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Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
Oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats
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Tingting Wang, Ling Zhao, Mengyu Liu, Fei Xie, Xuemei Ma *, Pengxiang Zhao, Yunqi Liu, Jiala Li, Minglian Wang, Zhaona Yang, Yutong Zhang
Beijing Environmental and Virus Cancer Key Laboratory, Beijing University of Technology, Beijing 100124, PR China College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China
ARTICLE INFO
Article history: Received 2 February 2014 Revised 7 June 2014 Accepted 9 June 2014 Available online 24 June 2014
Keywords:
Chlorpyrifos
Hydrogen
Neurotoxicity
Acetylcholinesterase
Oxidative stress
ABSTRACT
Chronic exposure to low-levels of organophosphate (OP) compounds, such as chlorpyrifos (CPF), induces oxidative stress and could be related to neurological disorders. Hydrogen has been identified as a novel antioxidant which could selectively scavenge hydroxyl radicals. We explore whether intake of hydrogen-rich water (HRW) can protect Wistar rats from CPF-induced neurotoxicity. Rats were gavaged daily with 6.75 mg/kg body weight (1/20 LD50) of CPF and given HRW by oral intake. Nissl staining and electron microscopy results indicated that HRW intake had protective effects on the CPF-induced damage of hippocampal neurons and neuronal mitochondria. Immunostaining results showed that the increased glial fibrillary acidic protein (GFAP) expression in astrocytes induced by CPF exposure can be ameliorated by HRW intake. Moreover, HRW intake also attenuated CPF-induced oxidative stress as evidenced by enhanced level of MDA, accompanied by an increase in GSH level and SOD and CAT activity. Acetylcholinesterase (AChE) activity tests showed significant decrease in brain AChE activity after CPF exposure, and this effect can be ameliorated by HRW intake. An in vitro study demonstrated that AChE activity was more intense in HRW than in normal water with or without chlorpyrifos-oxon (CPO), the metabolically-activated form of CPF. These observations suggest that HRW intake can protect rats from CPF-induced neurotoxicity, and the protective effects of hydrogen may be mediated by regulating the oxidant and antioxidant status of rats. Furthermore, this work defines a novel mechanism of biological activity of hydrogen by directly increasing the AChE activity.
© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Introduction
Organophosphate (OP) compounds are a group of insecticides widely used for the control of agricultural, industrial, home and public health pests (Aardema et al., 2008). Chlorpyrifos (CPF) is one of the most widely used OPs in agricultural settings, despite the restriction of some of its domestic applications by the United States Environmental Protection Agency in 2000 based on human health risk. An epidemiological study of 1000 individuals indicated that up to 82% of U.S. adults had detectable levels of the CPF metabolite (3,5,6,-trichloro-pyridinol) present in their urine (Hill et al., 1995). Although acute high-level exposure to OPs tends to cause severe "cholinergic syndrome" (Costa, 2006), repeated low-level exposure can also produce chronic neurological symptoms and deficits in neurobehavioral performance (Alavanja et al., 2004).
☆ This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303023).
* Corresponding author at: Beijing University of Technology, College of Life Science and Bio-engineering, No. 100, Pingleyuan, Chaoyang District, Beijing 100124, PR China. E-mail address: xmma@bjut.edu.cn (X. Ma).
Specifically, CPF has been shown to cause mild sensory neuropathy and some memory problems in adults (Kaplan et al., 1993). A recent study compared the neurological symptom of 57 CPF applicators for the cotton crop and 38 non-applicators. CPF applicators had impaired performance compared to controls on the majority of tests on the neurobehavioral battery developed for the study (Khan et al., 2014). Behavioral studies in rodents have identified hippocampus-dependent learning and memory as a target for the neurotoxic effects of repeated subthreshold CPF exposure (Prendergast et al., 1998; Terry et al., 2003).
In the human body, CPF is metabolically activated by oxidative desulfuration to CPF-oxon (CPO), a potent inhibitor of AChE. CPO and related OPs irreversibly inhibit AChE activity by phosphorylation of its active serine site, leading to accumulation of acetylcholine and overstimulation at cholinergic synapses. Other putative mechanisms have also been reported to play an important role in CPF toxicity. Among the additional mechanisms, the induction of oxidative stress has received tremendous focus and attention (Ogut et al., 2011; Saulsbury et al., 2009). Exposure to CPF modifies the endogenous antioxidant defense enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which leads to the development
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0041-008X/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
of oxidative stress (Bebe and Panemangalore, 2003; Chiapella et al., 2013; Geter et al.,2008).
A recent study has revealed that molecular hydrogen acts as a novel antioxidant which could selectively reduce •OH and ONOO— but not affect physiological reactive oxygen species (ROS) (Ohsawa et al., 2007). Subsequent studies have have confirmed that consumption of hydrogen reduces oxidative stress in a diverse range of disorders and organ systems including the nervous (Fu et al., 2009; Gu et al., 2010; Li etal., 2010), digestive (Chen et al., 2011; Zheng etal., 2009), cardiovascular (Hayashida et al., 2008; Nakao et al., 2010a) and respiratory systems (Mao et al., 2009). These studies strongly suggest the potential of molecular hydrogen as an effective therapeutic and preventive antioxidant. Drinking hydrogen rich water (HRW) has been considered a safe and convenient mode of delivery for molecular hydrogen. One of the advantages of HRW is its ability to cross the blood-brain barrier and therefore has potential to treat neurological diseases (Li etal., 2010).
In the present study, we hypothesized that oral intake of HRW might play a protective role in the CPF-induced neurotoxicity. We tested our hypothesis using an established adult rat model with oral administration of HRW after chronic exposure to subthreshold dose of CPF. The mechanisms underlying the protective effects of hydrogen have also been investigated.
Materials and methods
Animals. Eight week old male Wistar rats weighing 120-150 g were maintained under standard conditions at 22 °C to 25 °C with a 12 h light-dark cycle and were fed a normal diet. All procedures were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China). Before initiating the experiments, the animals were adapted to laboratory conditions. Rats were killed at 4 time points: 2, 4, 6, and 8 weeks. CPF (Sigma, St. Louis, MO, 99.2% purity) was prepared by corn oil (2 ml/kg) to obtain an effective concentration of 6.75 mg/kg body weight (1/20 LD50). CPF or corn oil was administered once daily by oral gavage for a period of 2-8 weeks. HRW or normal water was changed every morning and night and rats drank freely during the dosing period. At each time point, rats were randomly divided into three groups. Group I (C/oil): Rats in the control group gavaged with corn oil only and given normal water. Group II (CPF): Rats gavaged with CPF and given normal water. Group III (HRW): Rats gavaged with CPF and given HRW.
HRW preparation. Hydrogen-rich water (termed HRW) was prepared as described previously (Nakao et al., 2010b). Briefly, a plastic shelled product (termed hydrogen water stick) consisting of metallic magnesium (99.9% pure) and natural stones in polypropylene containers combined with ceramics (Doctor SUISOSUI®; Friendear Inc., Tokyo, Japan) was used to produce hydrogen. The hydrogen water sticks were placed into distilled water for more than 6 h before use at room temperature. The hydrogen concentration was monitored by using a needle-type Hydrogen Sensor (Unisense A/S, Aarhus, Denmark) every week. The hydrogen water sticks were replaced every two weeks to ensure maintenance of hydrogen concentration (more than 600 |jM).
In an in vitro study, hydrogen-rich water provided by Beijing Hydrovita Beverage Co., Ltd. (termed H-HRW) was used to determine the effect of hydrogen on AChE activity.
A ContrAA 700 high resolution continuum source atomic absorption spectrophotometer (AnalytikJena, Jena, Germany) was used for magnesium measurement. The instrument settings were: absorption line 285.2 nm and width 0.2 nm. The pH value was measured with an Orion model 420A pH meter (Boston, MA, USA).
Tissue preparation. At each time point, rats were anesthetized with isofluorane, the brain tissue was carefully excised and washed with ice-cold physiologic saline (0.9%, w/v). One-half of the brain was fixed
in 4% paraformaldehyde in 0.1 M phosphate buffer and embedded in paraffin for Nissl staining and immunochemistry. The hippocampus dissected from the other half of the brain was stored at — 80 °C until use for oxidative stress biomarkers and AChE activity analysis. Hippocampal tissues (30 mg) were homogenized in 0.3 ml RIPA buffer (Beijing Puli Lai Gene Technology Co., Ltd., Beijing, China) and then centrifuged at 8000 g for 10 min, the supernatant was used for the determination of oxidative stress biomarkers and AChE activity. For electron microscopy, rats were sacrificed via transaortic perfusion of 2.5% glutaraldehyde and their brains were removed. The hippocampus was dissected and cut into 1 mm x 2 mm x 1 mm blocks before being kept in the same fixative overnight.
Nissl staining and immunochemistry. Serial sections (8 |am) were cut coronally through the cerebrum containing the hippocampus. For Nissl staining, every subsequent 10th section (3 sections in each animal) was collected. Toluidine blue was used to stain the Nissl body in the neurons. Mounted slides were examined and photographed under an Olympus light microscope. Six random visual fields of the hippocampal cornu ammonis 1 (CA1), cornu ammonis 1 (CA3) and dentate gyrus (DG) were photographed in each section. The number of staining cells in each field was counted at high magnification (x400). The data were represented as the number of cells per high-power field. Alternatively, every subsequent 12th section (3 sections in each animal) was collected for GFAP staining. Sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases. The sections were incubated with 10% normal goat serum. After the blocking serum was removed, sections were immunostained overnight at 4 °C using a mouse monoclonal antibody against GFAP (Beyotime, China) to assess astrocyte activation, then with biotinylated secondary antibody at 37 °C for 20 min. The GFAP-positive cells in the hippocampus were detected using streptavidin-biotin complex (SABC) and DAB kits (Zhongshan, China). Six random high magnification fields (x400) per section were counted to evaluate for GFAP-positive cells. The GFAP-positive cell count per mm2 tissue area was calculated using Image-Pro Plus software.
Electron microscopy. Hippocampal regions of interest (CA1, CA3 and DG) were identified through light microscopy. The ultra thin sections were cut at 0.1 |jm and stained with uranyl acetate and lead citrate. A Hitachi H-7650 transmission electron microscope was used to capture images. Mitochondria per cell in the same hippocampal region in different treatment groups were quantified using grid counting by three blinded raters.
Measurement of oxidative stress biomarkers. Some biomarkers were determined to evaluate the oxidative stress status in brain. (1) Malondialdehyde (MDA) levels were measured using a thiobarbituric acid reactive species (TBARS) assay kit (Cayman, USA). The total protein concentration was determined by the Bradford Assay (Tiangen, China) using BSA as a standard. Results are expressed as [MDA] in nmol/mg of protein. (2) Glutathione (GSH) was determined according to the recycling system by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and GSH (Dojindo, Japan). Concentration of GSH in sample solutions was determined using a calibration curve, and expressed as nmol/mg protein. (3) SOD activity was measured with assay kits (Dojindo, Japan), according to the manufacturer's protocol. (4) CAT activity was assayed according to the method of Goth and expressed as mU/mg protein by the rate of decrease of hydrogen peroxide (Goth, 1991).
Determination of the AChE activity in vivo. The supernatant of the brain tissue homogenate was diluted with 0.1 M phosphate buffered saline (PBS, pH 7.4). The enzyme activity assay was carried out in a
96-well microtiter plate using the AMPLITE TM AChE assay kit (AAT Bioquest, Sunnyvale, CA) according to manufacturer's protocol.
Determination of the effect of hydrogen on AChE activity in vitro. Type V-S AChE (EC 3.1.1.7) and CPO (98% purity) was purchased from Sigma Chemical (St. Louis, MO, USA). CPO was dissolved in methanol. The activity of AChE was assayed by the method of Ellman (Ellman et al., 1961). Acetylthiocholine iodide (ATCh) was applied as the enzyme substrate with 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) as a chro-mogenic reagent. The product of enzymatic reaction is 5-thio-2-nitrobenzoate with absorption maximum at 410 nm and was detected optically.
To determine the effect of hydrogen on AChE inhibition in vitro, 0.6 mg AChE (827 U/mg) was diluted with 0.02 M Tris buffer (pH 7.5) to a concentration of 1 mg/ml (827 U/ml). The resulting solution was then diluted with normal water or H-HRW to 5 U/ml prior to the initiation of the reaction. DTNB was diluted with 50 mM Na2HPO4 (pH 7.0) to a concentration of 0.6 mM. ATCh was diluted to 1 mM with distilled water. 200 |al of the mixture of DTNB and ATCh (volume ratio: 1:1) was added to each well in the 96-well plate. 10 of AChE (5 U/ml) and 10 CPO (1 nM-1 mM) were added to the reaction simultaneously. The absorbance was measured immediately at 10-second intervals with a Victor 1420 Multilabel Counter (Wallac, Perkin-Elmer, Wellesley, MA, USA).
The apparent K (Appki) was determined according to the double reciprocal method of Hart and O'Brien (Barak et al., 1995; Hart and O'Brien, 1973). In brief, OD values are plotted against time for all the concentrations of CPO and the slopes of the tangents at each 10 s were obtained. Semilogarithmic plots of these slopes against time resulted in linear correlations for all the concentrations of CPO. The slopes (AIn v/At) were determined by linear regression. These values are related to the kinetic parameters of the inhibition process according to the expression:
-L = Kd 1 , 1 a = [Sl
kobs k2 [PX](1-a)+ k2 ' Km +[S]
where [PX] is the CPO concentration. Plotting At/AIn v against 1/[PX] (1 — a) yields the ratios Kd/k2 and 1/k2 as the slope and the ordinate intercept, respectively. The apparent bimolecular rate constants Appki were calculated from the ratio k2/Kd.
To determine the effect of hydrogen on AChE activity, AChE solution (1 mg/ml) was diluted to a concentration of 5 U/ml with different solvents, including H-HRW, boiled H-HRW, HRW, boiled HRWand normal water. The maximum velocity of substrate hydrolysis (Vmax) and the Michaelis-Menten constant (Km) were estimated by the double reciprocal method of Lineweaver and Burk (Lineweaver and Burk, 1934). AChE activity in different solvents was examined by the method of Ellman.
Statistical analysis. Statistical analysis was carried out in Prism (GraphPad) software. Comparison analysis between C/oil and CPF, or HRW and CPF groups were determined by one-way ANOVA followed by Tukey test. Data were shown as the mean ± s.e.m. Statistical significance was set at P < 0.05.
Results
The protective effect ofhydrogen on hippocampal neuron damage induced by CPF
To determine whether hydrogen can protect the hippocampal neurons from CPF-induced damage, hippocampal cytoarchitecture was evaluated using Nissl staining. The results showed that 8 weeks of CPF exposure induced dramatic damage in terms of cell death in the
subregions of the hippocampus, predominantly at the level of CA3 and distal CA3. Other parts of the hippocampus, including the CA1 and dentate gyrus (DG) region, demonstrated mild to moderate damage. Most of the damaged neurons showed shrinkage with condensed nuclei and sparse Nissl bodies. While in the HRW group, neurons had larger cell bodies compared with the CPF group, and most of the neurons had visible Nissl bodies and more mildly condensed nuclei than the CPF group (Fig. 1A). Quantitative data showed that the numbers of Nissl staining cells in all three hippocampal regions were dramatically decreased after CPF exposure, this effect can be attenuated by HRW intake (Figs. 1B-D). The CPF induced hippocampal neurons damage and the protective effects of HRW were also observed on week 2, 4, and 6 (data not shown).
To investigate the effects of CPF treatment and HRW intake on the mitochondria of hippocampal neurons, the ultrastructural changes in the mitochondria were examined by electron microscopy after 8 weeks of CPF exposure. CPF treatment induced significant mitochondrial damage in CA3 neurons, while the CA1 and DG region showed mild mitochondrial damage. Striking structural changes in mitochondria of CA3 neurons were observed after CPF treatment, including swelling, increases in matrix space, disorganization of cristae, and the appearance of amorphous matrix densities. In the HRW group, most of the mitochondria were intact with a dark matrix, a regular distribution of cristae, and no signs of swelling were observed. Rats given CPF showed significantly fewer mitochondria in both neurons than those in the C/oil group, but no such differences were observed in the HRW group and C/oil group (Fig. 2).
The effects of CPF treatment and HRW intake on hippocampal astrocytes
GFAP has been recognized as a special marker for astrocytes. Immu-nostaining results showed that the astrocytic density in all three hippo-campal regions, especially in DG region, was raised in CPF group after 2 weeks of CPF exposure. The numbers of GFAP-positive astrocytes in DG region in CPF group were significantly increased (156.54%) compared to C/oil group, and this effect was ameliorated by HRW intake (22.31%). While only a relatively smaller increase in the number of GFAP-positive astrocytes in CPF group was observed on weeks 4, 6 and 8. After 6 weeks of CPF exposure, the number of GFAP-positive astrocytes in CPF and HRW group was increased by 36.58% and 20.68% compared with that of C/oil group, respectively (Fig. 3).
HRW intake attenuates CPF-induced oxidative stress in the brain
Oxidative stress is an imbalance between tissue oxidants and antiox-idants. Indicators of oxidative stress, including MDAand GSH levels and SOD and CAT activity of the brain tissues were examined after 8 weeks of CPF exposure. The concentration of MDA, a biomarker of free radical mediated oxidative processes, was measured and showed that 8 weeks of CPF exposure caused increased MDA levels (58.29%) in the brain, which were significantly ameliorated by daily intake of HRW (35.08%) (Fig. 4A). CPF exposures were shown to produce an increase in GSH levels (87.81%) in the brain, and the effect was reversed by HRW intake compared to the control rats (21.59%) (Fig. 4B). The activity of CAT and SOD were elevated by 79.06% and 60.01% when compared with C/oil group, respectively, and these effects were also ameliorated by HRW intake (23.36% and 15.17%) (Figs. 4C and D).
Effect of HRW intake on the CPF-induced AChE inhibition in vivo
Since the measurement of AChE inhibition in organisms is widely used as a specific biomarker for exposure to OPs (Yang et al., 2008), the effects of CPF on AChE activity in brain tissues were determined. Eight weeks of CPF intake evoked a significant 173.9% reduction in AChE activity, relative to control levels. The AChE activity was significantly decreased (52.2%) in HRW group compared to the controls.
Fig. 1. HRW intake protects Wistar rats from CPF-induced damage in the hippocampus (n = 6). (A) Representative Nissl staining images showed the changes in the hippocampus after 8 weeks of CPF exposure. Row 1: CA3. Row2: distal CA3. Row 3: dentate gyrus (DG).Row4: CA1. Scale bar: 200 |jm (uppermost rows, low magnification) and 50 |jm (four lower rows, high magnification). Arrows indicate dying cells, empty spaces left by dead cells, and damaged neurons, identified by loss of Nissl substance. (B-D) The numbers of Nissl staining cells in hippocampal regions.
However, hydrogen treatment induced an increase of 79.9% in AChE activity compared to that of the CPF group (Fig. 5A).
Effect of hydrogen molecules on the AChE activity in vitro
We next evaluated the effects of hydrogen molecules on the CPO-induced AChE inhibition in an in vitro cell-free system. The Appki for CPO inhibition of AChE in H-HRW was similar to that in water (Figs. 5B and C). The Vmax for acetylthiocholine showed a marked increase of 20.4% in HRW relative to water. A significant elevation of Km for acetylthiocholine in H-HRW than in water was also observed (Fig. 5D).
We further evaluated the direct role of hydrogen on the modulation of AChE activity. AChE activity was determined in different solvents in the absence of CPO. H-HRW and HRW were boiled and cooled down to room temperature naturally to remove the hydrogen molecules. The hydrogen concentration was monitored and it was confirmed that no hydrogen was left in both boiled H-HRW and HRW. The pH values of H-HRW, HRW and normal water were measured at 6.0, 8.0 and 6.0, respectively. The magnesium concentration was 4.11 mg/L in HRW,
and that was too low to detect in both H-HRW and normal water. AChE activity in H-HRW was higher compared with that in the normal water (Fig. 5E). No difference of AChE activity was observed between boiled H-HRW and normal water (Fig. 5F). The increased AChE activity was also observed in HRW compared with that in normal water. The AChE activity in boiled HRW showed no significant difference from normal water (data not shown).
Discussion
It has been reported that the average daily intake of CPF by a 70 kg adult is estimated to be 0.005 |g/kg/day (Eaton et al., 2008). In the farm family study, the "absorbed daily dose" of CPF was estimated to be 0.27-1.96 |g/kg/day in 25 farm children (Curwin et al., 2007). A study on eight workers from a commercial pest control company showed that the mean estimated absorbed daily dose was 9.5 |g/kg/day (Fenske and Elkner, 1990). The majority of long-term toxicological investigations in laboratory animals have been conducted using the oral route of administration with doses in the range 0.05-25 mg/kg/day, which have been typically at least two orders of magnitude higher than those experienced
Fig. 2. HRW intake attenuated CPF-induced damage in the neuronal mitochondria (n = 3). The ultrastructural changes in the mitochondria were examined by electron microscopy after 8 weeks of CPF exposure. (A) Transmission electron microscopy of hippocampal neurons. M: mitochondria. N: nucleus. Scale bar: 1 |jm. (B) Mitochondrial counts. **P < 0.01 determined by one-way ANOVA test. Data are shown as the mean ± s.e.m.
during occupational exposures (Eaton et al., 2008). In this study, CPF was orally administered at a subthreshold dose level equivalent to 1/20 acute oral LD50 (6.75 mg/kg/day) for 2-8 weeks.
The results obtained here indicated that CPF exposure caused significant hippocampal neuron damage, especially in CA3 region, and this damage can be ameliorated by HRW intake. Similar to the findings in this study, previous studies have also found that repeated dermal application of CPF in sub-toxic doses (1/5 or 1/2 LD50) for 1 week or 3 weeks in mice can cause significant hippocampal neuron damage (Lim et al., 2011; Mitra et al., 2009). Astrocytes, a sub-type of glia in the CNS, provide guidance during neuronal migration, synaptogenesis and neuronal nutrition. Since it has been suggested that astrocytes can protect neurons from oxidative stress (Tanaka et al., 1999), and astroglial changes have been found following toxic insult to the CNS (Norenberg, 1994), we next examined the expression of GFAP, an astrocytic marker, to explore the effect of CPF and HRW treatment on astrocytes. The results showed that GFAP expression was significantly increased in CPF group compared to the controls and the increase can be ameliorated by HRW intake. An interesting finding of the study
was that 2 weeks of CPF exposure showed higher increase in GFAP expression compared to that for the extended periods. Similar to this study, previous studies reported that GFAP expression was significantly increased following 1 week of CPF application at both doses (1/10 and 1/5 dermal LD50) in mice (Limetal., 2011; Mitra, 2011). However, application for 3 weeks did not produce prominent visible changes in the expression of GFAP (Mitra, 2011). One possible explanation is that astrocytes may provide neuroprotective effects against CPF toxicity primarily at early stages.
A previous study demonstrated that CPF-induced toxicity may be mediated in part by the generation of oxidative stress, and the brain is more vulnerable to oxidative stress than other tissues (Bellissimo et al., 2001). Some indicators were measured to assess the oxidative stress status after CPF or HRW treatment. MDA is one of the major oxidation products of peroxidized polyunsaturated fatty acids and increased MDA content is an important marker of membrane lipid peroxidation. Previous studies have also shown that MDA significantly increased in various tissues of rats treated with OPs (Aly et al., 2010; Verma et al., 2007). In the present study, CPF treatment resulted in significantly
Fig. 3. The effect of HRW intake and CPF treatment on GFAP expression (n = 6). (A) Photomicrograph showing the immunohistochemical staining of GFAP expression in DG region of the hippocampus after 2 weeks and 6 weeks CPF exposure. Brown colored rounded cells are the astrocytes. Scale bars = 100 |jm. (B) The numbers of GFAP positive cell in DG region of the hippocampus after 2 weeks and 6 weeks CPF exposure. *P < 0.05, **P < 0.01 determined by one-way ANOVA test. Data are shown as the mean ± s.e.m.
Fig. 4. HRW intake attenuated CPF-induced oxidative stress in the brain of rats (n = 6). Tissue MDA (A) andGSH (B) levels 8 weeks post-CPF exposure. ComparisonsofCATactivity (C) and SOD activity (D) in the brain of rats. *P < 0.05, **P < 0.01 determined by one-way ANOVA test. Data are shown as the mean ± s.e.m.
Fig. 5. The effect of hydrogen molecules on AChE activity. (A) HRW intake attenuated CPF-induced AChE inhibition (n = 6). (B) Time course of hydrolysis of ATC in the presence of CPO. The data were used to calculate the pseudo-first-order kinetic rate constant kobs. (C) Double-reciprocal plot of kobs vs inhibitor concentration corrected for the presence of substrate (1 — a; where a = [S] / (Km + [S]) and the 1/slope represent the apparent bimolecular rate constant appki. CPO was used at a concentration of 0-10 |M. (D) Determination of the Kmand Vmaxfor acetylthiocholine (ATC) by acetylcholinesterase. To evaluate the direct role of hydrogen on the modulation of AChE, AChE activity was measured in different solvents, including (E) H-HRW and H2O and (F) boiled H-HRW and H2O. *P< 0.05, **P< 0.01 determined by one-way ANOVA test. Data are shown as the mean ± s.e.m.
increased MDA levels in rat brain. GSH plays a central role in the antiox-idant system and its content is a function of the balance between use and synthesis. It has been shown that exposure to compounds that can generate ROS can increase the content of GSH by increasing the rate of GSH synthesis (Dickinson and Forman, 2002). This response would allow the organisms to detoxify the pesticide, remove ROS and therefore increase tolerance to the pesticide (Kristoff et al., 2008). In this study, increased tissue GSH levels indicated that intracellular GSH may be robustly consumed and synthesized for scavenging by CPF-induced ROS in rat brain. CAT and SOD are the most important antioxidative enzymes against toxic effects of ROS. The increased activity of SOD is known to serve as protective responses to eliminate reactive free radicals (Celik and Suzek, 2009). Some studies have indicated that superoxide radicals can inhibit CAT activity and the increased H2O2 resulting from CAT inhibition could finally inhibit SOD activity (Gultekin et al., 2000). Previous studies have reported that CAT and SOD activities increased in rat tissues by OP exposure (Celik and Suzek, 2009; Goel et al., 2005; Sarabia et al., 2009; Uzun et al., 2010). This is consistent with our finding that CPF treatment significantly enhanced the CAT and SOD activity. The increased CAT and SOD activities might be associated with toxicity of CPF on rat brain tissues. The improvement in oxidative stress status in the brain following treatment with HRW, as evidenced by decreased levels of MDA compared with CPF group, accompanied by an increase in GSH level and in SOD and CAT activity, may be responsible for the attenuated hip-pocampal damage.
Another possible cause for the neuroprotective effect of hydrogen may be related to its high diffusibility. Hydrogen molecules can readily cross the blood-brain barrier and penetrate biomembranes smoothly to diffuse into the cytosol, nucleus and mitochondria. This is particularly important, as mitochondria is the major source of ROS and notoriously difficult to target. In this study, the damage to the neuronal mitochondria caused by CPF was attenuated after hydrogen treatment. The protective effect of hydrogen on the mitochondria has also been found in other studies. Ohsawa et al. (2007) reported that antimycin A treatment decreased the mitochondrial membrane potential and the mitochondrial ATP synthesis in cultured cells, and the mitochondrial damage can be protected by hydrogen treatment.
Since the primary target site of CPF is AChE, the effects of hydrogen on the activity of AChE were evaluated. The results showed that AChE activity in brain tissue was significantly decreased after CPF exposure and this effect can be attenuated by HRW treatment. We hypothesized that hydrogen molecules may have a direct effect on AChE inhibition. This is unusual, as hydrogen effects have been solely ascribed to exclusive removal of hydroxyl radicals in previous works (Itoh et al., 2009; Ohta, 2011). To prove this hypothesis, the effects of hydrogen molecules on AChE activity were evaluated in an in vitro cell-free system. The results showed that only a very short time pre-incubation of the enzyme with HRW could increase the activity greatly. The attenuated AChE activity inhibition induced by CPO after hydrogen treatment was also observed.
To further investigate whether hydrogen molecules have a direct effect on the AChE activity, we performed the enzyme activity tests in the absence of CPO. The results showed that AChE activity was enhanced in the presence of hydrogen and this effect was not observed when the hydrogen was removed by the boiling water treatment. Since AChE activity can be enhanced in some conditions, such as alkaline metal ions (Na+ and K+) and alkaline-earth metal ions (Ca2+ and Mg2+) (Hofer et al., 1984), both the magnesium concentration and pH value were measured and the results showed that these conditions may not be the causes for the increased activity. These findings indicated the possible direct interaction between hydrogen molecules and AChE enzyme. Belpassi et al. (2010) showed that water-H2 interaction is accompanied by charge transfer, which is shown to be a significant, strongly stereospecific component of the interaction. Water acts as an electron donor and acceptor in different orientations. The significantly
stronger interaction of H2O-H2 than would be expected 'disturb' the hydrogen bond networks and charge distribution in water, which might relate to the activation of the AChE enzyme (Belpassi et al., 2010).
Increasing numbers of studies have demonstrated that H2 has protective effects in various settings (Ohta, 2011). The diverse biological functions of hydrogen cannot be explained by the selective anti-oxidative effect alone (Ohta, 2012) and the direct interaction between hydrogen and enzymes provides insight into a novel mechanism for interpreting the physiological significance of hydrogen. AChE belongs to the superfamily of a/p-hydrolase fold proteins, which is one of the largest groups of structurally related proteins with diverse catalytic and non-catalytic functions (Lenfant et al., 2013). The family is characterized by the a/p-hydrolase fold, consisting of a central p-sheet surrounded by several a-helices and by a common catalytic triad formed of a catalytic nucleophile, a histidine and an acidic residue. To our knowledge, this is the first report of the direct interaction between hydrogen and enzyme molecules, which provides new insights into the biological functions of hydrogen molecules.
In conclusion, this study provides evidence that intake of HRW can protect Wistar rats from CPF-induced neurotoxicity, and the protective effects of hydrogen may be mediated by its anti-oxidative activity. Thus, these findings suggest the potential feasibility of HRW intake to protect people from OPs induced neurotoxicity. Furthermore, this work defines a novel mechanism of biological activity of hydrogen by directly increasing the AChE activity. The present results indicate that far more biochemical reactions may be affected by hydrogen molecules.
Conflict of interest
The authors declare that they have no actual or potential competing financial interests.
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