Abstract

Background: Malathion is an insecticide of the grouping of organophosphate pesticides (OPs), which shows strong insecticidal effects. In addition, vitamin E reacting to cell membrane site may prevent OP-induced oxidative injury.

Objectives: The aim of this study was to examine the protective function of vitamin E on toxicity of malathion, by measuring the activities of liver and liver mitochondrial superoxide dismutase (SOD), catalase (CAT),lipid peroxidation (LPO),and glutathione peroxidase (GPx) in rats.

Materials and Methods: The mitochondrial viability was determined in liver. ‎Effective doses of malathion(200 mg/kg/day) and vitamin E (alpha-tocopherylacetate [AT]; 15 mg/kg/day) were administered alone or in combination for 14 days. At the end of the experiment, the liver tissue and liver mitochondria of the animals were harvested and examined.

Results: In liver tissue, the activity of LPO and CAT was higher in the malathion group in comparison to controls. AT reduced malathion-induced LPO, SOD, CAT, and GPx in rat liver. Coadministration of AT with malathion improved LPO, SOD, and CAT levels in liver as well as CAT and GPx in liver mitochondria. Malathion-induced mitochondria toxicity was recovered by AT.

Conclusions: In conclusion, AT measurement can be beneficial for the safety or recovery of malathion-induced toxic injury in liver tissue and liver mitochondria.

Keywords: Malathion; Mitochondria, Liver; Oxidative Stress; Rats; Vitamin E

1. Background

Organophosphorus (OPs) pesticides are a major as well as the most diverse group of insecticides. The broad implication of OPs in public health and agricultural programs accompanies potentially dangerous impacts on humans, animals, plants, and environment (water, air, soil, and food) and causes acute and chronic poisoning (1, 2). OPs decrease the function of carboxylic ester hydrolases such as chymotrypsin, acetylcholinesterase (AChE), plasma or butyrylcholinesterase (BuChE), plasma and hepatic carboxylesterase (aliesterases), paraoxonases (PON1) and other esterases within the body (2-4). Malathion (O,O-dimethyl-S-[1,2-dicarboxyethyl] ethyl phosphorodithioate) is one of the main widely used Ops pesticides in agriculture and public health programs (5-7). Recently, it has been revealed that OPs produce oxidative stress in different tissues through the producing reactive oxygen species (ROS) (8, 9). Although ROS are part of normal oxidative metabolism, when produced in overload, they cause tissue injury including lipid peroxidation, DNA damage, and enzyme inactivation (10, 11). Moreover, oxidative stress is a process related to xenobiotic contact and different levels of environmental pollution (12). Hence, the mitochondrial respiratory chain serves as a main source of ROS derived from the disproportionate superoxide anions. Even though the presence of different antioxidants factors, e.g. ubiquinone and vitamin E, and antioxidant enzymes, and the mitochondria is the most powerful intracellular source of ROS; according to a hypothesis, the steady state concentration of O^.-2 in the mitochondrial matrix is about five-to ten-fold higher than that in the cytosol or nucleus. Consequently, mitochondria might also be a main target for the damaging function of ROS. The contact of diverse macromolecules with ROS may impair the role of these organelles and may directly influence cell viability and lead to prompt cell death (13, 14). In biological systems, vitamin E is a key antioxidant acting as a potent chain-breaking mediator during the scavenging of peroxyl radicals (15, 16). Moreover vitamin E decreases the chain reaction of lipid peroxidation in membranes. Hence, a lot of studies have been done to find out the defending properties of vitamin E in specific biological models of injury (17, 18).

2. Objectives

This study aimed to examine antioxidant effects of vitamin E on malathion-induced oxidative stress in rat liver homogenate and mitochondria.

3. Materials and Methods

Trichloroacetic acid (TCA), tetraethoxypropane (MDA), 2-thiobarbituric acid (TBA), n-butanol,malathion, sucrose, ethylenediaminetetraacetic acid (EDTA), Trishydrochloride acid, nitrobluetetrazolium (NBT), xanthine, xanthine oxidase, hydrogen peroxide (H₂O₂), Coomassie blue, bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were used in this study. All the chemicals were obtained from the Sigma Chemical Company (St. Louis, MO, USA).

The male Wistar rats (180-250 g) were obtained from the animal colony of the Pastor Institute, Iran. Animals were maintained under standard conditions with the temperature of 22℃ ± 1℃, humidity of 45%, and 12:12 hours light/dark cycle. Animals were randomly allocated to four groups of five animals and treated for two weeks. The groups were as follows: control group, malathion group, alpha-tocopherol acetate (AT) group, and malathion-AT group. Malathion was administered intraperitoneally (200 mg/kg/day) alone or in combination with subcutaneous AT (15 mg/kg/day). One group of animals received normal saline only and was considered as the control group. At the end of the treatment (24 hours after receiving the last treatment dose), animals were sacrificed; then, liver tissue was extracted and stored in liquid nitrogen.

The liver tissue was distincted with small cutters in a cold mannitol solution containing 0.225-mol D-mannitol, 75-mmol sucrose, and 0.2-mmol EDTA. The minced liver (30 g) was gently homogenized in a glass homogenizer with a Teflon pestle and then centrifuged at 700 ×g for ten minutes at 4°C to remove nuclei, unbroken cells, and other non-subcellular tissues. The supernatants were centrifuged at 7000 g for 20 minutes. These second supernatants were taken as the crude microsomal part and the pale loose upper layer of sediments, which was rich in swollen or broken mitochondria, lysosomes, and some microsomes, was washed away. The dark packed lower layer (heavy mitochondrial fraction) was resuspended in the mannitol solution and centrifuged twice at 7000 g for 20 minutes. Then weighty mitochondrial sediments were floated in Tris solution buffer (pH, 7.4) (19, 20).

Copper-zinc SOD (Cu/Zn SOD) was measured indirectly by assessing the reaction between SOD and the molecule NBT. The optical density (OD) changes at 560 nm over a five-minute period indicated the reduction of NBT by superoxide. The activity of total SOD was determined as the quantity of protein necessary for half-maximal inhibition of the NBT reaction (21).

In this method, the sample was added to a solution containing, glutathione (2 mmol), glutathione reductase (0.15 U/mL), sodium azide (0.4 mmol), reduced form of nicotinamide adenine dinucleotide phosphate (NADPH, 0.12 mmol), Tris (50 mmol, pH ¼ 7.5), EDTA (5 mmol), and dithiothreitol (DTT) buffer (1 mmol). The reaction was initiated by adding H₂O₂ and the diminish in absorbance at 340 nm/min over ten minutes was calculated (22).

The activity of CAT in the samples was measured by assessing the absorbance decreasing at 240 nm in a reaction medium containing H₂O₂ (10 mmol) and sodium phosphate buffer (50 mmol, pH 7.0). One unit of the enzyme was reported as 1 mol H₂O₂ as substrate consumed per minute, and the specific activity was reported as units per milligram protein (23).

In this method, the lipid peroxidation (LPO) product in the tissues was diluted by TBA reagent in acid heating reaction. Then, the samples were washed by adding 1.5 mL of 20% (w/v) TCA was added to 250 μL of samples and centrifuged in 3000 g for ten minutes. The precipitation was dissolved in sulfuric acid and 1.5 mL of the solution was added to 1.5 mL of 0.2% (w/v) TBA. Subsequent to incubation, 2 mL of n-butanol was added and the solution was centrifuged, cooled, and measured at 532 nm. Finally, the calibration curve of tetramethoxypropane standard solutions was used to verify the concentrations of TBA + MDA adducts in samples (24).

In this study, the protein content was measured using Bradford method. Strenuous Coomassie blue (G250) was diluted in 250 μL distilled water and then 750 μL of this diluted dye was added to 50 μL of the sample. The mixture was incubated at room temperature for ten minutes and an absorbance was measured at 595 nm by a spectrophotometer. Finally, a standard curve was constructed by using bovine serum albumin (BSA) as the standard (0.25 and 1 mg/mL) (25).

This assay is a quantitative colorimetric way to determine cell viability. It utilizes the yellow tetrazolium salt (MTT), which is metabolized by mitochondrial dehydrogenase enzyme from viable cells to yield a purple formazan reaction result at wavelength of 570 nm. Finally, the percentage of mitochondrial viability of each sample was measured (26).

Data were presented as mean ± SEM of at least three independent experiments with SPSS Version 16 (SPSS, Chicago, IL, USA). The one-way ANOVA, followed by a Tukey’s post hoc test, was used to compare multiple groups. The significance level was set to 0.05 (P < 0.05) for all comparisons.

4. Results

Malathion caused a significant raise in LPO when compared to the controls (P < 0.05). AT caused a significant decrease in LPO when compared to the malathion group (P < 0.05). AT caused a significant decrease in SOD activity in comparison to the malathion group (P < 0.05). In addition, coadministration of AT with malathion significantly reduced malathion-induced SOD activity (P < 0.05). Therefore, coadministration of AT with malathion significantly reduced malathion induced LPO (P < 0.05). In comparison to the malathion group, AT caused a significant decrease in GPx activity (P < 0.05). Malathion caused a significant increase in CAT activity in contrast to the control group (P < 0.05) while AT caused a significant decrease in CAT activity in comparison to the malathion group (P < 0.05), (Table 1).

Table 1. Oxidative Stress Biomarkers in Rat Liver and Liver Mitochondria a,b

4.2. Liver Mitochondria

Malathion and AT did not cause significant change on LPO in comparison to the other groups; however, malathion and AT did not cause significant change on SOD activity in contrast to the other groups. AT significantly reduced malathion-induced GPx activity (P < 0.05). Moreover, coadministration of AT with malathion significantly reduced malathion-induced CAT activity (P < 0.05). Administration of AT significantly decreased toxicity of the cells in comparison to the malathion group (P < 0.05). In addition, coadministration of AT with malathion significantly decreased malathion-induced mitochondria toxicity (P < 0.05), (Table 1).

5. Discussion

The main result of the present study was that malathion induced oxidative damages and mitochondrial dysfunction in rat liver. These results clearly indicate that malathion increased liver oxidative damage in rat by prompting of LPO, CAT, GPx, and SOD activities. Furthermore, AT was able to attenuate malathion-induced changes in almost all of the tested parameters. In addition, AT decreased the mitochondria toxicity in liver.The key function of mitochondria is producing energy, by oxidative phosphorylation and in the form of ATP, for cellular processes (27). This study reported the direct indications of damage in the liver mitochondria induced by malathion insecticide. It seems that the LPO byproducts induced a state of oxidative stress in mitochondria. Moreover, oxidative stress has been observed in some instinctive errors of metabolism (28), diseases (29-31), and pesticides poisoning (8, 9, 32, 33). In addition, both in vitro and in vivo studies showed that malathion toxicity is mediated by increased oxidants and oxidative injury (34), suggesting that malathion acts not only as a direct oxidant, but also as an initiator of oxidants (35). Latest studies have shown that the effects of acute and chronic exposure to malathion in oxidative stress induction is through the production of the ROS in the blood (36), liver (5), pancreas mitochondria (37), brain (38), and brain mitochondria (39) of rats. The results of previous studies showed that malathion poisoning results in oxidative stress condition, which is established by producing ROS, rise in cellular lipid peroxidation (LPO), and lastly, increase in level or activity of compensatory enzymatic and nonenzymatic antioxidant pathways (40). However, it has been revealed that malathion-induced oxidative stress modulates SOD and CAT activity in the liver (41). Our present results showed that malathion administration in rats caused significant induction in the activity of antioxidant enzymes, LPO, and increased mitochondria toxicity in the liver. In addition, malathion changed the antioxidant enzymes such as SOD, CAT, and GPx following subchronic exposure in animals (5, 42). Some studies have reported hepatotoxic effects of malathion exposure in both humans (43) and experimental animals (5, 42, 44); however, it has been confirmed that malathion-induced oxidative stress is due to the inactivation of mitochondrial respiratory complexes (39, 45). Our present results showed that malathion administration in rats caused significant mitochondrial toxicity.

Some studies have shown that mitochondria contains the highest concentration of AT (46), which accelerates ATP resynthesis in the ischemic heart tissue due to reperfusion (47). AT mostly acts as a chain-breaking antioxidant and radical scavenger, defending cell membrane against oxidative injury (48). Moreover, AT regulates production of ROS, maintains oxidative phosphorylation in the mitochondria, and accelerates damages of high-energy metabolites (49, 50). Finally, as evidenced by MTT assay, the present data verified that malathion-induced mitochondrial dysfunction could be modified by AT.

In conclusion, we reported that malathion induced a clear state of oxidative stress in liver that was in some way directly involved in the damage of hepatic mitochondria. In addition, AT was able to prevent the malathion-induced damage. These results proposed a possible mechanism to treat the oxidative injury caused by the OPs poisoning, in which the antioxidants such as AT may recover cell from injury. The present results showed that AT ‎could restore oxidant/antioxidant equilibrium in liver tissue and liver mitochondria and was also ‎capable of improving malathion-induced changes.

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