Introduction

Agricultural development continues to remain the most important objective of Indian planning and policy. Pesticides are substances or mixture of substances intended for preventing, destroying, repelling or mitigating any pest. A vast majority of the population in India is engaged in agriculture and is therefore exposed to the pesticides used in agriculture. Rampant use of these chemicals has given rise to several short-term and long-term adverse effects of these chemicals (Gupta, 2004). Pesticide exposure is a global public health issue.  Chlorpyrifos is one of the most extensively used broad-spectrum organophosphates and its phosphorus is linked to sulphur with a double bond (P = S). It is used throughout the world to control a variety of chewing and sucking insect pests and mites on a range of economically important crops. It is also registered for use  on  lawns,  Ornamental  plants,  animals,  domestic  dwellings  as  well  as  commercial  establishments (Thengodkar and Sivakami, 2010). However, indiscriminate use of insecticides has led to a widespread concern over the potential adverse effects of these chemicals on animal and human health as these chemicals interfere with the defense mechanisms of the host, which normally ensures its survival against invading pathogens (Sodhi et al., 2006).

Review of Literature

El-Hossary et al. (2009) investigated retinal and renal toxicity due to chlorpyrifos @ 63mg/kg bw and subsequent amelioration with vitamin D @ 1.35mg/kg bw for 30 days in Wistar rats. The experiment reveals. Histopathological changes such as shrunken gromeruli surrounded by wide Bowman’s space, pyknotic nuclei of epithelial cell, in renal tissue and vacuolated retinal pigment epithelium, distorted photoreceptors retinal tissues. While as treatment with vitamin D proved protective to renal and retinal tissue against chlorpyrifos induced toxic changes. Savithri et al. (2010) examined histopathological changes in liver of albino rats with oral sub-lethal (20 mg/kg bw) administration of chlorpyrifos as single, double and multiple doses with 48 hrs intervals and reported central venous congestion, sinusoidal haemorrhages, and focal necrotic areas in liver. Diffuse haemorrhagic areas were observed in the heart. Degenerative changes in the muscle layer, hypertrophy of goblet cells, and infiltration and hyperaemic changes in blood vessels in the intestines. Tripathi and Srivastav (2010a) studied liver profile of rats after long-term ingestion of different doses of chlorpyrifos administered orally @ 5 and 10 mg/kg b wt in rats. The histopathological changes noticed included were mainly hepatocytic vacuolation, degeneration of hepatocytes and their nuclei, hyperchromatic and hypertrophied nuclei at earlier stage of treatment, sinusoidal dilation and focal necrosis depending upon the doses and duration of the treatment. Tripathi and Srivastav (2010b) studied nephrotoxicity induced by long term (8 weeks) oral administration of chlorpyrifos in rats @ 5 and 10 mg/kg bwt and reported the shrinkage of glomerulus at initial stage of treatment, the tubular dilation, the glomerular hypercellularity, hypertrophy of tubular epithelium, degeneration of glomerulus and renal tubules, deposition of eosin-positive substances in the glomerulus and renal tubules and infiltration of leucocytes. Kammon et al. (2011) studied chronic toxicity (45 days) of chlorpyrifos in broilers and the protective effect of vitamin C. Oral administration of 0.8 mg/kg bw chlorpyrifos and gross lesions comprised of paleness, flaccid consistency and slightly enlargement of liver. Histopathologically, chlorpyrifos produced degenerative changes in various organs. Oral administration  of  100  mg/kg  bw  vitamin  C  partially  ameliorated  the degenerative changes in kidney and heart. Newairy and Abdou (2013) studied hepatotoxicty and brain damage due to CPF in male rats @ 6.8mg/kg for 28 days and reported dilation and vascular congestion in sinusoids and degenerated hepatocytes with pyknotic nuclei in liver and neuronal degeneration and shrinkage with pyknosis in brain sections. Shahzad et al. (2013) studied the immunotoxic effects of CPF @ 5, 10 and 20mg/kg bw  in broilers for 15 days and observed dose and time related pathological changes in bursa of Fabricius, spleen, and thymus. Histopathologically, bursa of Fabricius showed increased inter-follicular connective tissue proliferation, moderate cytoplasmic vacuolation, edema, and degenerative changes such as pyknosis and fragmentation of nuclei that depleted the follicles of lymphoid cells. Spleen showed disorganization of follicular patterns, severe congestion, cytoplasmic vacuolation, degenerative changes, and hyperplasia of reticular cells. The thymus in treated birds exhibited congestion, hyper-cellularity, and a presence of immature monocytes in the medullary region, as well as myoid cell necrosis.

Materials and Methods

Seventy two (72), zero (0) day old broiler birds of  either sex were procured from a private hatchery in Jammu and maintained on commercially available feed for birds obtained from Shalimar Feeds Pvt. Ltd., Bari Brahmana, Jammu (India). The birds were weighed and housed in battery brooders and supplied with feed and water ad libitum. After 21 days of brooding, birds were randomly divided into four groups of 18 chicks each. Group I served as control (no chlorpyrifos treatment) 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-1 body weight, respectively, orally in corn oil for a period of 6 weeks and 6 animals from each group were sacrificed at 2, 4 and 6 week’s interval. The experiment protocol used in this study was approved by Animal Ethical Committee   (IAEC   -862/ac/04/CPCSEA-16-12-2004).   After   exsanguinations   a   detailed   post-mortem examination was conducted and gross lesions were recorded and weights of different organs, liver, kidney, lungs, heart and brain, were recorded individually at every sacrifice interval and their relative weights were calculated as the per cent body weight. Representative pieces of tissue were collected in 10 per cent neutral buffered. Paraffin- embedded tissues were sectioned to 5 μ thickness and stained with haematoxylin and eosin for histopathological examination (Luna, 1968).  Data generated from relative body organ weight were presented as Mean + SE by employing two way ANOVA and a value of P = 0.05 was taken as significant employing Tukey’s descriptive statistical analysis (Snedecor and Cochran, 1994).

Relative Organ Weights

The data on average relative organ weights viz. liver, kidneys, lungs, heart, spleen, thymus, bursa of Fabricius and brain in birds of different groups taken at various intervals is presented in Table 1.

Table 1: Organ body weight ratio (%) (Mean±SE) in birds of different groups (n=6)

Mean bearing at least one common superscript (a, b, c 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).

Liver:  At sixth week Post experiment (PE), there was significant increase in liver weight in group II and III birds amongst each other and that of group I and IV birds.

Kidneys: At sixth week PE, group II and III birds showed non-significant changes amongst each other but significantly higher than group I and IV birds.

Brain: Brain showed non-significant increase in weight in all treatment group birds as compared to control group birds at second week PE.

Pathology and Histopathology

Patho-morphological studies revealed appreciable gross changes in the liver and kidneys at sixth week of sacrifice in all treatment group birds in dose and duration dependent manner. The liver in all the treatment group birds were pale enlarged with rounded borders with pale discoloration. Kidneys were enlarged, palor with smooth surface. No appreciable gross changes in brain and heart were observed in all treatment group birds.

Liver

Control group birds did not show any marked microscopic lesion of pathological significance and revealed prominent central vein, normal hepatic lobules with cord pattern and hepatocytes with vesicular nucleus and normal cytoplasm. Degeneration and necrosis of wall of central vein with perivascular hepatocytic degeneration was seen at second and fourth week PE in five birds (Plate 1).

Plate 1: Liver, group II (3.2 mg/kg), fourthWPE: Central vein showing degeneration and necrosis of wall with perivascular fibrosis and degeneration of hepatocyte.

At sixth week PE, focal area of hepatocytic necrosis encroached by MNC’s and severe hepatocytic degeneration in three birds (Plate 2).

Plate 2: Liver, group II (3.2 mg/kg), sixth WPE: Showing dilated and engorged portal vein with perivascular fibrosis and moderate degeneration of hepatocytes.

Kidneys

No microscopic lesions of any pathological significance was observed in kidney sections of control group birds and observed normal architecture. Severe degeneration of epithelial cells of PCT’s completely occluding the lumen was seen at fourth week PE in three birds (Plate 3). At sixth week PE, MNC’s infiltration in interstitium with fibrosis of Bowman’s capsule and hypercellularity of glomerular tuft was observed in three birds. Periglomerular MNC infiltration with presence of desquamated epithelial cells in lumen of PCT along with hypercellularity of glomeruli was seen at sixth week PE in two birds (Plate 4).

Group IV birds revealed no significant change at second week PE but at fourth week PE, interstitial haemorrhage and mild degeneration of PCT’s was revealed in one bird. At sixth week PE thickening of Bowman’s capsule and hypercellularity of glomerular tuft was observed in two birds.

Brain

Group I birds revealed no pathological change during the entire experimental period and included normal meninges and normal neuron with centrally located nucleus. At fourth week PE, MNC’s infiltration in meninges leading to leptomeningitis was observed in three birds (Plate 5).

Plate 5: Brain, group II (3.2mg/kg) sixth WPE: Showing MNC infiltration in meninges leading to leptomeningitis

Group III didn’t reveal any observable change up to fourth week PE. However, shrunken and deeply eosinophilic degenerated neurons with MNC’s infiltration in meninges were observed at sixth week PE in three birds (Plate 6). Group IV birds did not reveal any observable change up to fourth week PE. However, at sixth week PE, cerebral meningeal blood vessel engorgement was observed in two birds.

Plate 6: Brain, group II (3.2mg/kg) fourthWPE: Showing shrunken and deeply eosinophilic degenerated neurons.

Discussion

On microscopic examination, liver in  CPF administered groups revealed central  vein congestion, hepatocytic degeneration and necrosis, MNC’s infiltration, fibrosis in portal triad and also distortion of hepatic architecture at later stage. Liver being primary organ for xenobiotic metabolism gets acutely exposed to the toxins through portal as well as systemic circulation. Widespread systemic distribution has been reported with bioactivation occurring primarily by cytochrome P450 enzyme in liver (Blodgett, 2006). Similar changes have been reported by different workers in rats and poultry (Chaudhary et al., 2003; Tripathi and Srivastav, 2010a; Kammon et al., 2011). Chlorpyrifos has been reported to cause cytoplasmic vacuolation in hepatocytes due to disturbances in lipid inclusions and fat metabolism, necrosis, hepatitis and fibrosis (Solati et al., 2007; Tripathi and Srivastav, 2010a). In the present study, decrease in total serum protein, globulin and albumin levels, significant increase in ALT, AST and LPO levels and decreased SOD and Catalase levels with gross and histopathological alterations in liver are suggestive for hepatotoxicity in birds due to chlorpyrifos toxicity. Nephrotoxic effects of chlorpyrifos were characterized by  vascular congestion, glomerulo-tubular nephrosis, tubular degeneration with desquamation of lining epithelium of PCT’s, focal interstitial haemorrages and nephritis with mononuclear cell infiltration, and hypercellularity of glomerular tufts. Kidneys being central organ  are  target  sites  in  the  toxicological conditions and  have  been  reported  to  be  severely affected  in chlorpyrifos toxicity. Changes akin to present observation have been reported by various workers ( Ahmed et al.,2010; Mansour and Mossa, 2010; Tripathi and Srivastav, 2010b; Kammon et al., 2011). Tripathi and Srivastav (2010b) reported glomerular shrinkage and   hypercellularity as the main lesions with desquamation of tubular epithelium. The results showed that PCT’s were more affected than DCT’s. This could be due to the fact that PCT’s are the primary sites of reabsorption and active transport leading to the damage in the epithelial lining of these tubules. The observed gross and histopathological renal lesions indicated nephrotoxic potential of chlorpyrifos which was further supported by increase in AST, ALT and LPO levels and decrease SOD and catalase levels in the present study along with decrease in SOD and catalase enzymes.

On microscopic examination, brain showed engorgement of meningeal blood vessel and MNC’s infiltration, shrunken deeply eosinophilic neurons and necrosis. Similar changes were reported by El-Hossary et al. (2009) following administered of single oral dose of chlorpyrifos (@ 63 mg/kg) in rats. The authors opined that the pyknotic nerve cell nuclei were indicative of DNA damage leading to apoptosis (Latuszynsha et al., 2003). Parran et al. (2005) reported that chlorpyrifos induced disruption of the blood brain barrier leading to vascular disturbances and neurotoxic effects including degenerative changes. In the present study, decreased levels  of erythrocyte acetyl  cholinesterase, pseudo cholinesterase, increased LPO levels and  subsequently decrease in SOD and CAT levels in brain supported with gross and histopathological alterations i n brain were suggestive of neurotoxicity due to chlorpyrifos in bird.

Conclusion

From the present investigation it is concluded that Chlorpyrifos caused marked gross and histopathological changes in liver, kidneys and brain which were well marked at the end of experiment in dose and duration dependent manner. Thus, it is important to support the discontinuance of use of organophosphates in the vicinity of poultry houses.

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