Histopathological alterations in liver anatomy after ......Histopathological alterations in liver anatomy after exposure to chlorpyrifos in zebrafish ( Danio rerio) Bangeppagari Manjunatha

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Der Pharmacia Lettre, 2015, 7 (7):191-197

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ISSN 0975-5071

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Histopathological alterations in liver anatomy after exposure to chlorpyrifos in zebrafish (Danio rerio)

Bangeppagari Manjunatha1, 2*and Gundala Harold Philip1*

1Department of Zoology, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India

2Department of Life Sciences, Universidad de las Fuerzas Armadas-ESPE, Sangolqui, Quito, Ecuador, South America

_____________________________________________________________________________________________ ABSTRACT Chlorpyrifos is an organophosphate pesticide widely used in agriculture and aquaculture. This study investigated its effects on histopathology of zebrafish (Danio rerio) liver. For this six adult male and six adult female zebrafish were exposed to 200 µg/L of chlorpyrifos for 24h, 48h, 72h and 96hrs. Chlorpyrifos toxicity on liver histopathological changes were examined by light microscopy. Structural damage spotted in the liver were vacuolization and presence of sinusoid spaces were observed. The present study indicated that chlorpyrifos causes histopathological damage in liver tissue. Keywords: Chlorpyrifos, Zebrafish (Danio rerio), Liver histopathology. _____________________________________________________________________________________________

INTRODUCTION

The application of various pollutants like pesticides in the aquatic environment and their deposition in the biotic system is known to cause several structural and functional changes in the biota. A major part of the world’s food is being supplied from fish sources, so it is essential to secure the health of fishes [1]. In India as much as 70% of the chemical formulations employed in agricultural practices are believed to effect non-target organisms and to find their way to fresh water fish, ultimately polluting them [2]. Pesticides are extensively used to protect agricultural crops against the damages caused by pests. However, this may reach other ecological compartments as lakes, ponds and rivers through rain and wind, affecting many other organisms away from the primary target. Chlorpyrifos (O, O – diethyl O-3, 5, 6 trichloro-2-pyridyl-phosphorothioate) is used as broad-spectrum chlorinated organophosphate insecticide. Organophosphates (abbreviated as OP) are a common name for phosphoric acid esters. Numerous environmental issues have arisen so far due to the excessive use of this chemical compound [3], as it, among other consequences, causes toxic effects in non-target aquatic organisms, especially fish [4]. Fish are used to assess the health of aquatic environments and physiological changes occurring as a result of pollution and multiple studies have already established that chlorpyrifos has various detrimental effects on them [5-10], such as neurotoxicity via acetylcholinesterase inhibition [11], biochemical and histopathological alterations [12,13], oxidative stress [12,14], genotoxicity [15], and olfactory and neurobehavioral injuries [16]. It therefore poses a serious threat to aquatic organisms as well as to human health [17]. Alterations in the cellular morphology of pesticide treated fish [18] and their physiological functions upon exposure to different pesticide concentrations have been observed by Gupta and Saxena, [19]. The investigation of

Bangeppagari Manjunatha and Gundala Harold Philip Der Pharmacia Lettre, 2015, 7 (7):191-197 ______________________________________________________________________________


histopathology of various organs may, therefore, prove it is cost effective tool to determine the health of fish populations and reflecting the health of an entire aquatic ecosystem [20-22]. The study of fish liver is very important in the field of aquaculture induced by many problematic conditions and aquatic pollution [23].

Research carried out as per our literature survey on the effects of chlorpyrifos in zebrafish is reviewed here to show that there are only a few studies. Larvae of Danio rerio exposed to chlorpyrifos showed a significant increase in the percentage of individuals with morphological deformations [24], uncontrolled convulsion [25], significant concentration-dependent increase in the frequency of tail coilings, altered swimming activity and increase in mean turn angle [26] and dysregulation of genes required for neuronal maintenance and regeneration [16]. Developmental chlorpyrifos exposure caused a significant increase in startle responses in adult zebrafish [27], long-lasting behavioral impairments like spatial discrimination, dopamine depletion when tested as adults [28,29], also significant reductions in swimming activity [30] and AChE inhibition in adults [31,16]. Developmental exposure to different concentrations of chlorpyrifos was shown to cause significant persisting behavioral effects in adult zebrafish like decreased habituation, decreased escape dividing response, increased swimming activity and lower learning rate [32]. It was also shown to be highly toxic to the fish olfactory system [33,34], interfere with Hsp70 functioning, histopathology of organs [35] and metabolic enzymes [36] during early developmental stages. Widespread exposure of children to different organophosphorus (OP) compounds was also noticed earlier [37,38]. Low-level OP exposure has been linked to behavioral and cognitive problems in infants and school-aged children [39,40,41]. In comparison to adult children have much lower levels of essential enzymes needed to break down OPs [42]. This exposure to OPs during critical periods of development can have severe long term neurobehavioral. In rats also it was reported to be involved in multiple mechanisms causing genotoxicity, hepatic dysfunction, etc., [43]. To rescue the living world from the harmful effects of these chemical substances as well as to find some alternative ways to control the pests, it is very necessary to carry out works on diversity of organisms by applying varying concentration of pesticides. However, the reports on the effects of chlorpyrifos in zebrafish (Danio rerio) are still scanty. In the light of the above information and ideas, the present investigation is aimed to study the effect of environmentally relevant concentration of chlorpyrifos on liver of the zebrafish.

MATERIALS AND METHODS

Maintenance of zebra fish Zebrafish (Danio rerio) are maintained in our department aquarium facility for more than two generations with continuous breeding under defined conditions as described by Westerfield [44]. Fifty fish were housed in each 100L glass aquaria with continuous aeration at a temperature of 27 ± 1°C and 13:11 hr light:dark photoperiod. Fish were fed twice a day with alternating diet of freshly hatched brine shrimp (sanders brine shrimp co, Utah) and dry flake food (tetra brand). Preparation of stock solution Technical grade chlorpyrifos (99%) was obtained from the Nagarjuna Agri Chem Limited, Hyderabad. A Stock solution was prepared by dissolving 50 mg CP in 5ml acetone. This was stored at 4°C and from this daily requirements are taken and added to respective fish tanks. Experimental design Mature adult zebrafish in equal ratio (n=6) of male and female from our aquarium stock were kept in 20L glass aquaria with continuous aeration for four days. Later 200 µg/L of CP was added. Simultaneously control fish were maintained separately. Liver was collected after 24h, 48h, 72h and 96hrs from both male and female fish for conducting the experiments mentioned below. Collection of liver tissue After the stipulated time of exposure, liver tissue was gently rinsed in physiological saline (0.9% NaCl) and later fixed in Bouin’s fluid before processing for histological analysis.



Histology methodology Histological processing was done by adopting the procedure described by Humason [45]. Briefly the tissues were removed from fixative, washed in running tap water and processed for dehydration in an increasing percentage of ethyl alcohol. After this the tissues were cleaned in methyl benzoate and embedded in paraffin wax. Paraffin blocks were cut with a Leica manual rotary microtome at thickness of 5µ and were stained with Harris Hematoxylin [46] and counter stained with eosin. Photomicrographs were taken using Magnus (MLX) equipment.

RESULTS Effects of Chlorpyrifos on liver histology Two histological alterations were observed in liver of both sexes after exposure 200 µg/L of chlorpyrifos. They are vacuolization in male fish at all exposure periods (Fig. 1) and in female fish from 48h exposure period onwards (Fig. 2). Sinusoids spaces were also observed in male fish from 48h exposure period onwards and in female fish at all exposure periods.

Figure 1: (A) Section of control Male liver of zebrafish (Danio rerio). PN- Picnotic nuclei, H- Hepatocytes. Section of Male liver exposed

to chlorpyrifos Lesions observed after 24h (B), 48h (C), 72h (D) and 96h (E). SS- Sinusoids Space, H- Hepatocytes, V- Vacuoles



Figure 2: (A) Section of control Female liver of zebrafish (Danio rerio). PN- Picnotic nuclei, H- Hepatocytes. Section of Female liver exposed to chlorpyrifos Lesions observed after 24h (B), 48h (C), 72h (D) and 96h (E). SS- Sinusoids Space, H- Hepatocytes, V- Vacuoles

DISCUSSION

Aquatic ecosystems are often faced with problems caused by contaminants released into the environment [47]. Various non-target organisms, especially fish, are exposed to pesticides such as chlorpyrifos and this may cause many adverse effects, including biochemical alterations [48]. In order to add to risk assessment studies conducted thus far, we strived to obtain information about the effects of chlorpyrifos on zebrafish. Histopathological changes have been used as important biomarkers in environmental monitoring that allows examining specific target organs. The histological results observed in liver tissue of Danio rerio in the present study indicate that environmentally relevant concentrations caused moderate to severe alteration in liver architecture, which are an important organ performing a vital function like detoxification. Furthermore the alternations found in this organ are normally easier to identify than functional ones [49] and sever as warning signs of damage to animal health.



The organ most associated with the detoxification and accumulation process is liver and due to its function, position and blood supply, it is also one of the organs most affected by contaminants in the water [50], it also plays a prominent role in fish physiology, both in anabolism (protein, lipid, carbohydrate) and catabolism (glycogenolysis, detoxification) and it acts as storage center for many substances, mainly glycogen. The liver of the fish exposed to both low as well as high dose showed vacuolar degeneration, hypertrophy in the hepatocytes with nuclear pyknosis and karyopyknosis [51] due to apoptosis and fragmentation of the nucleus. These changes may be attributed to direct toxic effects of pollutants on hepatocyte as found in pesticide toxicity [52], because of it is the site of detoxification of all types of toxins and chemicals. The liver parenchyma showed sign of degeneration (cytoplasmic and nuclear), vacuolation of the hepatocytes [53], probably due to metabolic damage related to exposure to chlorpyrifos contaminated water. Histological studies of hepatic tissue are carried out as it is the major site of detoxification of toxic compounds and therefore is well known that liver is an organ that frequently undergoes changes when exposed to pesticides at sublethal doses. However the liver abnormalities observed in the present study were vacuolization and presence of sinusoid spaces in both male and female fish (Fig. 1 and 2). On the contrary chlorpyrifos caused much damage like appearance of pyknotic nucleus, protein precipitation, pancreatic acini with the loss of normal structure and necrosis of the hepatic tissue in freshwater fish C. catla and O. mossambicus [54,55]. In zebrafish another OP, Dimethoate caused pathological changes like swollen cells, vacuolizations of hepatocytes and slender sinusoids [20], 4-nitrofenol displayed swollen hepatocytes, binucleated cells and macrophages invasions into the intracellular space [56], TCDD triggered hypertrophy and lipidosis of hepatocytes [57] and Arsenic and mercury elicited decrease in number, dissociation and irregular shape of parenchymal cells [58, 59]. Hence it can be suggested that chlorpyrifos hepatotoxicity differ in different fish and in zebrafish it is not as hepatotoxic as other toxicants. Fish liver histopathology is an indicator of chemical toxicity and a useful way to study the effects of the exposure of aquatic animals to toxins present in the aquatic environment [60]. The effects of different pesticides on liver in various fish species have already been reported in other studies. Chlorpyrifos caused damages such as melanomacrophage aggregations, cellular atrophy, pyknotic nucleus, cytoplasmic vacuolation, cytoplasmic and nuclear degeneration, cellular rupture, necrosis, and nuclear and cellular hypertrophy in the liver tissues of the common carp, while phosalone induced histopathological changes such as nuclear degeneration, cytoplasmic vacuolation, hypertrophy, and congestion [61,62].

CONCLUSION

In this study, our results suggest that chlorpyrifos causes liver tissue histopathological damages under in vivo conditions, which proves that fish in both cultured and natural environments are sensitive to this pesticide and that chlorpyrifos contaminations would cause fish deaths. Therefore, stricter control must be applied to the use of this pesticide. Acknowledgement The author, Bangeppagari Manjunatha acknowledges the financial assistance rendered as Senior Research Fellow by the Department of Zoology (UGC-RFSMS Progamme), Government of India and in part of the National Secretariat of Higher Education, Science, Technology and Innovation (SENESCYT), Republic of Ecuador, South America.

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Histopathological alterations in liver anatomy after ......Histopathological alterations in liver anatomy after exposure to chlorpyrifos in zebrafish ( Danio rerio) Bangeppagari Manjunatha