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Toxicity of nano zinc oxide to mitochondria

Article in Toxicology Research · August 2012

DOI: 10.1039/C2TX20016C

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Toxicity of nano zinc oxide to mitochondria
Jia-han Li, Xiao-rong Liu, Yue Zhang, Fang-fang Tian, Guang-yuan Zhao, Qiu-li-yang Yu, Feng-lei Jiang*
and Yi Liu*
Received 27th February 2012, Accepted 24th June 2012
DOI: 10.1039/c2tx20016c

Zinc oxide nanoparticles (ZnO NPs) are increasingly applied in a diverse array of industrial and
commercial products. Therefore, it is urgently required to characterize their toxic behavior. ZnO NPs have
Published on 26 June 2012. Downloaded on 24/02/2014 14:07:27.

been reported to induce toxic effects at the levels of the individual organism, tissue, cell and DNA.
However, little is known about the potential impacts of ZnO NPs at a subcellular level. In the present
work, we investigated the toxicity of ZnO NPs to the isolated rat liver mitochondria. We found that
treatment of mitochondria with ZnO NPs resulted in collapse of mitochondrial membrane potential (Δψ),
swelling, depression of respiration, inner membrane permeabilization to H+ and K+, alterations of
ultrastructure, release of cytochrome c, generation of reactive oxygen species (ROS), and Zn2+ liberation
from ZnO NPs. These results suggested that ZnO NPs can increase the inner membrane permeability and
impair the respiratory chain, thus leading to energy dissipation, oxidative stress and even apoptosis. This
putative mechanism helps us learn more about the toxicology of this nanomaterial.

1. Introduction impairs mitochondrial function and leads to apoptosis in cancer

cells.11 Escherichia coli treated with ZnO NPs exhibited signifi-
Nanoparticles are sized between about 1 and 100 nm, showing cant DNA damage, including DNA unwinding and fragmenta-
many properties that are not seen in their bulk counterparts.1 They tion.12 However, little is known about the potential impacts of
are increasingly applied in a diverse array of industrial and com- ZnO NPs at a subcellular level.
mercial products, such as fillers, opacifiers, catalysts, semiconduc- Mitochondria, the primary source of cellular energy, are orga-
tors, cosmetics, microelectronics, drug carriers, clothes, and sports nelles found in almost all eukaryotic cells. Meanwhile, they play
goods.1–3 Especially, metal oxide nanoparticles are very impor- a determinant role in cell signaling and apoptosis.13–17 However,
tant, because they are widely used in water-treatment agents, because of their exquisite structures and complicated functions,
materials for solar batteries and automobile catalytic converters, mitochondria are the primary target for xenobiotics.18 Malfunc-
cosmetics, sunscreens, self-cleaning coatings and textiles.4 tioning mitochondria may present insufficient energy production,
However, recently it has been shown that many metal oxide nano- exothermic oxygen combustion and free radical emission.13,19 In
particles may pose potential risks to human health and other a word, mitochondrial dysfunctions lead to various diseases.15
organisms.4–6 Therefore, it is urgently required to understand how So far, most researches regarding the toxic effects of ZnO NPs
metal oxide nanoparticles can interact with biological system. on mitochondria were performed by tracking the release from or
Large amounts of ZnO nanoparticles (ZnO NPs) have been entrapment in mitochondria of fluorescent dyes within cultured
widely used in many fields, including sunscreen products, cos- cells.20 However, this technique has some limitations. For
metics, pigments, industrial coatings, plastic additives, semi- example, measurements must be taken within a relatively short
conductors, textiles, and antibacterial agents.6,7 Previous time.21 The direct effects of ZnO NPs on mitochondrial inner
toxicological studies showed that ZnO NPs are toxic at organ- membrane permeabilization, bioenergetics, ultrastructure and
ism, cell and molecular levels. For example, ZnO NPs showed swelling cannot be provided by this technology. In order to have
acute toxicity to zebrafish partly due to the release of zinc ions.8 a more thorough understanding of their toxicity on mitochondria,
Experiments using freshwater alga Pseudokirchneriella subcapi- it is necessary to conduct some experiments on isolated mitochon-
tata revealed that their toxicity was solely attributed to dissolved dria. Herein, we used isolated rat liver mitochondria to investigate
zinc.9 Oxidative stress may play a primary role in inducing the the immediate effects of ZnO NPs on mitochondrial functions and
cytotoxicity of ZnO NPs in human colon carcinoma cells.10 Dis- try to address the possible mechanisms of their toxicity.
solution of ZnO NPs results in elevated levels of Zn2+ which
2. Experiments
State Key Laboratory of Virology and Key Laboratory of Analytical
Chemistry for Biology and Medicine (Ministry of Education), College of 2.1 Chemicals
Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,
P.R. China. E-mail: fl[email protected], [email protected]; Fax: ZnO NPs (100 nm), bovine serum albumin (BSA), rotenone,
+86-027-68754067; Tel: +86-027-68756667 oligomycin, tetramethylrhodamine ethyl ester (TMRE),

This journal is © The Royal Society of Chemistry 2012 Toxicol. Res., 2012, 1, 137–144 | 137
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adenosine diphosphate (ADP), 2,4-dinitrophenol (DNP) and 2.6 Measurement of mitochondrial swelling
valinomycin were purchased from Sigma (St. Louis, MO). All
other reagents were of analytical reagent grade, and all solutions Mitochondrial swelling was measured by alterations of absor-
were prepared with deionized distilled (DI) water. DI water was bance at 540 nm.27 Mitochondria (0.5 mg ml−1) was suspended
prepared from a Milli-Q-RO4 water purification system in 2 ml solution C (200 mM sucrose, 10 mM Tris-Mops, 1 mM
(Millipore). Pi, 10 μM EGTA-Tris, 2 μM rotenone, and 3 μg ml−1 oligo-
mycin, pH 7.4). Spectra were recorded at room temperature on a
UNICO 4802 double beam spectrophotometer (Dayton, NJ).
2.2 Preparation of ZnO NPs suspension Ca2+ (1 μM) were added to the buffer before injecting ZnO NPs.
Similar experiments were conducted to test mitochondrial
A stock suspension was prepared by suspending 80 mg of ZnO inner membrane permeabilization to H+ and K+ in potassium
NPs in 8 ml of H2O containing 4 mg BSA.22,23 The stock acetate (KAc) and potassium nitrate (KNO3) medium, respec-
suspension was sonicated for 10 min before used.24 tively. The KAc medium contained 135 mM KAc, 5 mM
HEPES, 0.1 mM EGTA, 0.2 mM EDTA, 2 μM rotenone, and
2.3 Characterization of ZnO NPs 1 μM valinomycin ( pH 7.4), and the KNO3 medium contained
135 mM KNO3, 5 mM HEPES, 0.1 mM EGTA, 0.2 mM EDTA,
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For scanning electron microscopy (SEM), ZnO NPs were and 2 μM rotenone ( pH 7.4).28
untreated before scanned by a JSM-6700F FE-SEM (JEOL Ltd,
Tokyo, Japan).
For transmission electron microscopy (TEM), ZnO NPs were 2.7 Measurement of mitochondrial respiration
dispersed in H2O, drop-coated on copper TEM grids and
Oxygen consumption of isolated mitochondria was measured
scanned by a Tecnai G2 TEM at 200 kV (JEOL Ltd, Tokyo,
with a Clark-type oxygen electrode in a 1 ml thermostated,
Japan).
water-jacketed, closed chamber with magnetic stirring.29 The
incubation buffer contained 125 mM sucrose, 65 mM KCl,
2.4 Isolation of mitochondria 2.5 mM MgCl2, 5 mM KH2PO4, 5 mM HEPES, and 3 μM
rotenone ( pH 7.2). State 4 respiration was considered upon
Wistar rats (170–250 g) were purchased from Hubei Research addition of 5 mM succinate as the energizing substrate.
Centre of Experimental Animals (Wuhan, China). Liver mito- To induce state 3 respiration, 0.3 mM ADP was added sub-
chondria were isolated by differential centrifugation according to sequently. Finally, 1 μM FCCP was added to induce uncoupled
the literature with minor modifications.25 The fresh liver tissue respiration.
was chilled on ice bath, minced with scissors, and washed in
solution A (0.22 M mannitol, 0.07 M sucrose, 0.02 M HEPES,
2 mM Tris-HCl, and 1 mM EDTA, pH 7.4). About 5 g mince 2.8 Measurement of mitochondrial ultrastructure
were suspended in 100 ml solution A containing 0.4% BSA and
Using the techniques of ultrathin sectioning and transmission
homogenized with Dounce Tissue Grinders.
electron microscopy, the ultrastructure of mitochondria was
The homogenate was centrifuged at 3000 g for 2 min. Then,
studied. Mitochondria under various experimental conditions
the supernatant was centrifuged at 17 500 g for 3 min. The
were fixed for 30 min at 4 °C using glutaraldehyde at a final con-
resulting pellet was washed in solution A and centrifuged at
centration of 2.5% (v/v) in PBS buffer, centrifuged to micro-
17 500 g for 4 min. Subsequently, the pellet was resuspended in
pellets, postfixed with 1% (w/v) osmium tetroxide and
solution B (0.22 M mannitol, 0.07 M sucrose, 0.01 M Tris-HCl,
dehydrated. The ultrastructure of mitochondria was observed
and 1 mM EDTA, pH 7.4) and centrifuged at 17 500 g for
with a Tecnai G2 transmission electron microscope.30
4 min. The pellet was finally resuspended in solution B at a ratio
of 10 ml solution B per 10 g of starting material. All operations
were performed at 0–4 °C. 2.9 Cell viability assay
Mitochondrial protein concentration was measured by the
Biuret method using bovine serum albumin as the standard. The cytotoxicity of ZnO NPs was assessed by MTT assay.
Before experiments, we measured mitochondrial respiration with HeLa cells were used for toxicity evaluation and treated with
a Clark-type oxygen electrode and only mitochondrial suspen- various concentrations of ZnO NPs for 72 h. Optical density
sions with a RCR ≥ 3.0 were used. measurements were carried out using an ELISA reader at
450 nm (BioTech, China) and the IC50 value (50% inhibitory
concentration) was calculated by a four-parameter logistic
2.5 Measurement of mitochondrial membrane potential equation.
To monitor mitochondrial membrane potential (Δψ), fluor-
escence emission intensity of tetramethylrhodamine ethyl ester 2.10 Measurement of cytochrome c
(TMRE) was detected by a LS-55 fluorophotometer (Perkin-
Elmer, Norwalk, CT) equipped with a quartz cell of 1.0 cm path To confirm apoptosis caused by ZnO NPs, the release of cyto-
length at 25 °C (λex = 535 nm, λem = 590 nm). Mitochondria chrome c from mitochondria was detected by Cyt-C ELISA Kit
were dispersed in solution B with a final concentration of (Shanghai Hua Yi Bio Technology Co. Ltd, China) according to
0.5 mg ml−1.26 manufacture’s protocol.

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2.11 Determination of free zinc released from ZnO NPs

The level of free zinc released from ZnO NPs was measured by
IRIS Intrepid II XSP inductively coupled plasma-atomic emis-
sion spectroscopy (ICP-AES) (Thermo, USA). ZnO NPs dissolu-
tion was assessed using dialysis membrane of 3500 Da
molecular weight cutoff.

2.12 Lipid peroxidation

The effect of ZnO NPs on lipid peroxidation was evaluated by

oxygen consumption using a Clark-type electrode at 25 °C in
1 ml of a medium containing 175 mM KCl, 10 mM Tris-HCl,
3 μM rotenone, 1 mM adenosine diphosphate (ADP) and
0.1 mM Fe2+ (in the presence or absence of ZnO NPs) ( pH 7.2)
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(Fig. 10).28

3. Results
3.1 Characterization of ZnO NPs

The morphology and size of ZnO NPs were observed by SEM and
TEM. ZnO NPs were rod-shaped with a size of 100 nm (Fig. 1).

3.2 Changes of mitochondrial membrane potential

The effects of ZnO NPs on mitochondrial Δψ were measured

with a potentiometric fluorescent probe TMRE. TMRE can
accumulate in the mitochondrial matrix with the decrease of
fluorescence intensity. Upon collapse of mitochondrial Δψ,
TMRE is released into the buffer, causing an increase in fluore-
scence intensity. As shown in Fig. 2, mitochondrial Δψ
decreased with increasing concentrations of ZnO NPs.

3.3 Mitochondrial swelling induced by ZnO NPs

The effects of different concentrations of ZnO NPs on mitochon-

drial swelling were evaluated by the decrease in absorbance at
540 nm over 15 min. As shown in Fig. 3, ZnO NPs triggered
mitochondrial swelling in a time- and dose-dependent manner.
Taken together, data from the above experiments suggest that
ZnO NPs induced mitochondrial permeability transition (MPT).

3.4 Effects of ZnO NPs on inner membrane permeabilization to

H+ and K+

The ability of ZnO NPs to act on mitochondrial inner membrane

H+ conductance was examined by valinomycin-induced swelling
of de-energized mitochondria suspended in KAc medium. HAc
crosses the inner membrane and dissociates into Ac− and H+ in
the matrix, producing a H+ gradient which impairs mitochondrial Fig. 1 Characterization of ZnO NPs as measured by SEM (A, B) and
swelling. Thus, this sort of swelling can only be observed in the TEM (C).
presence of valinomycin which allows H+ to exchange with K+
entry.31,32 In the absence of ZnO NPs, mitochondria underwent a
slight swelling (Fig. 4A, trace a). In the presence of ZnO NPs The ability of ZnO NPs to permeabilize mitochondrial inner
(Fig. 4A, trace b, c, d, e), valinomycin-dependent mitochondrial membrane to K+ was tested by swelling of non-respiring mito-
swelling was notably increased, indicating a significant action on chondria in KNO3 medium. The inner membrane is freely
mitochondrial inner membrane H+ conductance. permeable to NO3−. So swelling can proceed in conditions of K+

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Fig. 2 Mitochondrial membrane potential measured by changes in

fluorescence emission intensity of TMRE. The traces represent typical
direct recordings representative of 4 experiments obtained from different
mitochondrial preparations. Traces a–e Mitochondrial suspensions
(0.5 mg ml−1) were added. c (ZnO NPs)/μg ml−1: 0, 5, 13, 25, 50.

Fig. 4 Effects of ZnO NPs on mitochondrial inner membrane permea-

bilization to H+ (A) and K+ (B). The traces represent typical direct
recordings representative of 4 experiments obtained from different mito-
chondrial preparations. A Traces a–d Mitochondrial suspensions (0.5 mg
ml−1) were added. c (ZnO NPs)/μg ml−1: 0, 10, 20, 30, 125. B Traces
a–e Mitochondrial suspensions (0.5 mg ml−1) were added. c (ZnO NPs)/
μg ml−1: 0, 15, 30, 45, 60.
Fig. 3 Mitochondrial swelling induced by ZnO NPs in the presence of
1 μM Ca2+. The traces represent typical direct recordings representative
of 4 experiments obtained from different mitochondrial preparations. (in the presence of ADP) indicates an intact respiratory chain
Traces a–e Mitochondrial suspensions (0.5 mg ml−1) were added. and ATP synthase.34 Therefore, the normal processes of ATP
c (ZnO NPs)/μg ml−1: 0, 5, 13, 25, 50. turnover and substrate oxidation can be maintained.35 As seen in
Fig. 5, the effect of ZnO NPs on state 4 respiration rate was
permeabilization.32 As shown in Fig. 4B, ZnO NPs enhanced biphasic: mitochondrial respiration was stimulated by low con-
the K+ conductance of the inner membrane with a dose-depen- centrations of ZnO NPs and inhibited by high concentrations of
ZnO NPs. However, the respiration rates at state 3 and uncoupled
dent effect.
state were significantly decreased by ZnO NPs with a dose-
dependent effect.
3.5 Effects of ZnO NPs on the respiration of isolated rat liver
mitochondria 3.6 Effects of ZnO NPs on mitochondrial ultrastructure

Fig. 5 shows the effects of ZnO NPs on mitochondrial respir- Mitochondrial ultrastructure was observed by a transmission
ation rate. In the absence of ZnO NPs, a low respiration rate at electron microscope. In the absence of ZnO NPs (Fig. 6A), most
state 4 (when ADP is exhausted) indicates an intact inner mem- of the isolated rat liver mitochondria maintained the structural
brane of isolated mitochondria.33,34 Thus mitochondria can integrity, as demonstrated by their normal shape with a well-
maintain a sufficiently high electrochemical proton potential to defined outer membrane, a narrow inter membrane space and
restrict electron transport.35 Meanwhile, the high rate at state 3 rich cristae.36 Because of a relatively high osmolarity of the

140 | Toxicol. Res., 2012, 1, 137–144 This journal is © The Royal Society of Chemistry 2012
 View Article Online

control conditions, ZnO NPs dissolved in the buffer to some

degree. When interacting with mitochondria, ZnO NPs released
far more Zn2+ with a dose-dependent effect.

3.10 Lipid peroxidation

Fig. 10 showed the effect of ZnO NPs on lipid peroxidation of

mitochondria. In the absence of ZnO NPs, it was easy to dis-
tinguish a two-step process in oxygen consumption. In the initial
lag phase, oxygen consumption was comparatively slow, which
might be related with the time required for the formation of ADP
– Fe3+–˙O2− from ADP, Fe2+ and O2. No sooner was a sufficient
amount of ADP – Fe3+–˙O2− generated than oxygen consump-
tion became rapidly, which was the result of lipid peroxidation.28
ZnO NPs effectively shortened the lag phase, but had little effect
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on the rapid oxygen consumption phase. These results suggested

Fig. 5 Effects of ZnO NPs on the respiration of isolated rat liver mito- that ZnO NPs enhanced the initiation of lipid peroxidation.
chondria. Respiration unit was represented as nmol O2/min mg−1
protein. Mitochondria (0.5 mg ml−1) were suspended at 25 °C in the
standard medium containing succinate (5 mM) as the respiratory sub- 4. Discussion
strate. Results are expressed as mean ± SD. of three independent
experiments. Mitochondria are the only cellular organelles that can build up a
Δψ of up to 180 mV, negative inside.15 The Δψ plays a key role
in regulating mitochondrial activity. Using this Δψ, mitochondria
medium, mitochondria were in the condensed configuration.37 can transport charged molecules and ions.39 Δψ is the major
Following MPT induced by ZnO NPs (Fig. 6B, C), mitochondria component of the electrochemical proton gradient, which is the
were obviously swollen, with decreased matrix electron density, primary form of energy generated in mitochondria. So, the fall
enlarged volume and inter membrane space, and ruptured cristae. of Δψ may result in a decrease in the electrochemical proton gra-
However, there was an interesting fact that low concentrations of dient, causing an immediate stimulation of oxygen consumption.
ZnO NPs (Fig. 6B) caused more severe damage to mitochondria If the electrochemical proton gradient is not dissipated by meta-
than high concentrations of ZnO NPs (Fig. 6C). bolic work (e.g. protein transport or ATP synthesis), the energy
of substrates is wasted in the form of heat emission.13 This study
demonstrates that ZnO NPs at low concentrations (less than
3.7 Cytotoxicity of ZnO NPs
25 μg ml−1) effectively collapse the mitochondrial Δψ (Fig. 2)
To evaluate the toxicity of ZnO NPs at the cellular level, a MTT and enhance oxygen consumption in state 4 respiration (Fig. 5),
colorimetric assay was conducted. As shown in Fig. 7, a 72 h confirming that ZnO NPs exert damaging effects on mitochon-
prolonged stimulation with ZnO NPs caused decreased cell via- drial energetics.
bility in a dose-dependent manner and the IC50 of ZnO NPs was To have a better understanding of mitochondrial bioenergetic
45.7 μg ml−1. function, the classic oxygen electrode experiments were
devised.35,40 In uncoupled respiration, when the permeability of
mitochondrial inner membrane is maximal, the activity of
3.8 Release of cytochrome c respiratory chain is determinant to the respiration rate.41 There-
fore, the inhibitory effect on uncoupled respiration suggests that
It is generally accepted that cytochrome c release from mitochon- ZnO NPs exert an inhibitory effect on mitochondrial respiratory
drial intermembrane space to cytosol is one of those apoptotic chain. In succinate-linked respiration, low concentrations of ZnO
features.38 To confirm that ZnO NPs induced apoptosis, the NPs cause increase in state 4 and decrease in state 3, suggesting
release of cytochrome c was detected. As shown in Fig. 8, in the that ZnO NPs at low levels affect both mitochondrial inner mem-
absence of ZnO NPs, only a small amount of cytochrome c was brane permeability and respiratory chain/ATP synthesis.34 High
released from mitochondria, probably due to mechanical damage concentrations of ZnO NPs cause decrease in both state 3 and
in isolation process. In experimental conditions, a large amount state 4. Addition of 100 μg ml−1 ZnO NPs even makes the rate
of cytochrome c was released from the organelle independent of of state 4 respiration lower than the control value. These results
concentrations of ZnO NPs. indicate that high levels of ZnO NPs cause damage to the
respiratory chain.42
3.9 Free Zn2+ released from ZnO NPs In fact, inhibition of mitochondrial respiration impairs mito-
chondrial energetics directly, while MPT dissipates the electro-
It is well known that Zn2+ had a great impact on mitochondria. chemical proton gradient, thus damaging mitochondrial
To discover the role of free Zn2+ released from ZnO NPs in the energetics. When MPT occurs, the mitochondrial inner
toxicity of nano materials, we measured the concentration of membrane is permeable to solutes with molecular mass up to
Zn2+ in the absence or presence of mitochondria, respectively. In 1500 Da.43,44 Thus, non-protein components of the

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Fig. 6 Effects of ZnO NPs on mitochondrial ultrastructure. Mitochondria were incubated in the standard medium with 0 (A), 5 (B) or 50
(C) μg ml−1 ZnO NPs.

Fig. 7 Cell viability of HeLa cells was determined by MTT assay after
72 h stimulation with various concentrations of ZnO NPs. Data were
shown as mean ± SD (n = 6). Fig. 8 Cytochrome c release from mitochondria induced by ZnO NPs.

142 | Toxicol. Res., 2012, 1, 137–144 This journal is © The Royal Society of Chemistry 2012
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Fig. 10 Effect of ZnO NPs on lipid peroxidation of mitochondria.

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Fig. 9 Free zinc released from ZnO NPs.

mitochondrial matrix will rapidly equilibrate across the inner

membrane. However, because the matrix proteins are at a higher
concentration than in the medium or cytosol, they will exert a
colloidal osmotic pressure, which causes mitochondrial matrix to
swell.45 MPT may also lead to apoptosis. As with Pandora’s
Box, mitochondria are full of potentially harmful compounds,
such as cytochrome c and apoptosis-inducing factors (AIFs).
During MPT, these compounds are released into the cytosol,
thus activating apoptotic cell death.46 In the present study, the
collapse of Δψ (Fig. 2), dose-dependent swelling (Fig. 3) and
alterations of mitochondrial ultrastructure (Fig. 6) caused by
ZnO NPs confirm that ZnO NPs can trigger MPT.27 Apart from
the energy dissipation mentioned above, MPT induced by ZnO Fig. 11 Proposed mechanism of mitochondrial dysfunction induced by
NPs may also lead to cell apoptosis (Fig. 7 and 8), which is con- ZnO NPs.
sistent with other reports.47,48
Under physiological conditions, the permeability of mitochon-
accepted that ROS is a major factor in the induction of MPT as
drial inner membrane to H+ and other ions must be fairly low, in
well as a consequence of MPT.58 Our findings suggested that
order to maintain a high efficiency of energy conservation.13
ZnO NPs favored increased ROS production in mitochondria
Flux of H+ and K+ into the matrix can induce Δψ decrease, thus
and enhanced the initiation of lipid peroxidation (Fig. 10).
causing MPT.49,50 Moreover, some reports reveal a close
Therefore, we tentatively hypothesize that release of Zn2+ and
relationship between mitochondrial K+ homeostasis and apopto-
production of ROS are central to ZnO NPs toxicity (Fig. 11).
sis. On the one hand, an enhanced mitochondrial K+ uptake
This study demonstrates a putative mechanism of the toxicity
leads to mitochondrial swelling, K+ accumulation and cyto-
of ZnO NPs to mitochondria. Due to release of Zn2+ and pro-
chrome c release. On the other hand, the increase in K+ conduc-
duction of ROS, ZnO NPs increase the inner membrane perme-
tance of inner membrane is responsible for an increased
ability and impair the respiratory chain, thus leading to energy
permeability of mitochondria to H+, leading to intracellular acid-
dissipation, oxidative stress and even apoptosis.
ification.49,51 As can be seen from Fig. 4, ZnO NPs can enhance
H+ and K+ conductance of mitochondrial inner membrane,
findings that are consistent with their ability to collapse Δψ,
Acknowledgements
trigger mitochondrial swelling, damage mitochondrial energetics
and even lead to apoptotic cell death. The authors gratefully acknowledge the financial support
As reported, there are two toxic mechanisms of ZnO NPs, from the Chinese 973 Program (grant no. 2011CB933600)
Zn2+ liberation and reactive oxygen species (ROS) production. and the National Natural Science Foundation of China (grant
Zn2+ can inhibit complexes of the respiratory chain or the tri- no. 21077081, 21173026).
carboxylic acid cycle, and stimulate MPT.52–55 In the present
study, ZnO NPs degradation was observed (Fig. 9), implying
that released Zn2+ was responsible for the toxic effects of ZnO References
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