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The toxic influence of paraquat on hippocampus of mice: Involvement of oxidative stress
NeuroToxicology 31 (2010) 310–316
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NeuroToxicology
The toxic influence of paraquat on hippocampus of mice: Involvement of oxidative stress Qing Chen, Yujie Niu, Rong Zhang *, Huicai Guo, Yanjie Gao, Yao Li, Rujun Liu Department of Toxicology, School of Public Health, Hebei Medical University, Zhongshan East Road 361, Shijiazhuang 050017, Hebei, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 November 2009 Accepted 26 February 2010 Available online 6 March 2010
Environmental paraquat (PQ) exposure has been suggested to be a potential risk factor for neurodegenerative disorders such as Parkinson’s disease (PD). The hippocampus plays an important role in the learning and memory abilities of the brain. This study aims to demonstrate the effect and mechanism of paraquat toxicity on the hippocampus of mice. Kunming mice were randomly divided into four groups (one control and three treatment groups) and the dosage levels were defined as 0, 0.89, 2.67 and 8 mg/kg body weight. Paraquat was given orally, once a day and for 28 consecutive days. After treatment with paraquat, the hippocampus cells were found to be irregular and the cytoplasm was found to be condensed. The nissl bodies were reduced and apoptotic or necrotic neuron was observed. Morris water maze tests showed that the response latency increased significantly in animals that were administered paraquat. The level of malondialdehyde (MDA) and generation of reactive oxygen species (ROS) in the hippocampus of mice increased significantly. The activities of total superoxide dismutase (SOD) in the hippocampus of mice decreased significantly after treatment with paraquat. An analysis of the energy metabolism of hippocampus showed that the concentration of adenosine-triphosphate (ATP) decreased significantly in the hippocampus after treatment with paraquat, which implied that the energy synthesis of mitochondria with hippocampal neurocytes declined. The level of 8-OHdG in mitochondrial DNA (mtDNA) increased significantly after treatment with paraquat, which indicated that the oxidative damage of mtDNA increased. This suggests that paraquat had a toxic influence on the hippocampus of mice, and that the mechanism of toxicity might be associated with the mitochondrial injury of hippocampal neurocytes induced by oxidative stress. ß 2010 Elsevier Inc. All rights reserved.
Keywords: Paraquat Hippocampus Oxidative stress ROS 8-OHdG
1. Introduction Paraquat (1,1-dimethyl-4,4-bipyridium dichloride, PQ), is one of the most widely used non-selective dipyridyl herbicides all over the world, especially in developing countries such as China and India. However, paraquat is also toxic to human and animals (Marry and Gibson, 1972). Many cases of acute poisoning and death from paraquat have been reported over the past decade in some countries. Paraquat is a toxin known to target the dopaminergic neurons whose structure is similar to 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) and could induce neurodegenerative diseases (Shimizu et al., 2003; Peng et al., 2005; Langston and Ballsrd, 1984). Paraquat is a well known environmental risk factor for Parkinson’s disease (Dinis-Oliveira et al., 2006; Di Monte et al., 2002). Daniela et al. reported that injections of paraquat into the hippocampus of rats produced limbic seizures and hippocampal cell damage (Melchiorri et al., 1998). Other
* Corresponding author. Tel.: +86 311 86265605; fax: +86 311 86265632. E-mail address: [email protected] (R. Zhang). 0161-813X/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2010.02.006
investigations indicated that paraquat produced selective neuronal injuries in the CA1 region of the hippocampus through organotypic cultures (James et al., 1998). Although detrimental effects of paraquat on the brain, and especially on the hippocampus, have been recorded extensively, the underlying molecular mechanisms of paraquat toxicity have not been well defined. Paraquat could induce dopaminergic cell death (Peng et al., 2009, 2007) and the damage of dopaminergic neurons in the nigrostriatum is related to oxidative stress caused by excessive ROS production (Schmuck et al., 2002; Thiruchelvam et al., 2003; Kim et al., 2004). Excessive ROS generation could be one of the causes of mitochondrial dysfunction (Bonneh-Barkay et al., 2005; McCarthy et al., 2004). Mitochondria easily succumb to diverse assaults generated either in situ or in the extracellular environment. Mitochondrial dysfunction results in a dwindling supply of cellular energy, the failure to maintain cellular homeostasis, and the activation of cell death (Beal, 2005; Banerjee et al., 2009). Some studies have indicated that mitochondria are involved in neurocyte apoptosis or necrosis that increases mitochondrial membrane permeability and causes the release of pro-apoptotic factors into the cytosol (e.g., procaspases and caspase activators). This leads to
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the apoptotic phenotype (Fleury et al., 2002). Furthermore, the proximity of mtDNA to ROS generated as a consequence of respiratory chain function likely renders mtDNA vulnerable to mutations (Richter et al., 1988; Ozawa, 1997). The mtDNA region which encodes 7 of the 49 protein subunits of the complex I enzyme is particularly vulnerable to mutations (Swerdlow et al., 1996). Mohammadi-Bardbori and Ghazi-Khansari (2008) reported that paraquat might have toxic effects on the mitochondria and that its dysfunction could be induced by oxidative stress. We hypothesize that exposure to paraquat impairs cognitive function and may act by altering the function of neurocyte mitochondria in mice. In the present study, we evaluated the detrimental effects of paraquat on the hippocampus in mice using a Morris water maze and histopathological examinations, and tested the hypothesis that the cognitive dysfunctions observed after paraquat exposure was associated with mitochondrial dysfunction. Our results will provide evidences for the mechanism of paraquat neurotoxicity in relation to mitochondrial dysfunction.
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guided to the platform by the experimenter and the maximum latency was scored. Phase-2 (spatial probe test): On day 4, the platform was removed from the tank and a 120 s probe test was conducted. The times of the animal passing the platform site were recorded for up to 120 s. 2.3. Hippocampus tissue pathological assay The animals were deeply anesthetized with Nembutal and perfused with 200 ml of 37 8C saline solution for 5 min. And then mice were perfused with 500 ml of ice-cold 4% paraformaldehyde in a phosphate buffer solution (PBS) for 2 h. The brains were separated and maintained in a fixative solution (4% paraformaldehyde) until they were embedded with paraffin. Paraffin sections (7 mm thickness) were prepared with routine methods, stained with thionine using standard procedures, cleared in xylene and mounted with cover-slips. Images were observed under a light microscope. 2.4. Lipid peroxidation assay
2. Materials and methods 2.1. Mice and paraquat treatment Kunming mice (aged 7 weeks, 20–23 g; held at the Centre of Experiment Animals, Hebei Medical University, China) were used in this study. The mice were adapted to their surroundings for 1 week before the experiment was initiated. Animals were housed in a room that was maintained at a constant temperature (18–22 8C) and humidity (40–50%). All animals were fed a diet and water ad libitum in stainless cages, and they received humane treatment in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Animals were assigned to four groups randomly: a control group (0 mg/kg) and three paraquat treatment groups with each group comprising 20 mice. The doses and time regimen were designed based on the previous researches (Thida et al., 2004; Thiruchelvam et al., 2003; Pawinee et al., 2002) and the LD50 (110–150 mg/kg in rats) of paraquat (Extension Toxicology Network). The doses of paraquat were 0.89, 2.67, 8 mg/kg body weight, respectively. Paraquat was administered orally in doses of 0.1 ml/10 g body weight, respectively, once a day and for 28 consecutive days. 2.2. Morris water maze test The Morris water maze test is used to map spatial orientation in mice (Nakagawa and Takashima, 1997). This test consists of a round water tank (98 cm in diameter and 55 cm in height) filled with opaque water (40 cm in depth) at a temperature of approximately 22 8C. The water tank was divided into four equal quadrants: south-west (SW), north-west (NW), north-east (NE) and south-east (SE). A rotundity platform (38 cm high and 10 cm in diameter) was submerged 2 cm below the water surface, located in the centre of the SW quadrant. The training procedure was divided into two phases (phase-1 and phase-2). Phase-1 (place navigation): Mice were given four training trials each day for 4 consecutive days. For each training trial, the mice were placed in the water facing the pool wall at one of four positions (at the north, south, east or west pole) in a different order each day, and were allowed to swim until they reached the platform. The time required to reach the platform was recorded for up to 120 s. The mice were kept on the platform for 10 s before removal. Mice that failed to reach the platform within 120 s were
The hippocampus was separated from the brain on an ice-plate, and the hippocampus tissue was then placed in a glass homogenizer filled with normal saline solution. The hippocampi were homogenized for 1 min on ice. The homogenates were made into suspensions of 5% and 1% (w/v), respectively. Finally, the activities of total SOD and the levels of MDA were detected according to the commercial kits manual (Nanjing Jiancheng Bioeng Inst., China) using a microplate reader (SynergyTM HT, BioTek Instruments, Inc. USA). The results were expressed per microgram of protein. Protein concentrations were measured using the bio-rad protein assay reagent using bovine serum albumin as a standard. 2.5. Measurement of ROS generation The production of ROS in the hippocampus was measured using the membrane permeable dye 20 ,70 -dichlorodihydrofluorescein diacetate molecule probes (DCFH-DA, Bryotime Institute of Biotechnology, China) and using a slight modification of the previously published method (Dilday and Leslie, 1989; Siraki et al., 2002). Briefly, hippocampal cell suspensions were made as follows: after mice decollation, the hippocampus was separated from the brain on the ice-plate. The hippocampus tissue was washed twice with ice-cold PBS (pH 7.4) and broken into pieces with iris scissors, and the cell suspension was then incubated in 2.5 mg/ml trypsin at 37 8C for 20 min. Afterwards, one-tenth volume of bovine serum was added to stop the reaction. Cells were collected by filtration in a 200-mesh sieve and centrifugation at 500 g for 5 min. The pellet was washed twice with PBS, resuspended in PBS containing 1 ml DCFH-DA, and incubated at 37 8C, 5% CO2 for 20 min. The cells were then washed three times with PBS and centrifuged at 500 g. The formation of the fluorescent-oxidized derivative of DCFH-DA was monitored using a FACSarie flow cytometer (Becton Dickinson, USA) at an emission wavelength of 525 nm and an excitation wavelength of 488 nm. Finally, ROS generation was quantified using the median fluorescence intensity of 10,000 cells. 2.6. Detection of mitochondrial 8-hydroxy-deoxyguanosine (8-OHdG) 2.6.1. Mitochondrial isolation and extraction of mtDNA Mice were sacrificed by decapitation after being anesthetized by ether and hippocampi were harvested immediately, rinsed in ice-cold PBS. And then mitochondrial isolation and extraction of mtDNA were carried out as described in previous studies with minor modifications (Choksi et al., 2007; Tuula et al., 1985). Briefly,
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the hippocampus tissue was minced with scissors and homogenized with homogenizer in 1 ml GTE buffer containing 50 mM sucrose, 10 mM EDTA and 25 mM Tris–HCl (pH 8.0). The disrupted cells were then centrifuged at 1000 g for 3 min at 4 8C. The supernatant was collected and centrifuged at 12,000 g for 10 min at 4 8C and the resulting pellet was considered to consist of mitochondria. NS buffer (200 ml) was added to the mitochondrial suspension (1% SDS, 0.2 M NaOH) and was shaken up and then stored at 80 8C for 2 min. Sodium acetate (150 ml 3 M; pH 4.8) was added to the suspension and the suspension was shaken up and kept on ice for 5 min before being centrifuged at 1000 g for 10 min at 4 8C. The supernatant was collected and extracted twice with phenol/chloroform/isoamylol (25:24:1). The mtDNA was precipitated with two volumes of 100% cold ethanol and centrifuged at 12,000 g for 10 min at 4 8C, and was then washed with 70% ethanol, air-dried and resuspended in TE (10 mM Tris– HCl, pH 8.0, 1 mM EDTA). The mtDNA samples were stored at 20 8C until analysis. 2.6.2. Detection of 8-OHdG contained in mtDNA The pH value of each mtDNA sample was adjusted to 5 with 20 ml of 20 mM sodium acetate buffer (pH 5.0). mtDNA was hydrolyzed to nucleotides with 5.7 U of nuclease P1 (NP1, Biolong biological and technology Co., Ltd., Shanghai, China) at 65 8C for 10 min. Thereafter, the pH value of each mtDNA sample was adjusted to 8 with 20 ml 1 M Tris–HCl buffer (pH 8.5), and was further hydrolyzed to nucleosides with alkaline phosphatase (AP1, Takara Biotechnology CO., Ltd., Dalian, China) at 37 8C for 1 h. Proteins were removed from the sample with centrifugal micropure filters. The amount of 8-OHdG (Sigma–Aldrich, USA) in DNA samples was determined using a high performance liquid chromatograph (HPLC; LC-20A; Shimadzu, Japan) equipped with a UV-detector. Samples were separated on a Hypersil BDS C18 HPLC column (250 mm 4.6 mm, 10 mm). Twenty microlitres of mtDNA digest were injected into the HPLC column. The mobile phase consisted of 10% methanol in 50 mM phosphate buffer solution (pH 5.5). The mobile phase was filtered through Millipore 0.22 mm Durapore membrane filters and ultrasound degassed prior to use. Chromatographic analyses were performed at 25 8C and the detection wavelength was 260 nm. The flow rate was 1 ml/ min. 2-dG (Sigma–Aldrich, USA) was eluted with a peak at 8.4 min, followed by 8-OHdG at 12.2 min. The levels of 8-OHdG were expressed as 8-OHdG/105 2-dG. 2.7. Detection of ATP, ADP and AMP in the hippocampus The hippocampus was separated as described previously and was weighed accurately before being homogenized in 1 ml 0.1 M perchloric acid. The concentration of homogenate was 5% (w/v) and centrifuged at 20,000 g for 20 min at 4 8C. The supernatant was collected, 0.5 M sodium carbonate solution was added to adjust the pH to 7–8 and the supernatant was again centrifuged at 20,000 g for 10 min at 4 8C. The supernatant was filtered through a 0.22 mm Millipore filter, and then ATP, ADP and AMP levels (Fluka, Switzerland) were measured using high performance liquid chromatography (LC-20AT, Shimadzu Corporation, Japan) with a UV-detector (SPD-20A). Twenty microlitres of the supernatant were injected into the HPLC column. Samples were separated on a Hypersil BDS C18 HPLC column (250 mm 4.6 mm, 5 mm). The mobile phase consisted of 20 mM phosphate buffer solution (pH 5.9). The mobile phase was filtered through Millipore 0.22 mm Durapore membrane filters and ultrasound degassed prior to use. Chromatographic analyses were performed at 25 8C. The flow rate was 0.9 ml/min. ATP, ADP and AMP was detected at a wavelength of 254 nm. ATP was eluted with a peak at 6.3 min, followed by ADP at 7.5 min and AMP at 15.8 min.
2.8. Statistical analysis All values were expressed as mean SD and the Statistical Package for Social Science 11.0 (SPSS/PC) was used for all analysis. A one-way analysis of variance (ANOVA) with LSD Post Hoc analysis was used to identify significant differences. Significance was as p < 0.05. 3. Results 3.1. The effect of paraquat on the learning and memory abilities of animals The time required for animals to reach the platform in the training trial of the place navigation test is shown in Fig. 1. The results indicated that paraquat increased the time required for animals to reach the platform. The time required for animals to reach the platform of the paraquat treatment groups (2.67 and 8 mg/kg) was significantly higher in comparison to the control group (p < 0.05), especially on days 3 and 4 (Fig. 1). The results of probe test indicated that paraquat significantly decreased the times required for animals to pass the site where the platform had been located originally (Fig. 2). In the 2.67 and 8 mg/kg treatment groups, the time required for animals to pass the platform site was significantly lower than those of the control group (p < 0.05). These results suggested that paraquat influenced the learning and memory abilities of mice. 3.2. The effect of paraquat on the histopathology of hippocampus tissue Histopathological examinations indicated that morphological changes occurred in the hippocampus of animals after paraquat
Fig. 1. The latency of animal reaching platform in Morris water maze on different days, 10 mice per group. Each column represents the mean SD, *p < 0.05 compared with the control.
Fig. 2. Times of animal passing platform site where the platform had located originally in probe test. After place navigation trial, a 120 s probe test was conducted on day 4, recorded the times of animal passing platform site. Ten mice per groups, each column represents the mean SD, *p < 0.05 compared with the control.
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Fig. 3. The effect of paraquat on structure of hippocampal CA1 region. Thionine staining, magnification, 100. A: control; B: 0.89 mg/kg; C: 2.67 mg/kg; D: 8 mg/kg. The neurons of paraquat treatment groups showed a sparse, irregular arrangement and pyknotic nucleus, compared with the control, the arrow shows neuron cell death.
exposure. In the control group, the hippocampal CAl pyramidal neurons presented a dense, regular arrangement and a large, round nuclei with light staining. However, in the paraquat treatment groups, especially in the 8 mg/kg group, the hippocampal CA1 pyramidal neurons presented a sparse, irregular arrangement and pyknotic nucleus (Fig. 3). In the hippocampal hilus region, the shape of the neurons in the control group was regular and the nissl bodies were found frequently. Whereas in the treatment groups (particularly the 8 mg/kg group), neurons were irregular arrangement, and cytoplasm was condensed, the nissl bodies were seldom found and apoptotic or necrotic neuron was found frequently (Fig. 4). The results indicated that the structure of hippocampal tissue in paraquat treatment groups presented morphological changes in comparison to the control. 3.3. The effect of paraquat on lipid peroxidation of the hippocampus Detection results showed a decreased activity of total SOD in the hippocampus of paraquat treatment groups (Fig. 5) whereas the concentration of MDA in hippocampus of the paraquat treatment group increased compared with the control (Fig. 6). In particular, the activity of total SOD and the concentration of MDA in the hippocampus of paraquat treatment groups (2.67 and
8 mg/kg) showed significant differences compared with the control group (p < 0.05 or p < 0.01). The results showed that paraquat reinforced lipid peroxidation in the hippocampus. 3.4. The effect of paraquat on oxidative stress in the hippocampus The levels of ROS and 8-OHdG were measured by the methods previously described to evaluate whether paraquat induced oxidative stress and damaged the hippocampus of mice. The results showed that the levels of ROS in paraquat treatment groups (2.67 and 8 mg/kg) increased significantly compared with the control group (p < 0.05; Fig. 7). These results indicated that paraquat could increase free radical generation in the hippocampus of mice. Previous research has suggested that the degree of oxidative damage to mtDNA was associated with the intra-mitochondrial accumulation of 8-OHdG (Richter, 1992, 1995). Therefore, 8-OHdG is often regarded as a biomarker of oxidative damage to mtDNA. In the present study, the levels of 8-OHdG generation in the hippocampus of mice in paraquat treatment groups increased with the paraquat dosage. ANOVA and Post Hoc LSD tests indicated that there were significant differences between the paraquat treatment groups (2.67 and 8 mg/kg) and the control group
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Fig. 4. The effect of paraquat on structure of hippocampal hilus region. Thionine staining, magnification, 400. A: control; B: 0.89 mg/kg; C: 2.67 mg/kg; D: 8 mg/kg. In the paraquat treatment groups, neuron shapes were irregular and cytoplasm was condensed, compared with the control. The arrow shows neuron cell death.
(p < 0.05; Fig. 8). These results suggested that paraquat could cause oxidative damage to hippocampal neuron mtDNA. 3.5. The effect of paraquat on the energy metabolism of mitochondria in the hippocampus
Fig. 5. The effect of paraquat on activities of total SOD in hippocampus of mice. Values were mean SD, 10 mice per group, **p < 0.01 compared with the control.
Fig. 6. The effect of paraquat on concentration of MDA in hippocampus of mice. Values were mean SD, 10 mice per group, *p < 0.05, **p < 0.01 compared with the control.
The major function of mitochondria is the synthesis of ATP through oxidative phosphorylation. The level of ATP generation reflects the status of mitochondria. Thus, concentrations of ATP, ADP and AMP in hippocampus tissue were measured by HPLC to evaluate the status of mitochondria. Our results showed that the concentrations of ATP in the paraquat treatment groups (8 mg/kg) decreased significantly. However, the concentration of AMP in the paraquat treatment groups (8 mg/kg) increased significantly in comparison to the control group (Fig. 9). These results suggested
Fig. 7. The effect of paraquat on the generation of reactive oxygen species (ROS) in hippocampus. Neurocyte was isolated from hippocampus tissues with methods described previously, ROS generation was measured by flowcytometer. Results were expressed as fluorescence intensity of ROS per 10,000 cells. Values were mean SD for 10 mice per group. *p < 0.05 compared with the control.
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Fig. 8. The effect of paraquat on the accumulation of 8-hydroxy-2-deoxyguanosine in mitochondria of hippocampus. The content of 8-OHdG was expressed as ratio of 8-OHdG/105dG, the generation of 8-OHdG was measured with HPLC. The results represent the level of mtDNA oxidative damage. Values were mean SD for 10 mice per group. *p < 0.05 compared with the control.
Fig. 9. The effect of paraquat on level of adenylic acid in hippocampus of mice. Value was given as mean SD, 10 mice per group, *p < 0.05 compared with the control.
that mitochondrial dysfunction induced by paraquat results in a decrease in energy synthesis. 4. Discussion Paraquat poisoning is a major cause of death and induces multisystem damage (Casey and Vale, 1994). Epidemiological studies indicate that long-term exposure to paraquat can be a risk factor in the incidence of Parkinson’s disease (Yang and TiffanyCastiglioni, 2005). Animal studies showed that paraquat is toxic to the dopaminergic neurons of the rat and mouse brain (Mangano and Hayley, 2008). James et al. (1998) reported that paraquat caused dose- and time-dependent injury through organotypic hippocampal culture when exposed to high concentrations or for long periods, and the CA1 region of the hippocampus was found to be the most vulnerable. In this study, we found that the time required to reach the platform was longer for paraquat treatment groups than for the control group during the training trial conducted on days 1–4 (Fig. 1). In the probe test, the amount of time required for the animals to pass the original platform was lower for paraquat treatment groups compared with the control group (Fig. 2). Our results demonstrated that paraquat could attenuate the learning and memory abilities of mice. The hippocampus is considered to be an important region of the brain for learning and memory functions. In the present study, a histopathological analysis showed that the structure of the hippocampus of mice treated with paraquat showed significant differences compared with the control mice (Figs. 3 and 4). Apoptotic or necrotic hippocampal neurons were observed. A previous study found that the microinfusion of paraquat into the pars compacta of the substantia nigra produced neuropathological changes culminating in neuronal necrosis. Similar neuropathological alterations were also observed in other no-dopaminergic
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areas (Calo et al., 1990). The neuropathologic lesions of hippocampus could result in dysfunction of the hippocampus. Previous studies have shown that paraquat can not only permeate the blood–brain barrier through neutral amino acid transporters (McCormack and Di Monte, 2003), but can also damage the nigrostriatal dopaminergic system (Peng et al., 2004; McCormack et al., 2002). Although the mechanism of paraquat neurotoxicity is not well understood, paraquat is known to damage the substantia nigra of the brain through oxidative stress and lipid peroxidation (Peng et al., 2004; Peskin and Winterbourn, 2000) and can then induce neuron cell death (Liou et al., 1997; McCarthy et al., 2004). Accordingly, the aim of this study was to determine whether paraquat could induce oxidative stress in hippocampal neurons. Accumulating evidence shows that paraquat causes oxidative stress by inducing the generation of ROS (Dinis-Oliveira et al., 2006). Excess production of ROS in the brain has been implicated as a common factor underlying the etiology of paraquat-induced neurotoxicity. Previous studies showed that the production of ROS was responsible for oxidative stress, and an increase in ROS induced dopaminergic cell death (Peng et al., 2009; Purisai et al., 2007). In the present study, we found that ROS generation in the hippocampus of paraquat treatment groups increased significantly compared with the control group (Fig. 7). Excess ROS generation can damage a wide variety of cellular constituents including DNA, RNA, proteins, sugars and lipids, thereby compromising cell viability. Typically, lipid peroxidation was the primary result of oxidative stress, and correlated effects on the levels of SOD and MDA were also observed. SOD is an antioxidant against free radicals, such as ROS, reactive nitrogen species (RNS) and so on, whereas MDA was a metabolite of the lipid peroxidation of membranes. The present study indicated that paraquat inhibited the activity of total SOD in the hippocampus and significantly increased the contents of MDA in the hippocampus (Figs. 5 and 6). These results suggested that paraquat could induce the excessive generation of ROS and could reinforce lipid peroxidation in the hippocampus, and thus affected the oxidation-antioxidation homeostasis. Previous studies implied that mitochondrial dysfunction induced by oxidative stress played an important role in paraquat neurotoxicity (Keeney et al., 2006). Mitochondria are candidate targets of paraquat toxicity in animal and plant tissues (Taylor et al., 2002) and are considered to be a major source of ROS in cells (Cadenas and Davies, 2000). Therefore, the mechanism of mitochondrial impairment induced by paraquat was related to the generation of ROS (Suntres, 2002). In the present study, the contents of 8-OHdG and ATP in the hippocampus tissues were measured with HPLC to explore the mechanism by which hippocampal damage was induced by paraquat. The level of 8OHdG is considered to be a reliable indicator of the oxidative damage of mitochondria (Svoboda et al., 2008). Our results showed that the levels of 8-OHdG in the hippocampus of mice in the paraquat treatment groups increased significantly compared with the control group (Fig. 8), which implied that paraquat could cause oxidative damage in mtDNA. Moreover, the total ATP production of the paraquat treatment groups significantly decreased in comparison to the control group. The AMP production of the paraquat treatment groups also increased significantly compared with the control group (Fig. 9). The contents of ATP and AMP can reflect not only energy metabolism but also the functional status of the mitochondrial electron transport chain. Therefore, the results clearly demonstrated that the energy generation of mitochondria in hippocampal neurocytes was affected by paraquat. Palmeira et al. (1995) reported that the mechanism of paraquat-induced damage of substantia nigra might be related to the effects of paraquat causing uncoupled oxidative phosphorylation by lipid
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peroxidation, which inhibited adenosine-triphosphate synthase activity. This study further demonstrated that mitochondrial damage induced by paraquat was likely the major cause of hippocampal neurocyte death. Therefore, we concluded that paraquat could damage the hippocampus of mice by oxidative stress, and that the injury was associated with hippocampal neuron mitochondrial dysfunction induced by mtDNA oxidative damage. Ultimately, the learning and memory abilities of mice might be affected. Significantly, this study demonstrated the effects of paraquat on the hippocampus for the first time and suggested that the effects of paraquat might be associated with oxidative stress in the hippocampus. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgment This work was supported by grants from the Hebei Education Department Foundation for Medical Emphases, No. 2008131. References Beal MF. Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 2005;58:495–505. Bonneh-Barkay D, Reaney SH, Langston WJ, Di Monte DA. Redox cycling of the herbicide paraquat inmicroglial cultures. Brain Res Mol Brain Res 2005;13:452–6. Banerjee R, Anatoly A, Starkov M, Flint B, Bobby T. Mitochondrial dysfunction in the limelight of Parkinson’s disease pathogenesis. Biochim Biophys Acta 2009; 1792:651–63. Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000;29:222–30. Casey P, Vale JA. Deaths from pesticide poisoning in England and Wales: 1945–1989. Hum Exp Toxicol 1994;13:95–101. Calo M, Iannone M, Passafaro M, Nistico G. Selective vulnerability of hippocampal CA3 neurones after microinfusion of paraquat into the rat substantia nigra or into the ventral tegmental area. J Comp Pathol 1990;103:73–8. Choksi KB, Nuss JE, Boylston WH, Rabek JP, Papaconstantinou J. Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes. Free Radical Biol Med 2007;43:1423–38. Dinis-Oliveira RJ, Remiao F, Carmo H, Duarte JA, Sanchez Navarro A, Bastos ML. Paraquat exposure as an etiological factor of Parkinson’s disease. Neurotoxicology 2006;27:1110–22. Dilday JE, Leslie SW. Ethanol inhibits NMDA-induced increase in free intracellular Ca2+ in dissociated brain cells. Brain Res 1989;499:383–7. Di Monte DA, Lavasani M, Manning-Bog AB. Environmental factors in Parkinson’s disease. Neurotoxicology 2002;23:487–502. Extension Toxicology Network. Pesticide information profiles. Paraquat; 1996, http:// extoxnet.orst.edu/pips/paraquat.htm (last accessed 5/20/08). Fleury C, Mignotte B, Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 2002;84:131–41. James J, Vornov JP, Ajit G, Thomas. Regional vulnerablility to endogenous and exogenous oxidative stress in organotypic hippocampal culture. Exp Neurol 1998; 149:109–22. Kim SJ, Kim JE, Moon IS. Paraquat induces apoptosis of cultured rat cortical cells. Mol Cells 2004;17:102–7. Keeney PM, Xie J, Capaldi RJ, Bennett JR. Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 2006;26:5256–64. Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen RC. Environmental risk factors and Parkonson’s disease: a case control study in Taiwan. Neurology 1997;48:1583–8. Langston JW, Ballsrd P. Parkonsinism induced by 1-methyl -4-phenyl-1,2,3,6 tetrahydropyridine(MPTP):implications for treatment and the pathogenesis of Parkonson’s disease. Can J Neurol Sci 1984;11:160–5. Marry RE, Gibson JE. A comparative study of PQ intoxication in rats, guinea pigs and monkey. Exp Mol Pathol 1972;17:317–25. Mangano E, Hayley S. Inflammatory priming of the substantia nigra influences the impact of later paraquat exposure: neuroimmune sensitization of neurodegeneration. Neurobiol Aging 2008;30:1361–78.
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NeuroToxicology
The toxic influence of paraquat on hippocampus of mice: Involvement of oxidative stress Qing Chen, Yujie Niu, Rong Zhang *, Huicai Guo, Yanjie Gao, Yao Li, Rujun Liu Department of Toxicology, School of Public Health, Hebei Medical University, Zhongshan East Road 361, Shijiazhuang 050017, Hebei, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 November 2009 Accepted 26 February 2010 Available online 6 March 2010
Environmental paraquat (PQ) exposure has been suggested to be a potential risk factor for neurodegenerative disorders such as Parkinson’s disease (PD). The hippocampus plays an important role in the learning and memory abilities of the brain. This study aims to demonstrate the effect and mechanism of paraquat toxicity on the hippocampus of mice. Kunming mice were randomly divided into four groups (one control and three treatment groups) and the dosage levels were defined as 0, 0.89, 2.67 and 8 mg/kg body weight. Paraquat was given orally, once a day and for 28 consecutive days. After treatment with paraquat, the hippocampus cells were found to be irregular and the cytoplasm was found to be condensed. The nissl bodies were reduced and apoptotic or necrotic neuron was observed. Morris water maze tests showed that the response latency increased significantly in animals that were administered paraquat. The level of malondialdehyde (MDA) and generation of reactive oxygen species (ROS) in the hippocampus of mice increased significantly. The activities of total superoxide dismutase (SOD) in the hippocampus of mice decreased significantly after treatment with paraquat. An analysis of the energy metabolism of hippocampus showed that the concentration of adenosine-triphosphate (ATP) decreased significantly in the hippocampus after treatment with paraquat, which implied that the energy synthesis of mitochondria with hippocampal neurocytes declined. The level of 8-OHdG in mitochondrial DNA (mtDNA) increased significantly after treatment with paraquat, which indicated that the oxidative damage of mtDNA increased. This suggests that paraquat had a toxic influence on the hippocampus of mice, and that the mechanism of toxicity might be associated with the mitochondrial injury of hippocampal neurocytes induced by oxidative stress. ß 2010 Elsevier Inc. All rights reserved.
Keywords: Paraquat Hippocampus Oxidative stress ROS 8-OHdG
1. Introduction Paraquat (1,1-dimethyl-4,4-bipyridium dichloride, PQ), is one of the most widely used non-selective dipyridyl herbicides all over the world, especially in developing countries such as China and India. However, paraquat is also toxic to human and animals (Marry and Gibson, 1972). Many cases of acute poisoning and death from paraquat have been reported over the past decade in some countries. Paraquat is a toxin known to target the dopaminergic neurons whose structure is similar to 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) and could induce neurodegenerative diseases (Shimizu et al., 2003; Peng et al., 2005; Langston and Ballsrd, 1984). Paraquat is a well known environmental risk factor for Parkinson’s disease (Dinis-Oliveira et al., 2006; Di Monte et al., 2002). Daniela et al. reported that injections of paraquat into the hippocampus of rats produced limbic seizures and hippocampal cell damage (Melchiorri et al., 1998). Other
* Corresponding author. Tel.: +86 311 86265605; fax: +86 311 86265632. E-mail address: [email protected] (R. Zhang). 0161-813X/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2010.02.006
investigations indicated that paraquat produced selective neuronal injuries in the CA1 region of the hippocampus through organotypic cultures (James et al., 1998). Although detrimental effects of paraquat on the brain, and especially on the hippocampus, have been recorded extensively, the underlying molecular mechanisms of paraquat toxicity have not been well defined. Paraquat could induce dopaminergic cell death (Peng et al., 2009, 2007) and the damage of dopaminergic neurons in the nigrostriatum is related to oxidative stress caused by excessive ROS production (Schmuck et al., 2002; Thiruchelvam et al., 2003; Kim et al., 2004). Excessive ROS generation could be one of the causes of mitochondrial dysfunction (Bonneh-Barkay et al., 2005; McCarthy et al., 2004). Mitochondria easily succumb to diverse assaults generated either in situ or in the extracellular environment. Mitochondrial dysfunction results in a dwindling supply of cellular energy, the failure to maintain cellular homeostasis, and the activation of cell death (Beal, 2005; Banerjee et al., 2009). Some studies have indicated that mitochondria are involved in neurocyte apoptosis or necrosis that increases mitochondrial membrane permeability and causes the release of pro-apoptotic factors into the cytosol (e.g., procaspases and caspase activators). This leads to
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the apoptotic phenotype (Fleury et al., 2002). Furthermore, the proximity of mtDNA to ROS generated as a consequence of respiratory chain function likely renders mtDNA vulnerable to mutations (Richter et al., 1988; Ozawa, 1997). The mtDNA region which encodes 7 of the 49 protein subunits of the complex I enzyme is particularly vulnerable to mutations (Swerdlow et al., 1996). Mohammadi-Bardbori and Ghazi-Khansari (2008) reported that paraquat might have toxic effects on the mitochondria and that its dysfunction could be induced by oxidative stress. We hypothesize that exposure to paraquat impairs cognitive function and may act by altering the function of neurocyte mitochondria in mice. In the present study, we evaluated the detrimental effects of paraquat on the hippocampus in mice using a Morris water maze and histopathological examinations, and tested the hypothesis that the cognitive dysfunctions observed after paraquat exposure was associated with mitochondrial dysfunction. Our results will provide evidences for the mechanism of paraquat neurotoxicity in relation to mitochondrial dysfunction.
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guided to the platform by the experimenter and the maximum latency was scored. Phase-2 (spatial probe test): On day 4, the platform was removed from the tank and a 120 s probe test was conducted. The times of the animal passing the platform site were recorded for up to 120 s. 2.3. Hippocampus tissue pathological assay The animals were deeply anesthetized with Nembutal and perfused with 200 ml of 37 8C saline solution for 5 min. And then mice were perfused with 500 ml of ice-cold 4% paraformaldehyde in a phosphate buffer solution (PBS) for 2 h. The brains were separated and maintained in a fixative solution (4% paraformaldehyde) until they were embedded with paraffin. Paraffin sections (7 mm thickness) were prepared with routine methods, stained with thionine using standard procedures, cleared in xylene and mounted with cover-slips. Images were observed under a light microscope. 2.4. Lipid peroxidation assay
2. Materials and methods 2.1. Mice and paraquat treatment Kunming mice (aged 7 weeks, 20–23 g; held at the Centre of Experiment Animals, Hebei Medical University, China) were used in this study. The mice were adapted to their surroundings for 1 week before the experiment was initiated. Animals were housed in a room that was maintained at a constant temperature (18–22 8C) and humidity (40–50%). All animals were fed a diet and water ad libitum in stainless cages, and they received humane treatment in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Animals were assigned to four groups randomly: a control group (0 mg/kg) and three paraquat treatment groups with each group comprising 20 mice. The doses and time regimen were designed based on the previous researches (Thida et al., 2004; Thiruchelvam et al., 2003; Pawinee et al., 2002) and the LD50 (110–150 mg/kg in rats) of paraquat (Extension Toxicology Network). The doses of paraquat were 0.89, 2.67, 8 mg/kg body weight, respectively. Paraquat was administered orally in doses of 0.1 ml/10 g body weight, respectively, once a day and for 28 consecutive days. 2.2. Morris water maze test The Morris water maze test is used to map spatial orientation in mice (Nakagawa and Takashima, 1997). This test consists of a round water tank (98 cm in diameter and 55 cm in height) filled with opaque water (40 cm in depth) at a temperature of approximately 22 8C. The water tank was divided into four equal quadrants: south-west (SW), north-west (NW), north-east (NE) and south-east (SE). A rotundity platform (38 cm high and 10 cm in diameter) was submerged 2 cm below the water surface, located in the centre of the SW quadrant. The training procedure was divided into two phases (phase-1 and phase-2). Phase-1 (place navigation): Mice were given four training trials each day for 4 consecutive days. For each training trial, the mice were placed in the water facing the pool wall at one of four positions (at the north, south, east or west pole) in a different order each day, and were allowed to swim until they reached the platform. The time required to reach the platform was recorded for up to 120 s. The mice were kept on the platform for 10 s before removal. Mice that failed to reach the platform within 120 s were
The hippocampus was separated from the brain on an ice-plate, and the hippocampus tissue was then placed in a glass homogenizer filled with normal saline solution. The hippocampi were homogenized for 1 min on ice. The homogenates were made into suspensions of 5% and 1% (w/v), respectively. Finally, the activities of total SOD and the levels of MDA were detected according to the commercial kits manual (Nanjing Jiancheng Bioeng Inst., China) using a microplate reader (SynergyTM HT, BioTek Instruments, Inc. USA). The results were expressed per microgram of protein. Protein concentrations were measured using the bio-rad protein assay reagent using bovine serum albumin as a standard. 2.5. Measurement of ROS generation The production of ROS in the hippocampus was measured using the membrane permeable dye 20 ,70 -dichlorodihydrofluorescein diacetate molecule probes (DCFH-DA, Bryotime Institute of Biotechnology, China) and using a slight modification of the previously published method (Dilday and Leslie, 1989; Siraki et al., 2002). Briefly, hippocampal cell suspensions were made as follows: after mice decollation, the hippocampus was separated from the brain on the ice-plate. The hippocampus tissue was washed twice with ice-cold PBS (pH 7.4) and broken into pieces with iris scissors, and the cell suspension was then incubated in 2.5 mg/ml trypsin at 37 8C for 20 min. Afterwards, one-tenth volume of bovine serum was added to stop the reaction. Cells were collected by filtration in a 200-mesh sieve and centrifugation at 500 g for 5 min. The pellet was washed twice with PBS, resuspended in PBS containing 1 ml DCFH-DA, and incubated at 37 8C, 5% CO2 for 20 min. The cells were then washed three times with PBS and centrifuged at 500 g. The formation of the fluorescent-oxidized derivative of DCFH-DA was monitored using a FACSarie flow cytometer (Becton Dickinson, USA) at an emission wavelength of 525 nm and an excitation wavelength of 488 nm. Finally, ROS generation was quantified using the median fluorescence intensity of 10,000 cells. 2.6. Detection of mitochondrial 8-hydroxy-deoxyguanosine (8-OHdG) 2.6.1. Mitochondrial isolation and extraction of mtDNA Mice were sacrificed by decapitation after being anesthetized by ether and hippocampi were harvested immediately, rinsed in ice-cold PBS. And then mitochondrial isolation and extraction of mtDNA were carried out as described in previous studies with minor modifications (Choksi et al., 2007; Tuula et al., 1985). Briefly,
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the hippocampus tissue was minced with scissors and homogenized with homogenizer in 1 ml GTE buffer containing 50 mM sucrose, 10 mM EDTA and 25 mM Tris–HCl (pH 8.0). The disrupted cells were then centrifuged at 1000 g for 3 min at 4 8C. The supernatant was collected and centrifuged at 12,000 g for 10 min at 4 8C and the resulting pellet was considered to consist of mitochondria. NS buffer (200 ml) was added to the mitochondrial suspension (1% SDS, 0.2 M NaOH) and was shaken up and then stored at 80 8C for 2 min. Sodium acetate (150 ml 3 M; pH 4.8) was added to the suspension and the suspension was shaken up and kept on ice for 5 min before being centrifuged at 1000 g for 10 min at 4 8C. The supernatant was collected and extracted twice with phenol/chloroform/isoamylol (25:24:1). The mtDNA was precipitated with two volumes of 100% cold ethanol and centrifuged at 12,000 g for 10 min at 4 8C, and was then washed with 70% ethanol, air-dried and resuspended in TE (10 mM Tris– HCl, pH 8.0, 1 mM EDTA). The mtDNA samples were stored at 20 8C until analysis. 2.6.2. Detection of 8-OHdG contained in mtDNA The pH value of each mtDNA sample was adjusted to 5 with 20 ml of 20 mM sodium acetate buffer (pH 5.0). mtDNA was hydrolyzed to nucleotides with 5.7 U of nuclease P1 (NP1, Biolong biological and technology Co., Ltd., Shanghai, China) at 65 8C for 10 min. Thereafter, the pH value of each mtDNA sample was adjusted to 8 with 20 ml 1 M Tris–HCl buffer (pH 8.5), and was further hydrolyzed to nucleosides with alkaline phosphatase (AP1, Takara Biotechnology CO., Ltd., Dalian, China) at 37 8C for 1 h. Proteins were removed from the sample with centrifugal micropure filters. The amount of 8-OHdG (Sigma–Aldrich, USA) in DNA samples was determined using a high performance liquid chromatograph (HPLC; LC-20A; Shimadzu, Japan) equipped with a UV-detector. Samples were separated on a Hypersil BDS C18 HPLC column (250 mm 4.6 mm, 10 mm). Twenty microlitres of mtDNA digest were injected into the HPLC column. The mobile phase consisted of 10% methanol in 50 mM phosphate buffer solution (pH 5.5). The mobile phase was filtered through Millipore 0.22 mm Durapore membrane filters and ultrasound degassed prior to use. Chromatographic analyses were performed at 25 8C and the detection wavelength was 260 nm. The flow rate was 1 ml/ min. 2-dG (Sigma–Aldrich, USA) was eluted with a peak at 8.4 min, followed by 8-OHdG at 12.2 min. The levels of 8-OHdG were expressed as 8-OHdG/105 2-dG. 2.7. Detection of ATP, ADP and AMP in the hippocampus The hippocampus was separated as described previously and was weighed accurately before being homogenized in 1 ml 0.1 M perchloric acid. The concentration of homogenate was 5% (w/v) and centrifuged at 20,000 g for 20 min at 4 8C. The supernatant was collected, 0.5 M sodium carbonate solution was added to adjust the pH to 7–8 and the supernatant was again centrifuged at 20,000 g for 10 min at 4 8C. The supernatant was filtered through a 0.22 mm Millipore filter, and then ATP, ADP and AMP levels (Fluka, Switzerland) were measured using high performance liquid chromatography (LC-20AT, Shimadzu Corporation, Japan) with a UV-detector (SPD-20A). Twenty microlitres of the supernatant were injected into the HPLC column. Samples were separated on a Hypersil BDS C18 HPLC column (250 mm 4.6 mm, 5 mm). The mobile phase consisted of 20 mM phosphate buffer solution (pH 5.9). The mobile phase was filtered through Millipore 0.22 mm Durapore membrane filters and ultrasound degassed prior to use. Chromatographic analyses were performed at 25 8C. The flow rate was 0.9 ml/min. ATP, ADP and AMP was detected at a wavelength of 254 nm. ATP was eluted with a peak at 6.3 min, followed by ADP at 7.5 min and AMP at 15.8 min.
2.8. Statistical analysis All values were expressed as mean SD and the Statistical Package for Social Science 11.0 (SPSS/PC) was used for all analysis. A one-way analysis of variance (ANOVA) with LSD Post Hoc analysis was used to identify significant differences. Significance was as p < 0.05. 3. Results 3.1. The effect of paraquat on the learning and memory abilities of animals The time required for animals to reach the platform in the training trial of the place navigation test is shown in Fig. 1. The results indicated that paraquat increased the time required for animals to reach the platform. The time required for animals to reach the platform of the paraquat treatment groups (2.67 and 8 mg/kg) was significantly higher in comparison to the control group (p < 0.05), especially on days 3 and 4 (Fig. 1). The results of probe test indicated that paraquat significantly decreased the times required for animals to pass the site where the platform had been located originally (Fig. 2). In the 2.67 and 8 mg/kg treatment groups, the time required for animals to pass the platform site was significantly lower than those of the control group (p < 0.05). These results suggested that paraquat influenced the learning and memory abilities of mice. 3.2. The effect of paraquat on the histopathology of hippocampus tissue Histopathological examinations indicated that morphological changes occurred in the hippocampus of animals after paraquat
Fig. 1. The latency of animal reaching platform in Morris water maze on different days, 10 mice per group. Each column represents the mean SD, *p < 0.05 compared with the control.
Fig. 2. Times of animal passing platform site where the platform had located originally in probe test. After place navigation trial, a 120 s probe test was conducted on day 4, recorded the times of animal passing platform site. Ten mice per groups, each column represents the mean SD, *p < 0.05 compared with the control.
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Fig. 3. The effect of paraquat on structure of hippocampal CA1 region. Thionine staining, magnification, 100. A: control; B: 0.89 mg/kg; C: 2.67 mg/kg; D: 8 mg/kg. The neurons of paraquat treatment groups showed a sparse, irregular arrangement and pyknotic nucleus, compared with the control, the arrow shows neuron cell death.
exposure. In the control group, the hippocampal CAl pyramidal neurons presented a dense, regular arrangement and a large, round nuclei with light staining. However, in the paraquat treatment groups, especially in the 8 mg/kg group, the hippocampal CA1 pyramidal neurons presented a sparse, irregular arrangement and pyknotic nucleus (Fig. 3). In the hippocampal hilus region, the shape of the neurons in the control group was regular and the nissl bodies were found frequently. Whereas in the treatment groups (particularly the 8 mg/kg group), neurons were irregular arrangement, and cytoplasm was condensed, the nissl bodies were seldom found and apoptotic or necrotic neuron was found frequently (Fig. 4). The results indicated that the structure of hippocampal tissue in paraquat treatment groups presented morphological changes in comparison to the control. 3.3. The effect of paraquat on lipid peroxidation of the hippocampus Detection results showed a decreased activity of total SOD in the hippocampus of paraquat treatment groups (Fig. 5) whereas the concentration of MDA in hippocampus of the paraquat treatment group increased compared with the control (Fig. 6). In particular, the activity of total SOD and the concentration of MDA in the hippocampus of paraquat treatment groups (2.67 and
8 mg/kg) showed significant differences compared with the control group (p < 0.05 or p < 0.01). The results showed that paraquat reinforced lipid peroxidation in the hippocampus. 3.4. The effect of paraquat on oxidative stress in the hippocampus The levels of ROS and 8-OHdG were measured by the methods previously described to evaluate whether paraquat induced oxidative stress and damaged the hippocampus of mice. The results showed that the levels of ROS in paraquat treatment groups (2.67 and 8 mg/kg) increased significantly compared with the control group (p < 0.05; Fig. 7). These results indicated that paraquat could increase free radical generation in the hippocampus of mice. Previous research has suggested that the degree of oxidative damage to mtDNA was associated with the intra-mitochondrial accumulation of 8-OHdG (Richter, 1992, 1995). Therefore, 8-OHdG is often regarded as a biomarker of oxidative damage to mtDNA. In the present study, the levels of 8-OHdG generation in the hippocampus of mice in paraquat treatment groups increased with the paraquat dosage. ANOVA and Post Hoc LSD tests indicated that there were significant differences between the paraquat treatment groups (2.67 and 8 mg/kg) and the control group
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Fig. 4. The effect of paraquat on structure of hippocampal hilus region. Thionine staining, magnification, 400. A: control; B: 0.89 mg/kg; C: 2.67 mg/kg; D: 8 mg/kg. In the paraquat treatment groups, neuron shapes were irregular and cytoplasm was condensed, compared with the control. The arrow shows neuron cell death.
(p < 0.05; Fig. 8). These results suggested that paraquat could cause oxidative damage to hippocampal neuron mtDNA. 3.5. The effect of paraquat on the energy metabolism of mitochondria in the hippocampus
Fig. 5. The effect of paraquat on activities of total SOD in hippocampus of mice. Values were mean SD, 10 mice per group, **p < 0.01 compared with the control.
Fig. 6. The effect of paraquat on concentration of MDA in hippocampus of mice. Values were mean SD, 10 mice per group, *p < 0.05, **p < 0.01 compared with the control.
The major function of mitochondria is the synthesis of ATP through oxidative phosphorylation. The level of ATP generation reflects the status of mitochondria. Thus, concentrations of ATP, ADP and AMP in hippocampus tissue were measured by HPLC to evaluate the status of mitochondria. Our results showed that the concentrations of ATP in the paraquat treatment groups (8 mg/kg) decreased significantly. However, the concentration of AMP in the paraquat treatment groups (8 mg/kg) increased significantly in comparison to the control group (Fig. 9). These results suggested
Fig. 7. The effect of paraquat on the generation of reactive oxygen species (ROS) in hippocampus. Neurocyte was isolated from hippocampus tissues with methods described previously, ROS generation was measured by flowcytometer. Results were expressed as fluorescence intensity of ROS per 10,000 cells. Values were mean SD for 10 mice per group. *p < 0.05 compared with the control.
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Fig. 8. The effect of paraquat on the accumulation of 8-hydroxy-2-deoxyguanosine in mitochondria of hippocampus. The content of 8-OHdG was expressed as ratio of 8-OHdG/105dG, the generation of 8-OHdG was measured with HPLC. The results represent the level of mtDNA oxidative damage. Values were mean SD for 10 mice per group. *p < 0.05 compared with the control.
Fig. 9. The effect of paraquat on level of adenylic acid in hippocampus of mice. Value was given as mean SD, 10 mice per group, *p < 0.05 compared with the control.
that mitochondrial dysfunction induced by paraquat results in a decrease in energy synthesis. 4. Discussion Paraquat poisoning is a major cause of death and induces multisystem damage (Casey and Vale, 1994). Epidemiological studies indicate that long-term exposure to paraquat can be a risk factor in the incidence of Parkinson’s disease (Yang and TiffanyCastiglioni, 2005). Animal studies showed that paraquat is toxic to the dopaminergic neurons of the rat and mouse brain (Mangano and Hayley, 2008). James et al. (1998) reported that paraquat caused dose- and time-dependent injury through organotypic hippocampal culture when exposed to high concentrations or for long periods, and the CA1 region of the hippocampus was found to be the most vulnerable. In this study, we found that the time required to reach the platform was longer for paraquat treatment groups than for the control group during the training trial conducted on days 1–4 (Fig. 1). In the probe test, the amount of time required for the animals to pass the original platform was lower for paraquat treatment groups compared with the control group (Fig. 2). Our results demonstrated that paraquat could attenuate the learning and memory abilities of mice. The hippocampus is considered to be an important region of the brain for learning and memory functions. In the present study, a histopathological analysis showed that the structure of the hippocampus of mice treated with paraquat showed significant differences compared with the control mice (Figs. 3 and 4). Apoptotic or necrotic hippocampal neurons were observed. A previous study found that the microinfusion of paraquat into the pars compacta of the substantia nigra produced neuropathological changes culminating in neuronal necrosis. Similar neuropathological alterations were also observed in other no-dopaminergic
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areas (Calo et al., 1990). The neuropathologic lesions of hippocampus could result in dysfunction of the hippocampus. Previous studies have shown that paraquat can not only permeate the blood–brain barrier through neutral amino acid transporters (McCormack and Di Monte, 2003), but can also damage the nigrostriatal dopaminergic system (Peng et al., 2004; McCormack et al., 2002). Although the mechanism of paraquat neurotoxicity is not well understood, paraquat is known to damage the substantia nigra of the brain through oxidative stress and lipid peroxidation (Peng et al., 2004; Peskin and Winterbourn, 2000) and can then induce neuron cell death (Liou et al., 1997; McCarthy et al., 2004). Accordingly, the aim of this study was to determine whether paraquat could induce oxidative stress in hippocampal neurons. Accumulating evidence shows that paraquat causes oxidative stress by inducing the generation of ROS (Dinis-Oliveira et al., 2006). Excess production of ROS in the brain has been implicated as a common factor underlying the etiology of paraquat-induced neurotoxicity. Previous studies showed that the production of ROS was responsible for oxidative stress, and an increase in ROS induced dopaminergic cell death (Peng et al., 2009; Purisai et al., 2007). In the present study, we found that ROS generation in the hippocampus of paraquat treatment groups increased significantly compared with the control group (Fig. 7). Excess ROS generation can damage a wide variety of cellular constituents including DNA, RNA, proteins, sugars and lipids, thereby compromising cell viability. Typically, lipid peroxidation was the primary result of oxidative stress, and correlated effects on the levels of SOD and MDA were also observed. SOD is an antioxidant against free radicals, such as ROS, reactive nitrogen species (RNS) and so on, whereas MDA was a metabolite of the lipid peroxidation of membranes. The present study indicated that paraquat inhibited the activity of total SOD in the hippocampus and significantly increased the contents of MDA in the hippocampus (Figs. 5 and 6). These results suggested that paraquat could induce the excessive generation of ROS and could reinforce lipid peroxidation in the hippocampus, and thus affected the oxidation-antioxidation homeostasis. Previous studies implied that mitochondrial dysfunction induced by oxidative stress played an important role in paraquat neurotoxicity (Keeney et al., 2006). Mitochondria are candidate targets of paraquat toxicity in animal and plant tissues (Taylor et al., 2002) and are considered to be a major source of ROS in cells (Cadenas and Davies, 2000). Therefore, the mechanism of mitochondrial impairment induced by paraquat was related to the generation of ROS (Suntres, 2002). In the present study, the contents of 8-OHdG and ATP in the hippocampus tissues were measured with HPLC to explore the mechanism by which hippocampal damage was induced by paraquat. The level of 8OHdG is considered to be a reliable indicator of the oxidative damage of mitochondria (Svoboda et al., 2008). Our results showed that the levels of 8-OHdG in the hippocampus of mice in the paraquat treatment groups increased significantly compared with the control group (Fig. 8), which implied that paraquat could cause oxidative damage in mtDNA. Moreover, the total ATP production of the paraquat treatment groups significantly decreased in comparison to the control group. The AMP production of the paraquat treatment groups also increased significantly compared with the control group (Fig. 9). The contents of ATP and AMP can reflect not only energy metabolism but also the functional status of the mitochondrial electron transport chain. Therefore, the results clearly demonstrated that the energy generation of mitochondria in hippocampal neurocytes was affected by paraquat. Palmeira et al. (1995) reported that the mechanism of paraquat-induced damage of substantia nigra might be related to the effects of paraquat causing uncoupled oxidative phosphorylation by lipid
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peroxidation, which inhibited adenosine-triphosphate synthase activity. This study further demonstrated that mitochondrial damage induced by paraquat was likely the major cause of hippocampal neurocyte death. Therefore, we concluded that paraquat could damage the hippocampus of mice by oxidative stress, and that the injury was associated with hippocampal neuron mitochondrial dysfunction induced by mtDNA oxidative damage. Ultimately, the learning and memory abilities of mice might be affected. Significantly, this study demonstrated the effects of paraquat on the hippocampus for the first time and suggested that the effects of paraquat might be associated with oxidative stress in the hippocampus. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgment This work was supported by grants from the Hebei Education Department Foundation for Medical Emphases, No. 2008131. References Beal MF. Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 2005;58:495–505. Bonneh-Barkay D, Reaney SH, Langston WJ, Di Monte DA. Redox cycling of the herbicide paraquat inmicroglial cultures. Brain Res Mol Brain Res 2005;13:452–6. Banerjee R, Anatoly A, Starkov M, Flint B, Bobby T. Mitochondrial dysfunction in the limelight of Parkinson’s disease pathogenesis. Biochim Biophys Acta 2009; 1792:651–63. Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000;29:222–30. Casey P, Vale JA. Deaths from pesticide poisoning in England and Wales: 1945–1989. Hum Exp Toxicol 1994;13:95–101. Calo M, Iannone M, Passafaro M, Nistico G. Selective vulnerability of hippocampal CA3 neurones after microinfusion of paraquat into the rat substantia nigra or into the ventral tegmental area. J Comp Pathol 1990;103:73–8. Choksi KB, Nuss JE, Boylston WH, Rabek JP, Papaconstantinou J. Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes. Free Radical Biol Med 2007;43:1423–38. Dinis-Oliveira RJ, Remiao F, Carmo H, Duarte JA, Sanchez Navarro A, Bastos ML. Paraquat exposure as an etiological factor of Parkinson’s disease. Neurotoxicology 2006;27:1110–22. Dilday JE, Leslie SW. Ethanol inhibits NMDA-induced increase in free intracellular Ca2+ in dissociated brain cells. Brain Res 1989;499:383–7. Di Monte DA, Lavasani M, Manning-Bog AB. Environmental factors in Parkinson’s disease. Neurotoxicology 2002;23:487–502. Extension Toxicology Network. Pesticide information profiles. Paraquat; 1996, http:// extoxnet.orst.edu/pips/paraquat.htm (last accessed 5/20/08). Fleury C, Mignotte B, Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 2002;84:131–41. James J, Vornov JP, Ajit G, Thomas. Regional vulnerablility to endogenous and exogenous oxidative stress in organotypic hippocampal culture. Exp Neurol 1998; 149:109–22. Kim SJ, Kim JE, Moon IS. Paraquat induces apoptosis of cultured rat cortical cells. Mol Cells 2004;17:102–7. Keeney PM, Xie J, Capaldi RJ, Bennett JR. Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 2006;26:5256–64. Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen RC. Environmental risk factors and Parkonson’s disease: a case control study in Taiwan. Neurology 1997;48:1583–8. Langston JW, Ballsrd P. Parkonsinism induced by 1-methyl -4-phenyl-1,2,3,6 tetrahydropyridine(MPTP):implications for treatment and the pathogenesis of Parkonson’s disease. Can J Neurol Sci 1984;11:160–5. Marry RE, Gibson JE. A comparative study of PQ intoxication in rats, guinea pigs and monkey. Exp Mol Pathol 1972;17:317–25. Mangano E, Hayley S. Inflammatory priming of the substantia nigra influences the impact of later paraquat exposure: neuroimmune sensitization of neurodegeneration. Neurobiol Aging 2008;30:1361–78.
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