Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00458.x |
Toxicity and interaction of titanium
dioxide nanoparticles with microtubule protein
Zahra Naghdi Gheshlaghi, Gholam
Hossein Riazi*, Shahin Ahmadian, Mahmoud Ghafari and Roya Mahinpour
Institute
of Biochemistry and Biophysics, Department of Biochemistry, University of
Tehran, Tehran 13145-1384, Iran
Received: May 18,
2008
Accepted: June 17,
2008
This work was
supported by a grant from the Institute of Biochemistry and Biophysics, Tehran University,
Tehran, Iran
*Corresponding
author: Tel, 98-21-61112473; Fax, 98-21-66404680; E-mail, [email protected]
Titanium dioxide
(TiO2) nanoparticles (NPs) are widely used in several
manufactured products. The small size of NPs facilitates their uptake into
cells as well as transcytosis across epithelial cells into blood and lymph
circulation to reach different sites, such as the central nervous system.
Different studies have shown the risks that TiO2 NPs in the
neuronal system and other organs present. As membrane-bound layer aggregates or
single particles, TiO2 NPs can enter
not only cells, but also mitochondria and nuclei. Therefore these particles
can interact with cytoplasmic proteins such as microtubules (MTs). MTs are
cytoskeletal proteins that are essential in eukaryotic cells for a variety of
functions, such as cellular transport, cell motility and mitosis. MTs in
neurons are used to transport substances such as neurotransmitters. Single TiO2 NPs in cytoplasm can interact with these proteins and affect
their crucial functions in different tissues. In this study, we showed the
effects of TiO2 NPs on MT polymerization
and structure using ultraviolet spectrophotometer and fluorometry. The
fluorescent spectroscopy showed a significant tubulin conformational change
in the presence of TiO2 NPs and the
ultraviolet spectroscopy results showed that TiO2 NPs affect tubulin polymerization and decrease it. The aim of
this study was to find the potential risks that TiO2 NPs pose to human organs and cells.
Keywords titanium dioxide; tubulin; microtubule;
protein interaction; nanoparticle
Titanium dioxide (TiO2)
nanoparticles (NPs) are used in numerous manufactured products, including
cosmetics, sunscreen, toothpaste and pharmaceuticals. However, there is
insufficient knowledge about the potential risks they posses [1–4]. The effect of NPs on the human body is of
increasing concern as these particles are found in an ever-expanding variety of
goods. Humans are exposed to NPs through inhalation, ingestion, dermal contact
and injection. The small size of NPs facilitates their uptake into cells as
well as transcytosis across epithelial cells into blood and lymph circulation
to reach sensitive target sites where they then persist. Previous studies have
observed the translocation of NPs along axons and dendrites of neurons as
well as access to the central nervous system and ganglia [5–7]. Therefore, the study of the potential
risks presented by NPs is immediately needed.
The lungs are continuously exposed
to environmental particles. Several studies have reported phagocytosis of
particles by lung epithelial cells. TiO2 NPs do not clear from the cells;
rather, they persist there, and their concentration increases. Analytical
transmission electron microscopy of different cell culture types showed single
TiO2
particles, small aggregates free and membrane-bound layer aggregates in
cytoplasm after exposure to TiO2 NPs. In another study, TiO2 (20–30 nm) was detected as free single particles
in cytoplasm. Exposure to TiO2 NPs leads to their accumulation in cells in
different organs where some vital proteins, such as microtubule, can interact
with them [8–10].
Although many studies have been
done on TiO2
toxicity in animal models and cell cultures [11–14],
there is little data on the interaction of TiO2 NPs with subcellular structures.
Evidence of oxidative stress responses after NPs endocytosis indicates that
research on possible cellular interactions between such particles and vital
proteins, such as microtubules (MTs), is urgently needed [15,16]. By studying
NPs’ biological and toxicological effects, its interactions with subcellular
structures, such as MTs, may be clarified.
MTs are cytoskeletal proteins that
are essential in eukaryotic cells for a variety of functions, such as cellular
transport, cell motility and mitosis. They are crucial in the
development and maintenance of cell shape; the transport of vesicles,
organelles and other components throughout cells; cell signaling; cell division
and mitosis. MTs in neurons are used to transport substances such as
neurotransmitters. MTs are formed from polymers of tubulin heterodimers (a– and b-tubulin)
that polymerize from end to end [17–19]. Agents
that interfere with MT’s assembly also interfere with their dynamics and
function, and inhibit all MT functions such as cell division and
neurotransmitter transportation [20–22].
To investigate the effects of TiO2 NPs’
intracellular interactions, we studied the effects of TiO2 NPs on
tubulin and MT protein by ultraviolet (UV) spectrophotometer and fluorescent
spectroscopy. To understand the mechanism of these reactions, repolymerization
tests were carried out. TiO2 NPs were added after tubulin polymerization
to discover whether TiO2 NPs disrupted the structure of MTs.
Fluorescent spectroscopy was used to study conformational changes in tubulin
that altered its function.
Materials and Methods
Materials
Ultrafine TiO2, with
particles averaging 20 nm, was a gift from Dr. Sarbolouki (University of
Tehran, Tehran, Iran). The particles were suspended in
piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer (Merck, Darmstadt,
Germany) to obtain an 8-mg/ml final concentration. Before use, KOH was added
to adjust the pH of the colloidal solution to 6.9. Particles were then
sonicated with a Bandelin sonicator (Bandelin, Berlin, Germany) for 3 min and
immediately added to the protein solution. EGTA, guanosine-5‘–triphosphate (GTP), ATP, glycerol and MgSO4 were acquired
from Sigma (Dorset, England).
Phosphocellulose P11 was obtained from Whatman (Florham Park, USA). All other
chemicals, such as NaCl, KOH and ANS (Merck) were of analytical grade and used
without further purification. All solutions were prepared with double distilled
water and were kept at 4 ºC before use.
Purification of tubulin
After homogenization in PEM buffer
(100 mM PIPES, pH 6.9, 1 mM EGTA, 2 mM MgSO4) and 1 mM MgATP, followed by two
cycles of temperature-dependent assembly-disassembly, MT proteins were
prepared from sheep brains. PMG (100 mM PIPES, pH 6.9, 2 mM MgSO4, 1 mM EGTA
and 3.4 M glycerol) was used as polymerization buffer. Microtubule-associated
proteins free tubulin was prepared by chromatography on phosphocellulose P11
with a slight modification of the method used by Weingarten et al [23].
Eluted tubulin fractions were stored at –70
ºC
for further study. The protein concentration was determined using the Bradford
reagent (Bio-Rad, Hercules, USA) with bovine serum albumin as standard
[24].
UV spectroscopy
Turbidimetric assay of MT and
tubulin was carried out by incubating the protein in PIPES buffer (with the
final concentration of 2 mg/ml) in cuvettes at 37 ºC in a thermostatically
controlled UV spectrophotometer (Varian, Melbourne,
Australia). Turbidity
change was measured at 350 nm. To examine the effect of NPs on polymerization,
the MT and tubulin proteins were pre-incubated with NPs at 4 ºC for 30
min, and polymerization was initiated with the addition of 1 mM GTP. The mixture
was warmed to 37 ºC.
Fluorescence spectroscopy
All fluorescence experiments were
carried out using a Varian eclipse spectrofluorometer equipped with a computer
to add and subtract spectra. Denaturation of tubulin was measured as
tryptophan emission after excitation at 295 nm. Interactions in the presence
of increasing concentrations of TiO2 NPs were carried out at 25 ºC. To
test conformational changes, 8-anilino-1-naphthalenesulfonic
acid (ANS) was used
to detect whether the TiO2-treated tubulin had an exposed hydrophobic
surface area. The excitation wavelength was 380 nm, and emission was monitored
between 450–550 nm. All measurements used 2 mM tubulin. All experiments were carried out
at 25 ºC.
Results
Inhibition of MT polymerization by TiO2 NPs
The effect of TiO2 NPs on the
polymerization of tubulin was measured as shown in Fig. 1. MT assembly
was clearly inhibited by different concentrations of TiO2 NPs in 2
mg/ml MT solution. Specifically, TiO2 NPs inhibited both the rate and
extent of MT assembly, and it had influence on the rate of MT nucleation by
increasing the time.
MT
repolymerization assay
Assembled MTs were disassembled by
cooling to 4 ºC; re-warming the solution to 37 ºC induced assembly, as in the
control. Repolymerization assays showed that depolymerized MTs made polymers
again after 30 min incubation in 4 ºC (Fig. 2).
Effect of TiO2 NPs on MT dynamics at steady
state
By adding TiO2 to a MT
solution at equilibrium point (Fig. 3, vertical arrow), a decrease in
turbidity was observed and the reaction reached a new equilibrium. The new
equilibrium was the same as the equilibrium observed for MTs treated with TiO2 NPs at zero
time (Fig. 1).
Intrinsic fluorescence spectra
To obtain structural information
at the tertiary level, intrinsic (tryptophan) fluorescence spectrum of tubulin
in the presence of different concentrations of TiO2 NPs was measured. Fluorescence
analysis indicated that the interaction of TiO2 NPs with tubulin resulted in fluorescence
quenching of surface-exposed tryptophans in tubulin. Fig. 4 shows that
the fluorescence intensity decreases with increasing quantities of TiO2 NPs.
Increasing of tubulin-bis-ANS fluorescence by TiO2 NPs
There are several low affinity sites
and one high affinity site for the apolar molecule ANS in tubulin. Tubulin-ANS
complex has a strong fluorescence and is extremely environmentally sensitive.
Therefore, it is a useful tool for probing the conformational state of the
tubulin dimer [25–27]. Tubulin-ANS fluorescence has
been used to determine the nature of interactions. Tubulin (2 mM) was incubated in the presence of various
concentrations of TiO2 NPs for 10 min at 4 ºC. ANS (50 mM final concentration) was added to the
tubulin-TiO2
NPs solution and incubated again for 7 min. Fig. 5 showed that TiO2 NPs made a
concentration-dependent increase in tubulin-ANS fluorescence. Furthermore,
incubation of tubulin with ANS before the addition of TiO2 generated
similar results (data not shown).
Discussion
Although TiO2 NPs are widely used in various
commercial products, there is insufficient knowledge about their side effects.
Studies have shown that some neurons exposed to TiO2
initiate a cellular process that can ultimately lead to cell death, and
according to Rahman et al, ultrafine TiO2 induces
apoptosis [14]. There are few reports about the cytotoxic and genotoxic effects
of TiO2 NPs. Some studies have shown an interaction between TiO2 NPs and some proteins, such as human plasma fibrinogen, but the
toxic effect of TiO2 NPs on MT protein has not yet been examined.
In this study, 0–50 mg/ml TiO2 NPs was used. We showed that TiO2 NPs inhibited tubulin polymerization and MT nucleation rate. The inhibition
of tubulin polymerization depended on the concentration of TiO2 NPs (Fig. 1). Assembled MTs were disassembled by cooling
and then reassembled by re-warming; MT repolymerization was observed (Fig. 2).
These results show that TiO2 NPs do not affect MT
repolymerization. While some agents at high concentrations induced irregular
aggregates of MTs, cooling did not appear to depolymerize these aggregates. As
such, TiO2 did not
induce aggregate formation even at high concentrations.
TiO2 NPs decreased tubulin
polymerization, and data showed that TiO2 NPs also induce MT
depolymerization and change the MT steady state equilibrium to a new
equilibrium (Fig. 3). Results showed approximately 35 mg/ml IC50 for TiO2 NPs. In the
presence of 20 mg/ml TiO2 NPs, the normal activity was 66%, and in 50
mg/ml TiO2
NPs, activity decreased to 33%. Results indicated that TiO2 affected
both soluble tubulin and tubulin in MT structure, suggesting that tubulin conformational
change led to decreased tubulin polymerization ability.
Using intrinsic fluorescent
spectroscopy, we identified changes in protein conformation involved in protein
function alteration. We used excitation wavelength at 295 nm so changing in
emission wavelength showed tryptophan environment changes. TiO2 NPs
modified the polarity in the vicinity of tryptophan residues, and we therefore
observed fluorescence quenching and the maximum blue shift of the emission
wavelength (Fig. 4). Fluorescence experiments with ANS demonstrated that
TiO2
NPs induce increases in fluorescence emission (Fig. 5). The tubulin ANS
complex’s increase in fluorescence may result from exposing some of intrinstic
tubulin’s hydrophobic pockets for ANS binding. Alternatively, binding may
induce a conformational change in tubulin leading to increased ANS binding or
tubulin-ANS fluorescence.
These results indicated that TiO2 NPs induce
conformational changes in tubulin that cause changes in tryptophan position,
moving them towards GTP binding sites in protein structures. GTP has
fluorescent quenching ability [28], so intrinsic fluorescence was decreased
and blue shift was observed. Conformational changes in protein allowed some
hydrophobic pockets to be reached, increasing tubulin-ANS fluorescence. Both
GTP and its binding site in tubulin have a crucial role in tubulin
polymerization. GTP should be hydrolyzed to guanosine-5‘-diphosphate
when tubulin wants to polymerize to MT form [18]. Fluorescence data showed
conformational changes in GTP binding sites, and turbidimetric assays
demonstrated that these changes result in the suppression of tubulin
polymerization.
In conclusion, our research has
shown that TiO2
NPs have an inhibitory effect on tubulin polymerization. In situ
experiments demonstrated that 2 mg/ml tubulin protein activity fell to 50% in
the presence of 35 mg/ml TiO2 NPs. Furthermore, the same
concentrations of TiO2 NPs that disrupted MT performance inhibited tubulin
polymerization. TiO2 NPs interact with both tubulin and MT protein, and change their
folding, which results in function alteration. The GTP binding site in tubulin
is also affected by TiO2 NPs, leading to tubulin function changes.
Tubulin is an important protein with a variety of functions, from transporting
neurotransmitters in nerve cells to cell division and cell shape maintenance
in other organs. TiO2 NPs can enter and persist in cells, where their concentration
increases and they interact with cytoplasmic proteins. Ultimately, long-term
exposure to NPs can be dangerous, and companies should be further researching
the hazards associated with NPs.
Acknowledgement
We would like to thank Dr.
Mohammad Nabi Sarbolouki of the Biomaterial Research Center (University of
Tehran, Tehran, Iran) for preparing TiO2 NPs.
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