Accepted Manuscript Interaction of phosphodiesterase 5 inhibitor with malathion on rat brain mito‐ chondrial-bound hexokinase activity Maryam Azadbar, Akram ranjbar, Azadeh Hosseini-Tabatabaei, Abolfazl Golestani, Maryam Baeeri, Mohammad Sharifzadeh, Mohammad Abdollahi PII: DOI: Reference:
S0048-3575(09)00101-1 10.1016/j.pestbp.2009.08.001 YPEST 3147
To appear in:
Pesticide Biochemistry and Physiology
Received Date: Revised Date: Accepted Date:
2 January 2009 4 July 2009 1 August 2009
Please cite this article as: M. Azadbar, A. ranjbar, A. Hosseini-Tabatabaei, A. Golestani, M. Baeeri, M. Sharifzadeh, M. Abdollahi, Interaction of phosphodiesterase 5 inhibitor with malathion on rat brain mitochondrial-bound hexokinase activity, Pesticide Biochemistry and Physiology (2009), doi: 10.1016/j.pestbp.2009.08.001
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Interaction of phosphodiesterase 5 inhibitor with malathion on rat brain mitochondrialbound hexokinase activity
Maryam Azadbar1, Akram ranjbar1, 2, Azadeh Hosseini-Tabatabaei1,3, Abolfazl Golestani4, Maryam Baeeri1, Mohammad Sharifzadeh, and Mohammad Abdollahi1
1
Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, and Endocrinology and Metabolism Research Center, Tehran University of Medical Sciences (TUMS), Tehran; 2School of Paramedical Sciences, Arak University of Medical Sciences, Arak; 3
Faculty of medicine, University of British Columbia, Vancouver, Canada; 4School of Medicine, TUMS, Tehran, Iran
Correspondence: Prof. Mohammad Abdollahi, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, and Endocrinology and Metabolism Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran. Tel/Fax: + 98 21 66959104, E-mail:
[email protected]
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Abstract The main objective of this study was to investigate the possible protective effect of pentoxifylline as a phosphodiesterase 5 inhibitor used and a cardiovascular medication on malathion-induced changes on rat mitochondrial-bound hexokinase activity. Animals in four various groups received moderate toxic dose of malathion (200 mg/kg/day), effective dose of pentoxifylline (50 mg/kg/day) alone and in combination, and the control group that received only vehicle. All administrations were done intraperitoneally for one week. At the end of the experiment, the brain was removed and the mitochondria were isolated. Hexokinase (HK) activity, cellular lipid peroxidation (LPO) and total antioxidant capacity (TAC) were analyzed in brain mitochondria. Malathion noticeably decreased TAC and increased HK activity and LPO in the mitochondria whereas pentoxifylline significantly restored malathion-induced changes in LPO, HK, and TAC. The results of the present study indicate that phosphodiesterase 5 inhibition remarkably protects brain mitochondria from malathion-induced changes on HK activity and oxidative stress. Key words: Malathion, Mitochondria, Hexokinase, Oxidative stress, Lipid peroxidation, Total antioxidant capacity, Rat
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1. Introduction Malathion [S-1,2 (bis-ethoxycarbonyl) ethyl O,O-dimethyl phosphorodithioate], an organophosphorus (OP) compound is being used as insecticide and acaricide in agricultral, veterinary, medical and public health practices. Its main toxicity is inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine (ACh) in synaptic terminals of nervious system. For many years, overstimulation of cholinergic system has been known as the main cause of OPs-induced-toxicity symptoms. In the recent years, studies have indicated that OP compounds especially malathion are able to induce oxidative stress by changing the status of oxidant-antioxidant balance of body [1, 2]. Now, this is known as a molecular mechanism of not only OPs’ neurotoxicity but also their toxic effects on many organs such as blood [3, 4], liver [5, 6] and muscles [7]. Any imbalance between production of mitochondrial reactive oxygen species (ROS) and cellular antioxidant capacity leads to oxidative stress, a condition that has been associated with a number of physiological and pathological events [8-12]. Since brain is the target of many degenerative and traumatic damages, and is inadequately equipped with scavenger antioxidant defense mechanisms, it is particularly vulnerable to oxidative stress [13-14]. The mitochondrial electron transport system in the body works as a major source of cellular ROS such as superoxide, hydroxyl, and hydrogen peroxide [15]. The charge of mitochondrial ROS production is dependent on mitochondrial membrane potential and inversely related to the accessibility of adenosine diphosphate (ADP) used to drive the adenosine triphosphate (ATP) synthesis [16]. Hexokinase (EC 2.7.1.1; HK) is the first enzyme in the glycolytic pathway that catalyzes the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP [17]. Four types of HK has been
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determined on the basis of their electrophoretic mobility that are present in various tissues. HK-1 constitutes the predominant form of the enzyme in the brain that is majorly associated with outer mitochondrial membrane [18]. Pentoxifylline (PTX) as a methylxanthine derivative has been widely used as a cardiovascular drug in ameliorating peripheral vascular diseases. PTX not only increases blood cells flexability and flow [6], but also possesses vasodilatory actions due to a nonselective inhibition on phosphodiesterase (PDE). Therefore, PTX increases generation of cyclic nucleotides such as cAMP and cGMP. In addition to this property, PTX is shown to have preventive effects on overproduction of superoxide and hydrogen peroxide species [19, 20]. Furthermore, it is revealed that PTX has inhibitory trait on cytokines production, especially TNF-α. This cytokine is a key factor in stimulating the generation of ROS from mitochondria [21, 22]. It is demonstrated that PTX is an inhibitor of xanthine oxidase, an enzyme involved in generation of ROS. It has been shown that PTX increases body total antioxidant capacity (TAC) whereas it decreases lipid peroxidation (LPO). Regarding above-mentioned evidences, the present study was set to examine protective effect of PTX in brain mitochondria of rats exposed to moderately toxic dose of malathion in a subacute model. Status of mitochondrial HK activity in relation to LPO and TAC were the key outcomes.
2. Materials and methods 2.1 Chemicals Dithiononitrobenzoic acid (DTNB), Tris base, 1,1, 3,3′-tetraethoxypropane (MDA), 2thiobarbituric acid (TBA), trichloroacetic acid (TCA), n-butanol, 2,4,6-tripyridyl-s-
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triazine (TPTZ), malathion, sucrose, ethylenediamine tetraacetic acid (EDTA), 3mercapto-1,2-propanediol [Thioglycerol (TG)], Coomassie blue, ATP, NADP, NADH, glucose-6-phosphate dehydrogenase, Tris-HCl, Dithiothreitol (DDT), and HEPES buffer from Sigma-Aldrich were used in this study.
2.2 Experimental design Adult male Wistar rats weighing 200-250 g maintained on a 12-h light/dark cycle with free access to tap water and standard laboratory chow were used. Twenty-four rats were randomly divided into four groups and treated for one week intraperitoneally (IP). One group was considered as control, which received no treatment but normal saline. Other three groups were Mal group which received malathion 200 mg/kg/day, PTX group which received PTX 50 mg/kg/day and PTX+Mal group in which PTX and malathione were administered in combination. At the end of the treatment, 24 h post the last dose of treatment, animals were anesthetized with ether inhalation and decapitated, brain tissue was separated and mitochondria was isolated quickly and kept frozen at -80ºC.
2.3 Isolation of brain mitochondria Briefly, brain tissue was rapidly transferred to an ice-cold isolation buffer containing sucrose (0.25 M), HEPES (10 mM), EDTA (100 mM), 3-mercapto-1,2-propanediol (Thioglycerol, 0.01 mM, pH 8). After three consecutive washes to remove contaminating blood, the tissues were sliced into little pieces in isolation buffer. Then they were manually homogenized in 20 volumes of thioglycerol buffer using a homogenizer. The homogenates were centrifuged at 6000 rpm for 15 min and the supernatants were
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removed and centrifuged again at 18000 g for 20 min. Supernatants were transferred into several microtubes for biochemical assays. Samples were kept at -80°C until analyses [23].
2.4 Assay of HK activity The basis of the method is measuring formation of NADH at 340 nm. The assay medium contained Trise base (10 mM, pH 8.5) including glucose (5 mM), MgCl2 (7.5 mM), glucose-6-phosphate dehydrogenase (G6PDH, 100 units/mL), HEPES (45 mM), Dithiothreitol (DDT, 0.1 M/25 ml), and NADP+ (32.5 mM) in a final volume of 1 mL. The reaction was started by adding ATP (2 mM) and the absorbance was measured in a double beam spectrophotometer. Data were reported as unit per mg protein (U/mg Pr) of tissue [24].
2.5 Assay of LPO The LPO products of tissues were determined by thiobarbituric acid reactive substances (TBARS) expressed as the extent of malondialdehyde (MDA) productions during an acid heating reaction. Briefly, the samples were diluted by TCA (20% w/v), centrifuged, and then, the precipitation was dissolved in sulfuric acid. The mixture was added by TBA (0.2% w/v), and then incubated for 1 h in a boiling water bath. Following incubation, nbutanol was added and the solution centrifuged and cooled. The absorption of the supernatant was recorded in 532 nm. Tetraethoxypropan was used as standard. Results were represented as nmol per mg protein of tissue (nmol/ mg Pr) [3].
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2.6 Assay of TAC Antioxidant capacity was determined by measuring the amount of reduction of Fe3+ to Fe2+ as named FRAP test. Briefly, in this test, the medium is exposed to Fe3+ and the antioxidants present in medium start to produce Fe2+ as an antioxidant activity. The reagent contained TPTZ dissolved in acetate buffer (pH 3.6) and FeCl3. The complex between Fe2+ and TPTZ gives a blue color with absorbance at 593 nm that is measured on the basis of a calibration curve obtained by different concentrations of FeCl3. Results were reported as nmol/mg Pr of tissue [3].
2.7 Assay of protein Concentration of protein was measured by the Bradford method. Concentrated Coomassie blue was diluted in distilled water, and added to the prepared homogenates. The mixture was incubated at room temperature for 10 min and the absorbance was measured at 595 nm by a spectrophotometer. Bovine serum albumin in the range of 0.251 mg/ml was used as standard. Results were expressed as mg/ml [25].
2.8 Statistical analysis Mean and standard error values were determined for all the parameters and the results were expressed as mean±SEM. All data were analyzed employing analysis of variance ANOVA followed by Newman Keuls test. A P value less than 0.05 was considered significant.
3. Results
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Figure 1 represents the effects of treatments on HK activity in brain mitochondria. Mean±SE values for control, Mal, PTX, and Mal+PTX groups were 1010±76, 2152±161, 1325±99, and 1700±128, respectively. Administration of malathion resulted in an increase in HK activity when compared to control rats. Treatment of animals with PTX increased HK activity when compared to control group. There was a significant improvement in terms of HK activity in PTX+Mal group when compared to Mal group and remained closer to control levels. Figure 2 shows the results of treatments on TBARS value in brain mitochondria. Mean±SE values for control, Mal, PTX, and Mal+PTX groups were 760±57, 1140±86, 496±37, and 697±52, respectively. There was a significant increase in TBARS value in Mal group as compared to controls. Also, TBARS value decreased in PTX group obviously when compared to control animals. Its level was reduced significantly in PTX+Mal groups in comparison with Mal group. Figure 3 shows the results of treatments in brain mitochonndria TAC of animals. Mean±SE values for control, Mal, PTX, and Mal+PTX groups were 283±21, 211±16, 333±25, and 260±20, respectively. Treatment of rats with malathion decreased TAC significantly as compared to control group. TAC was significantly increased by PTX as compared to controls. Administration of PTX in association with malathion enhanced TAC significantly when compared to MAL group and reached to control levels.
4. Discussion
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The results of this study indicate that PTX is almost able to ameliorate malathioneinduced toxicity in rat brain. In this experiment, PTX noticeably improved biomarkers of oxidative stress including TAC of tissue and cellular LPO and also improved HK activity. OPs are a group of pesticides, which are widely being used in agriculture. Their biodegradable characteristic has made them a good candidate for this purpose [26]. However, OP-induced poisoning is unavoidable. As mentioned before, their main mechanism of toxicity is inhibition of AChE. In addition to this effect, it has been proved that one major mechanism in OPs' acute and chronic toxicity is oxidative stress [27, 28]. The potential of malathion to induce oxidative stress in blood has been previously reported and confirmed by several studies [1, 2]. OPs induce LPO and decrease TAC, which are evidences confirming their oxidative stress-inducing toxicity [3, 29]. Changes in levels of antioxidant enzymes such as superoxide dismutase, gamma-glutamyl transpeptidase, glutathione reductase and catalase besides non-enzymatic antioxidants like tocoferol and ascorbic acid are some of the obsereved compensatory mechanisms of the host’s defence system [30]. Mentioning changes induced by malathion on LPO and TAC and HK in the present study, it is well understood that increase of HK activity is a reflex against oxidative stress status. To explain role of HK in this scenario, it is necessary to know the function of HK in brain in details. HK catalyzes the primary step in glycolytic pathway that is phosphorylation of glucose to form glucose-6-phosphate (G6P) [31]. The reversible binding of HK-1 to the outer mitochondrial membrane regulates its activity and compose over 80% of HK-1 activity in the brain [32-34]. The distribution of the enzyme between mitochondrially bound and dissociated forms has been found to be influenced by the level of certain
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metabolites, especially G6P [35]. Mitochondrially bound enzyme is more active than the soluble enzyme since the bound form can utilize more preferentially the ATP generated in mitochondria and is less sensitive to G6P inhibition [4, 5]. It is believed that intramitochondrial oxidative phosphorylation (IOP) which results in ATP generation is connected to glucose phosphorylation held by mitochondrially bound HK. In fact, IOP supplies substrate ATP for activity of HK. These two processes are shown to be linked to NADPH production. Previous studies have demonstrated that HK-1 protects cells against apoptosis by inhibiting cytochrome c release [31]. Another investigation has also proved this property for HK-2 [36, 37]. Tissues such as brain with low mitochondrial peroxidase activities are complemented by mitochondrial kinases acting as a defensive antioxidant system directed to inhibit production of ROS by sustaining an ADP re-cycling mechanism [38]. Our conclusion is that mitochondrial HK acts like an antioxidant in the brain of malathion-exposed rats. On the other hand, the increased metabolic flux in the mitochondria due to high glucose content, is coupled with reduction of NAD to NADH, resulting in increased formation of ROS such as superoxides, peroxinitrites, and highly reactive hydroxyl radicals [39]. Extra production of mitochondrial ROS needs to be detoxified tissue-specifically. The brain is much open to free radical-induced molecular damage because it is not covered with enough antioxidant defense elements [40]. Thus, in this scenario it is again concluded that HK acts as an indirect antioxidant. In agreement with this conclusion, the preventive antioxidant function of mitochondrial HK was recently established in rat brain by coupling the oxidative phosphorylation to G6P [16]. Explaining the mechanism of action of PTX, there is evidence that increased intracellular concentrations of cAMP and cGMP following use of specific PDE inhibitors can
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ameliorate cellular oxidative stress [20, 41-43]. The same potential in prevention of cellular LPO and damage has been reported for PTX as a PDE-5 inhibitor in oxidative stress-induced embryotoxicity [44]. There are additional evidences indicating the same benefit of PTX in oxidative stress status in diseases like diabetes [21] and colitis [22]. Alongside this property, the present study evaluated the beneficial effects of PTX in improvement of LPO. Resulted from oxidative stress, LPO was measured by measuring TBARS value. According to our results, PTX dramatically decreased LPO in brain mitochondria which confirms previous studies investigating this trait. Finally, this study showed that exposure to malathion decreases TAC of tissue significantly. PTX noticeably increases TAC in brain mitochondria. This finding is parallel to previous studies indicating this trait for PTX [41,45]. Taking collectively, the present results indicate that PTX can improve malathion-induced oxidative stress through activating of HK. Regarding high-energy consumption and needing high amounts of oxygen and glucose in the CNS comparing to other tissues, the pathways like oxidative phosphorylation involved in transduction of energy are highly active. Any imbalance in energy supply and demand in the brain mitochondria may lead to release of high amounts of ROS. It has been proved that malathion-poisioning results in oxidative stress condition which is demonstrated by generation of ROS, rise in LPO level and finally increase in level or activity of compensatory enzymatic and nonenzymatic pathways [4]. PTX may act against oxidative stress via blocking production of inflammatory mediators and the responsiveness of immunocompetent cells to inflammatory stimuli [20,41,43]. Proved with the results of this study, PTX serves some of its beneficial effects through
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improving mitochondrial HK activity, decreasing LPO and boosting TAC by increasing cyclic nucleotides content in brain mitochondria. The beneficial effects of PTX against malathion-induced toxicity by stimulating super oxide dismutase and other antioxidants has been recently found (45). In this study, a dose of 50/mg/kg and a duration of one week was chosen for treatment of animals with PTX. Future dose-duration studies are suggested to be done in order to find the optimum dose and time for treatment of malathion toxicity with PTX. Considering malathion toxicity on other targets like liver, kidney, uterus, blood and cardiovascular system [4, 30], further investigations may be done in future, on evaluating the beneficial effects of PTX on malathion-induced toxicity in those tissues. For sure, future clinical studies should be designed which focus on more susceptible people who are in direct contact with OPs such as pesticide formulating workers and farmers. Relative biomarkers of toxicity may be measured in blood samples and other objective tests like cognitive functions can be obtained by questionnaires. Finally, the observed results in this study and the previous findings indicate that PTX may find a place in ameliorating the oxidative injury following malathion exposure.
Acknowledgment: This work was supported by a grant from TUMS research council.
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Figure 1: HK activity in brain mitochondria of rats. aa significantly different from control group at P
