Article
Co-treatment of chlorpyrifos and lead induce serum lipid disorders in rats: Alleviation by taurine
Toxicology and Industrial Health 1–7 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714560394 tih.sagepub.com
Motunrayo G Akande1, Yusuf O Aliu2, Suleiman F Ambali3 and Joseph O Ayo4 Abstract The aim of this study was to investigate the effects of taurine (TA) on serum lipid profiles following chronic coadministration of chlorpyrifos (CP) and lead acetate (Pb) in male Wistar rats. Fifty rats randomly distributed into five groups served as subjects. Distilled water (DW) was given to DW group, while soya oil (SO; 1 mL kg1) was given to SO group. The TA group was treated with TA (50 mg kg1). The CP þ Pb group was administered sequentially with CP (4.25 mg kg1; 1/20th median lethal dose (LD50)) and Pb at 233.25 mg kg1 (1/20th LD50), while the TA þ CP þ Pb group received TA (50 mg kg1), CP (4.25 mg kg1), and Pb (233.25 mg kg1) sequentially. The treatments were administered once daily by oral gavage for 16 weeks. The rats were euthanised, and the blood samples were collected at the termination of the study. Sera obtained from the blood samples were analyzed for total cholesterol, high-density lipoprotein cholesterol, triglycerides, and malondialdehyde, and also the activities of serum antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase were analyzed. The low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and atherogenic index were calculated. The results showed that CP and Pb induced alterations in the serum lipid profiles and evoked oxidative stress. TA alleviated the disruptions in the serum lipid profiles of the rats partially by mitigating oxidative stress. It was concluded that TA may be used for prophylaxis against serum lipid disorders in animals that were constantly co-exposed to CP and Pb in the environment. Keywords Taurine, chlorpyrifos, lead, lipid disorders, oxidative stress, rats
Introduction Lipids are crucial for the generation of energy to carry out biological activities. Environmental contaminants such as organophosphorus (OP) insecticides and heavy metals are capable of inducing alterations to lipid profiles in biological systems. The disruptions in serum and tissue lipid profiles could be risk factors for atherosclerosis and coronary heart disease (Ibrahim and El-Gamal, 2003). OP compounds are currently the most commonly used pesticides in agriculture (El-Demerdash, 2011). They produce their insecticidal activity and their systemic toxicity in off-target species by inhibiting cholinesterase (Slotkin, 2011). Chlorpyrifos (CP) is a widely used OP insecticide that alters lipid profiles and increases the risk of inflammation, diabetes, and atherosclerosis (Ambali et al., 2011b).
Lead is a toxic heavy metal and a pervasive environmental and industrial pollutant (Newairy and Abdou, 2009). Pb alters lipid metabolism, and it 1
Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Abuja, Nigeria 2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria 3 Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, University of Ilorin, Ilorin, Nigeria 4 Department of Physiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria Corresponding author: Motunrayo G Akande, Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Abuja 900001, Nigeria. Email: [email protected]
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also increases the production of reactive oxygen species (ROS) (Ademuyiwa et al., 2009; Liu et al., 2010). Oxidative stress has been identified as a common molecular mechanism of toxicity of CP (Kammon et al., 2011) and Pb (Jackie et al., 2011). Oxidative stress may be evoked by ROS generation or failure of the cellular antioxidant system (Abdel Moneim et al., 2011). Consequently, modifications of critical cellular macromolecules such as membrane lipids, DNA, and/or protein occur in the body (Aly et al., 2010). Oxidative stress has been identified as the main factor in the rate of progression of atherosclerosis (Abdollahzad et al., 2009). It is notable that the body is endowed with numerous antioxidant molecules for the counteraction of oxidative stress. Taurine (2-aminoethanesulfonic acid; TA), an antioxidant, is present in high concentrations in many tissues (Issabeagloo et al., 2011). The antioxidant is capable of scavenging peroxyl radical, nitric oxide, and superoxide radicals (Oliveira et al., 2010). It is a safe food additive in commercial products, including energy drinks and infant formula (Yang et al., 2010). TA has been shown to inhibit lipid peroxidation, depress serum low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL) cholesterol levels, and elevate high-density lipoprotein (HDL) levels in hypercholesterolemic animals (Ito and Azuma, 2004). The aim of this study was to investigate the effects of TA on serum lipid profiles of male Wistar rats cotreated with CP and Pb.
Materials and methods Animals Fifty male Wistar rats, weighing between 150 and 200 g, were obtained from the animal house of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria. They were housed in cages in the Toxicology Laboratory of the Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria. The experimental animals were acclimatized for 2 weeks before the commencement of the study. They were given access to standard rat chow and tap water ad libitum. The study was conducted in accordance with the guidelines of the National Institute of Health Guide for Care and Use of Laboratory animals (Garber et al., 2011). The
approval for the study was granted by Ahmadu Bello University Research Ethics Committee.
Chemicals Commercial-grade CP marketed as Excel Termikill1 (Excel Crop Care Limited, Mumbai, Maharashtra, India) was purchased from an Agrochemical Company (Zaria, Nigeria). Excel Termikill is a 20% emulsifiable concentrate of CP. It was prepared by reconstitution in soya oil (SO; Grand Cereals and Oil Mills Limited, Jos, Nigeria) to make a 1% stock solution. Analytical grades of TA and lead acetate trihydrate (Pb) were obtained from Sigma Aldrich1 (Steinheim, Germany). Prior to daily administration, 100 mg of TA was reconstituted in distilled water (DW) to obtain 100 mg mL1 suspension, while 400 mg of Pb was dissolved in DW to obtain 400 mg mL1 suspension.
Toxicological study The Wistar rats were weighed and divided at random into five groups, with 10 rats in each group. DW was given to the DW group, while the SO group was administered with SO (1 mL kg1). The TA group was treated with TA only (50 mg kg1), while the CP þ Pb group was administered sequentially with CP (4.25 mg kg1, 1/20th median lethal dose (LD50)) and then Pb at 233.25 mg kg1 (1/20th LD50). The combination treatment group received TA (50 mg kg1), CP (4.25 mg kg1), and Pb (233.25 mg kg1) sequentially. The treatments were administered once daily by oral gavage for 16 weeks. The rats were observed for clinical signs of toxicity and weekly body weight changes during the study. At the end of the study, the rats were euthanised by severing the jugular veins after light ether anesthesia, followed by collection of 3 mL of blood samples into centrifuge tubes. Subsequently, the blood samples were incubated at room temperature for 30 min and then centrifuged at 1000g for 5 min to obtain sera samples.
Evaluation of serum lipid profile The serum lipid profile evaluated included total cholesterol (TC), HDL cholesterol (HDL-c), and triglycerides (TG). The parameters were assayed with an auto analyzer (Bayer Express Plus, Bayer, Germany). The LDL cholesterol (LDL-c) level, VLDL cholesterol (VLDL-c) level, and atherogenic index (AI) were calculated.
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Calculations The VLDL-c level was calculated using the following equation: VLDL-c ¼ 0:20 TG (Friedewald et al., 1972), while the LDL-c was calculated as follows: LDL-c mg dl1 ¼ TC HDL-c ð0:20 TGÞ (Friedewald et al., 1972). AI was calculated according to Lee and Nieman (1996) as follows: AI ¼
ðTC HDL cÞ HDL c
Statistical analysis
Figure 1. Effects of DW, SO, TA, CP þ Pb, and TA þ CP þ Pb on the dynamics of weekly body weight changes in Wistar rats (n ¼ 10). DW: distilled water; SO: soya oil; TA: taurine; CP: chlorpyrifos; Pb: lead acetate.
The data obtained were expressed as mean + standard error of the mean. The biochemical parameters were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison post hoc test. The statistical package used was Graphpad Prism version 4.00 for Windows (Graphpad software, San Diego, California, USA). Values of p < 0.05 were considered statistically significant.
Determination of serum MDA concentration The concentration of malondialdehyde (MDA) was evaluated in the serum. The method described by Draper and Hadley (1990) was used. The principle of the method was based on the spectrophotometric measurement of the color developed during the reaction of thiobarbituric acid with MDA. The solutions were cooled under tap water, and the absorbance was measured with an ultraviolet (UV) spectrophotometer (T80þ UV/visible Spectrometer1, PG Instruments Ltd., Leicestershire, United Kingdom) at 532 nm. The concentration of MDA in the samples was calculated by using the absorbance coefficient, MDA-TBA complex 1.56 105 cm1 M1.
Assays of serum antioxidant enzymes Superoxide dismutase (SOD) was assayed with the NWLSS™ SOD activity assay kit, based on the method described by Martin et al. (1987). Catalase (CAT) activity was measured by the NWLSS CAT activity assay kit, using the method described by Beers and Sizer (1952). Glutathione peroxidase (GPx) activity was measured with the NWLSS GPx activity assay kit (based on the method of Paglia and Valentine, 1967). The assay kits were purchased from Northwest Life Science Specialities, LLC, Vancouver, Washington, DC, USA.
Results Physical responses of the rats to the treatments Clinical observations. The Wistar rats in the DW, SO, TA, CP þ Pb, and TA þ CP þ Pb groups did not exhibit any clinical sign of toxicity, and there was no mortality during the study. Effects of the treatments on the body weights of the rats. The body weights of the rats in the DW, SO, and TA groups increased steadily during the study (Figure 1). The percentage changes in the body weights of the rats at week 0 compared with week 16 were as follows: DW (22%), SO (21%), TA (22%), CP þ Pb (14%), and TA þ CP þ Pb groups (23%). The lowest percentage change in body weight was recorded in the CP þ Pb group, while the highest percentage change in body weight was observed in the TA þ CP þ Pb group at week 0 compared to week 16.
Effects of the treatments on serum lipid profiles Effects of the treatments on TC concentration. There was no difference in the TC concentration between the groups (Figure 2). TA increased HDL-c concentration. There was an increase (p < 0.05) in the HDL-c concentration in the TA group compared with the CP þ Pb group (Figure 2). There was no difference in the HDL-c concentrations
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Figure 2. Effects of DW, SO, TA, CP þ Pb, and TA þ CP þ Pb on serum lipid parameters such as TC, HDL-c, TG, LDL-c, and VLDL-c of Wistar rats (n ¼ 10). *p < 0.05: CP þ Pb group versus TA group; #p < 0.01: CP þ Pb group versus SO group. DW: distilled water; SO: soya oil; TA: taurine; CP: chlorpyrifos; Pb: lead acetate; TC: total cholesterol; HDL-c: high-density lipoprotein cholesterol; TG: triglyceride; LDL-c: low-density lipoprotein cholesterol; VLDL-c: very low-density lipoprotein cholesterol.
Figure 4. Effects of DW, SO, TA, CP þ Pb, and TA þ CP þ Pb on serum MDA concentration. *p < 0.01: CP þ Pb group versus DW and SO groups, respectively; #p
Co-treatment of chlorpyrifos and lead induce serum lipid disorders in rats: Alleviation by taurine
Toxicology and Industrial Health 1–7 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714560394 tih.sagepub.com
Motunrayo G Akande1, Yusuf O Aliu2, Suleiman F Ambali3 and Joseph O Ayo4 Abstract The aim of this study was to investigate the effects of taurine (TA) on serum lipid profiles following chronic coadministration of chlorpyrifos (CP) and lead acetate (Pb) in male Wistar rats. Fifty rats randomly distributed into five groups served as subjects. Distilled water (DW) was given to DW group, while soya oil (SO; 1 mL kg1) was given to SO group. The TA group was treated with TA (50 mg kg1). The CP þ Pb group was administered sequentially with CP (4.25 mg kg1; 1/20th median lethal dose (LD50)) and Pb at 233.25 mg kg1 (1/20th LD50), while the TA þ CP þ Pb group received TA (50 mg kg1), CP (4.25 mg kg1), and Pb (233.25 mg kg1) sequentially. The treatments were administered once daily by oral gavage for 16 weeks. The rats were euthanised, and the blood samples were collected at the termination of the study. Sera obtained from the blood samples were analyzed for total cholesterol, high-density lipoprotein cholesterol, triglycerides, and malondialdehyde, and also the activities of serum antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase were analyzed. The low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and atherogenic index were calculated. The results showed that CP and Pb induced alterations in the serum lipid profiles and evoked oxidative stress. TA alleviated the disruptions in the serum lipid profiles of the rats partially by mitigating oxidative stress. It was concluded that TA may be used for prophylaxis against serum lipid disorders in animals that were constantly co-exposed to CP and Pb in the environment. Keywords Taurine, chlorpyrifos, lead, lipid disorders, oxidative stress, rats
Introduction Lipids are crucial for the generation of energy to carry out biological activities. Environmental contaminants such as organophosphorus (OP) insecticides and heavy metals are capable of inducing alterations to lipid profiles in biological systems. The disruptions in serum and tissue lipid profiles could be risk factors for atherosclerosis and coronary heart disease (Ibrahim and El-Gamal, 2003). OP compounds are currently the most commonly used pesticides in agriculture (El-Demerdash, 2011). They produce their insecticidal activity and their systemic toxicity in off-target species by inhibiting cholinesterase (Slotkin, 2011). Chlorpyrifos (CP) is a widely used OP insecticide that alters lipid profiles and increases the risk of inflammation, diabetes, and atherosclerosis (Ambali et al., 2011b).
Lead is a toxic heavy metal and a pervasive environmental and industrial pollutant (Newairy and Abdou, 2009). Pb alters lipid metabolism, and it 1
Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Abuja, Nigeria 2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria 3 Department of Physiology and Pharmacology, Faculty of Veterinary Medicine, University of Ilorin, Ilorin, Nigeria 4 Department of Physiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria Corresponding author: Motunrayo G Akande, Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Abuja 900001, Nigeria. Email: [email protected]
Downloaded from tih.sagepub.com at FRESNO PACIFIC UNIV on January 3, 2015
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Toxicology and Industrial Health
also increases the production of reactive oxygen species (ROS) (Ademuyiwa et al., 2009; Liu et al., 2010). Oxidative stress has been identified as a common molecular mechanism of toxicity of CP (Kammon et al., 2011) and Pb (Jackie et al., 2011). Oxidative stress may be evoked by ROS generation or failure of the cellular antioxidant system (Abdel Moneim et al., 2011). Consequently, modifications of critical cellular macromolecules such as membrane lipids, DNA, and/or protein occur in the body (Aly et al., 2010). Oxidative stress has been identified as the main factor in the rate of progression of atherosclerosis (Abdollahzad et al., 2009). It is notable that the body is endowed with numerous antioxidant molecules for the counteraction of oxidative stress. Taurine (2-aminoethanesulfonic acid; TA), an antioxidant, is present in high concentrations in many tissues (Issabeagloo et al., 2011). The antioxidant is capable of scavenging peroxyl radical, nitric oxide, and superoxide radicals (Oliveira et al., 2010). It is a safe food additive in commercial products, including energy drinks and infant formula (Yang et al., 2010). TA has been shown to inhibit lipid peroxidation, depress serum low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL) cholesterol levels, and elevate high-density lipoprotein (HDL) levels in hypercholesterolemic animals (Ito and Azuma, 2004). The aim of this study was to investigate the effects of TA on serum lipid profiles of male Wistar rats cotreated with CP and Pb.
Materials and methods Animals Fifty male Wistar rats, weighing between 150 and 200 g, were obtained from the animal house of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria. They were housed in cages in the Toxicology Laboratory of the Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria. The experimental animals were acclimatized for 2 weeks before the commencement of the study. They were given access to standard rat chow and tap water ad libitum. The study was conducted in accordance with the guidelines of the National Institute of Health Guide for Care and Use of Laboratory animals (Garber et al., 2011). The
approval for the study was granted by Ahmadu Bello University Research Ethics Committee.
Chemicals Commercial-grade CP marketed as Excel Termikill1 (Excel Crop Care Limited, Mumbai, Maharashtra, India) was purchased from an Agrochemical Company (Zaria, Nigeria). Excel Termikill is a 20% emulsifiable concentrate of CP. It was prepared by reconstitution in soya oil (SO; Grand Cereals and Oil Mills Limited, Jos, Nigeria) to make a 1% stock solution. Analytical grades of TA and lead acetate trihydrate (Pb) were obtained from Sigma Aldrich1 (Steinheim, Germany). Prior to daily administration, 100 mg of TA was reconstituted in distilled water (DW) to obtain 100 mg mL1 suspension, while 400 mg of Pb was dissolved in DW to obtain 400 mg mL1 suspension.
Toxicological study The Wistar rats were weighed and divided at random into five groups, with 10 rats in each group. DW was given to the DW group, while the SO group was administered with SO (1 mL kg1). The TA group was treated with TA only (50 mg kg1), while the CP þ Pb group was administered sequentially with CP (4.25 mg kg1, 1/20th median lethal dose (LD50)) and then Pb at 233.25 mg kg1 (1/20th LD50). The combination treatment group received TA (50 mg kg1), CP (4.25 mg kg1), and Pb (233.25 mg kg1) sequentially. The treatments were administered once daily by oral gavage for 16 weeks. The rats were observed for clinical signs of toxicity and weekly body weight changes during the study. At the end of the study, the rats were euthanised by severing the jugular veins after light ether anesthesia, followed by collection of 3 mL of blood samples into centrifuge tubes. Subsequently, the blood samples were incubated at room temperature for 30 min and then centrifuged at 1000g for 5 min to obtain sera samples.
Evaluation of serum lipid profile The serum lipid profile evaluated included total cholesterol (TC), HDL cholesterol (HDL-c), and triglycerides (TG). The parameters were assayed with an auto analyzer (Bayer Express Plus, Bayer, Germany). The LDL cholesterol (LDL-c) level, VLDL cholesterol (VLDL-c) level, and atherogenic index (AI) were calculated.
Downloaded from tih.sagepub.com at FRESNO PACIFIC UNIV on January 3, 2015
Akande et al.
3
Calculations The VLDL-c level was calculated using the following equation: VLDL-c ¼ 0:20 TG (Friedewald et al., 1972), while the LDL-c was calculated as follows: LDL-c mg dl1 ¼ TC HDL-c ð0:20 TGÞ (Friedewald et al., 1972). AI was calculated according to Lee and Nieman (1996) as follows: AI ¼
ðTC HDL cÞ HDL c
Statistical analysis
Figure 1. Effects of DW, SO, TA, CP þ Pb, and TA þ CP þ Pb on the dynamics of weekly body weight changes in Wistar rats (n ¼ 10). DW: distilled water; SO: soya oil; TA: taurine; CP: chlorpyrifos; Pb: lead acetate.
The data obtained were expressed as mean + standard error of the mean. The biochemical parameters were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison post hoc test. The statistical package used was Graphpad Prism version 4.00 for Windows (Graphpad software, San Diego, California, USA). Values of p < 0.05 were considered statistically significant.
Determination of serum MDA concentration The concentration of malondialdehyde (MDA) was evaluated in the serum. The method described by Draper and Hadley (1990) was used. The principle of the method was based on the spectrophotometric measurement of the color developed during the reaction of thiobarbituric acid with MDA. The solutions were cooled under tap water, and the absorbance was measured with an ultraviolet (UV) spectrophotometer (T80þ UV/visible Spectrometer1, PG Instruments Ltd., Leicestershire, United Kingdom) at 532 nm. The concentration of MDA in the samples was calculated by using the absorbance coefficient, MDA-TBA complex 1.56 105 cm1 M1.
Assays of serum antioxidant enzymes Superoxide dismutase (SOD) was assayed with the NWLSS™ SOD activity assay kit, based on the method described by Martin et al. (1987). Catalase (CAT) activity was measured by the NWLSS CAT activity assay kit, using the method described by Beers and Sizer (1952). Glutathione peroxidase (GPx) activity was measured with the NWLSS GPx activity assay kit (based on the method of Paglia and Valentine, 1967). The assay kits were purchased from Northwest Life Science Specialities, LLC, Vancouver, Washington, DC, USA.
Results Physical responses of the rats to the treatments Clinical observations. The Wistar rats in the DW, SO, TA, CP þ Pb, and TA þ CP þ Pb groups did not exhibit any clinical sign of toxicity, and there was no mortality during the study. Effects of the treatments on the body weights of the rats. The body weights of the rats in the DW, SO, and TA groups increased steadily during the study (Figure 1). The percentage changes in the body weights of the rats at week 0 compared with week 16 were as follows: DW (22%), SO (21%), TA (22%), CP þ Pb (14%), and TA þ CP þ Pb groups (23%). The lowest percentage change in body weight was recorded in the CP þ Pb group, while the highest percentage change in body weight was observed in the TA þ CP þ Pb group at week 0 compared to week 16.
Effects of the treatments on serum lipid profiles Effects of the treatments on TC concentration. There was no difference in the TC concentration between the groups (Figure 2). TA increased HDL-c concentration. There was an increase (p < 0.05) in the HDL-c concentration in the TA group compared with the CP þ Pb group (Figure 2). There was no difference in the HDL-c concentrations
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Figure 2. Effects of DW, SO, TA, CP þ Pb, and TA þ CP þ Pb on serum lipid parameters such as TC, HDL-c, TG, LDL-c, and VLDL-c of Wistar rats (n ¼ 10). *p < 0.05: CP þ Pb group versus TA group; #p < 0.01: CP þ Pb group versus SO group. DW: distilled water; SO: soya oil; TA: taurine; CP: chlorpyrifos; Pb: lead acetate; TC: total cholesterol; HDL-c: high-density lipoprotein cholesterol; TG: triglyceride; LDL-c: low-density lipoprotein cholesterol; VLDL-c: very low-density lipoprotein cholesterol.
Figure 4. Effects of DW, SO, TA, CP þ Pb, and TA þ CP þ Pb on serum MDA concentration. *p < 0.01: CP þ Pb group versus DW and SO groups, respectively; #p
