6 mice to paraquat

Toxicology Letters 195 (2010) 127–134

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Selective vulnerability of the striatal subregions of C57BL/6 mice to paraquat Min Jeong Kang a,b , Suk Ju Gil a , Jeong Eun Lee a , Hyun Chul Koh a,∗ a b

Department of Pharmacology, College of Medicine, Hanyang University, 133-791 Seoul, South Korea Department of Food & Nutrition, College of Human Ecology, Hanyang University, 133-791 Seoul, South Korea

a r t i c l e

i n f o

Article history: Received 8 December 2009 Received in revised form 9 February 2010 Accepted 15 March 2010 Available online 20 March 2010 Keywords: Paraquat Dopamine Striatum Glutathione Selective vulnerability

a b s t r a c t Paraquat (PQ) is a strong redox agent that contributes to the formation of reactive oxygen species (ROS) and induces toxicity of the nigrostriatal dopaminergic system. In this study, we investigated the effect of PQ on the dopaminergic system of four striatal subregions. Male C57BL/6 mice (aged 7 weeks and 23–25 g) were used for this study. The mice were administrated with normal saline or PQ (10 mg/kg i.p.) twice weekly for three consecutive weeks, and we evaluated changes in body weight and the performance of motor coordination. We also measured changes in tyrosine hydroxylase (TH) immunoreactivity, dopamine (DA) and its metabolites, reduced glutathione (GSH), and oxidized glutathione (GSSG) in the striatum. The body weight gain of PQ-treated mice was lower than that of control mice 2 weeks after PQ administration, and this lowering effect was sustained until 4 weeks after PQ administration. In the rotarod test, PQ had a significant effect on the time it took mice to fall from the rotating rod at 2 weeks after injection as compared to the control rats, and the effect was sustained up to 4 weeks after PQ administration. Additionally, the densities of TH-positive fibers were reduced in dorsal regions of both the striata and ventral subregion of the caudal striatum (RD, CD and CV subregions). The DA level however, decreased in four subregions of the striata. The rate of DA oxidation and O-methylation increased in the RD subregion. After PQ administration, GSH levels were significantly reduced in the RD and CV subregions, but GSSG levels in the RD and CD subregions increased compared to the control rats. The ratio of GSH/GSSG also decreased in the RD and CD subregions. We found that repeated PQ injection altered DA metabolism through the generation of oxidative stress in the striatum, and although the RD subregion showed the most prominent change, the dorsal region of the striatum may be more sensitive to PQ exposure. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Parkinson’s disease (PD) is a neurodegenerative disease whose main symptom is degeneration of the substantia nigra pars compacta (SNpc) and a subsequent loss of dopamine (DA) in the striatum. Increasing evidence supports the hypothesis that environmental factors contribute to PD development (McCormack et al., 2002; Drechsel and Patel, 2008). For example, the toxic precursor N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant in synthetic heroin, induces an acute parkinsonian syndrome in humans (Shimizu et al., 2003), and MPTP is thus, commonly used to create animal models of PD. The effects of MPTP increase with age (Irwin et al., 1994), which is also a risk factor in the human disease. But while MPTP is not found in the natural environment, paraquat (PQ), which bears structural resemblance to MPP+ , is widely used as a non-selective herbicide (Shimizu et al., 2003). PQ was reported to cause selective degeneration of dopaminergic neurons in the SNpc, reproducing a characteristic feature of PD (Fei et al., 2008). More-

∗ Corresponding author. Tel.: +82 2 2220 0653; fax: +82 2 2292 6686. E-mail address: [email protected] (H.C. Koh).

over, geographical regions of high PQ use overlap with regions of high PD incidence (Lanska, 1997; Ritz and Yu, 2000; Shimizu et al., 2003). There is substantial evidence indicating elevated oxidative stress during PQ poisoning including increased lipid peroxidation, diminished energy metabolism and decreased cytochrome oxidase activity (DiCiero Miranda et al., 2000). Dopaminergic neurons may be preferentially targeted by PQ because of their significant vulnerability to reactive oxygen species-mediated oxidative injury (Bonneh-Barkay et al., 2005). Compared to other neuronal cells, dopaminergic cells are much more sensitive to oxidative injury (Dinis-Oliveira et al., 2006; Lotharius and O’Malley, 2000). The cell bodies of dopaminergic neurons, located within the SN, send projections that terminate and release dopamine in the striatum. As a major cellular antioxidant, glutathione (GSH) participates in diverse enzymatic and non-enzymatic processes that protect cells against oxidative stress (Cohen, 1983; Rabinovic and Hastings, 1998). Importantly, GSH in the adult brain removes oxidants formed in normal metabolism, such as in mitochondrial electron transport and substrate oxidation and decreased GSH availability in the brain may lead to mitochondrial injury (Jain et al., 1991). Dopaminergic terminals in the central nervous system have high

0378-4274/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.03.011

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M.J. Kang et al. / Toxicology Letters 195 (2010) 127–134 cut into 40 ␮m coronal sections using a cryostat microtome (CM1850, Leica, Wetzlar, Germany) at −20 ◦ C. The tissue sections were stored in 50% glycerol (in PBS) at −20 ◦ C until the immunohistochemistry and in situ hybridization were performed. For DA (with its metabolites) and glutathione analyses of the mice (n = 5), after removing the whole brain from the skull, the striatum were rapidly isolated by using a rodent brain matrix (RBMS-200C, World Precision Instruments, FL, USA) and a standard mouse brain atlas (Paxinos and Watson, 1986). The coronal sections from two rostro-caudal levels through the striatum: a rostral striatum (+1.18 mm relative to bregma) and a caudal striatum (−0.46 mm relative to bregma) were divided into the dorsal and ventral subregions, respectively (Paxinos and Watson, 1986). The striatum was collected as a fragment of approximately 1 mm × 1 mm × 1 mm in size. The tissues were immediately stored at −80 ◦ C until the assay.

Fig. 1. Timeline and experiments. Mice received intraperitoneal treatment with paraqaut (PQ) at a dose of 10 mg/kg or an equivalent volume of normal saline twice a week for 3 weeks for a total of six injections. At the beginning of the study, animals were trained for a behavioral test with rota-rod test and were then tested every week for 4 weeks. One week after the last injection of PQ mice were sacrificed, and the striata was dissected for biochemical analysis.

ascorbic acid content that depend on the degree of GSH sufficiency (Rabinovic and Hastings, 1998). Early reductions in total glutathione in the SNpc occur in animal models of PD (Kaur et al., 2003). Although the brain contains low glutathione peroxidase activity, compared to activities in the kidney or liver, the ability of brain GSH levels to maintain cerebral function for the duration of a human life demonstrates the effectiveness of this antioxidant system (Pastore et al., 2003). In the present study, we investigated the effect of repeated PQ administration on the dopaminergic system of the striata in mice and the role of the GSH system in oxidative dopaminergic neurotoxicity. Additionally, we assessed the selective vulnerability to PQ-induced toxicity in four subregions of the striata. 2. Methods 2.1. Animals Male C57BL/6 mice (aged 7 weeks and 23–25 g; Japan SLC, Inc., Shizuoka, Japan) were used for this study. The mice were adapted to their surroundings for 1 week prior to the experiment. Animals were housed in a room maintained under constant temperature (18 ± 2 ◦ C) and humidity (50 ± 10%) conditions with an automatic 12/12 light/dark cycle. Food and water were available ad libitum. Animals were cared for and treated in accordance with guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. 2.2. Experimental design Mice performed the rota-rod test 7 days before PQ administration for behavioral test training and then were evaluated every 7 days after PQ administration. Additionally, body weight was checked prior to and 7 days after PQ administration. The mice were treated with PQ in normal saline by intraperitoneal (i.p.) injection twice weekly for three consecutive weeks (Thiruchelvam et al., 2003), and all drugs were injected in a volume of 0.2 ml with a 1 ml disposable syringe. Finally, 7 days after the last injection, animals were sacrificed for HPLC analysis of DA and its metabolites, and for immunostaining analysis (Fig. 1). In this study, we investigated the effect of PQ on the dopaminergic system of four striatal subregions (the whole striatum was divided into rostral and caudal striata and both striatal parts were subdivided into dorsal and ventral subregions). 2.3. Motor function test The rota rod test for assessing locomotor skills measures the time that an animal maintains balance on a moving Lucite rod. As a part of the test procedure animals were initially trained to maintain themselves on the rotating rod at 15 rpm for period of more than 3 min. Subsequently after a period of 24 h the animals were again screened for their ability to remain on the rotating rod for three successive trials of 3 min each. 2.4. Tissue preparation The mice were deeply anesthetized with pentobarbital sodium salt (50 mg/kg, i.p.; Sigma) at 1 week after the last injection of PQ. For TH immunostaining, mice (n = 5) were perfused intracardially with 0.1 ml of heparin (20 IU) and 20 ml of phosphate buffered saline (PBS; pH 7.4), followed by 50 ml of 4% paraformaldehyde (PFA) in 0.1 M PBS. The brain tissues were removed and post-fixed for 2 h in 4% PFA. Then, the fixed brain tissues were soaked in 30% sucrose over 2 days. The brain tissues were frozen in Tissue-Teck® (Sakura Finetek USA, Torrance, CA, USA) solution and

2.5. Tyrosine hydroxylase immunohistochemistry and density analysis Free-floating 40 ␮m sections were collected and processed for tyrosine hydroxylase (TH) immunohistochemistry. The sections were fixed in 100% methanol for 5 min and washed three times in PBS/BSA (0.1 M PBS containing 0.1% BSA). The sections were post-fixed in 4% PFA/0.15% picric acid in 0.1 M PBS for 20 min at room temperature (RT) and washed three times in PBS/BSA. After fixation, the sections were blocked with 10% normal goat serum (NGS; Gibco BRL, NY, USA) and 0.3% triton-X 100 in PBS/BSA for 1 h and incubated with the TH antibody (1:250; Chemicon International, Inc., Temecula, CA, USA) at 4 ◦ C overnight. After rinsing, sections were incubated in biotinylated anti-mouse IgG secondary (1:200; Pel-Freez Biological, Rogers, AR, USA) for 1 h at RT. After washing, the sections were incubated in horseradish peroxidase streptavidin (1:400, Vactor Laboratories, Inc., Burlingame, CA, USA) for 2 h at RT. The sections were washed three times and visualization was performed using a 3,3 -diaminobenzidine tetrahydrochloride (SK-4100 DAB kit; Vactor) for 40 s. Section development was stopped using distilled water. The sections were mounted on slides, air-dried, and dehydrated by passage through a decreasing ethanol series (70, 80, 90, 100, and 100% for 2 min each). The sections were cleared in xylene and coverslipped with permount (Fisher Scientific, Inc., IL, USA). The densities of TH immunoreactive (TH-IR) fibers in the striatum were determined using a computerized image analysis system (image analysis software). Images from sections were collected on a Leica DMRXE fluorescent microscope equipped with a F-View II camera. The densities of TH-IR fibers in the striatum were measured at the dorsal and ventral subregions of the rostral and caudal striatum. ‘Mean density’ values from the two areas were averaged. The optical density of the white matter (corpus callosum) was used as a background value and was subtracted from each mean density. The optical density value from the lesioned region was expressed as a percentage of the control (saline injection) group on the same section. 2.6. Dopamine and dopamine metabolites analysis by HPLC The levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) in the brain tissues were determined by a modified method (Mori et al., 2005). The tissue was mashed with a tip in 280 ␮l of 0.2 M perchloric acid containing 0.1 mM EDTA and 20 ␮l of 10 ␮M 3,4dihydroxyphenylalanine as an internal standard. Cell membranes were disrupted using a 50% duty cycle for 40 s in an ice bath with a sonicator (GE 50; Sonics & Materials Inc., Danbury, CT, USA) and spun down at 13,000 rpm for 10 min at 4 ◦ C using a centrifuge (MICRO 17TR, Hanil Science Industrial Co., Ltd., Inchun, Korea). The supernatants were filtered through centrifugal filter devices (Microcon YM-10, Millipore Co., Bedford, MA, USA) by centrifuging at 13,000 rpm for 15 min at 4 ◦ C. After filtration, 20 ␮l of the sample was injected directly into an injector (7725(i), Rheodyne, Cotati, CA, USA) and analyzed by a high-performance liquid chromatograph (HPLC) with electrochemical detection (Ossowska et al., 2005). The pellets were used for measurements of protein concentration. The HPLC system consisted of an electrochemical detector (ECD-300, EICOM, Kyoto, Japan), a solvent delivery system (515 HPLC-pump; Waters Co., Milford, MA, USA), a column oven (Waters Co., Milford, MA, USA), and a data processor dsCHROM-net (Donam Int., Seoul, Korea). The separation column was a reverse-phase C18 column (3.0 mm i.d. × 150 mm; EICOMPAK SC50DS; EICOM) and the guard column was a PREPAK (4.0 mm i.d. × 5 mm, EICOM). The appendance potential of ECD-300 (carbon electrode vs. Ag/AgCl reference electrode) was set at +750 mV. The mobile phase consisted of 90 mM sodium acetate–100 mM citric acid buffer (pH 3.5)/methanol (83:17, v/v) containing 190 mg/L of sodium-1octanesulfonic acid and 5 mg/L of 2Na EDTA. The flow rate was set at 0.5 ml/min at 30 ◦ C. 3,4-Dihydroxybenzylamine was used as the internal standard for the quantification of DA concentrations. DA levels were calculated using DA standard (3-hydroxydopamine; dopamine hydrochloride) and corrected by protein levels of the samples. Thus, the levels of DA and DA metabolites were expressed as ng/mg protein. 2.7. Reduced and oxidized glutathione assays Concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the striatum tissues were determined using a GSH assay kit (cayman 703002). The GSH and GSSG concentrations were measured according to the manufacturer’s instructions. Tissues were homogenized in 50 mM MES (Nmorpholinoethansulphonic acid) buffer (w:v, 1:10). Samples were deproteinated

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Fig. 2. The effects of paraquat (PQ) on body weight and behavioral activity in mice. (A) Changes in body weight after PQ exposure. Body weight was evaluated every week for 4 weeks. (B) Rota-rod performance was evaluated every week for 4 weeks of PQ exposure. Each plot represents the mean ± S.E.M., *p < 0.01 as compared to animals treated with normal saline alone. with 10% (w/v) MPA (metaphosphoric acid; Sigma) reagent. These samples can be stored for up to 6 months at −20 ◦ C without any degradation of GSH or GSSG. For GSSG quantification exclusive of GSH, GSH was derivatized with 10 ␮l of 1 M 2-vinylpyridine (in ethanol; Sigma). The absorbance in the well was measured at 405 nm using a microplate spectrophotometer (Spectra Max190, Molecular Devices, CA, USA) at 5 min intervals for 30 min (a total of six measurements). The protein concentration of the supernatant was determined by the Bradford method. Bradford (Sigma) assays are routinely performed at RT. Color development began immediately. The absorbance at 595 nm was recorded and the protein concentration was determined by comparison to a standard curve using bovine serum albumin (BSA; Sigma).

2.8. Statistical analysis For statistical analysis, the SPSS PC computer program (Statistical Package for Social Science 12.0) was used. Data were expressed as mean ± standard error of mean (S.E.M.), and the significance of differences was assessed using the unpaired Student’s t-test at a p value <0.05. The significance of differences among mean values was assessed using two-way ANOVA coupled with Duncan’s multiple range tests at p value <0.05.

3. Results 3.1. Effects of paraquat treatment on body weight and performance of motor coordination Body weight was monitored throughout the experiments. PQtreated and control mice gained body weight on average. However, the body weight gain of the PQ-treated group was lower than that of control groups 3 weeks after PQ administration, and this was sustained until 4 weeks after PQ administration (Fig. 2A). PQ treatment had no significant effect on the time it took the mice to fall from the rotating rod at 1 week after injection, as compared to control mice. It was interesting to note that PQ-treated mice stayed on the rotating rod for a longer period of time at 2 weeks after PQ treatment, as compared to the control group (Fig. 2B). The behavioral effects of PQ were sustained until 4 weeks after PQ administration (Fig. 2B).

Fig. 3. The effects of paraquat (PQ) on tyrosine hydroxylase (TH)-positive fibers in the rostral striatum of mice. Densities of TH-positive fibers were counted at 1 week after the last PQ (10 mg/kg) or normal saline injection. (A) Diagrams of the isolated brain tissues regions (Paxinos and Watson, 1986), including the dorsal subregion of the rostral striatum (RD) and the ventral subregion of the rostral striatum (RV) at the level +1.18 mm to bregma. (B) Representative TH immunohistochemical data (original magnification, 100×; scale bar 250 ␮m). The diagram represents a sample section, including the dorsal subregion of the rostral striatum (RD) and the ventral subregion of the rostral striatum (RV) at the level +1.18 mm to bregma. (C) Quantification of TH-positive fibers in RD and RV. Each column represents the mean ± S.E.M., *p < 0.05 as compared to animals treated with normal saline alone. Abbreviations: RD, dorsal subregion of the rostral striatum; RV, ventral subregion of the rostral striatum.

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Fig. 4. The effects of paraquat (PQ) on tyrosine hydroxylase (TH)-positive fibers in the caudal striatum of mice. Densities of TH-positive fibers were counted at 1 week after the last PQ (10 mg/kg) or normal saline injection. (A) Diagrams of the isolated brain tissues regions (Paxinos and Watson, 1986), including the dorsal subregion of the caudal striatum (CD), the ventral subregion of the caudal striatum (CV) at the level -0.46 mm to bregma. (B) Representative TH immunohistochemical data (original magnification, 100×; scale bar 250 ␮m). The diagram represents a sample section, including the dorsal subregion of the caudal striatum (CD) and the ventral subregion of the caudal striatum (CV) at the level −0.46 mm to bregma. (C) Quantification of TH-positive fibers in CD and CV. Each column represents the mean ± S.E.M., *p < 0.05 as compared to animals treated with normal saline alone. Abbreviations: CD, dorsal subregion of the caudal striatum; CV, ventral subregion of the caudal striatum.

3.2. Effects of paraqaut on tyrosine hydroxylase immunoreactivity in four subregions of the striata

3.5. Effects of paraqaut on dopamine catabolic pathways in four subregions of striatum

To determine the loss of TH-positive fibers in the striatum, we measured the densities of fibers in the rostral and caudal striata of mice (Figs. 3A and 4A). The PQ reduced the densities of TH-positive fibers in the dorsal subregion of the rostral striatum (n = 5, Fig. 3B and C). As shown in Fig. 4B and C, the densities of TH-positive fibers in the dorsal and ventral of caudal striata (CD and CV, respectively) were decreased by PQ treatment.

The oxidation pathway of DA catabolism and O-methylation only increased in the RD after administration of PQ (n = 5, Fig. 7A and C). However, the overall rate of metabolism in the RD subregion of the striatum, which is the sum of the activities in the two catabolic pathways, was not significantly increased by repeated PQ administration (n = 5, Fig. 7B).

3.3. Effects of paraqaut on dopamine and its metabolites in four subregions of striatum Levels of DA and its metabolites were analyzed 1 week after the last PQ injection. As shown in Fig. 5A, repeated administration of PQ significantly reduced the level of DOPAC in the CV subregion of the striatum (n = 5). PQ treatment also reduced DA levels in four subregions of the striata (n = 5, Fig. 5B). After PQ administration, the HVA level in the ventral subregion of the rostral striatum (RV), but not in the dorsal subregion was significantly reduced. In the caudal striatum, the levels of HVA were decreased by PQ treatment (n = 5, Fig. 5C). PQ administration did not significantly reduce the levels of 3-MT in the four subregions of the striata (n = 5, Fig. 5D). 3.4. Effects of paraqaut on reduced and oxidized glutathione levels in four subregions of striata GSH levels were significantly reduced in the dorsal subregion of the rostral striatum (RD) and CV subregion of the striatum (n = 5, Fig. 6A). GSSG levels in RD and CD subregions of the striata increased compared to that of the control group (n = 5, Fig. 6B). The PQ regimen depressed the redox state of glutathione, expressed as the ratio of GSH to GSSG (GSH/GSSG ratio) in the RD and CD subregions of the striata (n = 5, Fig. 6C).

4. Discussion We previously reported that repeated systemic administration of PQ to mice for 3 weeks led to a decrease of GSH levels with resultant loss of dopaminergic neurons in the SNpc (Kang et al., 2009). The present study demonstrated that PQ affects the striatal dopaminergic systems, and that systemic exposure of PQ shows a selective vulnerability of the striatal subregion to toxic response. To study the systemic effect of PQ, we examined the changes of body weight and behavioral performance at every week after PQ administration. We observed that PQ administration resulted in a loss of body weight. This is consistent with previous reports that showed a decrease in mouse body weight following exposure to PQ at different doses and time (Prasad et al., 2009). Decreased body weight indicates a general toxic effect of the chemical and has been associated with decreased food consumption and water intake (Prasad et al., 2009). Exposure to PQ improved the rotarod performance of mice suggesting improved motor coordination. Hong et al. (2007) however, reported that the dopaminergic neurotoxin MPTP decrease in rota-rod latency to fall compared to the normal mouse. Accordingly, we guess that the motor coordination improvement of PQ may be involved in the compensatory mechanisms of dopaminergic system or another mechanism. It has been proposed that these inconsistent data are due to the time of measurement post-dosing PQ, which is critical determinant of changes

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Fig. 5. Effects of paraquat (PQ) on dopamine (DA) and its metabolites in the striatum of mice. The mice were injected with PQ (10 mg/kg) twice a week for three consecutive weeks. (A) Levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in four subregions of the striatum. (B) Levels of DA in four subregions of striatum. (C) Levels of homovanillic acid (HVA) in four subregions of the striatum. (D) Levels of 3-methoxytyramine (3-MT) in four subregions of the striatum. Quantitative analyses of DA and its metabolites in the four subregions of the striatum were determinede at 1 week after the last PQ injection. Each column represents the mean ± S.E.M., **p < 0.01, *p < 0.05 as compared to animals treated with normal saline alone. Abbreviations: RD, dorsal subregion of the rostral striatum; RV, ventral subregion of the rostral striatum; CD, dorsal subregion of the caudal striatum; CV, ventral subregion of the caudal striatum; DOPAC, 3,4-dihydroxyphenylacetic acid; DA, dopamine; HVA, homovanillic acid; 3-MT, 3-methoxytyramine.

in the dopaminergic system. Additionally, there are several reports suggesting that another target site of PQ may be noradrenergic and serotonergic neurons (Shimizu et al., 2003; Ossowska et al., 2005; Kuter et al., 2007). The nigrostriatal tract connects the dopamine cells of the SNpc (A9 dopaminergic neuron) to the dorsolateral striatum, while the mesolimbic part of the tract connects the neurons of the VTA (A10 dopaminergic neuron) to the ventromedial striatum, nucleus accumbens, olfactory tubercle, and layer VI of the neocortex (van

Domburg and ten Donkelaar, 1991). Additionally, phenotypes of motor deficits show a difference pattern when A9/A10 dopaminergic neurons are degenerated (Moore et al., 2001). We found that PQ administration significantly decreased TH-positive immunoreactivities both in the rostrodorsal (RD) subregion and the whole caudal striata (CD and CV), with the exception of the rostroventral (RV) subregion. Despite controversy, evidence consistently supports a relationship between PQ exposure and striatal TH-positive immunoreactivity. Fernagut et al. (2007) reported

Fig. 6. Effects of paraquat (PQ) on glutathione of the four subregions in the striatum of mice. The mice were injected with PQ (10 mg/kg) twice a week for three consecutive weeks. (A) Levels of reduced glutathione (GSH) in four subregions of the striatum. (B) Levels of oxidized glutathione (GSSG) in four subregions of the striatum. (C) The ratio of GSH/GSSG in the striatum. (D) Total glutathione (GSH + GSSG) in the striatum. Each column represents the mean ± S.E.M., **p < 0.01, *p < 0.05 as compared to animals treated with normal saline alone.

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Fig. 7. Effects of paraquat (PQ) on the rate of total dopamine (DA) metabolism and on alternative catabolic pathways in the striatum of mice. (A) The index of the rate DA oxidation (MAO-dependent) ([DOPAC]/[DA]) × 100. (B) The index of the rate of O-methylation (COMT-dependent) ([3-MT]/[DA]) × 100. (C) The index of the rate of total DA catabolism ([HVA]/[DA]) × 100. Each column represents the mean ± S.E.M., **p < 0.01, *p < 0.05 as compared to animals treated with normal saline alone. Abbreviations: DOPAC, 3,4-dihydroxyphenylacetic acid; DA, dopamine; HVA, homovanillic acid; 3-MT, 3-methoxytyramine; RD, dorsal subregion of the rostral striatum; RV, ventral subregion of the rostral striatum; CD, dorsal subregion of the caudal striatum; CV, ventral subregion of the caudal striatum.

that exposure to PQ decreases TH-positive immunoreactivity in the dorsomedial region of the striatum. A similar report demonstrated that i.p. injection of 10 mg PQ showed a decrease in TH protein expression measured in a westernblot of whole striatal tissue (Prasad et al., 2009). Rojo et al. (2007) however, found no apparent changes in TH-positive immunoreactivities in the striatum. Thiruchelvam et al. (2000) also reported that PQ showed no apparent changes in TH-positive immunoreactivities in both the dorsal and ventral striatum of mice, and co-treatment of PQ with maneb however, produced a decrease of TH-positive immunoreactivities in the dorsal region of the striatum. In our experiments, repeated injections of PQ partly affected striatal TH-positive fibers in mice, and these results are the first evidence for a regional effect of PQ in the striatum. This study also showed that the loss of TH-positive fibers following PQ administration was accompanied by a decline in DA levels in four subregions of the striatum, whereas levels of DOPAC decreased in the CV subregion of the striatum. Additionally, HVA levels decreased in the RV, CD and CV, and 3-MT did not change in these four subregions of the striata. Thiruchelvam et al. (2000) reported that levels of striatal DA and DOPAC increased at 1 h and did not significantly change at 3 days but they decreased at 1 week after the last PQ injection. Although our PQ regimen (twice a week for 3 weeks) differs from that of Thiruchelvam et al. (2000; twice a week for 6 weeks), both results, which were both obtained 1 week after the last injection, are similar. Thiruchelvam et al. (2003) however, demonstrated that levels of striatal DA and DOPAC did not significantly change at 2 weeks after the last PQ injection (same regimen of ours). Thus the exposure time of PQ may be critical features for striatal DA loss. We were not able to determine the relationship between the levels of striatal DA including its metabolites and exposure time in these experiments, but should be a key determination for the future. Glutathione (GSH) peroxidase scavenges the H2 O2 that MAO (a mitochondrial enzyme) generates, to form glutathione disulfide (GSSG); hence changes in GSSG levels reflect the redox status of the dopaminergic nerve terminals (Spina and Cohen, 1989). In vivo, PQ forms reactive oxygen species (ROS), which may promote neurodegenerative disease (Schulz et al., 2000; Thiruchelvam et

al., 2003; Weinreb et al., 2004). Prasad et al. (2007) observed significant increase in ROS after single and repeated doses of PQ. Dopaminergic neurons experience a relatively high degree of oxidant stress because dopamine metabolism directly produces ROS (Halliwell, 1992; Hastings et al., 1996). The antioxidant GSH potentially detoxifies ROS, an important role in neuronal metabolism, and the resulting decline in GSH show a positive correlation with disease severity in PD (Schulz et al., 2000). A suppressed glutathione level may also serve as an early indicator of oxidative stress during the PD progression, preceding even the inhibition of respiratory chain complex I (Pastore et al., 2003). A high intracellular GSH level protects cells through a direct, non-enzymatic reaction with free radicals (Winterbourn and Metadiewa, 1994; Gabbay et al., 1996). In the preclinical stages of PD, Sofic et al. (1992) observed a decline in GSH levels in the SN. Our present results showed that treatment with PQ also decreased GSH levels in the CV and both rostral striatum (RD and RV) and increased GSSG levels in both the RD and CD subregions of the striata. We also found that GSH/GSSG ratios of RD and both caudal striatal subregions significantly decreased, and this data is consistent with a decrease of TH immunoreactivity in the same striatal subregion. The mechanism of PQ toxicity most likely involves a redox cycle that generates oxygen free radicals (Kadiiska et al., 1993; McCormack et al., 2002), which may in turn cause lipid peroxidation and promote cell death and apoptosis (Shimada et al., 1998; Yang and Sun, 1998). DA catabolism produces HVA by two metabolic pathways; (a) N-oxidation by monoamine oxidase type B (MAOB ) to form DOPAC within the neuron, and (b) O-methylated by catechol-O-methyl transferase (COMT), to form 3-MT externally (Antkiewicz-Michaluk et al., 2001). The DOPAC/DA ratio is therefore a measure of DA neuronal activity associated with DA release, reuptake, and oxidative metabolism (Westerink and Spaan, 1982; DeMaria et al., 1999). The possibility that shifting dopamine metabolism from N-oxidation towards the O-methylation pathway may protect DA neurons is supported by the neuroprotective effect of MAOB inhibition (Speiser et al., 1998; Stern, 1998). The HVA/DA ratio represents the external pool of methylated DA that does not undergo reuptake (Shepherd et al., 2006). An increase in the ratio of DA metabolites to DA may indicate the induction of a compensatory

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mechanism to increase DA release in the brains of PQ-treated animals (or conceivably in patient) (Liou et al., 1996, 2001). The neuron itself produces DOPAC as a metabolite, and derives a significant proportion of it from the pool of newly formed DA neurotransmitter (Pifl and Hornykiewicz, 2006). In the present study, exposure to PQ increased in DOPAC/DA and 3-MT/DA ratios of RD subregions, and decreased in HVA/DA ratio of both caudal subregions of the striata. These results demonstrate that PQ induced or accelerated neuronal damage by oxidative stress, and that it did induce a compensatory, neuroprotective response in the striatum especially in RD subregions. Although other subregions did not correspond to TH-positive immunoreactivities and GSH by PQ exposure, the RD subregion was more sensitive to PQ exposure with both the loss of TH positive fibers and the decrease of GSH levels. In conclusion, we have shown that repeated injection of PQ in mice affected the TH-positive immunoreactivities in the dorsal striatum through the generation of oxidative stress, and that the toxicity was related to GSH depletion in the striatum. Further, the RD subregion showed a selective vulnerability of the striatal dopaminergic system to PQ exposure, and the RV subregion relatively resisted a toxic response of PQ relative to other subregions. A comprehensive understanding of DA metabolism, the mechanism of PQ toxicity, and its role in environmental disease awaits further study. Conflict of interest statement The authors state that they no financial interest in the products mentioned within this article. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (2009-0091460) through the Medical Research Center at Hanyang University College of Medicine, Korea. References Antkiewicz-Michaluk, L., Michaluk, J., Mokrosz, M., Romanska, I., Lorenc-Koci, E., Ohta, S., Vetulani, J., 2001. Different action on dopamine catabolic pathways of two endogenous 1,2,3,4,-tetrahydroisoquinolines with similar antidopaminergic properties. J. Neurochem. 78, 100–108. Bonneh-Barkay, D., Reaney, S.H., Langston, W.J., Di Monte, D.A., 2005. Redox cycling of the herbicide paraquat in microglial cultures. Brain Res. Mol. Brain Res. 134, 52–56. Cohen, G., 1983. The pathobiology of Parkinson’s disease: biochemical aspects of dopamine neuron senescence. J. Neural. Transm. 19, 89–103. DeMaria, J.E., Lerant, A.A., Freeman, M.E., 1999. Prolactin activates all three populations of hypothalamic neuroendocrine dopaminergic neurons in ovariectomized rats. Brain Res. 837, 236–241. DiCiero Miranda, M., de Bruin, V.M., Vale, M.R., Viana, G.S., 2000. Lipid peroxidation and nitrite plus nitrate levels in brain tissue from patients with Alzheimer’s disease. Gerontology 46, 179–184. Dinis-Oliveira, R.J., Remião, F., Carmo, H., Duarte, J.A., Navarro, A.S., Bastos, M.L., Carvalho, F., 2006. Paraquat exposure as an etiological factor of Parkinson’s disease. Neurotoxicology 27, 1110–1122. Drechsel, D.A., Patel, M., 2008. Role of reactive oxygen species in the neurotoxicity of environmental agents implicated in Parkinson’s disease. Free Radic. Biol. Med. 44, 1873–1886. Fei, Q., McCormack, A.L., Di Monte, D.A., Ethell, D.W., 2008. Paraquat neurotoxicity is mediated by a Bak-dependent mechanism. J. Biol. Chem. 283, 3357–3364. Fernagut, P.O., Hutson, C.B., Fleming, S.M., Tetreaut, N.A., Salcedo, J., Masliah, E., Chesselet, M.F., 2007. Behavioral and histopathological consequences of paraquat intoxication in mice: effects of alpha-synuclein over-expression. Synapse 61, 991–1001. Gabbay, M., Tauber, M., Porat, S., Simantov, R., 1996. Selective role of glutathione in protecting human neuronal cells form dopamine-induced apotosis. Neuropharmacology 35, 571–578. Halliwell, B., 1992. Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609–1623. Hastings, T.G., Lewis, D.A., Zigmond, M.J., 1996. Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections. Proc. Natl. Acad. Sci. U.S.A. 93, 1956–1961.

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