Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 9 (september 2022) : 1090-1094

Changes in Biochemical Parameters, Antioxidative Enzymes and Histopathology of Liver Induced by Cadmium (Cd) and Chlorpyrifos (CPF) in Wistar Rats

Y. Ravikumar, D. Madhuri, M. Lakshman, A. Gopala Reddy, B. Kalakumar
1Department of Veterinary Pathology, College of Veterinary Science, Rajendranagar, Hyderabad- 500 030, Telangana, India.
Cite article:- Ravikumar Y., Madhuri D., Lakshman M., Reddy Gopala A., Kalakumar B. (2022). Changes in Biochemical Parameters, Antioxidative Enzymes and Histopathology of Liver Induced by Cadmium (Cd) and Chlorpyrifos (CPF) in Wistar Rats . Indian Journal of Animal Research. 56(9): 1090-1094. doi: 10.18805/IJAR.B-4178.
Background: Cd and CPF intoxication may occur directly through drinking water. Since the population tend to receive combination of multiple intoxicants through environment contamination, there is need for conducting studies to assess the impact of individual and combined environmental pollutants. The present research work was designed to study hepatotoxicity induced by Cd, CPF and their combination.

Methods: The experiment was carried out for 28 days in Wistar rats. G1: Control. G-2:CdCl2 @ 22.5mg/ kg b.wt / oral. G3: CPF @ 25 mg/ kg b.wt /per oral. G4:CdCl2@22.5 mg + CPF @ 25 mg/ kg b.wt /per oral. Biochemical parameters ware estimated from serum and liver samples were processed for tissue antioxidative parameters and histopathological examination. 

Result: Higher mean values of AST, ALT, ALP and lower liver GSH and SOD were observed in G2, 3 and 4 on 15th and 29th day when compared with G1. Liver in G2 and 3 showed mild degenerative changes, areas of necrosis and loss of architecture. In G4, lesions were moderate in severity. In addition, moderate perivascular fibrosis of portal triad was observed. The effects in combined group were severe than individual groups due to synergistic action of the combined pollutants.
There is growing evidence that long-term exposure to lower levels of heavy metals (Calderoni et al., 2005) and pesticides causes toxicity worldwide (Poulsen et al., 2008). Cadmium (Cd) and Chlorpyrifos (CPF) are the most common toxicants among all toxic compounds in the environment. The common sources of environmental Cadmium contamination are industrial, mining activities, plastic stabilizers and batteries which may result in widespread into environment and agricultural fields (Cheng et al., 2011). The Organo--phosphorus insecticides are extensively used for control of insects in home and agricultural practices. Chlorpyrifos (CPF) is one of the most commonly used organophosphate pesticides in domestic and agricultural applications throughout the world (Asperlin, 1994). Cd and CPF intoxication may occur directly through drinking water, indirectly through irrigation water and through feed ingredients of plant origin and also through inhalation of polluted air. Since the population tend to receive combination of multiple intoxicants through environment contamination, there is need for conducting induced toxicopathological studies to assess the impact of individual and combined environmental pollutants (Ravikumar et al., 2019). Cadmium induces oxidative stress (Rajender et al., 2011) and apoptosis (Henson et al., 2004). CPF causes deleterious effects through acetyl cholinesterase inhibition at synapse of central and peripheral nervous system (Gordon et al., 1997), thereby causing damage to various vital organs. Cd and CPF are known for damaging organs viz. liver, kidneys, heart, lungs, retina and bones in humans and experimental animals (Curcic et al., 2012). The present research work was designed to study hepatotoxicity induced by Cd, CPF and their combination in Wistar rats. 
Drugs and chemicals
CdClwas procured from Thermo Fisher Scientific India Pvt. Ltd. Mumbai. Chlorpyrifos was procured from Coromandel Fertilizers Pvt. Ltd. Vishakapatnam.
Experimental design

Male Wistar albino rats (48) were procured from Sanzyme Laboratories Ltd., Hyderabad. Rats were randomly divided into 4 groups consisting of 12 in each group. G-1 serves as control. G-2 rats were administered with Cdcl2 @ 22.5 mg/kg b.wt/per oral/day, G-3 rats were administered with CPF @ 25  mg/kg b.wt/per oral/day and G-4 rats were administered Cdcl2 @22.5 mg + CPF @ 25 mg/kg b.wt/per oral/day for 28 days of experiment.
Biochemical parameters
Blood was collected from retro-orbital plexus of rats, with the help of a capillary tube in to serum vaccutainers on 15th day and 29th day and serum was separated for estimation of biochemical parameters. Biochemical parameters viz, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were estimated in auto-biochemical analyzer by using Erba kits supplied by M/s Perala Agencies, Hyderabad.
Tissue antioxidative parameters
Liver was quickly removed after sacrifice, trimmed of extraneous tissue and washed with ice cold physiological saline solution. After that liver tissue was divided into different parts. Tissue homogenate (10%) was prepared in ice cold phosphate buffered saline for estimation of GSH (Moron et al., 1979) and SOD (Madesh and Balasubramanian, 1998).
Detailed necropsy was conducted on 15th and 29th day of the experiment and gross changes were noticed, if any. Pieces of liver were collected in 10% neutral buffer formalin (NBF). Samples were processed, sectioned (5 μm), stained with Hematoxylin and Eosin (H&E) as per the standard protocol (Luna, 1968).
Statistical analysis
Data obtained were subjected to statistical analysis by applying one way ANOVA using statistical package for social sciences (SPSS) version 16.0. Differences between means were tested by using Duncan’s multiple comparison tests and significance level was set at P<0.05 (Snedecor and Cochran, 1994).
Effect on biochemical parameters
Significantly (P<0.05) higher in aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) mean values (IU/L) were observed in rats of group 2, 3 and group 4 on 15th and 29th day respectively when compared with group 1. However, there was a significant (P<0.05) difference in mean values in the rats of combined toxic dose (i.e. group 4) than the rats of individual treated groups 2 and 3 (Table 1).

Table 1: Values (Mean±S.E.) of serum biochemical parameters and oxidative enzymes in experimental rats of different groups.

Effect on antioxidative parameters
The mean values of reduced glutathione concentration (GSH) and superoxide dismutase activity (SOD) were significantly (P<0.05) lower in group 2, 3 and group 4 compared with group 1 on 15th and 29th day of the experiment. Again, these values were significantly (P<0.05) lower in rats of group 4 compared to groups 2 and 3 (Table 1).
Histopathological findings in liver
At the end of the experiment animals were sacrificed and thoroughly examined for gross changes if any. Hepatomegaly with round edges was noticed on 15th and 29th day of experiment. The liver showed mild to moderate congestion in rats of groups 2 and 3 and moderate to severe in group 4 on 15th and 29th day of the experiment. Liver sections of control group showed normal architecture of hepatic parenchyma with central vein and portal triad (Fig 1a). The sections of liver in group 2 rats on 15th day showed increased sinusoidal spaces and mild degenerative changes (Fig 1b). On 29th day, vacuolar degeneration and areas of necrosis were observed (Fig 1c). The sections of liver on 15th day in rats of group 3 showed perivascular lymphocytic infiltration and disrupted cords with swollen hepatocytes (Fig 1d). On 29th day, the changes were more pronounced as well as focal areas of necrosis was evident (Fig 1e). In the rats of group 4, the liver sections showed similar type of lesion as noticed in groups 2 and 3 on 15th and 29th day but the intensity was moderate to severe (Fig 1f to 1i). In addition, moderate perivascular fibrosis of portal triad and shrunken, irregular hepatic cords were observed (Fig 1j).

Fig 1: Photomicrograph of liver.

Biochemical parameters revealed a significant (P<0.05) increase in the activity of AST, ALT and ALP in groups 2, 3 and 4 on 15th and 29th day of the experiment. An increased activity of AST, ALT and ALP in cadmium and CPF treated rats separately indicated liver dysfunction, which was accompanied by elevated levels of these hepatic marker enzymes in the blood stream. Elevated levels of ALP suggested biliary damage, which disrupts flow of blood to the liver. Similar findings were reported by Tomaszewska et al., (2015) and Nasim Babaknejad et al., (2015) in Cd treated rats. In the present study, these results are in accordance with the findings of Ambali et al., (2010) and Barski and Spodniewska (2018). Co-administration of both the toxicants produced further increase in these enzyme levels as compared to individual administration of either toxicant. The increased levels could be due to severe degeneration and necrosis of hepatocytes that result in release of transaminases in the blood. These findings can be correlated with the histological changes in the present study and are in agreement with the reports of Prabu et al., (2012) and Singh et al., (2016).
Significant (P<0.05) reduction in GSH and SOD levels in rats liver of combined group than those in the individual toxicity groups is suggestive of oxidative stress in the present study. El-Sharaky et al., (2007) observed that the increase in lipid peroxidation might be attributed to alterations in the antioxidant defence system. This defence system includes the glutathione peroxidase, thioredoxin reductase as well as the reduced glutathione (GSH), which normally protect the biological system against free radical toxicity. Sarkar et al., (1998) demonstrated that Cd modulates toxic effects through oxidative stress mechanisms. The changes in CdCl2 treated rats are in agreement with those of Renugadevi and Prabu (2010), Messaoudia et al., (2010), Pari and Shagirtha (2012) and Christian et al., (2016). The results in Group 3 indicated that CPF exposure inhibited GSH and SOD. These depletion might be due to the decreased synthesis of enzymes or oxidative inactivation of enzyme protein. The changes in CPF group were similar to the reports of Aly et al., (2010), Hassani et al., (2014) and Deng et al., (2016). In rats of group 4, a marked reduction in GSH levels compared to groups 2 and 3 indicated synergistic action of CdCl2 and CPF leading to higher oxidative damage.
The Cd induced hepatotoxicity was thought to be mediated through the cadmium metallothionein (Cd-Mt) complex, which is synthesized in the liver, released into circulation and taken up by renal proximal tubule cells (Dudley et al., 1985). In fact, when the synthesis of Mt becomes insufficient for binding all Cd ions in the liver, Cd not bound to Mt produce hepatocytes injury and caused different histopathological lesions. Similar lesions were also noticed by Jihen et al., (2008), Renugadevi and Prabu (2010) and Prabu et al., (2012). The histopathological changes are further corroborated by the decreased levels of GSH and SOD in rats exposed to CPF that might have caused membrane damage of cells resulting into degenerative to necrotic changes in liver. Similar changes were also reported by Savithri et al., (2010) and Singh et al., (2016). Irregular hepatic cords were observed. Similar observations were also noticed by Singh et al., (2016) and might be due to synergistic action of CdCl2 and CPF.
In conclusion, the adverse effects of combined CdCl2 and CPF group (Group 4) were more severe than the individual groups (Group 2 and 3) due to synergistic action of the combined pollutants.
The authors are thankful to the PV Narsimha Rao Telangana Veterinary University for providing support and necessary facilities to carry out the research work.
Author declare that there are no conflicts of interest to report.

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