Titanium Dioxide (P25) Produces
Reactive Oxygen Species in
Immortalized Brain Microglia (BV2):
Implications for Nanoparticle
T H O M A S C . L O N G ,‡N A V I D S A L E H ,§
R O B E R T D . T I L T O N ,|G R E G O R Y V .
L O W R Y ,§A N D B E L L I N A V E R O N E S I *, ⊥
Department of Environmental Sciences and Engineering,
School of Public Health, University of North Carolina,
Chapel Hill, North Carolina 27599-7431, Department of Civil
and Environmental Engineering, Department of Chemical
Engineering, and Department of Biomedical Engineering,
Carnegie Mellon University, Pittsburgh, Pennsylvania, 15213,
Neurotoxicology Division, National Health and
Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina, 27711
Concerns with the environmental and health risk of
widely distributed, commonly used nanoparticles are
increasing. Nanosize titanium dioxide (TiO2) is used in air
and water remediation and in numerous products
designed for direct human use and consumption. Its
effectiveness in deactivating pollutants and killing
microorganisms relates to photoactivation and the resulting
free radical activity. This property, coupled with its
multiple potential exposure routes, indicates that nanosize
TiO2could pose a risk to biological targets that are
sensitive to oxidative stress damage (e.g., brain). In this
study, brain microglia (BV2) were exposed to a physico-
chemically characterized (i.e., dispersion stability, particle
size distribution, and zeta potential) nanomaterial, Degussa
P25, and cellular expressions of reactive oxygen species
measured in cell culture media and physiological buffer
were -11.6 ( 1.2 mV and -9.25 ( 0.73 mV, respectively.
P25 aggregation was rapid in both media and buffer
with the hydrodynamic diameter of stable P25 aggregates
ranging from 826 nm to 2368 nm depending on the
concentration. The biological response of BV2 microglia
to noncytotoxic (2.5-120 ppm) concentrations of P25 was
a rapid (<5 min) and sustained (120 min) release of
reactive oxygen species. The time course of this release
suggested that P25 not only stimulated the immediate
“oxidative burst” response in microglia but also interfered
with mitochondrial energy production. Transmission
electron microscopy indicated that small groups of
nanosized particles and micron-sized aggregates were
aggregates after 6 and 18 h exposure to P25 (2.5 ppm).
Cell viability was maintained at all test concentrations (2.5-
120 ppm) over the 18 h exposure period. These data
indicate that mouse microglia respond to Degussa P25
with cellular and morphological expressions of free radical
The novel physical and chemical properties of engineered
nanoparticles make them attractive for use in medical,
Although hundreds of tons of nanoparticles enter the
environment annually, little is known of their interactions
are not completely benign to biological and environmental
targets (1). Such reports have the potential to transform the
aware of this possibility, has not only articulated the need
for research on the environmental and health effects of
testing should obviously begin with those that pose the
dioxide (TiO2) is a reasonable candidate for study since it is
widely used in manufacturing (5, 6), in the environment to
in consumer products (e.g., toothpastes, sunscreens, cos-
metics, and food products, etc.) (12, 13). Such widespread
use and its potential entry through dermal, ingestion, and
inhalation routes suggest that nanosize TiO2poses consider-
able exposure risk to humans, livestock, and eco-relevant
species (e.g., fish, daphnia, nematodes, and plankton).
Although exposure to different TiO2 particle sizes and
formulations (nanosize, pigment grade, and surface coated)
has produced only marginal results in rodents (14-16),
oxidative stress (OS)-mediated toxicity in diverse cell types,
(19), epithelia (20), skin fibroblast (21), liver (22), alveolar
macrophages (23-25), and Salmonella bacteria (26). The
potential neurotoxicity of TiO2 in culture has yet to be
Several studies report that inhaled or injected nanopar-
ticles enter systemic circulation (27-29) and migrate to
various organs and tissues, raising concern that they may
cause damage to biological systems through OS pathways
(30). The brain is especially vulnerable to OS damage, and
recent studies indicate that nanosize particles can cross the
(CNS) of animals (29, 32, 33). In the CNS, OS is largely
mediated by the microglia, a macrophage-like, phagocytic
damaging, exogenous stimuli (e.g., xenobiotics, chemicals,
of events that includes an increase in metabolic activity, a
change in cell shape and size, and cytoplasmic engulfment
(i.e., phagocytosis) of the offending stimuli. During phago-
cytosis, the plasma membrane of the phagocyte surrounds
* Corresponding author phone: (919) 541-5780; fax: (919) 541-
4849; e-mail: email@example.com.
†This paper is part of a focus group on Effects of Nanomaterials.
‡University of North Carolina.
§Department of Civil and Environmental Engineering, Carnegie
|Department of Chemical Engineering and Department of
Biomedical Engineering, Carnegie Mellon University.
⊥U.S. Environmental Protection Agency.
43469ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 14, 200610.1021/es060589n CCC: $33.50
2006 American Chemical Society
Published on Web 06/07/2006
the foreign substance, invaginates, and internalizes the
membrane bound material (i.e., phagosome). The signaling
mechanisms for the subsequent oxidative burst are not well
defined, but invagination of the plasma membrane appears
burst. This activation results in the immediate production
oxygen species (ROS) including hydrogen peroxide (H2O2),
hydroxyl radicals (OH•), and peroxynitrites that can destroy
the offending stimuli through OS pathways. The excess O2•-
and its dismutation product H2O2are either retained within
cytoplasmic granules, providing an immediate supply of
plasma membrane where they can potentially damage the
proteins, lipids, and DNA of neighboring cells, especially
neurons (36, 37). Microglia-mediated neuronal damage
through OS pathways has been proposed to underlie neu-
rodegenerative diseases such as Amyotrophic Lateral Scle-
rosis, Parkinson’s, and Alzheimer’s (38-40).
Although the oxidative burst is the major source of ROS
in the activated microglia, O2•-are also generated as
bioenergetics) in the quiescent cell. Mitochondria produce
cellular energy (ATP) by transferring electrons along a series
of enzymatic complexes (Complex I-IV) known as the
Electron Transport Chain (ETC) (41). During this transfer,
This is the fate of approximately 1% of all oxygen consumed
and defines the ETC as the major producer of ROS in
nonphagocytic cells and tissues. Although, the levels of O2•-
generated from ETC are relatively low and efficiently
neutralized by matrix-located antioxidant enzyme systems
(i.e., endogenous scavengers), the rate of O2•-generation
can be significantly increased if one or more of the ETC
enzyme complexes is inhibited.
is quickly reduced to the more stable H2O2 by superoxide
dismutase, an enzyme located in intracellular microsomes,
notably ferrous iron, superoxides can convert to the highly
reactive hydroxyl radical (OH•) via the Fenton reaction. The
hydroxyl radical is considered the primary agent of lipid
Several recent articles have prescribed a formal protocol
for nanotoxicy testing (4, 42) which suggests that physico-
chemically described nanoparticles should first be tested in
cultures of relevant target cells for the production of ROS.
This is followed by toxicity testing in more complex cul-
tures, in eco-relevant species, and ultimately in animals. In
the present study, we follow this format by first mea-
suring the ROS response of microglia (BV2) exposed to
were chosen in view of their responsiveness to xenobiotics
and their pivotal role in OS-mediated neurodegeneration.
The BV2 is an immortalized cell line that responds to
pharmaceutical agents, particulates, and environmental
chemicals with characteristic signs of OS (43-45). In the
present study, BV2 were exposed to noncytotoxic doses of
P25, and the immediate and prolonged release of ROS was
measured over a 120 min exposure period using various
used to document the phagocytic response of the microglia
to P25 exposure. Physicochemical properties of P25 (e.g.,
particle size distribution (PSD), zeta (?) potential, and
dispersion stability) were measured in culture media and
physiological buffer at time points relevant to the biological
Materials and Methods
Physicochemical Characterization. Commercial grade, nano-
size TiO2(Degussa Aeroxide P25) was a gift from Degussa
of P25 have been previously reported (6, 46, 47), they were
remeasured in the present study in the vehicles used to
Basic Salt Solution (HBSS) and Dulbecco’s Modified Eagle’s
Medium (DMEM). Both HBSS and DMEM are high ionic
strength solutions with high concentrations of divalent
cations that cause P25 to aggregate. DMEM also contains
low amounts of glucose and amino acids (Supporting
of P25 were measured in a slurry prepared from a 1 g/L
suspension of P25 in HBSS or DMEM. Slurry concentrations
(5-120 ppm) similar to those used to expose cells were used
immediately after 1 min sonication. The particle size
distribution was measured by dynamic light scattering
(Malvern Zeta Sizer Nano ZS, Southborough, MA) over a 3
h period to monitor the growth of the aggregates and to
determine a stable aggregate size. For calculating particle
size (volume average), the refractive index of the anatase
form of TiO2(nD) 2.49) (48) was assumed. The ?-potential
was determined in both HBSS and DMEM from electro-
phoretic mobility (EM) measurements of a 30 ppm slurry
(Malvern Zeta Sizer Nano ZS, Southborough, MA). The
Helmholtz-Smoluchowski equation was used to correlate
surface area was measured using a Nova 2200e BET surface
were degassed in helium for 1 h at 150 °C prior to analysis
(49). The dispersion stability, operationally defined as the
resistance to sedimentation, was determined by measuring
The optical density (λ ) 508 or 450 nm) of the suspension
was monitored for 18 h in a UV-visible spectrophotometer
(Varian, Palo Alto, CA). There was a linear relationship
between TiO2 concentration and UV response for all TiO2
Cell Culture Maintenance and Exposure. BV2 microglia
were grown in 225-cm2cell culture flasks in DMEM supple-
streptomycin (ATCC, Manassas, VA). After reaching 85%
confluency, cells were transferred to Corning 96-well plates.
To minimize light scatter during the spectrophotometric
readings, cells were plated in clear-bottom, black (fluores-
(Corning Inc., Corning, NY) and examined with a Molecular
Devices (Sunnyvale, CA) Spectramax Gemini EM (fluores-
cence) or Lmax II 384 plate reader (chemiluminescence).
measures of intracellular ATP taken after 1, 6, and 18 h
in 10× stock concentrations in HBSS and exposed to the
cells immediately before spectrophotometric readings were
Probes. Fluorescent and chemiluminescent probes were
chosen to measure the immediate generation of H2O2
resulting from the oxidative burst and that resulting from
interference with mitochondrial ETC (50). All probes were
purchased from Molecular Probes (Eugene, OR) except for
the chemiluminescent assay CellTiter-Glo (Promega, Madi-
son, WI). The concentrations and incubation times for each
legends. For each fluorescent assay, cells, exposed to the
fluorescent probe (i.e., “loaded”), were washed with HBSS
to remove any extracellular probe from the cell’s external
VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94347
environment. In this way, only intracellular levels of ROS
were measured. Because it has been reported that P25 can
be photoactivated by visible light (51), all cell culture
were done under darkroom ”safe-lights”.
The production of intracellular H2O2 generated from
the oxidative burst was measured at 3 5-min intervals with
Image-iT and at 5 15-min intervals with OxyBURST H2HFF
Green BSA. The production of H2O2resulting from interfer-
ence with the mitochondria’s ETC was measured using the
intervals over a 0-120 min exposure. Changes in the
mitochondrial membrane potential, an indicator of the
membrane’s net charge, were monitored with MitoTracker
Red over 20-min intervals. Data were collected over a 120
min period for each assay. Intracellular levels of ATP, an
index of cell viability, were measured at 1, 6, and 18 h
postexposure with a lucerifase-based chemiluminescence
Statistics. All data were collected using SoftMax Pro 4.8
software (Molecular Devices, Sunnvale, CA). Graphing and
statistics were performed with either Excel 2003 (Microsoft,
was graphed to show a time-course response. At least five
time points are depicted for each assay. Data were analyzed
using an unpaired two-tailed Student’s t-test to determine
the lowest statistically significant concentration relative to
time point at which a statistically significant difference was
in the figure legends.
Transmission Electron Microscopy (TEM). BV2 cells,
grown to 85% confluency in 6-well Costar plates (Corning,
Inc., Corning, NY), were exposed to P25 particles (2.5 ppm)
in 1% reduced serum DMEM for 6 and 18 h. After exposure,
cells were washed in warm HBSS to remove all noninter-
nalized particles and fixed overnight in cold 4% cacodylate-
buffered glutaraldehyde (Poly Scientific, Bayside NY). Cells
a Philips CM12 electron microscope.
Results and Discussion
as the N2-BET specific surface area, the ?-potential, and
particle aggregate size. These properties were measured in
cell culture media (DMEM) and the physiological buffer
with the manufacturer’s data and with previous reports (46,
47, 49, 53). The ?-potentials of P25, measured in DMEM (pH
) 7.5) and HBSS (pH ) 7.6) were -11.6 ( 1.2 mV and -9.25
( 0.73 mV, respectively. The negative surface charge at
physiological pH (pH ) 7.5-7.6) was consistent with the
commonly reported isoelectric point of pHIEP ) 6.4 for
Degussa P25 (47, 49, 53).
Degussa P25 is a mixture of the rutile and anatase forms
of TiO2 (70% anatase/30% rutile) with a reported primary
fluids with approximate ionic strengths of 155 mM and 166
mM, respectively, and both contain high concentrations of
the monovalent cations Na+and K+(160 mM) and the
divalent cations Ca2+and Mg2+(2 mM) as shown in Tables
S1 and S2 (Supporting Information). Aggregation continued
for 20-45 min after sonication (1 min), until a steady-state
stable aggregate size formed (Figure S1). The steady-state
aggregate size increased from 826 to 2368 nm as the
concentration increased from 5 to 120 ppm (Table 1). The
as these fluids had nearly identical ionic strength and
composition. The amino acids and serum present in re-
duced-serum DMEM (but not in HBSS) did not significantly
affect the rate of aggregation or the size of the formed
During the 6- and 18-h exposure times described in the
TEM studies, BV2 cells (which adhere to the bottom of the
cell culture plate/well), were exposed by particle diffusion
or sedimentation of aggregates. Such conditions are char-
acteristic of cell culture studies and could produce higher
particle exposures than that described by the concentration
(54). The sedimentation of P25 aggregates was measured in
low serum media at 6- and 18-h exposures to estimate the
fraction of particles that settled onto the cells during the
TEM exposure (Figure S2). By the end of the 18-h exposure
DMEM. The higher sedimentation rate seen at higher con-
at the higher test concentrations of P25 (see previous para-
and the concentrations/times needed for significant ROS
production is described in the Biological Effects section
Biological Effects. O2•-is an unstable molecule which is
quickly reduced to the more stable and measurable H2O2.
Kinetic analysis of H2O2 production measured both an
immediate production of ROS as generated by the oxidative
burst (Figure 1A and B) and by a later release caused by
disruption of mitochondrial ETC (Figure 2A). Microglia
responded to P25 (g10 ppm) with rapid (<3-5 min)
concentration-dependent formation of H2O2 as measured
with Image-iT and OxyBURST (Figure 1A and B). In contrast
to the rapid increases in H2O2 generated by the oxidative
burst (Figure 1A and B), significant release of ROS did not
occur until 60 min postexposure (g20 ppm) as measured by
MitoSOX (Figure 2A). MitoTracker Red is a potential-
dependent dye. Increases in MitoTracker Red staining
potential (i.e., hyperpolarization). The fluorescent MitoTrack-
er molecules, which are initially distributed throughout the
cytoplasm, accumulate on negatively charged (anionic)
membranes. The increased fluorescence suggests a steady
exposure concentration-dependent accumulation of net
(Figure 2B). This plausibly resulted from P25’s inhibition of
one or more of the ETC enzymatic complexes (Complex I
and III). Depolarization of the mitochondrial membrane is
normally associated with a reduction of membrane perme-
TABLE 1. Initial and Steady State Particle Geometric Mean
Hydrodynamic Diameter for Degussa P25 TiO2in HBSS or
hydrodynamic diameter (nm)
500 ( 3b
590 ( 3
865 ( 32d
992 ( 19
1570 ( 20
1350 ( 30
826 ( 69c
1164 ( 85
1284 ( 57d
1316 ( 68
2090 ( 180
2368 ( 163
aThe chemical composition of HBSS and DMEM is provided in the
Supporting Information.bErrors represent one standard deviation
based on 3 replicate measurements.cAverage particle size measured
after reaching steady state (∼30 min). Error bars represent 1 standard
deviation of these values.dMeasured in DMEM.
43489ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 14, 2006
the initiation of necrotic or apoptotic pathways (36, 41).
in response to P25. This was supported by measures of
intracellular ATP (Figure S4) which were maintained at all
confirming that all cellular expressions of ROS release were
measured in actively respiring and viable microglia.
A correlation between the size of P25 aggregates (Figure
S2) and the “effective” exposures (i.e., earliest time point
and lowest concentration resulting in significant ROS pro-
duction) was examined. ROS generated from the oxida-
tive burst (Image-iT, OxyBURST) indicated that signifi-
cant increases occurred at 3-5 min in response to g10 ppm
P25. At this time and concentration, P25 aggregate size was
∼750-800 nm. Significant increases in ROS generated from
the mitochondrial ETC occurred >60 min exposure, in
nm. The relationship between “effective” exposures and
toxicity studies since aggregate size can have a profound
impact on the cell’s uptake of particles and their response
to that uptake.
TEM indicated that after 6 h exposure, P25 aggregates
were phagocytized in small clusters and internalized within
the microglia’s cytoplasm where they aggregated into
electron-dense ∼0.5-2 micrometer clumps (Figure 3A and
B). The appearance of small (100-300 nm) and large
(800-2000 nm) particle aggregates was consistent with the
recorded particle size distribution data (Figure S2). Phago-
cytes (e.g., macrophages, microglia) preferentially engulf
particles in the 1-3 µm range (55). In contrast, nanosize
particles (<100 nm) are not phagocytized in the strict sense,
rather, they are engulfed through other, nonspecified mech-
interactions (56). Ultrastructural evidence of swollen and
disrupted mitochondria lying in close proximity to the
aggregates of P25 was recorded after 18 h exposure (Figure
that the mitochondrial membrane permeability transition
pore has begun to open and apoptotic or necrotic signals
initiated (36, 37). Still, the microglia remained viable in
response to P25 exposure, since ATP intracellular levels
In summary, these data demonstrate that nanosize P25
particles stimulate microglia to produce ROS through the
ETC. Nevertheless, the microglia remained viable at all
to ROS damage (38, 57). Whether the microglia’s release of
in this study, but pilot data indicate that P25 stimulates
apoptotic pathways in cultured neurons at concentrations
(59, 60), but in agreement with others (20), photoactivation
of P25 does not appear necessary to stimulate ROS in
iT and OxyBURST. (A) Cells were incubated (30 min, 37 °C) in 25
µM Image-iT + HBSS/Ca2+/Mg2+, washed, and exposed to P25
(5-120 ppm). Significant (p < 0.05) increases of fluorescence
(measured at 495/529 nm) relative to its baseline occurred in cells
exposed to g10 ppm P25 at 3 min, and continued throughout the
30 min recording time. (B) Cells were incubated (30 min, 37 °C) in
10 µg/mL OxyBURST in reduced-serum media, washed, and then
exposed to P25 (2.5-120 ppm). Significant (p < 0.05) increases of
fluorescence (measured at 508/528 nm) occurred in cells exposed
to g10 ppm at 5 min and continued to increase throughout the 120
min recording period.
FIGURE 2. (A) Increases in O2-were measured by the fluorescent
probe MitoSOX Red. Cells, incubated in 2 µM MitoSOX (10 min, 37
°C) showed significant (p < 0.05) increases in fluorescence
(measured at 510/580 nm) after 60 min exposure to g20 ppm P25,
and fluorescence continued to increase for 150 min postexposure.
(hyper-polarization) relative to its baseline control after 20 min
exposure to g80 ppm P25 which continued for >100 min.
VOL. 40, NO. 14, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY94349
and surface area) of “incidental” nanosize particles such as
toxicity. It is plausible that similar physical properties of
engineered nanoparticles could affect biological targets
through OS or more novel toxicity pathways (4, 61, 62).
Defining the causal mechanism(s) linking those physical
properties with their biological effects should be a primary
focus of nanotoxicity studies. Studies on the inflammatory
toxicity associated with nanosize particulate matter offer
some insights into possible mechanisms. Nanosize PM
particles appear to mediate toxicity through OS pathways.
has been related to the large surface area of the ultrafine
correlated with the negative surface charge carried by the
the membrane of microglia and macrophages are possible
target sites that could react to negatively charged nanopar-
receptors by the negative surface charge carried on PM
particles stimulates OS inflammatory pathways and the
subsequent release of inflammatory cytokines from both
respiratory epithelial cells (65) and BV2 microglia (66).
Scavenger receptors, found on microglia and macrophages
(67), are also sensitive to repeating patterns of charge that
may be found on ordered crystalline metal oxide nanopar-
ticles. Such receptors have been implicated in mediating
cytotoxicity in alveolar macrophages exposed to TiO2(68).
In the present study, the negative zeta potential of P25
particles and the ordered arrangement of charged O-sites
on their surface could be activating either type of receptor
will examine the causal relationship between the surface
charge of nanoparticles and OS mediated events in neurons
and other cell types to better understand how the physical
We acknowledge the kind gift of P25 from Degussa Inc.
(Parsippany, NJ) and also thank Dr. John Hong, National
Institute of Environmental Health Sciences, RTP, NC for the
of Martin Schneider, Chapel Hill, NC, and the expert
preparation of electron microscopy samples by Wallace
Ambrose, School of Dentistry, University of North Carolina,
Chapel Hill, NC. This document has been reviewed by the
National Health and Environmental Effects Research Labo-
that the contents reflect the views of the Agency, nor does
mention of trade names or commercial products constitute
the endorsement of recommendation for use.
Supporting Information Available
Additional information on the composition of HBSS and
DMEM (Tables S1 and S2) and the physicochemical mea-
surements of P25 (Figures S1-S3); viability data (ATP
intracellular measures) taken over an 18 h exposure period
(Figure S4). This material is available free of charge via the
Internet at http://pubs.acs.org.
FIGURE 3. (A) Quiescent microglia are large, 8-10 micrometer phagocytic cells with many oval-shaped mitochondria visible in their
cytoplasm. (B) An early (6 h exposure) response of microglia to 2.5 ppm P25 was the elaboration of numerous pseudopodia which engulfed
small groups of electron-dense particles (*). (C) Within 18 h postexposure, multiple vacuoles containing P25 aggregates were seen in
proximity to pale-staining, swollen mitochondria. (D) Higher magnification showed swelling and disruption of mitochondria lying in close
proximity to the aggregates. Image magnification: (A) 4400×; (B) 11 400×; (C) 4400×; (D) 18 000×.
43509ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 14, 2006
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Received for review March 13, 2006. Revised manuscript
received April 28, 2006. Accepted May 3, 2006.
43529ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 14, 2006