authored by Oyewo EB*
Abstract
Background and Objective: Chlorpyrifos [O, O-diethyl-O-(3, 5, 6-trichloro-2-pyridyl)-phosphorothioate] (CPF) is a broad-spectrum Organophosphate insecticide that are used in many farms and homes. Despite the several reported toxicity in humans, there has been virtually no alternative effective insecticide. Thus, the amelioration of the toxicities seems the best option in alternative medicine. This study, therefore, investigated the effects of methanol extract of Diospyros chloroxylon leaf (MEDCL) on the brain and heart of rats exposed to CPF.
Materials and Methods: Twenty-four rats were randomized into four groups of 6 rats each, and treated separately with distilled water (Control), CPF (5 mg/kg), MEDCL (100 mg/kg) and MEDCL (100 mg/kg) + CPF (5 mg/kg), following 7 days of acclimatization. After 4 weeks of treatments, the rats were sacrificed, and the levels of Superoxide dismutase (SOD), Catalase (CAT), Malondialdehyde (MDA), reduced glutathione (GSH), Glutathione peroxidase (GPx), glutathione S-transferase (GST) and DNA fragmentation were spectrophotometrically assessed in the brain and heart, while Acetylcholinesterase (AChE) activities were assessed in the serum and brain of the rats.
Results: The results showed that CPF significantly reduced the levels of SOD, CAT, GSH, GPx and GST, while that of MDA was elevated in brain and heart, compared with controls. Treatment with CPF significantly lowered the activities of AChE in serum and brain by 94% and 48% respectively, while the level of DNA fragmentation was significantly elevated in the CPF-treated rats. Supplementation with MEDCL significantly ameliorated the changes in the rats.
Conclusion: From the foregoing, the suppressive potential of methanol extract of Diospyros chloroxylon leaf is marked indicated in brain and cardiac redox imbalance induced on exposure to Chlorpyrifos.
Keyword: Chlorpyrifos; Diospyros chloroxylon; Oxidative stress; Acetylcholinesterase; DNA fragmentation
Introduction
Chlorpyrifos [O, O-diethyl-O-(3,5,6-trichloro-2-pyridyl)- phosphorothioate] (CPF) is an organophosphate insecticide, acaricide and miticide used in protection of various crops and ornamental plants [1]. Chlorpyrifos becomes introduced into the environment via direct application on crops, lawns, domesticated animals, as well as in homes and workplaces. However, volatilization is the major way in which this organophosphate is dispersed in the environment, after application. In the environment, it becomes decomposed through the sunlight, bacterial and chemical processes [2]. Mackay, et al. [3] has observed the atmospheric formation of Chlorpyrifos oxon from Chlorpyrifos. The hepatic biotransformation of Chlorpyrifos has been reported to involve cytochrome P-450 dependent desulfuration, to form Chlorpyrifos oxon [4,5]. This oxon is rapidly hydrolyzed to 3, 5, 6-trichloro-2-pyridinol (TCP) through the activity of Aryl- esterase. Both bioactivation and detoxification of Chlorpyrifos have been suggested to occur very rapidly, since TCP was detected as the only metabolite in the hepatic effluent under steady-state conditions 4. The TCP has been noted to be, in several orders of magnitude, less toxic than either Chlorpyrifos or its oxon form [6,7].
Some earlier studies by Bakke, et al. [8] and Nolan, et al. [9] had indicated that the hydrolysis of Chlorpyrifos oxon by A-esterase could probably be a common route of detoxification, since TCP or its conjugate is the major metabolite detected in rodents and humans. A kinetic study of the relative rates of deulfuration and detoxification of Chlorpyrifos by Chambers and Chambers [10] suggested a gender-dependence, which may explain its higher toxicity in female rats than male ones. Various mutagenicity studies using Chorpyrifos revealed that it could cause metaphasic chromosomal aberrations in mouse spleen cell culture [11], sister chromatid exchange in human lymphoid cells [12] and induction of micronuclei, chromosomal lesions, and DNA damage in many organisms [13-15]. However, the USEPA [16] reported the nonmutagenicity of Chlorpyrifos in both bacterial and mammalian cells but did noticed slight genetic aberrations in yeast and DNA in bacterial cells. Tumor developments in mammalian organs, such as prostate [17], breast [18,19] and rectum [20] have been reported to be caused by Chlorpyrifos exposure.
The hepatic cytochrome P-450 dependent metabolism of testosterone and estradiol has been noticed to be inhibited Chlorpyrifos exposure [21,22]. Furthermore, this organophosphate insecticide has been reported to cause decrease in testicular testosterone biosynthesis, and low productions of major steroidogenic enzymes, steroidogenic acute regulatory (StAR) protein and luteinizing hormone receptor stimulated cAMP as investigated by Viswanath, et al. [23].
Diospyros choloroxylon is a widely distributed shrub, belonging to the Diospyros species of the family, Ebenaceae [24]. This shrub and some other members of the species have been used in orthodox medicine all over the world in treatments of several ailments and diseases [25-27]. Studies have shown a possible link between the medicinal potential of D. chloroxylon and the presence of secondary metabolites, such as, alkaloids, flavonoids, tannins, saponins, triterpenoids and phenolics [28]. An important triterpenoid, betulinc acid, present in Diospyros species [29], has been reported to possess several biological properties [30-35]. In the recent time, methanol extract of D. chloroxylon leaf has been reported potent against some environmental toxicants [36,37]. In the present study, the hypothesis was that methanol extract of Diospyros chloroxylon leaf could attenuate redox-induced injuries in the brain and heart of rats exposed to Chlorpyrifos.
Materials and Methods
Duration and place of study
Both experimental work and data analysis were carried out between the months of February and June 2018, in the Department of Biochemistry, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria.
Chemicals
Glutathione, Epinephrine, 5, 5 dithiobis-(2-nitrobenzoate) (DTNB) and hydrogen peroxide were purchased from the Sigma chemical Co. Saint Louis, MO, USA. Trichloroacetic acid, 2-thiobarbituric acid, Triton X-100 and Diphenylamine were purchased from the British Drug House (BDH) Chemical td, Poole, U.K. All other reagents were of good analytical grades.
Collection and extraction of Plant material
The leaves of Diospyros chloroxylon were bought in February 2018, from a local herb seller in Ogbomoso, and authenticated at the Department of Biology, Botany Unit, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria. The leaves were washed with distilled water, air-dried, and pulverized with an electrical grinder. The leaf powder was soaked in methanol for 72 hours. The extraction was repeated twice, and the extract was collected, filtered, and concentrated under vacuum using rotary evaporator at 45oC. The resulting crude extract was stored under refrigeration at 4oC.
Experimental animals and design
Twenty-four (24) male Wistar rats (140.09±9.61 g) were bought from the Animal house of the Institute for Advanced Medical Research and Training (IAMRAT), University of Ibadan, Nigeria. The rats were later brought to the Animal house of the Department of Biochemistry, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria. They were randomized into four (4) groups (6 rats each) and housed in plastic cages and fed on rat pellets and drinking water (ad libitum) for 7 days of acclimatization, under 12-h light/dark cycle and temperature of 29±2oC. The 4 groups of rats were separately treated as follows: distilled water (Control), CPF (5 mg/kg), methanol extract of D. chloroxylon leaf (MEDCL) (100 mg/kg), and CPF (5 mg/kg) + MEDCL (100mg/kg). CPF and MEDCL were administered twice per week and every other day, respectively for 4 weeks.
Collection of blood and organs
After 4 weeks, the rats were fasted overnight. Blood was collected into non-heparinized bottles by ocular bleeding and animals were sacrificed by cervical dislocation. Blood was allowed to clot and then centrifuged at 3000xg for 10 minutes to obtain serum. Brain and heart were excised, washed in ice-cold 1.15% potassium chloride solution to remove blood stains. Each organ was divided into 2 portions, one portion was homogenized with phosphate buffer (pH 7.4) using a Teflon homogenizer and centrifuged using a high speed refrigerated centrifuge (HITACHI) at 10,000xg for 10 minutes to obtain homogenate used for antioxidant and Acetylcholinesterase assays. The other portion of organs was kept for DNA fragmentation assay.
Biochemical assays
Determination of protein level: Protein levels of brain, heart and serum were determined as described by Lowry, et al. [38] using Bovine serum albumin as the standard.
Determination of malondialdehyde level: Malondialdehyde (MDA) levels of brain and heart were estimated as described by Ohkawa, et al. [39]. The absorbance of the clear pink supernatant was measured spectrophotometrically against a reference blank at 532 nm. The MDA concentration was calculated using a molar extinction coefficient (Ɛ) of 1.56 x 105 M-1cm-1.
Determination of superoxide dismutase activity: Superoxide dismutase activities of brain and heart were measured by the epinephrine method described by Misra and Fridovich [40]. The increase in absorbance of the assay reaction at 480 nm was monitored spectrophotometrically at 30 seconds intervals for 150 seconds. The specific activity of SOD was expressed in units/mg protein.
Determination of catalase activity: Catalase activities of brain and heart were assayed according to the method of Aebi [41]. The method is based on the ability of catalase to promote decomposition of hydrogen peroxide in a reaction mixture. The change in absorbance 240 nm was monitored spectrophotometrically at 60 seconds intervals for 180 seconds. Catalase activity was expressed as units/mg protein.
Determination of glutathione peroxidase activity: Glutathione peroxidase (GPx) activities of brain and heart were determined using the method described by Andersen, et al. [42]. The assay is based on the reaction of organic peroxide in a reaction mixture and oxidation of reduced glutathione (GSH) to form disulfide glutathione (GSSG). The GSSG is later reduced to GSH by glutathione reductase and NADPH. The decrease in absorbance at 412 nm is directly proportional to the GPx activity, which is expressed in μmol/mg protein/min.
Determination of reduced glutathione level: Reduced Glutathione (GSH) levels of brain and heart were determined using the method of Mitchell, et al. [43]. The assay is based on the oxidation of GSH by sulfhydryl reagent DTNB, to form a yellow derivative, 51-thio-2-nitrobenzoic acid, with an absorbance at 412 nm. GSH level is proportional to absorbance at 412 nm. Values were expressed as U/ mg protein.
Determination of glutathione-s-transferase activity: Glutathione-S-transferase (GST) activities of brain and heart were assayed according to the method of Habig, et al. [44]. The method is based on the ability of GST to catalyse the conjugation of L-glutathione and CDNB to form a conjugate, GS-DNB, with an absorbance at 340 nm. The rate of increase in absorbance at 340 nm is directly proportional to GST activity. Specific activities were expressed as μM/mg protein/min.
Determination of acetylcholinesterase activity: Acetylcholinesterase (AChE) activities of serum and brain were determined using the method described by Ellman, et al. [45], with acetylthiocholine iodide as a substrate. In this method AChE hydrolyzes acetylthiocholine iodide into thiocholine and butyric acid. The thiocholine reacts with 5, 5-dithiobis-2- nitrobenzoic acid (DTNB) to form 5-thio-2-nitrobenzoic acid to form a yellow product whose absorbance is measured spectrophotometrically at 412 nm.
Determination of DNA fragmentation level: Finally, a spectrophotometric method described by Wu et al.46 was used to determine the percentage fragmented DNA. Briefly, the brain and heart were homogenized in Tris-HCl-EDTA (lysis) buffer and centrifuged at 27,000 x g for 10 mins to separate the intact DNA (pellet) from the fragmented DNA (supernatant). Both the pellet and supernatant were treated with freshly prepared DPA reagent for colour development. The mixtures were incubated at 370C for 20-24 hours. The absorbance was read spectrophotometrically at 620 nm. The percentage fragmented DNA was calculated using the formula:
Statistical Analysis
All values were expressed as the mean±standard deviation of six rats per group. Data were analysed using the Graph Pad Prism 6.0 package. Level of significance among the groups was evaluated using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. P values of < 0.05 were considered significant.
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