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 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 »  References
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SHORT COMMUNICATION
Year : 2012  |  Volume : 44  |  Issue : 4  |  Page : 512-515
 

Endosulfan induced early pathological changes in vital organs of rat: A biochemical approach


1 Department of Pathology, KVG Medical College & Hospital, Sullia, Karnataka, India
2 Department of Physiology, KVG Medical College & Hospital, Sullia, Karnataka, India
3 Blood Bank, MVST Trust-Government Wenlock District zospital, Mangalore, Karnataka, India

Date of Submission17-Jun-2011
Date of Decision03-Sep-2011
Date of Acceptance30-Apr-2012
Date of Web Publication3-Aug-2012

Correspondence Address:
Urban J. A. D'Souza
Department of Physiology, KVG Medical College & Hospital, Sullia, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7613.99335

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 » Abstract 

Aim: To evaluate the pathogenesis in heart and liver by the early induction of biochemical and antioxidant derangements in rats exposed to endosulfan.
Materials and Methods: Wistar rats were gavaged with endosulfan (0.5, 1 and 1.5 mg/kg body weight in sunflower oil) for a period of 21 days (single dose at 24 h interval). Control and sunflower oil control groups were also maintained simultaneously. Rats were sacrificed on the 22 nd day posttreatment. Blood samples, heart and liver were collected and different biochemical parameters such as total protein, cholesterol, triglycerides, amino acids and antioxidant and lipid peroxidation level were measured. Statistical analysis was carried out by one way ANOVA, followed by Bonferroni' post-hoc test.
Results: Endosulfan induced a significant increase in the serum levels of total protein, amino acids, triglyceride, total cholesterol, free fatty acid and phospholipid levels in a dose-dependent manner. In the heart and liver, lipid peroxidation was increased significantly in a dose-dependent manner and the antioxidant levels such as superoxide dismutase (SOD), glutathione S-transferase, glutathione peroxidase, and catalase were significantly decreased in a dose-dependent pattern.
Conclusion: Exposure to endosulfan results in a significant derangement in the biochemical parameters with a decrease in antioxidant levels in the heart and liver. This is an early indication of pathogenesis in the vital organs of rats.


Keywords: Antioxidants, biochemical, catalase, cholesterol, endosulfan, free fatty acids, superoxide dismutase


How to cite this article:
Alva S, Damodar D, D'Souza A, D'Souza UJ. Endosulfan induced early pathological changes in vital organs of rat: A biochemical approach. Indian J Pharmacol 2012;44:512-5

How to cite this URL:
Alva S, Damodar D, D'Souza A, D'Souza UJ. Endosulfan induced early pathological changes in vital organs of rat: A biochemical approach. Indian J Pharmacol [serial online] 2012 [cited 2020 Aug 10];44:512-5. Available from: http://www.ijp-online.com/text.asp?2012/44/4/512/99335



 » Introduction Top


Organochlorine insecticide endosulfan is a broad spectrum compound used widely to contain a variety of pests in agriculture and has produced a lot of panic in Kerala state, especially the surrounding places of Kasaragod district. It is commonly used to control the insects on cocoa, coffee, cashew, tobacco, and cotton plants. Its common trade name is thiodan, which was introduced in the year 1950 mainly as an aquatoxic compound. On terrestrial use it is generally used as a spray and its degradation product endosulfan sulfate remain persistent in soil for a long duration and presumed to be a major threat in the environment. [1] Amidst the evidences of its negative health effects, endosulfan is still employed, though many other organochlorine pesticides were banned. Chlorinated cyclodienes, which are well-known blockers of neuronal GABA A -gated chloride channels. [2] Food contaminated with its degradation products are of main concern to the animal and human health and xenobiotics are able to produce weak estrogenic effects and interfere in the normal endocrine functions, which in turn may derange metabolic, biochemical, and regulatory functions of body. [3] Main concerns of estrogen mimicking effects of these chemicals are interference in the normal metabolic activity in the liver and adverse sexual functions and maturation. [4] Since this chemical is used in spray forms, its presence in the atmosphere in the vicinity of farming area is a major public concern. Estrogen-sensitive cells in human were reported to be sensitive to the estrogenic insults of endosulfan and thereby disruption in the normal functions of endocrine glands. [5],[6],[7] The major problem faced in Kerala state during the past decade due to the spraying of this acaricide on cashew and rubber plantations and untoward health problems faced by the local people is an example of live man-made calamity due to its adverse toxicity in the neighborhood. Since the vital organs such as heart and liver play a pivotal role in the existence of life, early pathogenesis of endosulfan toxicity need to be investigated in terms of chemical pathology. There are few reports in the literature stating the adverse effects of endosulfan but a systematic study of different vital organ toxicity in preclinical animal model on exposure to low doses of endosulfan is less reported. Hence in this study, different biochemical parameters including lipid peroxidation and antioxidant levels were estimated to confirm the pathological and biochemical basis of endosulfan toxicity in vital organs such as heart and liver.


 » Materials and Methods Top


Adult virgin male Wistar rats (180-200 g weight) from the animal house of KVG Medical College were maintained under standard laboratory conditions on a natural day/night cycle at a room temperature of around 24°C. Standard food pellet and tap water was available freely Ad libitum. All animal studies were carried out in accordance with the Institutional ethical committee for the Care and Use of Laboratory Animals for medical research.

Test chemical

Endosulfan (6, 7, 8, 9, 10, 10-Hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6,9-methano-2, 4, 3-benzodioxathiepine-3-oxide- C 9 H 6 Cl 6 O 3 S) was the organochlorine test compound used in this study. The test chemical was prepared in sunflower oil (vehicle) and three dose levels-0.5, 1 and 1.5 mg/kg body weights based on a previous report [5] were selected for the study.

Experimental design

After two weeks of acclimatization period, rats were randomly divided into five groups; experimental (n = 6 each) and control/sunflower oil control (n = 6 each group) groups. Rats in experimental group were divided into three groups and received 0.5, 1 and 1.5 mg/kg body weight (n = 6 each group). Each group was gavaged with different dose levels of endosulfan in sunflower oil (2 ml) for a period of 21 days with a single dose daily administered at 10 AM. The control group received 2 ml saline and sunflower oil control received only sunflower oil (2 ml) for the same duration as that of the endosulfan groups. First treatment day was considered as treatment day 1. The exact dosage for each rat was corrected for individual body weight on every second day by appropriate volume adjustments. Rats were sacrificed on the 22 nd day after the 21 successive treatments by cervical dislocation. Blood samples were collected for serum and a laporatomy was performed and the vital organs, -heart and liver, were removed carefully for the estimation of lipid peroxidation and different antioxidant levels. Heart and liver tissues were homogenized separately in Tris buffer (pH 7.4).

Blood samples were used for the estimation of total protein, [8] free amino acids, [9] free fatty acids, [10] total cholesterol, [11] triglycerides, [12] lactate dehydrogeneae, [13] and phospholipids. [14] The supernatant of homogenates of heart and liver were used for the estimation of the activity of superoxide dismutase (SOD), [15] glutathione peroxidase (GPoxd), [16] glutathione S-transferase (GST), [17] and catalase activity. [18]

Statistical analysis

The data was expressed as Mean ± SE, analyzed by using oneway ANOVA, followed by Bonferroni' posthoc test. Statistical package of social sciences (SPSS) version 16 was used and values of P < 0.05 were considered as statistically significant.


 » Results Top


As a measure of toxicity of endosulfan, body weights of rats have significantly decreased following the treatment of endosulfan in a dose-dependent response [Table 1]. The maximum dose of the study (1.5 mg/kg) has produced a reduction of around 12% in the body weight and in the scientific limits of toxicity indication. Serum biochemical markers such as total protein, amino acids, and lactate-dehydrogenase levels have reached a significant increase compared with the control with a dose-related response with an injury to the cellular associations. Endosulfan has a significant dose-dependent effect on different serum biochemical parameters of this study. Total protein, amino acid, triglyceride, total cholesterol levels, free fatty acid and phospholipid levels have significantly increased in the serum (P < 0.05, 0.01) [Table 1] and [Table 2]. The lipids such as triglycerides, total cholesterol, free fatty acids and phospholipids have also showed a significant increase on exposure to endosulfan for a period of 21 days. The lipid markers such as triglycerides, total cholesterol and free fatty acids, though showed a significant increase, there was no dose-dependent response [Table 2]. But majority of the biochemical responses studied have exhibited a dose-dependent effect with endosulfan. Lipid peroxidation, the total lipid peroxidation (TBARS) have a dose-dependent increase in the heart muscle, so also the antioxidant enzymes such as SOD, GST, GPoxd, and catalase have significantly (P < 0.05, 0.01) decreased with endosulfan in a dose-dependent pattern [Table 3].
Table 1: Body weight and serum biochemical changes in endosulfan-treated rats (n = 6)

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Table 2: Serum lipid parameters in endosulfan-treated rats (n = 6)

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Table 3: Antioxidant enzyme-levels and lipid peroxidation in the heart tissue in endosulfan-treated rats (n = 6)

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Simultaneously, a similar trend in the lipid peroxidation and antioxidant enzyme levels was followed in liver, as that was evident in the cardiac tissue. Most of the antioxidant levels such as SOD, GST, GPoxd and catalase significantly (P < 0.05, 0.01) decreased in a dose-dependent pattern [Table 4]. Overall, endosulfan has resulted in a total derangement in the lipid peroxidation, antioxidant status in both the vital organs of this study and an overall biochemical derangement indicating an early pathogenic sequelae that may have an adverse effect.
Table 4: Antioxidant enzyme-levels and lipid peroxidation in liver-of endosulfan-treated rats (n = 6)

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 » Discussion Top


Endosulfan usage is a major health hazardous pesticide reported to cause neurobehavioral, musculoskeletal, endocrine and reproductive-related adverse health effects in human beings. [19],[20],[21],[22],[23] In the state of Kerala, Kasaragod district, untoward health hazardous effects were reported regularly by the daily newspaper, which is an eye-opener for the government bodies and public in particular to curtail the use of this deadly chemical. There is enough scientific data on adverse health effects of pesticides in literature, a recent report on endosulfan-induced lipid peroxidation in rat brain and ameliorating effects of vitamins C and E are interesting [24] but early pathogenic toxicity on vital organs were not highlighted. Irrespective of few scientific data, a systematic analysis of its adverse effects on important vital organs such as heart and liver were not well reported using a very low dose in animal models. Since our institution is located in the neighborhood of Kasaragod district, a preclinical study using animal model was proposed to study the biochemical basis of low dose of endosulfan toxicity as an early sign of pathogenesis in rat model. In the present study a systematic dose-dependent decline in body weight was significant compared with the control group suggestive of general toxicity in the animal model of this study.

Different serum biochemical parameters also have showed a significant increment in a dose-dependent manner. Increase in total protein, amino acid and free acid level is due to the overall derangement in metabolic activity. Dose-dependent increase in different biochemical parameters such as protein, cholesterol, triglycerides, phospholipids and amino acids may be due a generalized leakage of cell membrane and intercellular spaces in addition to a metabolic derangement in the liver. Along with a significant rise in lipid peroxidation level, the oxidative stress has resulted in the pathogenesis of cell death and as a preliminary step there may be a leakage of different amino acids, free fatty acids, and phospholipids into the extracellular fluid, which were evident significantly in the plasma. Different antioxidants such as catalase, GPoxds, SOD activities were significantly decreased, an evidence of increase in lipid peroxidation. A dose-dependent significant decrease in GST activity may be due to the loss of protection against the reactive oxygen species in the intracellular vital tissues, which is in correspondence with the lipid peroxidation. Since the vital organs such as heart and liver play a pivotal role in the regular vital activity long-term exposure to this chemical may result in hepatocellular and cardiac cell death thereby inducing irreversible changes and subsequent fatality. In a recent study with fresh water fish, spotted murrel, channa punctatus, endosulfan produced alteration in liver histology with a fall in ascorbic acid levels. [25] Increase in lactate dehydrogenase activity with a concurrent rise in lipid peroxidation level is an indicator of cell membrane leakage and dissociation of intercellular connections due to the oxidative stress on exposure to endosulfan. Similar results have been reported earlier with insecticide aldrin, dieldrin, and endosulfan in culture cells with fractions of their lethal dose. [26] Hexachlorocyclohexane and lindane also have shown similar effects with GPoxd, glutathione reductase and GST activity. [27] A significant dose-dependent rise in cholesterol level is an indicator of impairment in steroid production mimicking weak estrogenic and anti-androgenic activities of endosulfan. In a cell culture study it was confirmed that, methoxychlor pesticide inhibits P-450 cholesterol side chain cleavage resulting in a decline in androgen production, [28] our finding with an increase in cholesterol indirectly suggests that, there may be a decrease in the diversion of cholesterol for the production of androgen due to the inhibition of enzyme by endosulfan and a subsequent increase in the cholesterol levels in the plasma. It also directs for the need of a detailed study of testicular functions and spermatogenesis to confirm any derangement in the reproductive functions in male. Hence overall this study concludes that, endosulfan induces a dose-dependent increase in lipid peroxidation in the heart and liver, which may be detrimental to the normal harmony and functions of vital cells. Further a detailed histological assessment with markers of cell integrity and apoptosis are required to conclusively arrive at the endosulfan-induced cellular anomalies.

 
 » References Top

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27.García-Fernández AJ, Bayoumi AE, Pérez-Pertejo Y, Romero D, Ordóñez C, Reguera RM, et al. Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells. Xenobiotica 2002;32:1007-16.  Back to cited text no. 27
    
28.Akgul Y, Derk RC, Meighan T, Rao KM, Murono EP. The methoxychlor metabolite, HPTE, directly inhibits the catalytic activity of cholesterol side-chain cleavage (P450scc) in cultured rat ovarian cells. Reprod Toxicol 2008;25:67-75.  Back to cited text no. 28
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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