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Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease

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Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease

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Evidence continues to accumulate that pesticides are the leading candidates of environmental toxins that may contribute to the pathogenesis of Parkinson’s disease. The mechanisms, however, remain largely unclear. According to epidemiological studies, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl) which are commonly used in China and detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS) function. Our results reveal that all the nine studied pesticides induce morphological changes of mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them (paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad) caused a significant dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance for selection of pesticides in China. Moreover, our results demonstrate a potential link between inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism.
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Int. J. Mol. Sci. 2017, 18, 2507; doi:10.3390/ijms18122507 www.mdpi.com/journal/ijms
Article
Effects of Commonly Used Pesticides in China on the
Mitochondria and Ubiquitin-Proteasome System in
Parkinson’s Disease
Tingting Chen
1
, Jieqiong Tan
1
, Zhengqing Wan
1
, Yongyi Zou
1
,
Henok Kessete Afewerky
1,2,3
, Zhuohua Zhang
1
and Tongmei Zhang
1,2,3,
*
1
State Key Laboratory of Medical Genetics, Xiangya Medical School, Central South University,
Changsha 410078, China; chentingting@sklmg.edu.cn (T.C.);
jieqiong.tan@gmail.com (J.T.); wanzhengqing@sklmg.edu.cn (Z.W.);
zouyongyi@gmail.com (Y.Z.); henokessete@hust.edu.cn (H.K.A.);
zhangzhuohua@sklmg.edu.cn (Z.Z.)
2
Department of Physiology, School of Basic Medicine, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, China
3
The Institute of Brain Research, Huazhong University of Science and Technology, Wuhan 430030, China
* Correspondence: zhangtongmei1202@gmail.com; Tel.: +86-731-8480-5358; Fax: +86-731-8447-8152
Received: 18 October 2017; Accepted: 20 November 2017; Published: 23 November 2017
Abstract: Evidence continues to accumulate that pesticides are the leading candidates of
environmental toxins that may contribute to the pathogenesis of Parkinson’s disease.
The mechanisms, however, remain largely unclear. According to epidemiological studies, we
selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan,
fenpyroximate, tebufenpyrad, trichlorphon and carbaryl) which are commonly used in China and
detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS)
function. Our results reveal that all the nine studied pesticides induce morphological changes of
mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan,
fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them
(paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad) caused a significant
dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce
mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine
pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation
or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance
for selection of pesticides in China. Moreover, our results demonstrate a potential link between
inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism.
Keywords: Parkinson’s disease; pesticides; mitochondria; ubiquitin-Proteasome system; China
1. Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder affecting
approximately 2% of people aged over 65. It is pathologically characterized by pronounced loss of
dopaminergic neurons in the substantia nigra of the midbrain and formation of ubiquitin-positive
Lewy body aggregates [1,2]. Epidemiological studies indicate both environmental neurotoxins and
genetic predisposition as potential risk factors for PD. Evidence continues to accumulate that
pesticides are the leading candidates of environmental toxins that may contribute to the
pathogenesis of PD [3,4]. Several pesticides such as rotenone, paraquat, dieldrin and maneb have
been used to develop PD models, with pathological features of the degeneration of dopaminergic
neurons [5–9].
Int. J. Mol. Sci. 2017, 18, 2507 2 of 16
Although the mechanisms of loss of dopaminergic neurons in PD remain unclear, there is
compelling evidence that mitochondria and ubiquitin-Proteasome system (UPS) dysfunction represent
critical events [10,11]. Identification of mutants in PINK1, DJ-1, PRKN and LRRK-2 genes, which
participate in oxidative stress and mitochondrial dysfunction, affirms the hypotheses of dopaminergic
neuronal degeneration in PD [12]. Mutants of α-synuclein, Ubiquitin C-Terminal Hydrolase L1
(UCH-L1) and parkin support the involvement of UPS dysfunction in PD [12–16]. Furthermore,
various Parkinsonian toxicants have been shown to impair mitochondria and UPS function. Rotenone,
an inhibitor of mitochondrial complex I, demonstrates many features of PD including selective
dopaminergic degeneration, increased oxidative damage, Lewy body-like inclusions formation and
α-synuclein aggregation [9,17,18]. In addition, exposure to proteasome inhibitors in rats causes motor
dysfunction, loss of dopaminergic neurons and formation of Lewy bodies [19]. All these findings are
suggestive of the critical role of mitochondria and UPS dysfunction in PD.
Previous studies have linked individual pesticides to mitochondrial dysfunction [20–24] or UPS
dysfunction [25–29]. For example, paraquat, rotenone, pyridaben, fenpyroximate, fenazaquin and
tebufenpyrad have been reported to directly inhibit complex I [24,30]. Rotenone, ziram,
diethyldithiocarbamate, endosulfan, benomyl, cyanazine, dieldrin, metam, propargite, triflumizole
and dieldrin showed inhibitory effects on proteasome activities [26,27,29]. However, the correlation
between mitochondrial dysfunction and UPS dysfunction for pesticides treatment is not very clear.
In the present study, we have characterized the toxic potency of nine (paraquat, rotenone,
chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl)
commonly used pesticides that belong to different chemical groups and defined their toxicity
mechanism. Our results showed that paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan,
fenpyroximate and tebufenpyrad induce mitochondria fragmentation. Furthermore, some of them
(paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad) cause a significant
dose-dependent decrease of intracellular ATP. However, two out of the nine pesticides, namely
trichlorphon and carbaryl, were found not to induce mitochondrial fragmentation or functional
damage, nor inhibit the activity of the proteasome. Interestingly, our results suggest that the
pesticides that induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. We
also herewith establish a potential link between mitochondria and the UPS.
2. Results
2.1. Pesticides Caused Dose-Dependent Apoptotic Cell Death in SH-SY5Y Cells
Environmental factors are closely related to the occurrence of PD, and hence researchers have
been conducting extensive screening of environmentally predisposing factors in PD, and have found
that exposure to pesticides, drinking well water and farming may increase the risk of PD [31].
Among the environmental factors, pesticides, which are widely used throughout the world, are the
common factors. Pesticides can be grouped in different ways; according to purposes, pesticides can
be divided into insecticides, rodenticides, fungicides, herbicides and so on, and in terms of chemical
structures, they can be divided into organophosphate, carbamate, organochlorine, pyrethroid and
pseudo chrysanthemum bug [32]. In reference to former epidemiological studies, for the current
study, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin,
endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl), which are commonly used in
China, and have analyzed their possible toxicological mechanisms to cause cell death.
The toxicities of pesticides are classified by their lethal concentrations according to WHO
classification [33], so we treated SH-SY5Y cells with the proper concentration according to the
toxicity of the different pesticides. After being treated with pesticides in different concentrations for
24 h, SH-SY5Y cells were stained with annexin V-FITC and propidium iodide, followed by FACScan
flow cytometer analysis. Results showed that these nine pesticides can lead to dose-dependent
apoptosis, whereby the lethal concentrations of these pesticides are different, presumably due to the
differences in the mechanisms of their toxicity. In general, a low concentration of these pesticides
was found to have a toxic, but not lethal, effect on cells. Therefore, in order to study the toxicological
Int. J. Mol. Sci. 2017, 18, 2507 3 of 16
mechanism of these pesticides, we chose lower concentrations to carry out the following
experiments (Figure 1).
Figure 1. Pesticides caused dose-dependent apoptotic cell death in SH-SY5Y cells. SH-SY5Y cells were
treated with different concentrations of pesticides for 24 h. Cell apoptosis (AI) was detected using
annexin V-FITC/PI assay and analyzed by one-way ANOVA and Dunnett’s test (n = 4), and camptothecin
(J) was the positive control. Error bars represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.
2.2. Pesticides Induced Morphological Changes of Mitochondria
Several pesticides are known to cause neurotoxicological effects in humans. For example,
exposure to the most common pesticides, namely rotenone and paraquat, induces behavioral and
pathological changes that characterize PD in animal models and humans [24,34]. Moreover, the
potential mechanism of these pesticides may increase the risk of PD through disruption of
mitochondrial function [35]. Recent studies revealed that the mitochondrial morphology is an
important determinant of mitochondrial function [36,37]. To investigate the effect of pesticide on
mitochondrial morphology, HeLa cells were treated with different concentrations of various
pesticides for 24 h. Cells were then fixed and labeled with a specific antibody against mitochondrial
outer membrane protein TOM20, and immunolocalization was visualized by laser confocal
microscopy. Mitochondria demonstrated a tubular and filamentous morphology in control HeLa
cells. We measured mitochondrial aspect ratio to get the changes of the length of mitochondria.
Aspect ratio (an index of mitochondrial branch length) means the ratio between the bigger (major)
and the smaller (minor) side of each mitochondrial fragment. The results showed that seven out of
Int. J. Mol. Sci. 2017, 18, 2507 4 of 16
nine representative pesticides, including paraquat (500 μM), rotenone (1 nM), chlorpyrifos (200 μM),
pendimethalin (50 μM), endosulfan (50 μM), fenpyroximate (1 μM) and tebufenpyrad (20 μM),
induced mitochondrial fragmentation to varying degrees (Figure 2A–H,K). However, trichlorphon
(200 μM) and carbaryl (100 μM) caused mitochondrial elongation (Figure 2I–K). As for nuclear
shape, no difference was noted between pesticide-treated and control cells. These results suggest
that pesticides induce morphological changes of mitochondria prior to cell death.
Figure 2. Pesticides induced morphological changes of mitochondria. HeLa cells were treated with
different concentrations of pesticides or control solvent DMSO (A,a) for 24 h. Mitochondria (red) and
cell nuclei (blue) are shown. High-magnification pictures (aj) of cells are also shown to exemplify the
mitochondria. Mitochondria were fragmented after exposure to 500 μM paraquat (B,b); 1 nM rotenone
(C,c); 200 μM chlorpyrifos (D,d); 50 μM pendimethalin (E,e); 50 μM endosulfan (F,f); 1 μM
fenpyroximate (G,g) and 20 μM tebufenpyrad (H,h). Conversely, mitochondria were elongated after
exposure to 200 μM trichlorphon (I,i) and 100 μM carbaryl (J,j); bar = 10 μM. Mitochondrial aspect
ratios in control or different pesticide-treated cells were measured by ImageJ and analyzed by one-way
ANOVA and Dunnett’s test (K). The aspect ratios of mitochondria were analyzed by one-way ANOVA
and Dunnett’s test (n = 50 cells); error bars represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.
Int. J. Mol. Sci. 2017, 18, 2507 5 of 16
2.3. Paraquat, Rotenone, Chlorpyrifos, Fenpyroximate and Tebufenpyrad Induced a Significant
Dose-Dependent Decrease of Intracellular ATP
We determined the effect of pesticides on intracellular ATP concentration as a functional
parameter of mitochondria. Cells were exposed to low concentrations of pesticides for 12 h and ATP
levels were determined using a luciferase-based assay. Dose-dependent ATP depletion was detected
in SH-SY5Y cells in response to five pesticide treatments, including paraquat, rotenone, chlorpyrifos,
fenpyroximate and tebufenpyrad (Figure 3A–C,F,G). Rotenone was the most potent, with ATP
depletion observed at concentrations as low as 10 nmol/L, whereas pendimethalin, endosulfan,
trichlorphon and carbaryl were found not to influence cellular ATP (Figure 3 D,E,H,I).
Figure 3. Paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad induced a significant
dose-dependent decrease of intracellular ATP. SH-SY5Y cells were exposed to low concentrations of
paraquat (A); rotenone (B); chlorpyrifos (C); pendimethalin (D); endosulfan (E); fenpyroximate (F);
tebufenpyrad (G); trichlorphon (H); and carbaryl (I) for 12 h, followed by ATP-level detection using
a luciferase-based assay and analysis by one-way ANOVA and Dunnett’s test (n = 4). Error bars
represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.
2.4. Paraquat, Rotenone and Chlorpyrifos Inhibited NADH Dehydrogenase Activity
NADH dehydrogenase is the first enzyme (complex I) of the mitochondrial electron transport
chain, which translocate four protons across the inner membrane per molecule of oxidized NADH,
helping to build the electrochemical potential used to produce ATP. As paraquat, rotenone,
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chlorpyrifos, fenpyroximate and tebufenpyrad caused a significant dose-dependent decrease of
intracellular ATP, we examined their effect on NADH dehydrogenase activity. The results showed
that NADH dehydrogenase activity was decreased in paraquat- (0.5 mM), rotenone- (100 nM) and
chlorpyrifos (500 μM)-treated cells as compared to controls (Figure 4). It is known that chlorpyrifos
causes oxidative stress and neurotoxicity in humans and animals [38,39], which may be attributed to
the activity disorder of NADH dehydrogenase. Dichlorvos, another organophosphate, caused the
same effect through the same mechanism [40]. Paraquat and rotenone are classical complex I
inhibitors [24,41]. On the other hand, trichlorphon (200 μM), carbaryl (100 μM), fenpyroximate (2.5
μM), pendimethalin (50 μM), endosulfan (50 μM) and tebufenpyrad (50 μM) had no significant
influence on NADH dehydrogenase activity (Figure 4).
Figure 4. Paraquat, rotenone and chlorpyrifos inhibited NADH dehydrogenase activity. SH-SY5Y
cells were exposed to low concentrations of paraquat (0.5 mM), rotenone (100 nM), chlorpyrifos (500
μM), pendimethalin (50 μM), endosulfan (50 μM), fenpyroximate (2.5 μM), tebufenpyrad (50 μM),
trichlorphon (200 μM) and carbaryl (100 μM) for 24 h, then NADH dehydrogenase activity was
detected and analyzed by one-way ANOVA and Dunnett’s test (n = 5). Error bars represent SEM.
* p < 0.05; ** p < 0.01.
2.5. All Present-Study Pesticides Except Trichlorphon and Carbaryl Inhibited 26S Proteasome Activity
UPS dysfunction is an important pathogenic factor in PD [11,42]. Exposure to pesticide may
increase the risk of developing PD by inhibiting the UPS. The effects of pesticides on proteasome
activity were examined in SH-SY5Y cells overexpressed with a GFP-conjugated proteasome
degradation signal, GFPU [43]. The product of GFPU was continuously degraded and kept at very
low levels under normal conditions. Compromised proteasome function reduced the clearance
capacity of UPS and increased the steady-state GFPU level. Pesticides were administered to the
cultures for 24 h. Expression levels of GFPU were determined by immunoblotting. Our results
showed that seven out of nine representative pesticides, namely paraquat, rotenone, chlorpyrifos,
pendimethalin, endosulfan, fenpyroximate and tebufenpyrad, had inhibitory effects on proteasome
activities at low concentrations (Figure 5A,B). Trichlorphon and carbaryl had no significant
influence on proteasome activity (Figure 5A,B). In order to prove that the increased GFPU level with
pesticide treatment was the consequence of inhibition of proteasome activities, cells were exposed
to pesticides with proteasome inhibitor MG132. Our results showed that there was no further
increase of GFPU levels with pesticide treatment (Figure 5C). This indicated that the increased
GFPU level with pesticide treatment was due to the inhibition of proteasome activities.
Int. J. Mol. Sci. 2017, 18, 2507 7 of 16
Figure 5. All present-study pesticides except trichlorphon and carbaryl inhibited 26S proteasome
activity. SH-SY5Y cells stably expressing GFPU were exposed to low concentrations of pesticides for
24 h, and GFP expression levels were detected by immunoblotting (A); relative levels of GFP were
measured by ImageJ and analyzed by one-way ANOVA and Dunnett’s test (n = 4) (B); error bars
represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. β-tubulin was detected as a loading control. (C)
SH-SY5Y cells stably expressing GFPU were exposed to pesticides (500 μM paraquat, 1 nM rotenone,
50 μM chlorpyrifos, 50 μM pendimethalin, 50 μM endosulfan, 0.1 μM fenpyroximate, 10 μM
tebufenpyrad, 100 μM trichlorphon, 10 μM carbaryl) with 0.1 μM MG132 for 24 h, and GFP
expression levels were detected by immunoblotting.
2.6. All Present-Study Pesticides Except Trichlorphon and Carbaryl Inhibited 20S Proteasome Activity
To explore the mechanism by which pesticides may inhibit proteasome function, we examined
the action of the pesticides on 20S proteasome. SH-SY5Y cells were treated with different
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concentrations of pesticides for 24 h. Cell lysates were then collected. After collection, the
proteasome activity was monitored by the fluorogenic substrate LLVY. Our results showed that
seven pesticides, namely paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan,
fenpyroximate and tebufenpyrad, had an inhibitory effect on the 20S proteasome activity (Figure 6).
However, there was no inhibition owing to trichlorphon or carbaryl.
Figure 6. All present-study pesticides except trichlorphon and carbaryl inhibited 20S proteasome
activity. SH-SY5Y cells were exposed to low concentrations of paraquat (A); rotenone (B);
chlorpyrifos (C); pendimethalin (D); endosulfan (E); fenpyroximate (F); tebufenpyrad (G);
trichlorphon (H); and carbaryl (I) for 24 h; MG132 (J) was the positive control. The 20S proteasome
activity was measured with addition of substrate LLVY-AMC and analyzed by one-way ANOVA
and Dunnett’s test (n = 5). Error bars represent SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.
2.7. Trichlorphon and Carbaryl Did Not Affect Mitochondrial and UPS Functions
According to the above results, five out of the nine representative pesticides, including
paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad, induced mitochondrial
fragmentation and also caused a decrease of intracellular ATP. However, interestingly, among these
five pesticides, only paraquat, rotenone and chlorpyrifos inhibited NADH dehydrogenase activity.
Moreover, of the nine pesticides, only pendimethalin and endosulfan affected the mitochondrial
Int. J. Mol. Sci. 2017, 18, 2507 9 of 16
morphology without affecting the intracellular ATP and NADH dehydrogenase activity. It is noted
that the pesticides that affected the morphology and function of mitochondria also inhibited the
activity of the 20S and the 26S proteasome.
Most importantly, among these nine presently studied pesticides, trichlorphon and carbaryl
were exceptionally found not to cause mitochondrial fragmentation. Furthermore, these two
pesticides were determined not to have an effect on the intracellular ATP and NADH
dehydrogenase activity in the half-lethal concentration treatments, nor inhibit the proteasome
activity. These results suggest that trichlorphon and carbaryl have low toxicity for mitochondrial
and UPS functions (Table 1).
Table 1. Summary of the effects of pesticides on mitochondria and the ubiquitin-Proteasome system.
Name Chemical Class Mitochondria Ubiquitin-Proteasome System
Morphology ATP Complex I 26S 20S
Paraquat Dipyridyl compounds Fragmentation Depletion Decrease Inhibition Inhibition
Rotenone Rotenoids Fragmentation Depletion Decrease Inhibition Inhibition
Chlorpyrifos Organophosphates Fragmentation Depletion Decrease Inhibition Inhibition
Pendimethalin Dinitroanilines Fragmentation No influence No influence Inhibition Inhibition
Endosulfan Organochlorides Fragmentation No influence No influence Inhibition Inhibition
Fenpyroximate Acaricides Fragmentation Depletion No influence Inhibition Inhibition
Tebufenpyrad Acaricides Fragmentation Depletion No influence Inhibition Inhibition
Trichlorphon Organophosphates Elongation No influence No influence No influence No influence
Carbaryl Carbamates Elongation No influence No influence No influence No influence
3. Discussion
The etiology of PD is complex and diverse. Some mutations have been described to lead to
familial PD. However, the most common PD cases are sporadic and suspected to be attributed to
environmental rather than genetic factors. Epidemiologic studies show that daily habits, dietary
factors, drugs, medical history and exposure to toxic environmental agents are associated with risks
of PD [31,44,45]. As part of the environmental factors, several pesticides contribute to PD with
unknown mechanisms.
Pesticides are a ubiquitous component of our environment and have been widely used in
China. In 1999, over 5.6 billion pounds of pesticides were applied worldwide, resulting in detectable
levels of pesticides in human bodies [32]. Pesticides can be grouped by their functional class of
organisms (e.g., herbicides or insecticides) or their chemical structures. In our study, we have chosen
the representative members that cover a wide range of pesticides commonly used in China,
including dipyridyl compounds, rotenoids, organophosphates, dinitroanilines, organochlorides,
acaricides and carbamates. Epidemiological studies indicate that exposure to pesticides has
profound effects on the nervous system, in which high-level exposure to the pesticide is associated
with neurodegenerative symptoms as well as deficits in neurobehavioral performance and
abnormalities [46,47]. Chronic low-level exposure to pesticides is also associated with increased risk
of neurologic disease [32]. The farmers exposed to multiple pesticides have been reported to have a
broad range of nonspecific symptoms, such as headache, insomnia and confusion [48,49].
Nevertheless, the neurotoxic mechanisms of chronic low-level exposure have remained unclear.
Therefore, our study of the mitochondria and UPS inhibition with a low concentration of pesticide
treatment may give enlightenment to the problem.
In the current study, we examined the neurotoxic mechanism of commonly used pesticides in
China. The results revealed that pesticides induce morphological changes of mitochondria and
dose-dependent apoptotic cell death. Some of the pesticides caused a significant dose-dependent
decrease of intracellular ATP and NADH dehydrogenase activity. Moreover, most pesticides
inhibited 26S and 20S proteasome activity. Many research findings on sporadic and familial PD cases
show that an impairment of mitochondrial function results in age-related neurodegeneration.
In addition, several studies indicate that dysfunction of mitochondrial dynamism [50,51], complex I
deficiency [52,53], mtDNA mutations [54,55] and blocked mitophagy [56,57] contribute to the
pathogenesis of PD. Dysfunctional UPS also contributes to protein aggregation [11,58,59], oxidative
stress [60,61] and dopaminergic cell death in Parkinson’s disease pathogenesis. Therefore, our
Int. J. Mol. Sci. 2017, 18, 2507 10 of 16
findings suggest that mitochondria and UPS inhibition constitute a potential mechanism for
pesticide-induced Parkinsonism.
An exposure to pesticides and mitochondrial dysfunction are increasingly implicated in the
pathogenesis of PD. In our study, paraquat, rotenone, chlorpyrifos, fenpyroximate and
tebufenpyrad induced mitochondrial fragmentation and ATP decline; however, only paraquat,
rotenone and chlorpyrifos inhibited NADH dehydrogenase activity. The research of paraquat
toxicity in mouse strains and rats show that it can induce PD-like lesions. The underlying
mechanisms are directly or indirectly related to reactive oxygen species [62,63]. Rotenone,
fenpyroximate and tebufenpyrad have also been demonstrated to displace 3H-dihydrorotenone
(DHR) from complex I and inhibit complex I activity [24,34]. Trichlorphon and carbaryl increase the
length of mitochondria or the ratio between the bigger (major) and the smaller (minor) side for each
mitochondrial fragment, but do not induce mitochondrial functional changes (ATP, NADH
dehydrogenase activity and mitochondrial membrane potential). This suggests that trichlorphon- or
carbaryl-induced cell toxicity is not mainly through damaging mitochondria. However, the
mechanism of trichlorphon- or carbaryl-induced mitochondrial morphology changes needs to be
further elucidated.
Interestingly, pesticides that induced mitochondrial fragmentation and ATP decline also
inhibited UPS function. It might be due to the 26S proteasome, which is the main component in the
UPS, that functions in an ATP-dependent manner [64]. One group of byproducts of aerobic
metabolism in mitochondria are reactive oxygen species, and the accumulation of these oxidized
proteins is associated with age-related diseases [65,66]. Oxidative stress not only damages the
proteins, which are degraded, but also impairs the composition of the UPS itself [67]. Hence, altered
UPS function might occur as a secondary response to cell death. On the other hand, the UPS is a part
of the surveillance network which controls the mitochondrial protein quality and mitochondrial
integrity [64,68]. The UPS itself is also a key cellular event responsible for degeneration, as our
results revealed that pendimethalin and endosulfan do not cause a decrease of ATP while inhibiting
20S and 26S proteasome activity. Epidemiological studies suggest that exposure to
organophosphorus, carbamate and organochlorine insecticides, as well as dithiocarbamate
fungicides, might be associated with PD [7,69,70]. However, except for organophosphorus, the
detailed neurotoxic mechanism had remained unclear. The current study elucidates the mechanism
of neurotoxicity for these pesticides associated with PD. Although acaricide, fenpyroximate and
tebufenpyrad caused neurotoxicity through a similar mechanism, chlorpyrifos and trichlorphon
(both organophosphorus) were found to induce cell death in a different manner, indicating that
specific pesticides induce different neurotoxicological pathways.
Our results show that, of the nine examined pesticides in this study, exclusively trichlorphon
and carbaryl do not inhibit the activity of mitochondria and the ubiquitin-Proteasome system.
Trichlorphon and carbaryl are commonly used insecticides in China [71]. Trichlorphon is a member
of the organochloride insecticides inhibiting acetylcholinesterase, and has long been used as a
pesticide for agricultural plant protection and as a repellent for the treatment of parasitic diseases.
Trichlorphon has also been proposed for the treatment of Alzheimer’s disease [72]. As for carbaryl, it
is a member of the carbamate family, which are slowly reversible inhibitors that disrupt the
cholinergic nervous system and cause death [73]. In general, both trichlorphon and carbaryl may
have low toxicity for mitochondrial and UPS functions, and also may cause cell apoptosis through
other mechanisms. However, further studies are needed to demonstrate these hypotheses.
In conclusion, our study reveals that some pesticides commonly used in China induce
mitochondrial as well as proteasome dysfunction, which might constitute a potential mechanism of
the neurodegeneration in Parkinsonism. Hence, our findings provide significant guidance on the
subject of pesticide selection for agricultural, industrial and domestic applications. Further studies,
including in-vivo animal experiments, are necessary for the determination of the actual risk of
pesticides to human health.
Int. J. Mol. Sci. 2017, 18, 2507 11 of 16
4. Materials and Methods
4.1. Materials
SH-SY5Y and HeLa cells were obtained from American Type Culture Collection (ATCC) and
maintained as suggested by the provider. HeLa cells were cultured in Dulbecco’s High Glucose
Modified Eagles Medium (GE Healthcare HyClone, Logan, UT, USA, SH30022) containing 2 mM/L
L-glutamine (Life Technologies, Waltham, MA, USA, 25030) and 1 mM/L sodium pyruvate (Life
Technologies, Waltham, MA, USA, 11360) supplemented with 10% fetal bovine serum (GE
Healthcare HyClone, Logan, UT, USA, SH30084). SH-SY5Y cells were cultured in DMEM: F12 (GE
Healthcare HyClone, Logan, UT, USA, SH30023) containing 1 mM/L Non-Essential Amino Acids
(Thermo Fisher Scientific, Waltham, MA, USA, 11140050), 200 mM L-glutamine (GIBCO, Thermo
Fisher Scientific, Waltham, MA, USA, 25030) and 100 mM sodium pyruvate (Thermo Fisher
Scientific, Waltham, USA, 11360070) supplemented with 10% fetal bovine serum (GE Healthcare
HyClone, Logan, UT, USA, SH30084). The nine tested pesticides, namely paraquat (36541); rotenone
(R8875); chlorpyrifos (90047); pendimethalin (36191); endosulfan (PS81); fenpyroximate (31684);
tebufenpyrad (46438); trichlorphon (45698); and carbaryl (32055), were all from Sigma-Aldrich
(Billerica, MA, USA). Most pesticides were dissolved in dimethyl sulfoxide (DMSO), except for
paraquat in H2O. DMSO final concentration was not more than 0.1% in the culture medium. A stable
cell line expressing GFPU was generated as described [74] and maintained in 200 μg/mL G418.
4.2. Cell Apoptosis Assay
Cell apoptosis was quantified using an Annexin V-FITC Apoptosis Detection Kit according to
manufacturer’s instruction (Sigma-Aldrich, Billerica, MA, USA, APOAF-20TST). Briefly, after
pesticides exposure, cells collected by trypsin digestion were stained by mixing with 3 μL annexin
V-FITC and 1 μL propidine iodide (PI), followed by incubation at room temperature in darkness for
15 min. Apoptotic cells were immediately analyzed using a FACScan flow cytometer (Becton
Dickinson, SanJose, CA, USA) with excitation at 488 nm and emission at 530 nm (FITC) and 610 nm
(PI). Each experiment was repeated three times.
4.3. Immunofluorescence and Analysis of Mitochondrial Morphology
Cells were cultured on glass coverslips for 24–48 h, followed by fixation with 4%
paraformaldehyde for 10 min and permeabilization with 0.1% Triton X-100 in PBS for another 10
min at room temperature. After blocking with 3% BSA for 30 min, cells were stained with mouse
anti-Tom20 monoclonal antibody (Santa Cruz, Dallas, TX, USA, sc-17764) and then incubated with
the Goat Anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Invitrogen,
Waltham, MA, USA, A-11003). Following this procedure, cell nuclei were counterstained with DAPI
(Thermo Fisher Scientific, Waltham, MA, USA, D1306), and images were acquired under a Leica
confocal microscope (Buffalo Grove, IL, USA, TCS SP5) with appropriate excitation and emission
filter pairs. For mitochondrial morphology analysis, aspect ratio (an index of mitochondrial branch
length) means the ratio between the bigger (major) and the smaller (minor) side for each
mitochondrial fragment. The images were processed to maximize the signal/background ratio using
the automatic adjustment of brightness/contrast of the ImageJ software. Finally, the major and the
minor of each fragment were determined using the “analyse particles” function of ImageJ. Analyses
were performed on at least 50 cells for each condition [75].
4.4. Immunoblotting
Cells were lysed with SDS sample buffer (63 mM Tris-HCl, 10% glycerol, 2% SDS). After
supernatant collection, protein concentration was determined using a BCA protein assay kit
(Thermo Fisher Scientific, Waltham, MA, USA, 23225). Sample proteins were separated on
SDS–PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5%
non-fat dry milk in 0.1% Triton X-100/PBS buffer for 1 h and then were incubated with appropriate
Int. J. Mol. Sci. 2017, 18, 2507 12 of 16
primary antibodies in blocking solution overnight at 4 °C. Following rinse with 0.1% Triton
X-100/PBS buffer, the membranes were re-incubated with appropriate secondary antibodies for 1 h
and immunoreactive bands were detected via enhanced chemiluminescence kit according to the
manufacturer’s instruction (Thermo Fisher Scientific, Waltham, MA, USA, 32106). The GFP (632381)
and β-tubulin (T8328) antibody were purchased from Clontech (Mountain View, CA, USA) and
Sigma-Aldrich (Billerica, MA, USA), respectively.
4.5. ATP Measurements
Cellular ATP levels in SH-SY5Y cells exposed to pesticides were determined using the
CellTiter-GloH Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA, G7570). In brief,
cells were grown in 24-well fluorimeter plates and exposed to various concentrations of the
pesticides. Following removal of media, cells were washed with cold PBS and then ATP levels were
determined as described in the manufacturer’s protocol. Then, the determined ATP levels were
normalized by total protein concentration. Each experiment was repeated three times.
4.6. Mitochondrial Complex I Activity Assay
Mitochondrial complex I activity in SH-SY5Y cells exposed to pesticides was determined via
Mitochondrial Complex I Activity Assay Kit (Sigma-Aldrich, Billerica, MA, USA, AAMT001). In
brief, cells were grown in 10 cm dishes and exposed to pesticides, and then collected and lysed with
Mammalian Tissue Lysis/Extraction Reagent (Sigma-Aldrich, Billerica, MA, USA, C3228). Protein
concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA,
USA, 23225). Mitochondrial complex I activity, determined as described in the manufacturer’s
protocol, was defined as a change in absorbance at 450 nm per minute for each amount of sample.
Each experiment was repeated three times.
4.7. Determination of Cellular 26S Proteasome Activity
A stable cell line overexpressing GFPU in SH-SY5Y cells was established as a cellular model to
test 26S proteasome activity. The reporter gene, which consists of a short tag CL1, is fused to the
C-terminus of GFP. CL1, encoding a fragment of amino acids (ACKNWFSSLSHFVIHL), was shown
to be a degradation substrate for the ubiquitin-Proteasome system [43]. The expression levels of
GFPU after exposure to pesticides were determined by immunoblotting.
4.8. Determination of 20S Proteasome Activity
20S proteasome activity was determined using the Proteasome Activity Assay Kit (BioVision,
Milpitas, CA, USA, K245). Briefly, cells were grown in 6-well fluorimeter plates and exposed to
various concentrations of pesticides. Following removal of media, cells were washed with cold PBS
and 20S proteasome activity was determined according to the manufacturer’s protocol. The
determined 20S proteasome activity was then normalized by total protein concentration. Each
experiment was repeated three times.
4.9. Statistical Analysis
Statistical analyses were performed using GraphPad Prism 5 software. The data are presented
as mean ± SEM. Statistical significance between treatment groups against their controls was derived
using one-way analysis of variance (ANOVA) followed by Dunnett’s tests. Two-tailed Student’s
t-test was used to determine the significance of difference between 2 groups.
5. Conclusions
Our research reveals that some pesticides commonly used in China induce mitochondrial as
well as proteasome dysfunction, which might constitute a potential mechanism of the
neurodegeneration in Parkinsonism.
Int. J. Mol. Sci. 2017, 18, 2507 13 of 16
Acknowledgments: This work was supported by the National Natural Science Foundation of China (No.
31500832). We greatly thank Minzi Deng (State Key Laboratory of Medical Genetics, Xiangya Medical School,
Central South University, Changsha) for providing language help on manuscript.
Author Contributions: Tingting Chen and Tongmei Zhang conceived and designed the experiments;
Tingting Chen, Jieqiong Tan, Zhengqing Wan and Yongyi Zou performed the experiments; Tingting Chen and
Tongmei Zhang analyzed the data and wrote the manuscript; Henok Kessete Afewerky and Zhuohua Zhang
helped to draft the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
PD Parkinson’s Disease
PI Propidine Iodide
UPS Ubiquitin-proteasome System
WHO World Health Organization
DHR [3H]dihydrorotenone
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... Therefore several studies have examined pesticides residues in food reported by [ 20;21;22;23] Effects on human Health Memory Lack: Decrease ability pay attention and poor of bedding addition continues forgetfulness for workers in the production and storage of pesticides, who are exposed during work in factory and farms where exposure is constantly , where increase in current years the phenomenon of repeated forgetfulness and memory lack may lead to Alzheimer disease therefore [24 ], indicated to exposure for Organophosphorus pesticides and Organochlorines was found to effect the enzyme acetylcholine esterase ], which is responsible for the nervous connection and transmission for the nerve impulses in the central nervous system . In other study [25 ] confirmed that exposure to herbicides and insecticides , which works to weaken the activities of energy structure mitochondria in the cells organs , also works to impede the access of oxygen to the metabolic process , thus works on the emergence of Alzheimer disease due to the lack of oxygen to the cerebral blood vessels . Pregnant women: Pesticide is severe damage to pregnant woman as depend on the type of pesticides, period of exposure and the degree of toxicity. ...
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The aquatic environment is affected by pesticides toxins through the drainage irrigation water contamination with pesticides .The affects the water quality and becomes an unsuitable environment for aquatic organisms to live .Those workers in the fields of plant and environment protection from agricultural pestes and disease-carrying insects are exposed to the hazards of phosphorous and chlorinated pesticides , where the health of farmers who work in pesticide spraying is affected including breathing ,loss of memory and blood variables , in addition to its effect on pregnant and fetuses .
... However, Azevedo et al. [110] also observed that the higher concentration of pyriproxyfen (0.1 µg/mL) reduced the Ca 2+ uptake into the cell interior, which could be related to a loss of mitochondrial membrane potential, or a damage of the ER caused by the pesticide. These results are in line with the extensive evidence pointing to the mitochondria as one of the targets of neurotoxic action exerted by pesticides [116][117][118][119]. ...
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Pesticides of different chemical classes exert their toxic effects on the nervous system by acting on the different regulatory mechanisms of calcium (Ca2+) homeostasis. Pesticides have been shown to alter Ca2+ homeostasis, mainly by increasing its intracellular concentration above physiological levels. The pesticide-induced Ca2+ overload occurs through two main mechanisms: the entry of Ca2+ from the extracellular medium through the different types of Ca2+ channels present in the plasma membrane or its release into the cytoplasm from intracellular stocks, mainly from the endoplasmic reticulum. It has also been observed that intracellular increases in the Ca2+ concentrations are maintained over time, because pesticides inhibit the enzymes involved in reducing its levels. Thus, the alteration of Ca2+ levels can lead to the activation of various signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. In this review, we also discuss some proposed strategies to counteract the detrimental effects of pesticides on Ca2+ homeostasis.
... Therefore several studies have examined pesticides residues in food reported by [ 20;21;22;23] Effects on human Health Memory Lack: Decrease ability pay attention and poor of bedding addition continues forgetfulness for workers in the production and storage of pesticides, who are exposed during work in factory and farms where exposure is constantly , where increase in current years the phenomenon of repeated forgetfulness and memory lack may lead to Alzheimer disease therefore [24 ], indicated to exposure for Organophosphorus pesticides and Organochlorines was found to effect the enzyme acetylcholine esterase ], which is responsible for the nervous connection and transmission for the nerve impulses in the central nervous system . In other study [25 ] confirmed that exposure to herbicides and insecticides , which works to weaken the activities of energy structure mitochondria in the cells organs , also works to impede the access of oxygen to the metabolic process , thus works on the emergence of Alzheimer disease due to the lack of oxygen to the cerebral blood vessels . Pregnant women: Pesticide is severe damage to pregnant woman as depend on the type of pesticides, period of exposure and the degree of toxicity. ...
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Full-text available
The aquatic environment is affected by pesticides toxins through the drainage irrigation water contamination with pesticides .The affects the water quality and becomes an unsuitable environment for aquatic organisms to live .Those workers in the fields of plant and environment protection from agricultural pestes and disease-carrying insects are exposed to the hazards of phosphorous and chlorinated pesticides , where the health of farmers who work in pesticide spraying is affected including breathing ,loss of memory and blood variables , in addition to its effect on pregnant and fetuses .
... The main pathological hallmarks of PD include pronounced degeneration of dopaminergic neurons in the midbrain substantia nigra and aggregation of neuronal Lewy Bodies (Marino et al. 2020). Research studies suggest that environmental toxins, such as certain pesticides, that induce mitochondrial dysfunction are the major contributing factors of PD (Wirdefeldt et al. 2011;Bragoszewski et al. 2017;Chen et al. 2017). However, the PD pathogenesis mechanisms remain unclear, and there is no definitive PD-modifying therapy to date. ...
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