2
NPs induced dam-
age in such organs as spleen, liver, kidney, lung, brain or heart
(Chang et al., 2013; Shi et al., 2013). However, there are also stud-
ies showing lack of TiO
2
NPs toxicity, thus further study is neces-
sary to univocally determine the possible impact of TiO
2
NPs on
human health.
In this review we attempt to summarize the knowledge on the
toxicity of TiO
2
NPs on central nervous system, both in vivo and
in vitro experimental design.
2. Exposure routes and biodistribution of TiO
2
NPs
TiO
2
NPs enter the body by different exposure routes and then
are distributed to the important organs throughout the body.
Gastrointestinal absorption seems a prevalent exposure route, as
TiO
2
NPs are used as an additive in toothpaste, cachou, capsule,
drug carriers, etc. Wang et al. (2007a) showed that 2 weeks after
oral administration to TiO
2
NPs titanium content was significantly
elevated in the brain, as compared to controls. Nonetheless tita-
nium mainly accumulated in the liver, kidneys, spleen and lung
of mice. However, more recent research showed that TiO
2
NPs
were not significantly accumulated after oral administration, likely
due to the minimal absorption from the gut (Cho et al., 2013).
As TiO
2
NPs are commonly used in cosmetics another important
gate of entry seems to be dermal absorption. In a study by Kertész
et al. (2005) human skin xenografts taken from different patients
were exposed to Anthelios XL F60, a cosmetic containing micro-
nised TiO
2
NPs. The result showed that TiO
2
NPs penetrated to
the disjunctum part of the stratum corneum. In two cases TiO
2
NPs penetrated also to the stratum granulosum.Gamer et al.
(2006) have investigated in vitro penetration of TiO
2
NPs in
cosmetic formulations through porcine skin. TiO
2
NPs were
detected on the outermost surface of the stratum corneum, but
did not penetrate through porcine skin. Penetration of four types
of rutile TiO
2
NPs into intact and damaged Yucatan micropig skin
in vitro was also studied by Senzui et al. (2010). TiO
2
NPs did not
penetrate through skin irrespective of the NPs size and coating,
even if the stratum corneum was damaged. It should be noted that
after hair removal some NPs penetrated into vacant hair follicles,
although they were not detected in dermis or viable epidermis.
In yet another study, a homemade static diffusion cell was used
for in vitro evaluation of the cutaneous penetration of TiO
2
NPs
through the porcine skin. It was shown that the titanium was
deposited mainly at the surface and stratum corneum, but no tita-
nium was detected in permeate fluid. The result was the same,
even if skin was damaged, irradiated or damaged and irradiated
(Miquel-Jeanjean et al., 2012). The study aimed on determination
of the influence of subchronic exposure of TiO
2
NPs on the hairless
rat skin showed that nanoparticles were not located in the viable
skin areas. TiO
2
NPs were observed only in the stratum disjunctum
and keratinized layer of the follicular infundibulum. Moreover, no
penetration to major organs was observed, despite the significant
increase in titanium content in lung after 8 weeks exposure. That
was, however, rather due to the inhalation of TiO
2
NPs than to
the direct absorption from the skin (Adachi et al., 2013). On the
contrary, Wu et al. (2009) demonstrated that after exposure of por-
cine ear skin to formulation containing 5% TiO
2
NPs for 30 consec-
utive days, TiO
2
NPs were observed in the stratum corneum,stratum
granulosum, prickle and basal cell layer. The effect was size depen-
dent and the penetration capacity increased with the decreased
TiO
2
NPs size. Furthermore, the authors showed that TiO
2
NPs pen-
etrated through the skin and entered into various organs and
caused pathological changes, mainly in the skin and liver.
However, the presence of titanium in the brain was almost
negligible.
Inhalation is an another portal of TiO
2
NPs entry into the body.
Inhaled nanoparticles are deposited in three different regions of
the respiratory tract: nasopharyngeal, tracheobronchial and alveo-
lar region, and can be translocated to the central nervous system,
mainly through sensory nerves (Simkó and Mattsson, 2010).
Wang et al. (2007b) investigated distribution of TiO
2
NPs in the
olfactory bulb of mice after nasal inhalation. After one month
exposure the microbeam SRXRF mapping analysis showed that
TiO
2
NPs were taken up by the olfactory bulb via the primary olfac-
tory neurons and accumulated in the olfactory nerve layer, olfac-
tory ventricle and granular cell layer. The concentration and
distribution area of titanium in the olfactory bulb increased with
increasing particles size. In the consecutive studies, Wang et al.
(2008a,b) showed that intranasally installed TiO
2
NPs translocated
to the central nervous system and accumulated mainly in the
hippocampus.
Geiser et al. (2005) analyzed the intrapulmonary distribution of
inhaled TiO
2
NPs in rats. They found that nanoparticles were local-
ized in the lung tissue compartment (air-filled spaces, epithelium/
endothelium, connective tissue and capillary lumen), especially in
the airspace. Further investigations detected that distribution of
TiO
2
NPs within the four compartments of rat lung was not random
and depended on time after exposure. Nanoparticles were trans-
ported from the airspaces to the connective tissue and subse-
quently released into the systemic circulation. However, the
majority of TiO
2
NPs were still observed within the airspace
(Mühlfeld et al., 2007). In concordance, Li et al. (2010) indicated
that after intratracheally instillation of TiO
2
NPs once per-week
for 4 consecutive weeks, NPs might translocated to the blood circu-
lation and then to extrapulmonary tissues, and they were able to
pass through the blood–brain barrier and induced to brain damage.
In the latest study by Baisch et al. (2014), rats were exposed to TiO
2
M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052 1043
NPs by intratracheal instillation or whole body aerosol inhalation.
The authors did not detect the TiO
2
NPs in the blood at 24 h or
7 days post exposure. Furthermore, they demonstrated that com-
partmentalization pattern of deposited NPs in respiratory tract
was the same regardless the way of delivery NPs.
Krystek et al. (2014) determined distribution of titanium from
TiO
2
NPs in tissues of Wistar rats administrated by injection.
Fourteen days after exposure the highest titanium content was
found in liver and spleen. A small amount of titanium was also
determined in lung, kidney, heart, brain and muscle.
3. Toxicity in vitro: Inhibition of proliferation and cell death
Many in vitro studies have shown that TiO
2
NPs are cytotoxic for
nervous cells and can induce apoptosis. Li et al. (2009) observed
time and dose dependent TiO
2
NPs cytotoxicity in cultured murine
microglia N9 cells. Inhibition of cell proliferation, changes in cell
morphology and apoptosis was also observed in human U373
and rat C6 glial cells (Márquez-Ramírez et al., 2012) and human
astrocytoma U87 cells (Lai et al., 2008). Also the study by Liu
et al. (2010) on rat PC12 cells treated with TiO
2
NPs resulted in
induction of apoptosis. Interestingly, the viability of PC12 cells
was significantly higher and apoptosis was inhibited by pre-
incubation with ROS scavenger N-(mercaptopropionyl)-glycine
(N-MPG) that suggest the involvement of ROS in the TiO
2
NPs
induced neurotoxicity and apoptosis. Also Wu et al. (2010) investi-
gated the cytotoxicity and the mode of cell death of PC12 cells
exposed to TiO
2
NPs. The authors confirmed the occurrence of
apoptosis, likely mediated by ROS-dependent induction of JNK/
p53 signaling pathway. More recently, Sheng et al. (2015a) showed
that TiO
2
NPs were cytotoxic for rat primary cultured hippocampal
neurons and caused typical apoptotic features, such as mitochon-
drial swelling, carina disappearance, nucleus shrinkage, anomalous
nuclear membrane and chromatin marginalization. In addition,
TiO
2
NPs treatment resulted in dilation of endoplasmic reticulum,
increased level of intracellular Ca
2+
, reduction of mitochondrial
membrane potential and alterations of apoptosis-related cytokines
expression. These findings suggested that TiO
2
NPs-induced neu-
ronal apoptosis may be associated with mitochondria- and endo-
plasmic reticulum-mediated signaling pathway.
Long et al. (2006, 2007) examined neurotoxicity of Aeroxide P25
NPs on mouse microglia BV2 cells, rat dopaminergic neurons (N27
cells) and primary cultures of rat striatum. Rapid, prolonged
release of ROS was observed in BV2 cells upon Aeroxide P25 expo-
sure, accompanied by increased caspase 3/7 activity and apoptotic
loss of nuclear material. Also, primary cultures of rat striatum
exposed to Aeroxide P25 showed reduction of the neuron number
and evidence of neuronal apoptosis after 6 h. On the contrary, in
the N27 neurons Aeroxide P25 did not induce apoptosis nor cyto-
toxicity, despite the activation of caspase 3/7 activity and the fact
that numerous Aeroxide P25 aggregates and single NP were seen
membrane-bound and in the cytoplasm. These results suggest that
Aeroxide P25 is non toxic to N27 neurons, and the neurotoxicity
observed in primary striatum cultures might be microglia-
dependent effect rather than direct influence on neurons.
Activation of microglia was also observed by Shin et al. (2010) in
the inflamed, but not in normal, brain of mice exposed to ultrafine
TiO
2
. Ability of activated microglia to induce death of target cells
was studied by Xue et al. (2012) in co-culture with PC12 cells.
Supernatant from TiO
2
NPs treated microglia caused significant
cytotoxicity in PC12 cells. The authors suggested that TiO
2
NPs
stimulated microglia produced inflammatory factors, which caused
PC12 cells cytotoxicity.
Valdiglesias et al. (2013) showed that TiO
2
NPs exposure did not
reduce the viability of SHSY5Y cells, although nanoparticles were
effectively internalized by neuronal cells. However, TiO
2
NPs
induced dose-dependent alterations in cell cycle and apoptosis.
Changes in the mitochondrial membrane potential suggest that
apoptosis was induced by mitochondrial pathway. Contrast
between results of cell viability and apoptosis tests was explained
by different sensitivity of the methods. The authors examined also
a genotoxic potential of TiO
2
NPs, but obtained confounding results
that revealed dose-dependent formation of micronuclei, but no
increase in number of
c
HA2X foci, both markers of DNA double
strand breaks induction. However, the alkaline comet assay
showed an increase of oxidative DNA damage that suggest that
micronuclei formation might be associated with single strand
breaks (SSB) or alkaline-labile sites formation or incomplete exci-
sion-repair. The effects of in vitro exposure to TiO
2
NPs are summa-
rized in Table 1.
4. In vivo toxicity in nonmammalian vertebrates
Majority of data for TiO
2
NPs neuronal toxicity in nonmam-
malian eukaryotes are available for a variety of fish species. A study
on rainbow trout (Oncorhynchus mykiss) showed that after semi-
static exposure to TiO
2
NPs for 14 days, concentration of titanium
in brain of treated fishes was higher than in the untreated control,
but the effect was not dose dependent. Histological examination
showed that there were no gross pathological changes in the fore,
mid and hind brain. Moreover, there were no evidence of gross
inflammation, cranial bleeding, blood vessel abnormality, neither
vacuolation, oedema, cellular atrophy nor necrosis of the brain tis-
sue (Federici et al., 2007). Another study on rainbow trout showed
increase of titanium concentration in the brain of fishes dietary
exposed for 8 weeks to TiO
2
NPs. The brain of exposed animals
showed transient increase of titanium concentration at week 4
and at the end of experiment (Ramsden et al., 2009). This findings
suggest that titanium does not clear quickly from the brain tissue.
On the other hand, Boyle et al. (2013) reported no increase in
titanium concentration in the brain of rainbow trout after for
14 days waterborne exposure to TiO
2
NPs, despite the increasing
concentration of titanium in the gill tissue. Histological observa-
tion showed also that the level of pathologies observed in brain
was minimal, though blood vessels on the surface of the cerebel-
lum were enlarged after TiO
2
NPs treatment. Exposure to TiO
2
NPs increased locomotor activity of rainbow trout, but rather to
compensate respiratory hypoxia resulted from the gill injury than
due to behavioral changes resulted from the damage to brain.
Recent study by Chen et al. (2011) showed that after a long-
time exposure TiO
2
NPs were accumulated in a time-dependent
manner in the brain of zebrafish (Danio rerio). But histological
examination of brain showed lack of any significant histological
alteration, vacuolation, oedema and cellular atrophy. Similar
results were obtained by Ramsden et al. (2013). Their study were
aimed at examination of the sub-lethal effect of 14 days exposure
to TiO
2
NPs on the physiology and reproduction of zebrafish. At
the end of exposure no histological changes in brain were observed
and the architecture of the brain was normal. Recently, Sheng et al.
(in press) evaluated the TiO
2
NP-induced neurotoxicity in zebrafish
after low dose and subchronic exposure to TiO
2
NPs. Their study
indicated that TiO
2
NPs can be translocated to the brain or neu-
ronal cell and cause brain injury.
A study by Federici et al. (2007) focused also at the analysis of
major tissue electrolytes and trace elements in brains of TiO
2
NPs
exposed rainbow trout. The authors conclude that semi-static
exposure to TiO
2
NPs for 14 days have no influence on Na
+
and
Ca
2+
levels in the brain. However, a statistically significant
increases in K
+
and Mn concentrations were observed. The K
+
level
showed a transient rise, but had recovered by the end of
1044 M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052
experiment. Levels of Cu and Zn decreased about twice at day 7,
nonetheless Cu and Zn concentrations were higher than in the con-
trol at the end of experiment. A slight and insignificant decrease in
the activity of brain Na
+
K
+
-ATPase activity was also observed. Lack
of changes in the brain Na
+
K
+
-ATPase activity upon treatment with
TiO
2
NPs was further confirmed by Boyle et al. (2013) on rainbow
trout and Ramsden et al. (2013) on zebrfish. Interestingly this
group previously reported 50% inhibition of the brain Na
+
K
+
-
ATPase activity in rainbow trout treated with TiO
2
NP for 4 weeks,
accompanied by a decrease in Cu level and a transient increase in
Zn level. No significant changes in Na
+
,K
+
,Ca
2+
, Mn levels were
observed (Ramsden et al., 2009).
Recently, Wang et al. (2014b) examined the effects of exposure
to TiO
2
NPs on zebrafish embryonic development and the neuroge-
nesis. Embryos was treated at one- to four-cell stage until 72 h
postfertilization. Results of the study showed that TiO
2
NPs aque-
ous exposure at a low dose did not affect the survival nor gross
development of embryos, and did not alter the migration of macro-
phages into the brain and retina during embryonic development.
What is more, onset of neurogenesis and neuronal differentiation
were not disrupted by the TiO
2
NPs exposure.
5. In vivo toxicity in mammals
Animal studies showed that TiO
2
NPs accumulated in the mouse
brain. Wang et al. (2008a,b) compared titanium concentration in
the whole brain and sub-brain regions homogenates of mice intra-
nasally installed with TiO
2
NPs. After 30 days of exposition
titanium concentration in the brain of exposed animals was higher
than untreated controls. The SRXRF analysis of brain sections
revealed that titanium accumulated preferably in the cerebral cor-
tex, thalamus and CA1 and CA3 region of hippocampus. However,
after exposure to TiO
2
NPs no pathological changes of neurons in
cerebral cortex and cerebellum was observed, as revealed by
Nissl staining method, but the pyramidal cell soma was enlarged
and elongated. In addition, in the neurons of CA1 region of hip-
pocampus of exposed animals a scattered Nissl bodies pattern
was observed, accompanied by the large cell stomata, an irregular
appearance of neurons and a drastic neuronal loss, in parallel with
increased number of activated astrocytes (Wang et al., 2008a). In a
complementary study degeneration of hippocampal neurons and
irregular arrangement of neuron cells in the olfactory nerve layers
were observed in intranasally instilled mice (Wang et al., 2008b).
Zhang et al. (2011) assessed the effect of rutile TiO
2
NPs exposure
on the murine brain. Investigators found that titanium accumu-
lated in the cerebral cortex and in the striatum, however the dam-
aged cell were found only in the cerebral cortex, CA1 region and
denate gyrus of hippocampus of exposed mice.
In concordance, in the intranasally administered female mice Ze
et al. (2014b,c) found that titanium accumulated in the brain in
dose-dependent manner causing loss of brain weight.
Furthermore, histopathological observation revealed overprolifera-
tion of glial cells, accompanied by tissue necrosis. Similar results
were obtained in a complementary study on male mice (Ze et al.,
2013). In the further study this group assessed damage of mouse
hippocampus induced by subchronic exposure to intranasally
Table 1
The effect of in vitro exposure to TiO
2
.
Size and coating Model Concentration/dose Time of
treatment
Outcome References
21 nm (Aeroxide
Ò
TiO
2
P25, Evonik
Degussa)
Murine BV2
microglia
2.5–120 ppm 1, 6 and 18 h Release of ROS, cell viability was maintained Long et al.
(2006)
Rutile 1–1.3
l
, anatase
<25 nm
U87 astrocytoma Up to 100
l
g/mL Up to 72 h Decrease in survival, changes in cellular morphology, apoptosis Lai et al.
(2008)
Commercial TiO
2
NPs
and larger TiO
2
particles
Murine microglia
N9 cells
4, 8, 16, 32, 64 and
125
l
g/mL
24 h Decreased cell viability, cell morphology altered and induced
apoptosis
Li et al.
(2009)
21 nm (Aeroxide
Ò
TiO
2
P25, Evonik
Degussa)
Rat PC12 cells 1, 10, 50 and
100
l
g/mL
6, 12, 24 and
48 h
Decreased cell viability, ROS production, induction of apoptosis Liu et al.
(2010)
20 nm Rat PC12 cells 25, 50, 100 and
200
l
g/mL
6 and 24 h Decreased cell viability, induction of apoptosis, activation of
oxidative stress, disturbing cell cycle, triggering JNK- and p53-
mediated signaling pathway
Wu et al.
(2010)
21 nm (Aeroxide
Ò
TiO
2
P25, Evonik
Degussa)
Murine BV2
microglia
25, 50, 100 and
200
l
g/mL
24 h Enhanced TNF-
a
production and augmented NF-kB binding
activity
Shin et al.
(2010)
20 nm Primary
microglia, rat
PC12 cells
0.25 mg/ml and
0.5 mg/mL
24 h (micro-glia)
or 24 h and 48 h
(PC12 cells)
Induced microglial activation, release of proinflammatory
factors; dysfunction and cytotoxicity in PC12 cells
Xue et al.
(2012)
Anatase (96%) and
rutile (4%), 4–
200 nm
Human U373
and rat C6
astrocytoma cell
lines
Up to 40 mg/cm
2
24, 48 and 24 h Inhibited cell proliferation, induced morphological changes,
induces apoptosis. The authenticity of U373 cell line available
from ATTC was recently questioned (see http://www.
lgcstandards-atcc.org/Global/FAQs/C/F/U373%20MG%20ATCC%
20HTB17-1055.aspx)
Márquez-
Ramírez
et al. (2012)
25 nm Human SHSY5Y
neuronal cells
20, 40, 60, 80, 100,
120 and 150
l
g/mL
3, 6 and 24 h No decrease in viability despite the visible NPs uptake, no
morphological alterations, cell cycle alterations, apoptosis by
intrinsic pathway, no oxidative damage, genotoxicity not
related with DSB induction
Valdiglesias
et al. (2013)
Anatase (96%) and
rutile (4%), 50 nm
Humn U373 and
rat C6
astrocytoma cell
lines
20 mg/cm
2
2, 4, 6 and 24 h Oxidative stress and mitochondrial damage. Lipid
peroxidation. Induction of antioxidant enzymes. The
authenticity of U373 cell line available from ATTC was recently
questioned (see http://www.lgcstandards-atcc.org/Global/
FAQs/C/F/U373%20MG%20ATCC%20HTB17-1055.aspx)
Huerta-
García et al.
(2014)
5–6 nm Primary cultured
hippocampal
neurons
5, 15 and 30
l
g/mL 24 h Decreased cell viability, induction of apoptosis Sheng et al.
(2015a)
M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052 1045
administered TiO
2
NPs. ICP-MS and Raman spectroscopy revealed
TiO
2
NPs deposits in hippocampus sections. Histopathological
examinations showed dose-dependent hippocampal damage,
including disperative replication of pyramidal cells, rarefaction
and edema of glial cells, nuclear irregularity, decreased of cell vol-
ume, degeneration, nuclear pyknosis, cytoplasm feosin in pyrami-
dal cells (Ze et al., 2014a). Similar determinations were carried
by Ze et al. (in press). In this study mice were exposed to TiO
2
NPs by the intranasal administration every day for 9 months.
Histopathological examination of the hippocampus revealed
dose-dependent severe pathological changes, such as edema of
pyramidal neuron, hemorrhage and proliferation of all glial cells.
In the study by Ma et al. (2010), titanium accumulated in a dose
dependent manner in mouse brain 14 days after injection of TiO
2
NPs to the abdominal cavity. The titanium content in brain was
higher for anatase TiO
2
NPs then bulk TiO
2
, when same doses were
applied. The highest administered doses caused a significant
reduction of brain weight, accompanied by neuronal degeneration
and inflammatory response. A dose dependent titanium accumula-
tion in brain, lost of brain weight and the brain tissue abnormality
was also observed after 60 days of intragastric administration of
TiO
2
NPs (Hu et al., 2010, 2011).
On the contrary, 60 days chronic dermal exposure to different
forms of TiO
2
NPs showed that TiO
2
NPs did not pass through the
blood–brain barrier with exception of Degussa P25 NPs, which
accumulated in the brain. However histopathological analysis
showed no differences between treated and control animals (Wu
et al., 2009). Very little brain damage was also observed in a study
by Wang et al. (2007a), when mice were treated with a single dose
TiO
2
NPs suspension by an oral gavage. The latest study by Younes
et al. (in press) showed that titanium accumulated in the brain of
rats after 10 injections (20 mg/kg body weight) every 2 days for
20 days, but no significant differences were observed in the coeffi-
cients of the brain to body weight nor the morphology of neurons.
The mechanism of brain cell death after in vivo TiO
2
NPs expo-
sure was investigated in mice by Hu et al. (2011). The authors
found that intragastric administration of TiO
2
NPs for 60 days led
to hippocampal apoptosis. Protein expression analysis revealed
elevated levels of proapoptotic proteins, whereas expression of
Bcl-2 protein was significantly suppressed. Hippocampus cell
apoptosis was also observed in female mice after a long–term nasal
administration of TiO
2
NPs (Ze et al., 2014a,b).
The brain effects caused by in vivo exposure to TiO
2
NPs are
summarized in Table 2.
6. Inflammatory response
Several in vitro and in vivo studies demonstrated that exposure
to TiO
2
NPs induced proinflammatory response, however pub-
lished results are unclear and further study are necessary to solve
many discrepancies. Intranasally installed 155 nm TiO
2
NPs
increased the levels of TNF-
a
in brain and IL-1bin serum of
exposed mice, whereas no effect was observed for 80 nm TiO
2
NPs (Wang et al., 2008b). On the contrary, Shin et al. (2010)
reported no increase of TNF-
a
and IL-1blevels in brains of mice
treated with fine and ultrafine TiO
2
NPs, despite the significant
increase of TNF-
a
and IL-1bmRNA level in brains of LPS pretreated
mice. This was further confirmed in vitro on LPS-stimulated BV2
microglial cells, but the effect was not dose-dependent. A release
of monocyte chemoattractant protein-1 (MCP-1) and macrophage
inflammatory protein-1
a
(MIP-1
a
) from microglia upon exposure
to TiO
2
NPs was also observed by Xue et al. (2012). Increased pro-
duction of inflammatory mediators due to the exposure to TiO
2
NPs
is likely driven through the stimulation of NF-
j
B signaling path-
way, which is a major transcriptional factor of inflammation-
related gene induction. Indeed, TiO
2
NPs augmented NF-
j
B binding
in normal (Xue et al., 2012) and in LPS-stimulated microglial cells
(Shin et al., 2010). Recently, Ze et al. (2014c) also showed that
exposure to TiO
2
NPs resulted in hippocampal neuroinflammation
in the intranasally administered mice. They found that TiO
2
NPs
exposure induced Toll-like receptor 2 and 4 (TLR2 and TLR4) genes
and their protein expression, as well as inflammation-related
genes and their proteins expression, i.e. NF-
j
B, TNF-
a
, NF-
j
BP52,
NF-
j
BP65, NF-
j
B-inducible kinase, I
j
B kinase and IL-1b. In addi-
tion, the I
j
B mRNA level was significantly down-regulated and
the level of I
j
B protein was reduced in the hippocampus after
TiO
2
NPs exposure.
In addition to proinflammatory cytokines, TiO
2
NPs treatment
stimulated NOS activity and nitric oxide (NO) production.
Injection of TiO
2
NPs to the abdominal cavity caused increase of
NOS activity and elevated the NO level in brain tissue (Ma et al.,
2010). Also in vitro treatment of microglia cells with TiO
2
NPs
caused increase of NO production through an iNOS-dependent
pathway (Xue et al., 2012).
7. Other responses
Changes in gene expression in BV2 microglia exposed to
Degussa P25 TiO
2
NPs were studied by Long et al. (2007). Several
signaling pathways were upregulated, including genes involved
in B-cell receptor signaling, calcium signaling, death receptor sig-
naling, apoptosis, inflammation, oxidative stress response, cell
cycling, whereas genes involved in adaptive change and key energy
production pathways were down regulated. Down-regulated were
also pathways associated with peroxisomes and Nrf2-mediated
oxidative stress, pathways triggered by response to low oxygen
availability and associated with mitochondrial dysfunction (Long
et al., 2007). In the study on gene expression in brains from TiO
2
NPs-exposed female mice, Ze et al. (2014b) found that the expres-
sion of 424 genes were obviously altered, including those associ-
ated with memory and learning, DNA repair, lipid metabolism,
immune response, energy metabolism, apoptosis, oxidative stress,
brain development, protein metabolism, signal transduction.
Sheng et al. (in press) examined the gene expression in brain from
zebrafish after subchronic exposure to low dose TiO
2
NPs. They
found that expressions of learning and memory behavior-related
genes were disturbed.
Dopamine metabolism was studied in PC12 cells cocultured
with TiO
2
NPs stimulated microglia. Expression of tyrosine hydrox-
ylase gene was suppressed by TiO
2
NPs exposure, resulting in a
lower conversion of tyrosine to dopamine. However, no changes
in vesicular monoamine transporter-2 gene expression was
observed, suggesting that TiO
2
NPs exposure did not affected the
dopamine transport system (Xue et al., 2012). TiO
2
NPs decreased
glutamate (Glu) level and AChE activity when injected to the
mouse brain (Ma et al., 2010). On the contrary, after 30 days of
intranasal administration of TiO
2
NPs the AChE activity and glu-
tamic acid concentration in brain of mice increased significantly
compared with the control (Wang et al., 2008a). Hu et al. (2010)
found that 60-days of intragastric administration of TiO
2
NPs caused inhibition of brain Na
+
/K
+
-ATPase, Ca
2+
-ATPase,
Ca
2+
/Mg
2+
-ATPase activity, but elevation of AChE and total nitric
oxide synthase (TNOS) activity. In addition, the level of acetyl-
choline, Glu and NO were higher, whereas the level of monoamine
neurotransmitter and related metabolites (such as norepinephrine
(NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid, 5-hydrox-
ytryptamine (5-HT) and 5-hydroxyindoleacetic acid) were lower
(Hu et al., 2010). Similar results were obtained by Sheng et al. (in
press) who examined the neurotoxicity of TiO
2
NPs in zebrafish
after an aquatic administration with low dose TiO
2
NPs for 45
1046 M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052
Table 2
Brain effects caused by in vivo exposure to TiO
2
NPs.
Size Model Dose Treatment Outcome References
22 nm Rats 4–5
l
g 1 h inhalation TiO
2
NPs were found on the luminal side of airways and alveoli as
well as within each tissue compartment of the lung
Geiser et al.
(2005)
21 nm (Aeroxide
Ò
TiO
2
P25,
Evonik Degussa)
Rainbow trout
(Oncorhynchus mykiss)
0.1, 0.5 or 1.0 mg/L 14 days Semi-static exposure No histological alterations in the brain, biochemical disturbances Federici et al.
(2007)
22 nm Rats 4–5
l
g 1 h inhalation The distribution of TiO
2
NPs was not random. The residence time
of NPs in each tissue compartment of the respiratory system
depends on the compartment and the time after exposure
Mühlfeld et al.
(2007)
25, 80 and 155 nm Mice 5 g/kg Single dose, oral gavage TiO
2
NPs mainly retained in the liver, spleen, kidneys and lung
tissues, minority present in the brain
Wang et al.
(2007a)
25, 80 and 155 nm Mice 0.1 g/ml 25
l
l suspension once a day for first
5 days, 10
l
l suspension once every
other day for month. Nasal inhalation
TiO
2
NPs translocated to the olfactory bulb through the olfactory
nerves
Wang et al.
(2007b)
80 and 155 nm Mice 500
l
g 30 days intranasally Morphological changes of hippocampal neurons, Ti accumulation
in the hippocampus, induction of oxidative stress
Wang et al.
(2008a)
80 and 155 nm Mice 500
l
g 30 days intranasally Pathological changes in the hippocampus and olfactory bulb,
induction of oxidative stress and immune response of brain
Wang et al.
(2008b)
21 nm (Aeroxide
Ò
TiO
2
P25,
Evonik Degussa)
Rainbow trout
(Oncorhynchus mykiss)
10 or 100 mg/kg dry weight
feed
8 weeks. Dietary exposure Accumulation in the brain, disturbance in Cu and Zn levels and
inhibition of Na
+
K
+
-ATPase activity
Ramsden et al.
(2009)
25–70 nm Mice 0.1
l
g On gestational days 6, 9, 12, and 15
subcutaneous injection
Alterations of gene expression of the offspring Shimizu et al.
(2009)
25–70 nm Mice 0.1 mg 3, 7, 10 and 14 days postcoitum
subcutaneous injection
TiO
2
NPs administrated to pregnant mice are transferred to and
affected cranial nerve system of the offspring
Takeda et al.
(2009)
4, 10, 25, 60 nm and 21 nm
(Aeroxide
Ò
TiO
2
P25,
Evonik Degussa)
Domestic pigs, BALB/c
hairless mice
400
l
g/cm
2
30 days (pigs) or 60 days (mice).
Dermal exposure
TiO
2
NPs penetrated through the skin, accumulated in different
tissues and induced diverse pathological lesions in several major
organs
Wu et al. (2009)
21 nm (UV-titan L181) Mice 42 mg/m
3
1 h/day on gestation days 8–18,
inhalation
Gestationally exposed offspring displayed moderate
neurobehavioral alterations. Cognitive function was unaffected
Hougaard et al.
(2010)
5 nm Mice 5, 10 and 50 mg/kg 60 days, intragastric exposure Spatial recognition memory impairment, disturbance of the
homeostasis of trace elements, enzymes and neurotransmitters
Hu et al. (2010)
3 nm Mice Total dose of 13.2 mg/kg Once per-week for 4 consecutive
weeks. Intratracheal instillation
NPs pass through the blood–brain barrier (BBB), and induce the
brain injury through oxidative stress response
Li et al. (2010)
5 nm Mice 5, 10, 50, 100 and 150 mg/
kg
14 days, abdominal cavity injection Translocation and accumulation in the brain, brain injury,
increased oxidative stress
Ma et al. (2010)
21 nm (Aeroxide
Ò
TiO
2
P25,
Evonik Degussa)
Mice 40 mg/kg (1 mg/mouse) Single dose intraperitoneal injections No inflammation induction in the normal brain; increased
proinflammatory cytokine mRNA levels, increased expression of
IL-1bprotein, enhanced ROS production, increased activation of
microglia in the inflamed brain
Shin et al. (2010)
>10 nm Zebrafish (Danio rerio) 1, 2, 4, 5 and 7 mg/L 2, 4, 6 months, dietary exposure Accumulation in the brain, but without histological alteration in
the brain
Chen et al. (2011)
>25 nm Rats 100 mg/kg From prenatal day 21 to postnatal day
2; or from postnatal day 2 to day 21.
Oral exposure of mothers
Developmental exposure could affect synaptic plasticity in
offspring’s hippocampal DG area
Gao et al. (2011)
6.5 nm Mice 5, 10 and 50 mg/kg 60 days, intragastric exposure Accumulation in hippocampus, hippocampal apoptosis, spatial
recognition memory impairment, increased oxidative stress
Hu et al. (2011)
10 nm 40 nm,
10 nm 50 nm, 50 nm
Mice 500
l
g 30 days, intranasally Accumulation in the cerebral cortex and striatum, morphological
changes of neurons, significant disturbance of the monoamine
neurotransmitter levels
Zhang et al.
(2011)
17 nm Skin of Hairless Wistar Yagi
rats
0.4 mg/cm
2
Once a day, for 56 consecutive days.
Dermal exposure
NPs did not penetrate the skin Adachi et al.
(2013)
21 nm (Aeroxide
Ò
TiO
2
P25,
Evonik Degussa)
Rainbow trout
(Oncorhynchus mykiss)
1 mg/L 14 days. Waterborne exposure No accumulation in the brain, increased oxidative stress defence,
lack of the differences in the Na
+
K
+
-ATPase and
acetylcholinesterase activities
Boyle et al. (2013)
21 nm (ABC Nanotech Co.,
Ltd., Daejeon, Korea)
Rats 260.4, 520.8 and 1041.5 mg/
kg/day
13 weeks, oral administration TiO
2
NPs were not significantly increased in sampled organs
including the brain
Cho et al. (2013)
(continued on next page)
M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052 1047
Table 2 (continued)
Size Model Dose Treatment Outcome References
21 nm (Aeroxide
Ò
TiO
2
P25,
Evonik Degussa)
Zebrafish (Danio rerio) 0.1 or 1.0 mg/L 14 days, dietary exposure No changes in Na
+
K
+
-ATPase activity in brain, elevated total
glutathione level, no histological changes in the brain
Ramsden et al.
(2013)
5–6 nm Mice 2.5, 5 and 10 mg/kg 90 days, intranasally Accumulation in the brain, overproliferation of spongiocytes and
hemorrhage, activation of the P38-Nrf-2 signaling pathway,
increased oxidative stress
Ze et al. (2013)
25 nm (AEROXIDE P25
powder, Evonik,
Germany)
Rats 45
l
g or 200
l
g Single dose (0.5 s) or 4 h repeated
exposure over 4 consecutive days).
Intratracheal instillation or whole
body aerosol inhalation
No translocation to blood and secondary organs, majority of TiO
2
NPs associated with lavaged cells or lung parenchyma
Baisch et al.
(2014)
22 and 110 nm Rats 2.3 mg/mL Single dose injection Ti concentrations were >4
l
g/g tissue in spleen, liver and lung
tissue
Krystek et al.
(2014)
10 nm Rats 100 mg/kg On gestation days 2–21, intragastric
exposure
Reduced cell proliferation in the hippocampus, impaired learning
and memory of the offspring
Mohammadipour
et al. (2014)
21 nm, P-25 type Zebrafish (Danio rerio)
embryos
1 mg/L Until 72 h postfertilization, aqueous
exposure
Neurogenesis and neuronal differentiation were not disrupted,
embryonic development was normal
Wang et al.
(2014b)
5–6 nm Mice 2.5, 5 and 10 mg/kg 90 days intranasally Accumulation in the hippocampus, hippocampal lesion,
alterations of gene and protein expression, spatial recognition
impairment
Ze et al. (2014a)
5–6 nm Mice 2.5, 5 and 10 mg/kg 90 days intranasally Translocation and accumulation in the brain, increased oxidative
stress, overproliferation of all glial cells, tissue necrosis,
hippocampal cell apoptosis, alterations in the gene expression
Ze et al. (2014b)
5–6 nm Mice 2.5, 5 and 10 mg/kg 90 days intranasally Accumulation in the hippocampus, overproliferation of all glial
cells, hippocampal lesion, altered expression of genes and
proteins involved in the signaling pathway (Toll-like receptors
and inflammatory cytokines), neuroinflammation, spatial memory
impairment
Ze et al. (2014c)
5–6 nm Mice 1.25, 2.5 and 5 mg/kg 9 months intranasally Hippocampal injuries, impairment of glutamic metabolism,
reduction of glutamate receptors expression in the hippocampus
Ze et al. (in press)
6.5 nm Zebrafish (Danio rerio) 5, 10, 20, and 40
l
g/L 45 consecutive days. Waterborne
exposure
TiO
2
NPs translocated to the brain, brain injury, reduction of
spatial recognition memory, alterations of gene expression,
disturbance in levels of neurotransmitters
Sheng et al. (in
press)
20–30 nm Rats 20 mg/kg Every 2 days for 20 days,
intraperitoneal injection
Accumulation in the brain, but without significant changes in
brain weight and morphology of neurons; alteration of emotional
behavior
Younes et al. (in
press)
1048 M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052
consecutive days. They found that level of monoamine neurotrans-
mitter and their metabolites, including NE, DA and 5-HT in the
brain was reduced, whereas the level of NO was increased.
Significant disturbance of monoamine neurotransmitter levels in
the murine sub-brain regions (hippocampus, cerebral cortex, cere-
bellum and striatum) was also after intranasal exposure to TiO
2
NPs was also reported by Zhang et al. (2011) in mice.
In concordance with previous reports (Wang et al., 2008a; Hu
et al., 2010), Ze et al. (in press) described a significantly elevated
Glu content in the hippocampus of mice after 9-month intranasal
exposure to TiO
2
NPs. TiO
2
NPs exposure resulted also in increase
of phosphate-activated glutaminase activity and reduction in
glutamine formation and glutamine synthetase activity in the
hippocampus. Significant inhibition of the expression of
N-methyl-D-aspartate receptor subunits and metabotropic
glutamate receptor 2 in the hippocampus were also observed.
In addition to biochemical changes in brain, TiO
2
NPs exposure
affected also animal behavior. Intragastric or intranasal adminis-
tration of TiO
2
NPs damaged the spatial recognition memory, as
indicated by the Y-maze or Morris water maze tests (Hu et al.,
2010, 2011; Ze et al., 2014a,c). Younes et al. (in press) found that
intraperitoneal injection of TiO
2
NPs caused serious effects on the
emotional behavior. The results of the elevated plus maze test
showed that TiO
2
NPs treatment increased the anxious index of
exposed rats. Moreover TiO
2
NPs exposed mice learnt slower than
control mice and had significantly reduced long-term synaptic
plasticity (LTP) of hippocampal denate gyrus (HDG) area (Ze
et al., 2014a). Recently, Sheng et al. (in press) also done the Y-maze
test on zebrafish after low dose and subchronic exposure to TiO
2
NPs. They found reduction of spatial recognition memory and loco-
motor activity, which were associated with brain injury and alter-
ations of memory behavior-related gene expression. Experimental
data suggest also that TiO
2
NPs exposure during lactation, but not
during the pregnancy, resulted in the weakness of basic synaptic
transmission in HDG area. Furthermore, the TiO
2
NPs exposure
during lactation reduced the LTP of HDG area, suggesting that
exposure to TiO
2
NPs during lactation may impact newborn brain
development and affect the physiological functions of hippocam-
pus (Gao et al., 2011). Wang et al. (2014a) found that co-exposure
of TiO
2
NPs and a persistent organic pollutant BDE-209 (poly-
brominated diphenyl ether congener) caused a decrease in locomo-
tion activity and downregulation of the expression of genes
involved in the development of central nervous system of zebrafish
larvae.
8. The role of oxidative stress – induced toxicity and DNA
damage
An in vitro studies showed that exposure to TiO
2
NPs stimulated
ROS generation. Exposure to 21 nm Aeroxide P25 TiO
2
NPs stimu-
lated BV2 microglia to immediate ROS production through the
oxidative burst and further continuous release of ROS due to inter-
ference with mitochondrial electron transport chain (ETC) (Long
et al., 2006, 2007). The TiO
2
NPs dose-dependent generation of
ROS was also observed in PC12 cells (Liu et al., 2010; Wu et al.,
2010), and in U373 and C6 astrocytoma cell lines accompanied
by mitochondrial damage and induction of antioxidant enzymes
(Huerta-García et al., 2014). On the other hand, Valdiglesias et al.
(2013) indicated that TiO
2
NPs treatment did not induce oxidative
damage in neuronal SHSY5Y cells.
The first evidence of in vivo ROS production after exposure to
TiO
2
NPs was observed in the brain of nonmammalian vertebrates.
The concentration of total glutathione was elevated in the brains of
TiO
2
NPs exposed zebrafish (Ramsden et al., 2013) and rainbow
trout (Boyle et al., 2013). TiO
2
NPs exposure also elevated level of
thiobarbituric reactive substances (TBARS) in brain of rainbow
trout (Federici et al., 2007; Boyle et al., 2013).
A dramatic increase of the ROS levels (O
2
,H
2
O
2
), accompanied
by the decreased levels of activity/concentration of ROS scavengers
(SOD, CAT, APx, GSH-Px, GSH and ascorbic acid) was observed in
the hippocampus of orally exposed mice (Hu et al., 2011). The
dose-dependent increase in production of O
2
and H
2
O
2
, and higher
levels of lipid, protein and DNA peroxidation products was also
observed in brain of intranasally exposed mice (Ze et al., 2013,
2014a). This was accompanied by increased expression levels of
cytokines and activation of oxidative stress response signaling
pathways (p38, c-Jun N-terminal kinase, NF-
j
B, Nrf-2) (Ze et al.,
2013). ROS production was also significantly increased in the cor-
tex and hippocampus of septic brain, 24 h after ultrafine TiO
2
administration (Shin et al., 2010).
A transient, short term increase of the activity/concentration of
the major antioxidants (GSH-Px, GST, SOD, GSH) was also observed
after intranasal administration of TiO
2
NPs, but it normalized at the
end of experiment (30 days) (Wang et al., 2008b). A further study
confirmed no changes in the activity of GSH-Px and GST, and
GSH content 30 days after intranasal administration of the rutile
or anatase TiO
2
NPs. However, the activity of CAT increased,
whereas SOD activity decreased. In addition, exposure to TiO
2
NPs caused increase in malondialdehyde and protein carbonyl
levels and NO production (Wang et al., 2008a).
Enhanced production of O
2
and H
2
O
2
in the brain was also
observed after TiO
2
NPs delivery to the abdominal cavity of mice.
This was further accompanied by decreased ratio of ascorbic acid
to dehydroascorbic acid and GSH to GSSG, and decrease of the
enzymatic activity of SOD, CAT, APx and GSH-Px. Moreover, the
total antioxidant capacity of the mouse brain was significantly
reduced with increasing doses of TiO
2
NPs (Ma et al., 2010).
9. Offspring effect
The study on the effect of maternal exposure to TiO
2
NPs on the
offspring showed that subcutaneously administration of TiO
2
NPs
to pregnant mice affected development of a cranial nerve system
of the male offspring. TiO
2
NPs was detected in the olfactory bulb
and the cerebral cortex of a newborn. Furthermore, numerous cas-
pase-3 positive, apoptotic cells were found in the olfactory bulb,
suggesting that TiO
2
NPs translocated from pregnant mice to brain
of their offspring, and caused a various nervous system degenera-
tion (Takeda et al., 2009). Transmission of TiO
2
NPs through pla-
centa was further confirmed by Gao et al. (2011), who showed
by ICP-MS analysis that prenatal oral administration of rats to
TiO
2
NPs resulted in the presence of titanium in the hippocampus
of the offspring. The prenatal exposure of mice to TiO
2
NPs resulted
also in the alternations in brain gene expression of the developing
fetuses and newborn. Functional analysis of data revealed that the
mostly affected were genes associated with apoptosis, brain devel-
opment, motor activity, glial cell differentiation, cell death, oxida-
tive stress, neurotransmitter, affectivity, brain related disorders
and anatomy of mitochondria and synapses (Shimizu et al.,
2009). Prenatal exposure to TiO
2
NPs affected also behavior of
the offspring. Although, the Morris water maze test revealed no
changes in learning skills and memory of prenatally exposed off-
spring, the dry water maze pool clearly indicated alternations in
the activity of prenatally exposed adult mice. In addition, the sib-
ling of exposed females displayed enhanced prepulse inhibition,
indicating that prenatally exposed animals may display neurobe-
havioral alterations in adulthood (Hougaard et al., 2010).
Recently, Mohammadipour et al. (2014) also determined the
effects of exposure to TiO
2
NPs during pregnancy on hippocampal
cell proliferation and the learning and memory of offspring. Results
M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052 1049
of ICP-MS analysis showed that titanium accumulated in the hip-
pocampus of prenatally exposed rats. Further analysis revealed
that the brain coefficients of rat offspring exposed to TiO
2
NPs dur-
ing development were significantly higher as compared to controls
and cell proliferation in the hippocampus was significantly
reduced. Behavioral tests detected alterations in memory and
learning affected by developmental exposure to TiO
2
NPs.
In the study by Hougaard et al. (2010) mice were exposed to
rutile form of TiO
2
NPs modified by Zr, Si, Al, and Na and coated
with complex polyalcohols. The results revealed that offspring of
the treated animals displayed neurobehavioral alterations.
However, the authors assumed that degradation or release of the
coatings, or leaching or dissolving of metals from the nanomaterial
were responsible for the observed effects rather than TiO
2
NP itself.
Consequently, future studies are needed to investigate the effect of
pure and coated particles to elucidate the effects of rutile form and
role of the particle surface in toxicity.
10. Implications of TiO
2
NPs physicochemical properties in their
toxicity
It is well known that physicochemical properties of NPs, such as
size, crystalline structure, surface area, surface coating or shape
influence the activity of NPs and play an important role in their
toxicity. In addition, TiO
2
NPs possess a photocatalytic activity that
is also modulated by their physicochemical properties.
The size dependency of TiO
2
NPs toxicity has been frequently
demonstrated, however such relationship with reference to neu-
ronal cells is limited. Wu et al. (2010) using PC12 cells found that
20 nm TiO
2
NPs were more cytotoxic than micrometer particles of
TiO
2
. However, the majority of reports discussed in this review
dealt with only one size TiO
2
NPs (e.g. Shin et al., 2010; Federici
et al., 2007; Ramsden et al., 2009; Boyle et al., 2013; Ze et al.,
2013, 2014a,b,c; Ma et al., 2010; Hu et al., 2010, 2011). Even, if sev-
eral NPs were investigated, they usually varied in size and the crys-
tal structure (Wu et al., 2009; Wang et al., 2007a,b, 2008a,b), thus
differentiation between the size-dependent effects and other
effects was impossible.
TiO
2
occurs naturally in three crystal structures: tetragonal ana-
tase and rutile, and orthorhombic brookite. However, only anatase
and rutile crystals play important role in the applications of TiO
2
NPs (Diebold, 2003). Although, it seems that anatase TiO
2
NPs are
more toxic than rutile ones in vitro and in vivo, studies regarding
association between the crystal structure of TiO
2
NPs and their
neurotoxicity are very scarce. The majority of studies related only
to anatase form of TiO
2
NPs (Adachi et al., 2013; Chen et al., 2011;
Sheng et al., in press; Ze et al., 2013, 2014a,b,c; Ma et al., 2010; Hu
Fig. 1. Mechanism of TiO
2
NPs toxicity on central nervous system.
1050 M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052
et al., 2010, 2011; Gao et al., 2011; Takeda et al., 2009; Shimizu
et al., 2009; Mohammadipour et al., 2014) or mixture of 80% ana-
tase and 20% rutile forms (Liu et al., 2010; Shin et al., 2010; Long
et al., 2006; Cho et al., 2013; Federici et al., 2007; Ramsden et al.,
2009, 2013; Boyle et al., 2013). The only report describing the cyto-
toxicity of the rutile and anatase TiO
2
NPs, while controlling the
primary nanoparticle size, surface area and coating, was published
by Wu et al. (2010). They found that anatase TiO
2
NPs were more
toxic and more prone to induce the oxidative stress than rutile
ones. Moreover, anatase TiO
2
NPs induced apoptosis and necrosis,
whereas rutile TiO
2
NPs initiated only apoptosis. Nevertheless,
both forms disturbed the balance between the pro- and antioxi-
dant processes and, as a consequence, induced intracellular oxida-
tive stress in neuron cells. This was in concordance with the former
study by Wang et al. (2008b), who investigated effects of 80 nm
rutile and 155 nm anatase form of TiO
2
NPs. Although, anatase
TiO
2
NPs were larger than rutile ones, exposure to anatase TiO
2
NPs resulted in the higher inflammatory response and oxidative
damage. In an another study, Valdiglesias et al. (2013) assessed
the effects of 100% anatase TiO
2
NPs and the mixture of 80% ana-
tase and 20% rutile forms. The effects of exposure to both types
of NPs were comparable. Both were effectively internalized by
the SHSY5Y neuronal cells and induced dose-dependent cell cycle
alterations, apoptosis, and genotoxicity not related with double
strand break production nor with oxidative damage production.
However, the analysis of cellular uptake showed that the 100% ana-
tase TiO
2
NPs were more readily uptaken then the mixture, and
consequently 100% anatase TiO
2
NPs were more effective in induc-
ing cytotoxicity and cell cycle alterations.
Surface area and modification is another important issue in TiO
2
NPs toxicity. Hydrophilic TiO
2
NPs easily translocated across the
cerebral cortex and striatum, whereas the hydrophobic TiO
2
NPs
accumulated only in the striatum of intranasally exposed mice.
In addition, hydrophilic TiO
2
NPs caused much greater lesions on
the murine brain than hydrophobic ones (Zhang et al., 2011). On
the contrary, Wang et al. (2008a, b) found that hydrophobic
80 nm rutile TiO
2
NPs accumulated mostly in the hippocampus
and cerebellum. This discrepancy, however, might be caused by
the different shape and size of NPs used in these studies.
Study by Zhang et al. (2011) demonstrated that shape is also a
very important factor influenced on neurological effects of NPs.
Short rod-like TiO
2
NPs were more neurotoxic than needle-like
TiO
2
NPs, both NPs were rutile, hydrophilic and have similar parti-
cle size.
11. Summary
Fig. 1 summarizes the present stage of knowledge about toxicity
of TiO
2
NPs in central nervous system reviewed in this work. A
clear picture of the effects of TiO
2
NPs exposure on nervous system
is blurred by differences in physicochemical properties, dosage,
route and time of exposure or the use of different cellular models.
Although, differences arising from the use of different cellular/an-
imal models or modes of exposure should be expected, the main
difficulty seems to be insufficient NPs characterization, especially
in the older papers. Even, if NPs physicochemical properties are rel-
atively well characterized, any examination of the biological pur-
ity, e.g. by LPS contamination, is very uncommon. In addition,
many studies lack a positive control that may give the readers an
idea about the extent of observed results, despite the statistical
analysis revealed their significance. Considering all of the studies
conducted so far, the apparent need for systematic approach is
clearly visible, enabling better understanding of the influence of
the TiO
2
NPs physicochemical properties on its expressed toxicity,
and in consequence better assessment of the human risk from the
exposure to TiO
2
NPs. The majority of published in vitro studies
focused on TiO
2
NPs toxicity, usually understood as the estimation
of cellular ability to reduce MTT, which is not necessarily the same.
Thus, further studies are necessary to give a deeper insight to the
mechanisms behind the observed effects, including analysis of cel-
lular signaling pathways, changes in gene expression and
epigenetics.
Nevertheless, while discrepancies between the experimental
results might hamper conclusion drawing, it is clear that TiO
2
NPs accumulate in the brain, especially in the cortex and hip-
pocampus, and cause brain damage and neurotoxicity. TiO
2
NPs
exposure triggers different signaling pathways that lead to the
apoptotic processes through mitochondria-dependent pathway.
The key mediator of cell damage and apoptosis seems to be ROS
production. Moreover, TiO
2
NPs exposure induces microglial acti-
vation, that in turn causes release of proinflammatory cytokines,
further neurodegeneration and brain injury. It is noteworthy that
exposure during pregnancy and lactation period may have delete-
rious effects on offspring. However, further study are necessary,
especially in regard to long-term chronic exposure and the effects
of acute prenatal exposure on further generations, to fully under-
stand the impact of TiO
2
NPs exposure on living organisms and
their nervous system.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
The Transparency document associated with this article can be
found in the online version.
Acknowledgement
This work was financed by the National Science Centre Fund
Projects Nos. DEC-2013/09/B/NZ7/03934 (MC, KS, SP, LKS), DEC-
2013/11/N/NZ7/00415 (KSi) and 3165/B/P01/2011/40 (MK).
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1052 M. Czajka et al./ Toxicology in Vitro 29 (2015) 1042–1052
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Titanium dioxide nanoparticles (TiO2 NPs) are widely used in the chemical, electrical and electronic industries. TiO2 NPs can enter directly into the brain through the olfactory bulb and can be deposited in the hippocampus region; therefore, we determined the toxic effect of TiO2 NPs on rat and human glial cells, C6 and U373, respectively. We evaluated some events related to oxidative stress: 1) redox-signaling mechanisms by oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA); 2) peroxidation of lipids by cis-parinaric acid; 3) antioxidant enzymes expression by PCR in real time; and 4) mitochondrial damage by MitoTracker Green FM staining and Rh123. TiO2 NPs induced a strong oxidative stress in both glial cell lines by mediating changes in the cellular redox state and lipid peroxidation associated to a rise in the expression of glutathione peroxidase (GPx), catalase and superoxide dismutase 2 (SOD2). TiO2 NPs also produced morphological changes, damage of mitochondria, and an increase of mitochondrial membrane potential, indicating toxicity. TiO2 NPs had a cytotoxic effect on glial cells; however, more in vitro and in vivo studies are required to ascertain that exposure to TiO2 NPs can cause brain injury and be hazardous to health.
Titanium dioxide nanoparticles (TiO2 NPs) are widely used in the chemical, electrical and electronic industries. TiO2 NPs can enter directly into the brain through the olfactory bulb and can be deposited in the hippocampus region; therefore, we determined the toxic effect of TiO2 NPs on rat and human glial cells, C6 and U373, respectively. We evaluated some events related to oxidative stress: 1) redox-signaling mechanisms by oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA); 2) peroxidation of lipids by cis-parinaric acid; 3) antioxidant enzymes expression by PCR in real time; and 4) mitochondrial damage by MitoTracker Green FM staining and Rh123. TiO2 NPs induced a strong oxidative stress in both glial cell lines by mediating changes in the cellular redox state and lipid peroxidation associated to a rise in the expression of glutathione peroxidase (GPx), catalase and superoxide dismutase 2 (SOD2). TiO2 NPs also produced morphological changes, damage of mitochondria, and an increase of mitochondrial membrane potential, indicating toxicity. TiO2 NPs had a cytotoxic effect on glial cells; however, more in vitro and in vivo studies are required to ascertain that exposure to TiO2 NPs can cause brain injury and be hazardous to health.