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Indexed and abstracted in Science Citation Index Expanded and in Journal Citation Reports/Science Edition
Bratisl Med J 2019; 120 (7)
516–522
DOI: 10.4149/BLL_2019_083
EXPERIMENTAL STUDY
Mitochondrial toxicity of aluminium nanoparticles
in comparison to its ionic form on isolated rat brain
mitochondria
Arab-Nozari M
1,2, Zamani E3, Latifi A1,2, Shaki F1,2
Pharmaceutical Science Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical
Sciences, Sari, Iran. fshaki.tox@gmail.com
ABSTRACT
OBJECTIVES: The aim of this study was to evaluate the toxic effect of AlNPs on rat brain mitochondria and
compare it with that of aluminium’s ionic form.
METHODS: Mitochondria were isolated from rat brain. Isolated mitochondria were treated with normal saline
(Control) and different concentrations of aluminium ions (AlIs) and AlNPs (50, 100 and 200 μM). Then, the ef-
fect of AlNPs on electron transport chain complexes as well as various endpoints such as mitochondrial oxida-
tive damage (reactive oxygen species, lipid peroxidation, glutathione, and protein carbonyl) and mitochondrial
function were assessed. Also, apoptosis was evaluated by cytochrome c release, mitochondrial membrane
potential and swelling.
RESULTS: When compared to the control group, the exposure to AlNPs showed a marked elevation in oxida-
tive stress markers and inhibition of complex III which was accompanied by disturbance in mitochondrial func-
tion. Also, AlNPs induced a signifi cant collapse of mitochondrial membrane potential, mitochondrial swelling,
and cytochrome c release.
CONCLUSIONS: The comparison of mitochondrial toxicity markers between both forms of aluminium revealed
that the toxic effect of AlNPs on isolated brain mitochondria was substantially greater than that that caused by
AlIs, which can probably be ascribed to its higher reactivity (Tab. 1, Fig. 8, Ref. 45). Text in PDF www.elis.sk.
KEY WORDS: aluminium, nanoparticle, isolated brain mitochondria, toxicity, oxidative stress.
1Pharmaceutical Science Research Center, Hemoglobinopathy Institute,
Mazandaran University of Medical Sciences, Sari, Iran, 2Department of
Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran Uni-
versity of Medical Sciences, Sari, Iran, and 3Department of Toxicology
and Pharmacology, Faculty of Pharmacy, Guilan University of Medical
Sciences, Rasht, Iran
Address for correspondence: F. Shaki, PhD, Khazarabad road, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. postal
code: 48471-93696.
Phone: +98.9112559051
Introduction
The use of nanomaterials in various applications is growing
because of their physical, chemical and biological novel proper-
ties such as large specifi c surface area and high reaction activity
(1). Aluminium oxide nanoparticles are one of the most plenti-
fully produced nanoparticles (NPs) used in different fi elds such
as catalysis, ceramics, polymer modifi cation, heat transfer fl uids,
and waste water treatment. In addition, aluminium nanoparticles
(AlNPs) have shown vast biological applications in biosensors,
biofi ltration, drug delivery and antigen delivery for immunization
purposes (2). Therefore, there is a potential risk for discharging
AlNPs into environment. Nowadays, neurotoxicity of aluminium
(Al) has gained an increasing attention because of clinical and ex-
perimental evidence in humans and rodents (3). It has been shown
that Al can accumulates in all regions of rat brain following chronic
exposure, while reaching its maximum in the hippocampus, which
is the site of memory and learning (4, 5). Also, neurological dys-
functions that were reported in dialysis patients were probably
related to high Al concentrations in the dialysis fl uid as well as to
the use of phosphate-binding gels containing Al (6). In addition,
several studies exhibited that Al-contaminated drinking water is
a risk factor for Alzheimer’s disease (7–9).
It has been shown that the ability of NPs to induce oxidative
stress is one of the most important mechanisms involved in their
toxic effects. In fact, NPs can induce reactive oxygen species
(ROS) directly via exposure to acidic environment of lysosomes
(10) or via interacting with oxidative organelles such as mito-
chondria (11).
Previous studies showed that Al exposure is associated with
the impairment of mitochondrial functions both in vitro (12) and
in vivo (13). Mitochondria are the major source of ROS and energy
production in cells. Toxic materials can affect the electron transport
chain (ETC) and lead to increased ROS production and oxidation
of mitochondrial DNA, proteins and lipids. These events lead to
the opening of mitochondrial permeability transition (MPT) pores
and thus initiate the cell death signalling (14).
Arab-Nozari M et al. Mitochondrial toxicity of aluminium nanoparticles…
xx
517
The present study aims to provide a comprehensive analysis
of the toxic effect of AlNPs on isolated rat brain mitochondria and
compare it with that of aluminum ionic form.
Material and methods
Animals
Male Wistar rats (250–300 g), were provided from Laboratory
Animals Research Center, Mazandaran University of Medical Sci-
ences, Sari, Iran. Animals were housed in an air-conditioned room
with controlled temperature of 22±2 ºC and maintained on a 12/12-h
light/dark cycle with free access to food and water. All experimen-
tal procedures were conducted according to the ethical standard
and protocols approved by the Committee of Animal Experimen-
tation of Mazandaran University of Medical Sciences, Sari, Iran.
All efforts were made to minimize the number of animals used
and their suffering.
Mitochondrial preparation
Mitochondria were prepared from the whole brain of Wistar rats
using the differential centrifugation technique (15). Protein concen-
trations were determined by the Coomassie blue protein-binding
method using bovine serum albumin as standard (16). The isolation
of mitochondria was confi rmed by measuring succinate dehydroge-
nase (17). Isolated brain mitochondria were prepared fresh for each
experiment and used within 4 h after isolation and all steps were
strictly operated on ice. Mitochondrial protein concentration of 1
mg/ml was used for normalization of all experiments. Isolated brain
mitochondria were incubated with different concentrations of AlNPs
and aluminium ions (AlIs) (50, 100 and 200 M) for 1 h at 37 °C.
Measurement of reactive oxygen species
The ROS level was measured using dichlorofl uorescin-di-
acetate (DCFH-DA) through a Shimadzu RF5000U fl uorescence
spectrophotometer at 485 nm excitation and 520 nm emission
wavelength (18) .
For fi nding the source of ROS production in the electron trans-
fer chain in mitochondria, malate/pyruvate (substrate complex
I) and succinate (substrate complex III) were used in incubation
medium as two different substrates, while in some samples, 2
mM rotenone (inhibitor complex I), 2 mM antimycin A (inhibitor
complex III) were added (15).
Measurement of Lipid peroxidation (LPO)
The content of malondialdehyde (MDA), as a marker of lip-
id peroxidation, was determined using the dithiobarbituric acid
(TBA) method (19).
Measurement of glutathione content
The glutathione (GSH) content was determined by 5,5’-dithio-
bis-2-nitrobenzoic acid (DTNB) as an indicator. (20).
Measurement of protein carbonyl
The determination of protein carbonyl was performed by us-
ing guanidine hydrochloride reagent (21).
Assessment of mitochondrial toxicity
Mitochondrial toxicity was assessed by measuring the reduc-
tion of tetrazolium salt (MTT). (22).
Determination of the mitochondrial membrane potential (MMP)
Mitochondrial uptake of the cationic fl uorescent dye, rhoda-
mine 123, has been used to estimate the mitochondrial membrane
potential. The fl uorescence was monitored using Schimadzou
RF-5000U fl uorescence spectrophotometer at the excitation and
emission wavelength of 490 nm and 535 nm, respectively (23).
Determination of mitochondrial swelling
The mitochondrial swelling in the isolated brain mitochon-
dria was estimated through changes in light scattering monitored
spectrophotometrically at 540 nm (30 °C) (24).
Cytochrome c release assay
The concentration of cytochrome c was determined by using
the Quantikine rat/mouse cytochrome c immunoassay kit provided
by R & D Systems, Inc. (Minneapolis, Minn.).
Statistical analysis
All results are expressed as mean ± SEM. The distribution of
our data follows a normal pattern. The signifi cance of difference
between two groups was evaluated using unpaired and paired
Student’s t-test. For multiple comparisons, one-way analysis of
variance (ANOVA) was used. When ANOVA showed a signifi -
cant difference, Tukey’s post-hoc test was applied. The statistical
signifi cance was regarded when p < 0.05.
Results
Effects of AlNPs and AlIs on ROS production
As presented in Figure 1, the addition of both AlNP and AlI
to isolated brain mitochondria resulted in an increase in ROS
generation in a concentration-dependent manner. Next, the effect
of AlNPs on mitochondrial electron transfer chain was evaluated
by measuring ROS generation with complex substrates I and III.
As shown in Table 1, ROS generation was increased after adding
AlNPs in the presence of malate/pyruvate (complex substrate I)
and succinate (complex substrate III). As a result, AlNP-induced
ROS production increased more in succinate-supported mito-
chondria than that observed after adding malate/pyruvate. The
ROS production was also measured alternatively with complex
Treatment + malate/pyruvate + succinate
Control 58±6.2 54±7.1
AlNP 108±11.5* 120±8.4*
AlNP 10mM+Antimycin A 146±9.7#165±11.6#
AlNP 10mM+Rotenone 125.3±8#128.3±7.4
Values are expressed as mean±SD for three rats in each group. * Signifi cantly differ-
ent when compared to the control (p < 0.05), # signifi cantly different when compared
to AlNP-treated mitochondria (p < 0.05).
Tab. 1. Effect of complex substrate I and III and inhibitors on AlNP
-induced ROS production.
Bratisl Med J 2019; 120 (7)
516–522
518
inhibitors I and III being either present or absent.. The addition
of antimycin A (inhibitor complex III) elevated the AlNP-induced
ROS production and in contrast to the latter complex, the addition
of rotenone showed no signifi cant effect on AlNP-induced ROS
production in succinate-supported mitochondria. However, in the
presence of AINPs, rotenone induced a high rate of ROS produc-
tion in malate/pyruvate-supported mitochondria.
Effects of AlNPs and AlIs on lipid peroxidation
As shown in Figure 2, the exposure of brain mitochondria to
AlIs and AlPs increased the MDA values to a signifi cantly higher
level than was that measured in the control group (p < 0.05). Also,
the administration of AlNPs to brain mitochondria increased the
MDA values to a signifi cantly higher level than was that deter-
mined in AlI group (p < 0.05).
Fig. 1. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on reactive oxygen species (ROS) production in isolated brain
mitochondria. Data were expressed as mean ± standard error. The re-
active oxygen species production was evaluated by dichlorofl uorescein
as an indicator as described in Material and Methods. ** Signifi cantly
different from control group (p < 0.01), *** signifi cantly different from
control group (p < 0.001), ### signifi cantly different from AlI group.
Fig. 2. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on lipid peroxidation in isolated brain mitochondria. Data
were expressed as mean ± standard error. Lipid peroxidation was eval-
uated by malondialdehyde (MDA) level as an indicator as described in
Material and methods. ** Signifi cantly different from control group
(p < 0.01), *** signifi cantly different from control group (p < 0.001),
## signifi cantly different from AlI group (p < 0.01), ### signifi cantly dif-
ferent from AlI group (p < 0.001).
Fig. 3. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on glutathione (GSH) level in isolated brain mitochondria.
Data were expressed as mean ± standard error. GSH was evaluated
by DTNB level as an indicator as described in Material and Methods.
***Signifi cantly different from control group (p < 0.001), # signifi cantly
different from AlI group (p < 0.05).
Fig. 4. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on protein carbonyl concentration in isolated brain mito-
chondria. Data were expressed as mean ± standard error. Protein
carbonyl was determined by reading the absorbance at 365 nm wave
length as described in Material and Methods. **signifi cantly different
from control group (p < 0.01), ***signifi cantly different from control
group (p < 0.001), ##signifi cantly different from AlI group (p < 0.01)
Arab-Nozari M et al. Mitochondrial toxicity of aluminium nanoparticles…
xx
519
Effects of AlNPs and AlIs on glutathione levels
The GSH concentration was found to be decreased in conse-
quence of ROS formation in groups treated with AlIs and AlNPs.
However, the depletion of GSH was signifi cant after the admin-
istration of AlIs and AlNPs at all concentrations. Also, as shown
in Figure 3, AlNPs (at 200 M) signifi cantly affected the oxide
GSH level in isolated brain mitochondria as compared to that in
AlI group (p < 0.05).
Effects of AlNPs and AlIs on protein carbonyl production
In this study, a high concentration of AlIs (200 M) signifi -
cantly raised the protein carbonyl concentration in isolated brain
mitochondria (p < 0.05). The raising effect on the latter concentra-
tion can be induced also by lower concentrations of AlNPs (100
and 200 M), When comparing the effects of the same concentra-
tions of AlNPs and AlIs, namely that of 200 M, the promotion
of protein carbonyl induced by the former agent was signifi cantly
higher (p < 0.05) (Fig. 4).
Effects of AlNPs and AlIs on mitochondrial function
As compared to the control group, the administration of Al-
NPs and AlIs at concentrations 100 M and 200 M signifi cantly
decreased the function of brain mitochondria in a concentration-
dependent manner (p < 0.05). As to the comparison of the effects
of AlNPs and AlIs, we found out that at concentration of 200 M,
the former agent was signifi cantly more effective in attenuating
the mitochondrial function (Fig. 5).
Effects of AlNPs and AlIs on mitochondrial membrane potential
As shown in Figure 7, AlNPs signifi cantly increased the MMP
in a concentration-related manner (p < 0.05). Additionally, there
is a signifi cant difference between AlNP and AlI at 100 and 200
M concentrations (Fig 6).
Effects of AlNPs and AlIs on mitochondrial swelling
The data in Figure 7 indicate that whereas the addition of AlNP
at medium and high concentrations (100 and 200 M) to mito-
chondrial suspensions led to a signifi cant level of mitochondrial
swelling in a concentration-dependent manner, the suppressing
effect of AlNPs on mitochondrial absorbance at 540 nm was not
as profound. The latter effect occurred to be signifi cant only at the
high concentration of AlNPs.
Effects of AlNP and AlI on cytochrome c release
As to the quantity of cytochrome c, there were statistical-
ly signifi cant differences between the control mitochondria and
Fig. 5. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on mitochondrial function. Data were expressed as mean ±
standard error. Mitochondrial function determined by measuring the
reduction of MTT as described in Material and Methods. ** signifi -
cantly different from control group (p < 0.01), *** signifi cantly dif-
ferent from control group (p < 0.001), ## signifi cantly different from
AlI group (p < 0.01).
Fig. 6. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on mitochondrial membrane potential (MMP). Data were ex-
pressed as mean ± standard error. MMP determined by Rhodamine
123 as an indicator as described in Material and methods. * signifi -
cantly different from control group (p < 0.05), *** signifi cantly dif-
ferent from control group (p < 0.001), # signifi cantly different from AlI
group (p < 0.05), ### signifi cantly different from AlI group (p < 0.001).
Fig. 7. Effects of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on mitochondrial swelling. Data were expressed as mean ±
standard error. Mitochondrial swelling was determined by reading
the absorbance at 540 nm wave length as described in Material and
Methods. * signifi cantly different from control group (p < 0.05), ***
signifi cantly different from control group (p < 0.001).
Bratisl Med J 2019; 120 (7)
516–522
520
AlNP- and AlI-treated mitochondria, namely at concentrations of
100 and 200 M (p < 0.05). We also found out that following the
administration of AlIs and AlNPs, the average concentration of ex-
pelled cytochrome c from mitochondrial fraction was amplifi ed in
a concentration-dependent manner. In addition, the administration
of AlNPs to isolated mitochondria showed a signifi cant statistical
difference between AlNP and AlI groups, namely at concentrations
of 100 and 200 M (Fig. 8).
Discussion
This study provides a detailed evaluation of mitochondrial
toxicity of AlNPs at different concentrations. It determines their
toxic effects on isolated brain mitochondria by measuring the mi-
tochondrial oxidative damage endpoints and cell death pathway.
At the same time, it compares the effects of AlNPs with those of
aluminium’s ionic form. Nowadays, there are several reports ex-
pressing health and environmental concerns related to the use of
nanoparticles (25). AlNPs are among the most intensively produced
nanomaterials. Their use is gaining momentum in a wide range
of industries and biological applications (26). Previous studies
showed increased incidence of neuro-degeneration diseases follow-
ing exposure to Al (27). Also, oxidative stress and mitochondrial
dysfunction is the most important mechanism suggested to take
place in Al toxicity (27–29). It has been well shown that the mecha-
nism of oxidative stress is commonly responsible for toxic effects
of nanoparticles (30, 31). Mitochondria are known to be the main
source of ROS generation in cells and various studies showed that
nanoparticles of various sizes and chemical compositions could be
preferentially localized in mitochondria while promoting ROS pro-
duction (32, 33). For example, Wang et al reported the deposition
of CuO nanoparticles in the mitochondria of human lung epithelial
cells, which subsequently led to enhanced ROS production (34).
As shown in results, the exposure to both AlNPs and AlIs in-
duced ROS generation in a concentration-dependent manner. Previ-
ous studies revealed that complexes I and III are the main sources
of ROS production in the mitochondrial respiratory chain (35). As
evident in results, the addition of AlNPs signifi cantly increased
ROS production in both succinate and malate/pyruvate-supported
brain mitochondria. It is worth mentioning that ROS production
in succinate-supported mitochondria was more pronounced than
in the malate/pyruvate-supported group. Also, the addition of an-
timycin A (an inhibitor of electron transport at the ubiquinone-cy-
tochrome b region) induced an elevation of ROS production while
rotenone did not inhibit the succinate-supported ROS production.
Evidence suggests that the ubiquinone-cytochrome b region in
complex III can be considered as the main site of ALNP-induced
ROS production in isolated brain mitochondria.
On the other hand, the brain has a high mitochondrial con-
tent and low level of antioxidant enzymes. Therefore, any agent
that disrupts the brain mitochondrial function can lead to exces-
sive ROS production. Therefore, at fi rst, we evaluated the mito-
chondrial function after the exposure to both forms of aluminium
(nanoparticles and ions) by means of measuring MTT. As shown
in results, AlNPs induced mitochondrial toxicity in all concentra-
tions and it was more pronounced than that induced by AlIs, thus
demonstrating high mitochondrial toxicity of AlNPs.
In addition, the mitochondrial electron transfer chain is in-
volved in energy as well as ROS production in cells. Interestingly,
the release of cytochrome c from mitochondria can be the start-
ing point of programmed cell death (apoptosis) signalling (36). It
is well known that xenobiotics can promote ROS production via
disruption of mitochondrial electron transfer chain. In addition,
the brain is highly dependent on oxidative phosphorylation as its
energy source compared to other cells in the body. Therefore, any
agent that impairs the mitochondrial electron transfer can disturb
ATP production as well as the normal pathway of electrons in mi-
tochondria, which leads to increased ROS production and fi nally
incurs oxidative damage to neurons (37). Also, several studies
reported the important role of mitochondrial dysfunction in many
neurodegenerative diseases (38, 39). Results indicated that AlNPs
have a more toxic effect than AlIs on isolated brain mitochondria
in inducing oxidative stress parameters such as ROS, LPO, and
protein carbonyl formation, including GSH oxidation.
It was previously reported that AlNP can promote oxidative
stress in various biological systems. For example, M’rad et al
showed that Al2O3 nanoparticles could induce oxidative damage
in the hipocampus by increasing he levels of MDA as well as by
decreasing the levels of antioxidant defence enzymes such as su-
peroxide dismutase and gluthation peroxidase (40).
In fact, it is believed that compared to the bulk material, NPs
could induce more ROS production as a consequence of their large
surface area (41). Also, several studies evaluated the ability of
metal oxide NPs to induce oxidative stress in various experimen-
tal models, in which the amounts of ROS observed in metal oxide
NPs’ suspensions were higher than in ionic formulations (42).
Fig. 8. Effect of aluminium ions (AlIs) and aluminium nanoparticles
(AlNPs) on cytochrome c release. Cytochrome c release was measured
using cytochrome c assay kit as described in Material and Methods.
Values represented as mean± standard error. * signifi cantly different
from control group (p < 0.05), *** signifi cantly different from control
group (p < 0.001), # signifi cantly different from AlI group (p < 0.05),
## signifi cantly different from AlI group (p < 0.01).
Arab-Nozari M et al. Mitochondrial toxicity of aluminium nanoparticles…
xx
521
In this study, the exposure of isolated brain mitochondria to
both Al forms signifi cantly increased the lipid peroxidation. It has
been shown that oxidation of lipid membrane results in disrup-
tion of mitochondrial membrane and consequently to the collapse
of MMP and cytochrome c release (43). On the other hand, GSH
oxidation was observed after exposure to different concentrations
of AlNP in isolated brain mitochondria. Not only are the reduced
levels of GSH in mitochondria considered as the main antioxidant,
they are also crucial for the maintenance of the reduced form of
thiol groups in mitochondrial membrane proteins (44) Oxidation
of these thiol groups facilitates conformational changes occurring
in the pore complex that induces the mitochondrial permeability
transition (MPT). The opening of MPT pores permits unlim-
ited movement of water and solutes and mitochondria undergo
structural changes referred to as mitochondrial swelling (15).
Accordingly, we investigated the effect of AlNPs on MMP and
mitochondrial swelling. As shown in the results, AlNPs decreased
MMP and induced mitochondrial swelling in a concentration-de-
pendent manner. Interestingly, AlNPs showed signifi cantly more
toxic effects in inducing MMP and mitochondrial swelling than
AlIs. The induction of MPT can be associated with the release of
apoptogenic factor such as cytochrome c into cytosol which then
initiates both necrosis and apoptosis mechanisms (45). Accord-
ingly, in comparison with AlIs, the AlNPs induced a signifi cantly
higher release of cytochrome c from isolated brain mitochondria.
The latter fact emphasises the confi rmed mitochondrial toxicity
of these nanoparticles.
Conclusion
This study showed that the mechanism of AlNPs’ toxicity in
isolated brain mitochondria takes place via the inhibition of elec-
tron transfer chain. Also, AlNPs increased the inner membrane
permeability and mitochondrial membrane potential dissipation,
thus leading to a release of apoptogenic factors. In overall, most of
the mitochondrial toxicity endpoints in the present study showed
that the effect of AlNPs was more toxic than that of aluminium’s
ionic form, which can be explained by high reactivity of NPs due
to their large surface area.
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Received February 24, 2019.
Accepted April 8, 2019.
