The present investigation delineates role of free radicals in inducing tissue damage in the vital organs viz. the liver, kidneys and brain in chlorpyrifos toxicity in broilers. A total of 72 experimental birds were selected for present study. These experimental birds were divided into 4 groups viz. Group І (Control), Group II, III and IV, comprising of 18 broiler birds each. Chlorpyrifos in corn oil was administered orally on daily basis to each group 2Qof birds @ 3.2, 1.6 and 0.64 mg/kg respectively, for a period of 6 weeks. The liver, kidneys and brain tissues of these experimental birds were evaluated to reveal the free radical damage due to chlorpyrifos. Significant increase in Lipid peroxidation (LPO) and decrease in levels of superoxide dismutase (SOD) and catalase (CAT enzymes) in these organs indicated cell injury due to oxidative stress caused by free radicals in chlorpyrifos toxicity.
Organophosphate (OP) compounds are the most widely used insecticides accounting for 50% of global insecticidal use, Casida and Quinstad (2004). Organophosphorus insecticides have been widely recognized as a health hazard due to their widespread use and release into the environment Al-Haj et al., (2005). Besides fatalities, caused by high dose, exposure of animals to low doses of organophosphorus insecticides has been found to have widespread effect on body including organ specific lesions in central nervous system Lengyl et al., 2005, liver Gomes et al., 1999, kidneys Kossmann et al., 1997 and generalized effects like immunosuppression, teratogenesis, carcinogenesis and metabolic disorders. Among organophosphates, chlorpyrifos (CPF) has been principally used as a pesticide in agricultural sector; domestically it is extensively used in home gardens as well as indoors to get rid of fleas and flies Abbas (2015). It has also been used to control termites in chicken houses Singh et al., 2016. United States Environmental Protection Agency (USEPA) has restricted some of its domestic uses due to its severe toxicity. USEPA has also categorized CPF as class II toxin Eisler (2000). Despite this, CPF remains one of the most widely used OP insecticides. It is an active ingredient of various preparations used against ecto-parasites of dogs, cats and cattle Blangburn et al., 2001. Chlorpyrifos, unlike other insecticides remains in the soil for long time. It reaches animal when it is smeared on their body surface to eradicate lice and fleas affecting livestock and poultry as well as for treatment of dog kennels. It is also detected in poultry egg, meat and cow milk and milk products Rawat et al., 2003. Despite all of its detrimental effects it is used widely.
Review of Literature
Verma et al., 2007 reported that rats administered with 100mg CPF/kg bw /day for three days, caused increase in the levels of lipid peroxidation (LPO) in the liver, kidney, spleen and brain tissue. Reduced glutathione (GSH) levels declined in all the tissues tested and oxidized glutathione (GSSG) concomitantly increased thus decreasing the GSH/GSSG ratio was also reported. Mansour and Mossa (2009) studied chlorpyrifos induced oxidative stress and lipid peroxidation in erythrocytes of male and female rats administered chlorpyrifos orally @ 6.75 mg/kg bw for 28 consecutive days and the ameliorative effect of zinc (227mg/l) in drinking water. It was revealed that zinc showed significant alteration in the activity of AChE in rats treated with chlorpyrifos and an insignificant effect with respect to lipid peroxidation, glutathione-s-transferase (GST), superoxide dismutase (SOD) and catalase activity.
Demir et al., 2010, studied effects of chlorpyrifos in the rat erythrocyte antioxidant system and evaluated the ameliorating effects of catechin and quercetin on the oxidative damage induced by chlorpyrifos. Wistar rats were given chlorpyrifos (5.4 mg/kg, 1/25 of the oral LD50), for 4 weeks, which resulted in increased levels of malondialdehyde (MDA) and decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities compared with the control group in rat erythrocytes. Bharathi et al. (2010) studied chlorpyrifos-induced oxidative stress and toxicity in poultry in relation to antioxidant effect of certain herbs. Broilers were fed Chlorpyrifos @ 100 ppm in feed for the 4 weeks and thereafter, administered with different herbs and their combinations for remaining two weeks. The blood and Sera samples were collected at fourth week and sixth week for the estimation of Erythrocytic Superoxide Dismutase (SOD), Catalase, Alanine Transaminase (ALT) and Serum Creatinine respectively. The birds were sacrificed at the end of experiment and tissues were collected for the assay of GSH and (Thiobarbituric Acid Reactive substances) TBARS in liver and brain homogenates. The activity of SOD, Catalase, ALT, and the concentration of TBARS and serum creatinine were increased significantly, while the concentration of total proteins and tissue GSH decreased significantly in all the groups. It was concluded that chlorpyrifos induces toxicity by generating reactive oxygen species and antioxidant herbs are useful in treating the chlorpyrifos-induced toxicity. Kalender et al. (2012) reported increase in levels of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT), while decrease in glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in Wistar rats exposed to CPF @ 5.4 mg/kg for 4 weeks. Gayal et al. (2012) studied that following a multiple oral dose of chlorpyrifos @15 mg/kg BW at an interval of 24hr for 7 days in poultry birds resulted in reduction of Hb, PCV, TEC and TLC. Kavitha et al. (2013) conducted experimental studies on 72 hens aged 56 weeks and divided into four groups. Groups 2, 3 and 4 were administered chlorpyrifos @ 350mg/kg BW s/c. in divided doses over a period of 24 hrs. To prevent death due to cholinergic toxicity, atropine and 2-PAM were administered. In group 3, vitamin E was administered @ 50mg/kg BW p/o 10 days prior to administration of CPF and in group 4 phenytoin @ 50 mg/kg p/o was administered for 5 days prior to CPF. It was concluded that there was a decline in AChE activity with increased activity of SOD, catalase and also decline in glutathione levels was observed. The above parameters were significantly lowered with prior administration of vitamin E. Nisar et al. (2013) reported that oral administration of chlorpyrifos in Wistar rats @ 5.5mg/kg in corn oil for 98 days resulted in significant decrease in catalase and superoxide dismutase (SOD) with significant increase in lipid peroxidation levels.
Ahmad et al. (2015) investigated the deleterious effects of chlorpyrifos (CPF) in experimentally exposed broiler birds @ 5, 10 and 20 mg/kg body weight (BW) for 14 days through the stomach tube. Birds exposed to high dose (20 mg/kg BW) showed signs of toxicity like salivation, lacrimation, gasping, convulsions, frequent defecation and tremors. Significantly (P ≤ 0.05) decreased hematological parameters i.e. total erythrocyte counts, hemoglobin concentration, hematocrit and total leukocyte were observed in the high dosed group as compared to control and other low dosed fed birds. Serum protein and albumin showed a significant (P ≤ 0.05) increase in high dosed CPF fed birds. The acetylcholinestrease (AChE) activity was significantly (P ≤ 0.05) decreased in blood, serum and plasma in CPF fed birds compared to control birds. Significantly (P ≤ 0.05) higher levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were found in CPF fed birds as compared to control birds. Necrotic and degenerative changes were observed on histopathological investigations of spleen, kidneys, bursa of Fabricius, thymus and brain tissues in CPF exposed birds thus suggesting that chlorpyrifos induced toxicopathological effects on health biomarkers of broiler chicks.
Material and Methods
The study was conducted on 21 day old broiler birds in the Division of Veterinary Pathology, FVSc and A.H, SKUAST-J, R. S. Pura, Jammu in the year 2013. A total of 72 birds of either sex were selected for present study. All the procedures, conducted during the study in the experimental animals were duly approved by the Institutional Animal Ethical Committee (IAEC -862/ac/04/CPCSEA-16-12-2004). These birds were procured from a private hatchery in Jammu region and maintained on commercially available feed for birds obtained from Shalimar Feeds Pvt. Ltd., Bari Brahmana, Jammu (India).
Housing and Management
Day old birds on arrival were weighed and examined for any abnormality or overt ill health. These birds were given a series of vaccines and reared under strict hygienic conditions in the divisional animal house. Before housing the chicks, the experimental room, brooder and other equipments were thoroughly cleaned with 2.5% phenol followed by fumigation with formaldehyde (35ml of commercial formalin and 17.5gm potassium permanganate per hundred cubic feet area).
The chicks were fed on basal control diet i.e. commercial feed procured from local market and this feed along with clean tap water was provided to the birds ad libitum throughout the experiment.
After acclimatization for 21 days, the birds were randomly divided into four groups of 18 birds each. Group I served as control while group II, III and IV birds were given chlorpyrifos (commercially obtained from Tata Rallis India Ltd., Mumbai as Tafaban) @ 3.2, 1.6 and 0.64 mg/ kg body weight, respectively, orally in corn oil for a period of 6 weeks. The dose selection criteria for the chlorpyrifos in the present study was factually based on the 1/10th , 1/20th and 1/50th of the oral lethal (LD50 i.e. 32mg/ kg body weight) dose of chlorpyrifos in birds keeping in view the body weight and daily water consumption. Six (6) animals from each group were sacrificed at 2, 4 and 6 week’s interval by humane method.
Assessment of Oxidative Stress
Estimations of different oxidative stress-related biochemical parameters in liver, kidneys and brain were carried out using a UV spectrophotometer for recording the absorbance of the test samples.
Preparation of Liver, Kidneys and Brain Homogenate
Samples (200 mg) of liver , kidneys and brain were weighed and taken in 2 ml of ice-cold PBS [NaCl (8 g), KCl (0.2 g), KH2PO4 (0.2 g) and Na2HPO4 (0.94 g) in about 800 ml of distilled water and the volume was then made to 1 litre with distilled water. The samples were homogenized with tissue homogenizer under ice-cold condition and were centrifuged for 10 min at 3000 rpm. The supernatants were stored at -20oC until assay.
Protein Estimation in Tissue Homogenates
Protein content in liver, kidneys and brain homogenate was determined by the method of Lowryet al., 1951 and Shafiq-U-Rehman (1984).
Lipid Peroxidation (LPO)
The extent of LPO was evaluated in terms of malondialdehyde (MDA) production and determined by the thiobarbituric acid (TBA) method Lowry et al., 1951. The amount of LPO was expressed as nM of MDA formed per gram of wet tissue. The absorbance was read at 535 nm.
Activity of catalase enzyme in liver, kidneys and brain homogenate was estimated by spectrophotometric method as described by Aebi (1983) and was expressed as K/g of wet tissue [K = mM H2O2 utilised / min/ g of wet tissue]. The optical density was recorded at every 30 sec for 3 min at 240 nm against distilled water (blank).
Superoxide Dismutase (SOD)
Superoxide dismutase (SOD) was estimated as per the method described by Marklund and Marklund (1974). The data generated was analyzed using two-way analysis of variance Snedecor and Cochran (1994).
Results and Discussion
Clinical signs were observed after 4 weeks post experiment, which included inappetence, dullness, weakness, depression, lethargy, decrease in feed intake and body weight, ruffled feathers, change in behaviour, disinclination to move, muscle contractions and staggering, the severity of which was dose and duration dependent. All treatment group birds revealed significant decrease in feed consumption, body weight, body weight gain and increase in FCR with that of control group birds in dose and duration dependent manner.
Haematobiochemistry and Histopathology
Significant haematobiochemical observations included anaemia, leucopenia, lymphocytopenia, hypoproteinemia, hypoalbuminemia with decrease Albumin:Globulin ratio and RBC acetyl cholinesterase and pseudo cholinesterase concentration and increased serum ALT and AST levels. Grossly, liver and kidneys were pale and enlarged. Relative weights of liver, kidneys and brain were significantly increased. Histopathologically, liver of all the treatment group birds showed hepatocytic degeneration and necrosis, MNC’s infiltration and fibrosis in portal area, degeneration and swelling of epithelial cells of PCT’s obliterating lumen and presence of MNC’s in kidneys and engorgement of cerebral blood vessel with increased perivascular space, shrunken deeply eosinophilic neurons, MNC’s infiltration leading to leptomeningitis in brain.
The average LPO, SOD and CAT levels in liver of birds of different groups observed at different intervals is summarised in Table 1. All toxin administered group birds showed significantly elevated LPO levels amongst each other in comparison with control (group I) at all sacrifice intervals. While the treatment group birds showed significant decrease in SOD and CAT concentration amongst each than control group birds from second to sixth week PE at the end of the experimental trial.
Table 1 shows the average LPO levels in birds of different groups observed at different intervals indicating that all treatment group birds showed significantly elevated LPO levels amongst each other as compared with control group at all sacrifice intervals. There was a significant decrease in SOD as well as CAT concentration in toxin treated groups amongst each other than control group birds from second to sixth week PE at the end of trial.
All the toxin treated groups showed significantly elevated LPO levels amongst each other in comparison with control group at all sacrifice intervals. This variation in LPO levels at different interval is summarised in Table 1. There was a significant decrease in SOD concentration amongst toxin treated groups amongst each other than control group birds at second, fourth as well as sixth week sacrifice. CAT concentration followed the same trend as that seen in SOD concentration at the end of trial.
Oxidative stress is caused by over production of Reactive Oxygen Species (ROS). A balance between ROS and primary antioxidant defense such as GSH and antioxidant scavenging enzymes like superoxide dismutase (SOD) and catalase (CAT) is needed in preventing damage by oxidative stress. It is basically defined as an alteration in the steady-state balance between oxidant and antioxidant agents in the cells. Previous studies have shown a correlation between inhibition of AChE and lipid peroxidation following sub-chronic and chronic exposure to organophosphates Akhgari et al., 2003. Lipid peroxidation has been considered one of the molecular pathways involved in the toxicity of organophosphates Datta et al., 1992.
Table 1: Oxidative Stress Parameters (Mean ± SE) in Birds of Different Treatment Groups (n=6)
|2nd||3.50 ± 0.35dA||9.05 ± 0.91aA||6.53 ± 0.24bA||5.27 ± 0.42cA|
|4th||6.76 ± 0.15dB||10.75 ±0.09aA||8.80 ± 0.46bA||7.21 ± 0.42cB|
|6th||11.22 ± 0.81dC||21.45 ± 0.63aB||15.41 ± 0.24bC||11.37 ± 0.41cC|
|SOD (U/mg)||2nd||83.54 ± 1.54aA||53.89 ± 0.57dA||61.65 ± 0.38cA||74.85 ± 1.52bA|
|4th||100.69 ± 3.17aB||58.20 ± 1.06dA||71.14 ± 2.13cB||84.89 ± 1.43bB|
|6th||111.22 ± 4.06aB||67.80 ± 1.57dB||86.92 ± 0.54cC||97.87 ± 0.54bC|
|CAT (KA/g)||2nd||281.16 ± 0.55aA||207.98 ± 2.34dA||220.78 ± 3.83cA||231.19 ±2.15bA|
|4th||294.80 ±0.37aB||231.60 ± 0.81dB||250.00 ± 3.53cB||265.40 ±1.86bB|
|6th||308.80 ± 0.58aC||244.80 ± 0.63dC||262.80 ± 2.08cC||273.20± 1.46bC|
|2nd||1.43 ± 0.16dA||4.10 ± 0.34aA||3.90 ± 0.31bA||2.37 ± 0.15cA|
|4th||3.07± 0.20dB||7.78 ± 0.29aB||4.66 ± 0.26bB||3.88 ± 0.47cB|
|6th||6.78 ± 0.24dC||12.44 ±0.71aC||8.65 ± 0.29bC||7.60 ± 0.26cC|
|SOD (U/mg)||2nd||72.33 ± 1.76aA||48.03 ± 1.60dA||58.23 ± 1.06cA||63.88 ± 0.71bA|
|4th||85.07 ± 1.63aB||58.89 ± 0.62dB||65.68 ± 1.21cB||72.84 ± 0.73bB|
|6th||95.52 ± 1.15aC||63.29 ± 0.54dC||72.74 ± 0.63cC||83.80 ± 1.06bC|
|CAT (KA/g)||2nd||183.00 ± 0.70aA||143.78 ± 0.45dA||156.97 ± 0.86cA||173.09 ±0.78bA|
|4th||195.79 ± 1.02aB||156.40 ± 0.50dB||166.40 ± 0.50cB||179.43± 1.31bB|
|6th||205.20 ±1.62aC||173.23 ±0.84dC||181.83 ± 1.79cB||190.60 ±0.81bC|
|2nd||3.50 ± 0.35dA||9.05 ± 0.91aA||6.53 ± 0.24bA||5.27 ± 0.42cA|
|4th||6.76 ± 0.15dB||10.75 ± 0.09aA||8.80 ± 0.46bA||7.21±0.42cB|
|6th||11.22 ± 0.81cC||21.45 ± 0.63aB||15.41 ± 0.24bC||11.37 ± 0.41cC|
|SOD (U/mg)||2nd||80.16 ± 0.76aA||55.31 ± 1.54dA||65.46 ± 1.56cA||72.23 ± 0.63bA|
|4th||84.34 ± 0.60aA||62.34 ± 0.60dB||74.00 ± 0.54cB||77.80 ±0.37bB|
|6th||91.87 ± 0.67aB||68.20 ± 0.73dC||77.40 ± 0.40cB||86.20 ± 1.15bC|
|CAT (KA/g)||2nd||127.51 ± 0.38aA||101.44 ± 0.67dA||118.50 ± 0.79cA||123.17 ±0.83bA|
|4th||136.81 ± 0.94aB||116.95 ± 0.59dB||122.00 ± 0.70cB||127.22± 0.57bB|
|6th||151.79 ± 0.96aC||127.92 ± 0.33dC||132.59 ± 1.03cB||139.40 ±0.68bC|
*Mean bearing at least one common superscript (a, b, c, d and A, B, C) do not differ significantly between groups and weeks (P<0.05), respectively. Group I (control) Group II (3.2 mg/kg), Group III (1.6mg/kg), Group IV (0.64mg/kg).
Malondialdehyde (MDA) is an end point of lipid peroxidation process which may be defined as an oxidative deterioration of polyunsaturated lipids. Several enzymatic antioxidant defenses designed to scavenge reactive oxygen species (ROS) in the eukaryotic cells protect them from oxidative injury. A fine balance between several antioxidant species and ROS appears to be more important for the overall protection of cells Galaris and Evangelou (2002). Lipid peroxidation has been measured by quantifying the thiobarbituric acid reactive substances. It is considered an important feature of the cellular injury brought about by free radical attack. The enhanced susceptibility of membrane to LPO can lead to loss of adenosine triphosphatases (ATPases) activity and changes in cell functions Ohnishi et al., 1982. ATPases are lipid dependent membrane bound enzymes, which are involved in active transport, maintenance of cellular homeostasis and in neurotransmission process Garcia and Corredor (2003).
Repeated oral administration of chlorpyrifos significantly caused increased lipid peroxidation levels from second week PE in liver and kidneys of treatment group birds than the control group. The results are in agreement with studies by Gao et al. (2003), Verma and Srivastava (2003) in rats and Mevlüt Şener Ural (2013) in fish. Some more investigations also indicate that accumulation of lipid peroxides had resulted after exposure to acute dose of chlorpyrifos in rat liver Bagchi et al., 1995 and kidneys Oncu et al., 2002. The end products of LPO like melondialdehyde (MDA) could also cause tissue injury by interacting with bio macromolecules Mylonans and Kouretas (1999). Superoxide Dismutase (SOD) is the first line of defence against the action of O2– and other reactive oxygen species (ROS). Superoxide radicals are produced in mitochondria and endoplasmic reticulum as a consequence of auto-oxidation of electron transport chain components. These superoxide free radicals are generated during monovalent reduction of oxygen and are toxic to biological systems. The major enzyme that protects against superoxide production in the body is superoxide dismutase which disproportionates the superoxide to hydrogen peroxide and oxygen McCord and Fridovich (1969). Reactive oxygen species are produced by the univalent reduction of dioxygen to superoxide anion (O2−), which in turn disproportionate to H2O2 and O2 either spontaneously or through a reaction catalyzed by superoxide dismutase McCord and Fridovich (1969). Endogenous H2O2 may be converted by catalase (CAT) to H2O Kehrer (1993) or otherwise it may generate the highly reactive free hydroxyl radical (OH−) by the Fenton reaction McCord and Dary (1978), which is widely believed to be mainly responsible for oxidative damage Halliwell and Gutteridge (1990). A significant decrease in SOD levels was observed in liver and kidneys of treatment group birds. These findings were in consonance with the studies of Bharathi et al., 2010 and Abd El-Aziz-A et al., 2012 in CPF treated rats. Decreased SOD activity in the present investigation suggests of excess free radical generation which impair natural defence mechanism of this enzyme. Catalase significantly declined from second week PE in liver and kidneys of all toxin treated group birds. These findings were in agreement with previous studies of Gultekin et al., 2001 and Verma et al.,2007 in rats. The low levels of CAT following the CPF treatment could possibly be contributed to the consumption of this enzyme in converting the H2O2 to H2O Goel et al., 2005. It has been also shown that CAT activity was inhibited by free radicals, such as singlet oxygen and superoxide and peroxyl radicals Bharathi et al., 2010. Therefore, CAT may be inhibited by both CPF itself and increased ROS induced by CPF Gultekin et al., 2006.
The LPO levels in brain significantly increased in toxin fed group birds from second week PE. This finding was consistent with studies of Gultekin et al., 2001 and Verma and Srivastava (2001). A dose dependent increase in the level of MDA in all regions of brain on exposure to CPF has been reported by Verma and Srivastava (2001). Gultekin et al., 2001 and Verma et al., 2007 in their studies showed a significant decrease in SOD and catalase levels in the brain of rats, in the present study also, SOD and catalase levels in brain were significantly decreased in all toxin fed birds. Several cellular features of the brain make it highly sensitive to oxidative stress e.g. the highest oxygen metabolic rate than any organ in the body Maiese (2002). Further the brain possesses inadequate defence system against oxidative stress such as significantly lower catalase activity in the brain than other body organs Floyd and Carney (1992). Hence, the increased MDA levels well correlated with the damaging effect of CPF in liver, kidneys and brain due to free radicals which was confirmed by reduced levels of SOD and CAT concurrent with decreased AChE levels in blood and plasma along with gross and histopathological changes in liver, kidneys and brain, which were represented by the clinical signs the birds showed.
Sub lethal doses of chlorpyrifos accumulates in liver, kidneys and brain tissues, of broiler birds following oral administration for a period of six weeks hampering lipid peroxidation as well as other enzymes of these organs. Moreover, all the treatment group birds showed significantly elevated LPO levels and decreased SOD and CAT levels in liver, kidneys and brain homogenate in comparison with control group birds in dose and duration dependent manner indicating that the cell injury in chlorpyrifos toxicity was mainly mediated through free radicals.
The authors thank department of Veterinary Pathology SKUAST for providing necessary facilities to undertake the research. We are grateful to all those who provided us experimental birds as well as those who provided us technical assistance.