Physiological Dysfunction in Fish After Insecticides Exposure

A fundamental contributor to the Green Revolution has been the development and application of insecticides for the control of a wide variety of insectivorous and herbaceous pests which would otherwise diminish the quantity and quality of food production (Ecobichon, 2001). Unfortunately, in spite of its advantages, chemistry has great disadvantages as well. Insecticides are threatening the long-term survival of major ecosystems by disruption of ecological relationships between organisms and loss of biodiversity. On the other hand, agriculture, as the largest consumer of freshwater and as a major cause of reduction of surface and groundwater resources through erosion and chemical runoff, are close related to the loss of water quality (Wauchope, 1978). The processing industry associated to agriculture is also a significant source of organic pollution in most countries. Conventionally, in most countries, all types of agricultural practices and land use, including animal farming, are treated as nonpoint sources. The main characteristics of non-point sources are not simply determined or controlled directly and therefore, are difficult to regulate their impacts on ecosystem health (Garcia et al., 2001). Non-point source pollutants are mainly transported overland and through the soil by runoff (Dubus et al., 2000). These pollutants ultimately find their way into groundwater, wetlands, rivers and lakes and, finally, to oceans in the form of sediment and chemical loads carried by rivers (Albanis et al., 1998; EPA, 2003; Aydin and Koprucu, 2005). The major insecticides that are usually applied in agriculture and public healthy sections include organophosphate, organocholorines, pyrethroids and carbamate. Contamination of water by insecticides is mainly due to intensive agriculture combined with surface runoff and subsurface drainage, usually within a few weeks after application (Banaee et al., 2011) Most of insecticides have been known to be highly toxic to non-target organisms that inhabit natural environments close to agricultural fields. Several studies reported that some of surface waters


Introduction
A fundamental contributor to the Green Revolution has been the development and application of insecticides for the control of a wide variety of insectivorous and herbaceous pests which would otherwise diminish the quantity and quality of food production (Ecobichon, 2001). Unfortunately, in spite of its advantages, chemistry has great disadvantages as well. Insecticides are threatening the long-term survival of major ecosystems by disruption of ecological relationships between organisms and loss of biodiversity. On the other hand, agriculture, as the largest consumer of freshwater and as a major cause of reduction of surface and groundwater resources through erosion and chemical runoff, are close related to the loss of water quality (Wauchope, 1978). The processing industry associated to agriculture is also a significant source of organic pollution in most countries. Conventionally, in most countries, all types of agricultural practices and land use, including animal farming, are treated as nonpoint sources. The main characteristics of non-point sources are not simply determined or controlled directly and therefore, are difficult to regulate their impacts on ecosystem health (Garcia et al., 2001). Non-point source pollutants are mainly transported overland and through the soil by runoff (Dubus et al., 2000). These pollutants ultimately find their way into groundwater, wetlands, rivers and lakes and, finally, to oceans in the form of sediment and chemical loads carried by rivers (Albanis et al., 1998;EPA, 2003;Aydın and Köprücü, 2005). The major insecticides that are usually applied in agriculture and public healthy sections include organophosphate, organocholorines, pyrethroids and carbamate. Contamination of water by insecticides is mainly due to intensive agriculture combined with surface runoff and subsurface drainage, usually within a few weeks after application  Most of insecticides have been known to be highly toxic to non-target organisms that inhabit natural environments close to agricultural fields. Several studies reported that some of surface waters and surrounding environments were contaminated with different insecticides (Arjmandi et al., 2010;Bagheri et al., 2000;Ghassempour et al., 2002;Rahiminezhhad et al., 2009;Tarahi Tabrizi, 2001). The continuous presences of insecticides are the consequence of application (timing, rate, frequency) and the rainfall during the application period ( Since fishes are important sources of proteins and lipids for humans and domestic animals, so health of fishes is very important for human beings. Fish like other aquatic organisms may be exposed to a great range of insecticides during the course of their life cycle. In fish, different insecticides can be absorbed through gills, skin or alimentary ducts (Schlenk, 2005;Banaee, 2012). Fishes are particularly sensitive to environmental contamination of water. Hence, pollutants such as insecticides may significantly damage certain physiological and biochemical processes when they enter into the organs of fishes . So, the effects of insecticides on fishes are of great concern.
Recently, many studies have been conducted to determine the mechanisms of insecticides' damage in fishes, with the ultimate goal of monitoring, controlling and possibly intervening in xenobiotics exposure and its effects on the aquatic ecosystem. The main mechanism of action of organophosphate and carbamate insecticides is block of enzyme acetylcholinesterase action that results in signs and symptoms of intensive cholinergic stimulation. Organochlorines are neurotoxins which effect on sodium and potassium channels in neurons. Decrease of potassium permeability and inhibition of cadmodulin, Na/K and Ca-ATPase activity occur by organochlorine insecticides. Pyrethroids can block Na channel and effect on the function of GABA-receptors in nerve fiber. Oxidative stress is another mechanism for toxicity of insectisides resulting in cell death includescellular necrosis and apoptosis and dysfunction in cellular physiology include alterations in metabolic and vital functions of the cells.Hence, fish should be able of managing environmental exposure by detoxifying these xenobiotic. In order to do this task, fish like aerobic animals have evolved complex of detoxification system, composed of two main parts including enzymatic and non-enzymatic components. Theroles of enzymatic and non-enzymatic detoxification system of animal's body lessen the potential damages caused by the toxicity of environmental pollutants. Although it is possible that reactive oxygen species (ROS) produced during the insecticides detoxification process in liver tissue may react with vital macromolecules such as lipid, protein, carbohydrate and nucleic acid and result in oxidative damage to aquatic organisms (Üner et al., 2006). ROS derived damage to natural and structure cellular components are generally considered a serious mechanism involved in the physiological and pathological disorders (Sepici-Dinçel et al., 2009).So, much literature suggests an association between impaired detoxification and disease such as oxidative stress, histopathological damages, hormonal dysfunctions, reproductive disorders, immunosuppression syndrome and etc.
Hematological and biochemical studies, along with histopathology, are the major means to learn a toxicants mode of action. Hematological and clinical chemistry parameters can be detected rapidly and hence can be used for prediction and diagnosis of insecticides toxicity. Alterations in these parameters show toxic stress in the treated animals especially on blood and blood-forming organs. Alterations in blood biochemical parameters as important diagnostic tool can be used for the detection of abnormalities in the liver and other tissues . This chapter describes some of the important hematological, biochemical and histopathological changes that occur in fish exposed to different insecticides. An initial description is given of the detoxification process, and then the other physiological dysfunction in various biological systems occurring in fish exposed to different insecticides will be discussed.

Metabolism of insecticides
In fish, insecticides are readily converted into more polar compounds through biotransformation and stored in the bile until their excretion. In general, detoxification process involves two phase, functionalization (phase I) and conjugation (phase II). The first biotransformation step (phase I) adds oxygen to the insecticides structure and the second step (phase II) conjugates the oxygenated product with and endogenous water-soluble small molecule present in the cell. The final products are highly water-soluble conjugates that are easier to eliminate than the parent compounds.Although, recently, anti-porter activity (p-glycoprotein or multidrug resistance) has been defined as the Phase III detoxification system, there is no exactly information about this detoxification system in fish.Classic detoxification pathways of insecticides in aquatic organisms are presented in Table 1.

Phase I
The phase I detoxification system, composed over 10 families of enzymes which played important role in the metabolism of various xenobiotic. The phase I detoxification system, composed mainly of the cytochrome P450 supergene family of enzyme, that are present in all eukaryotes and some prokaryotes and is generally the first enzymatic defense system against xenobiotic.A great diversity of cytochrome P450 enzymes in fish has been recognized (Stegeman and Hahn, 1994), and CYP1A, CYP2B, CYP2E1, CYP2K1 and CYP3A have been recently identified in liver of some freshwater fish (Nabb et al., 2006) which play an important role in the detoxification of organophosphate and carbamate insecticides (Ferrari et al., 2007). The common pathways of biotransformation of different kinds of insecticides include three cytochrome P450 (CYP) mediated reactions: O-dealkylation, hydroxylation, and epoxidation of insecticides (Keizer et al., 1995;Kitamura et al., 2000;Straus et al., 2000;Behrens & Segner, 2001;Nebbia, 2001). Most insecticides are metabolized through phase I biotransformation. In general, CYP450 enzymes mediated reactions by using oxygen and NADH, as a cofactor, lead to detoxification and subsequent excretion of xenobiotic. The CYP450 enzymes can also facilitate dealkylation, dearylation, aromatic ring hydroxylation, thioether oxidation, and deamination (Table 1.). However, CYP450 enzymes mediated metabolism can also cause formation of reactive metabolites which is far more dangerous than parental compounds. For example, oxidative group transfer of certain organophosphorous insecticides to the toxicorganophosphate, e.g. conversion of parathion to paraoxon,oxidative dechlorination of chloroform to phosgene,activation of ethyl carbamate to urethan. However, many of these same chemicals are also detoxified by cytochrome P450 by conversion to less toxic metabolites. In some cases, the same enzyme may catalyze activation and detoxification reactions for a given chemical. The resulting toxic effect of a xenobiotic chemical is thus due to a balance between metabolic activation and deactivation (Casarett and Doull, 1996).

Phase II
In phase II reactions, metabolites produced in phase I detoxification often conjugate with glutathione, uridyl-diphosphate glucose (UDPG), uridyl-diphosphate-glucuronic acid (UDPGA), amino acid derivatives and sulfate derivatives and can readily excrete from the fish body (Keizer et al., 1995;Kitamura et al., 2000;Straus et al., 2000;Behrens & Segner, 2001;Nebbia, 2001). In fact, this results from enzymatic oxidation and hydrolysis that produce metabolites with OH, COOH and NH2, SH functional groups. These functional groups are then subject to conjugation with carbohydrates, glutathione, sulfate, andamino acids, and then, the final metabolites may also be excreted from the body of fish through the skin, gills, genital products, urine as sulphated and glucuronidated metabolites and stool as glutathione conjugated metabolites (Kitamura et

Behavioral response
Behavioral alterations and the change of body's color pattern of fish or darkness of skin and mucosa increase to the skin and gill surface, as well as bleeding around the eyeball and the base of pectoral fins and also, the volume increase of the liver and the gall bladder in fish exposed to insecticides were the main symptoms evidenced in the toxicology studies.
Behavioral changes are the most sensitive indicators of potential toxic effects. Most insecticides affect the behavioral patterns of fish by interfering with the nervous systems and sensory receptors and consequently it can lead to disorders in the fish response to environmental stimuli. The effect of certain insecticides on the activity of acetylcholinestrase may also lead to a decreased mobility in fish (Banaee, 2012). Several studies have demonstrated that insecticides are metabolized in liver to toxic derivate via cytochrome P450 mono oxygenases (Fujii and Asaka, 1982;Hamm et al., 2001;Schlenk, 2005) and finally, these metabolites were hydrolyzed in microsomes (Keizer et al., 1993;Keizer et al., 1995) and excreted from the body. Nevertheless, rainbow trout was very sensitive to organophosphate insecticides toxicity due to a lack of esterase activity and a very sensitive acetylcholinesterase activity to OPs inhibition (Keizer et al., 1995 (Koprucu et al., 2006). Disorder in γ-aminobutyrate (GABA) system in brain of rainbow trout exposed to sub-lethal lindane was reported by Aldegunde et al., (1999

Oxidative stress
During the detoxification process, ROS are produced (Üner et al., 2006;Isik and Celik, 2008) and they can indiscriminately attack and damage cellular macromolecules -lipids, proteins and DNA-in living cells resulting in serious disturbances in physiological cell processes (Sureda et al., 2006;Tejada et al., 2007). Li et al. (2010c, d;2011b) believed that cellular antioxidant responses could be used as potential biomarkers for monitoring residual xenobiotic present in aquatic environment. For example, Salvo et al. (2012) found that endosulfan at the sub-lethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Similar results was observed in gar (Atractosteus tropicus), Tilapia (Oreochromis niloticus), tropical reef fish (Acanthochromis polyacanthus) exposed to ethorophos (Mena Torres et  Since an increase of GPx activity is necessary to eliminate the excess of H 2 O 2 and lipid hydroperoxides produced in hepatocytes of fishes exposed to. The increased activity of GPx accelerates the utilization of GSHto GSSG. This increased GSSG, indicative of a more oxidized state, may explain the decreased levels of total antioxidant capacity in liver cells of fish after exposure to pesticide. A decrease in GPx activity to basal values is probably related to decreasing cellular GSH levels on the days 14 and 28, although it cannot be discarded a direct effect of diazinon on the biosynthesis of the enzyme. Similar alterations in GPx activity were observed in different tissues of C. carpio exposed to diazinon (Oruç and Usta, 2007). Decreased GPx activity in gills, muscle, liver and brain of treated fishes with parathion were also reported, by Monterio et al. (2006).
The SOD enzymes are enzymes that catalyse the dismutation of superoxide into hydrogen peroxide and oxygen whereas CAT catalyzes the decomposition of hydrogen peroxide to water and oxygen. The increased SOD and CAT activities in hepatocytes of fishes exposed to diazinonmight be biochemical responses to over production of superoxide radicals and H 2 2008) reported in rainbow trout exposed to diazinon and methyl parathion a decrease in SOD activities in liver, gills and muscle tissues separately.
Banaee et al., (2012) found that the levels of total antioxidant capacity in hepatocytes of fishes exposed to both concentrations of diazinon were significantly decreased. Similar results were observed in carps exposed to sub-lethal concentrations of cyfluthrin (Sepici-Dinçel et al., 2009). The overproduction of free radicals during pesticide detoxification may be associated with the decrease in the hepatic total antioxidant capacity (Monterio et al., 2006). Impairment in the synthesis of enzymatic and non-enzymatic antioxidant may be the most important factor in reducing levels of cellular total antioxidant. Therefore, the decline in the hepatic total antioxidant levels make the fish cells more vulnerable to oxidative stress damage.
Glutathione S-transferases (GSTs), a family of cytosolic multifunctional enzymes, are detoxifying enzymes that are present in different tissues of fish. They catalyze the conjugation of glutathione with a variety of reactive electrophilic compounds, thereby neutralizing their active electrophilic sites and subsequently making the parent compound more water soluble. For example, the toxicity of diazinon can be decreased by the action of carboxylesterase enzyme which catalyses the hydrolytic degeneration of diazinon and by the action of glutathione Stransferase which catalyses the formation of excrete-able conjugate (Keizer et al., 1995). In addition to catalytic functions, the GSTs can also bind covalently/non-covalently to a wide number of hydrophobic compounds, such as insecticides.
Thioltransferase catalyzes the reversible thiol-disulfide interchange reactions. The enzyme has a major role in maintaining intracellular thiol in the reduced state and functions in this capacity by coupling to glutathione and glutathione reductase. Thioltransferase also has a role in the cellular regulation by catalyzing the reversible modification of proteins by thiol-disulfide interchange (Bernstein et al., 1982).
Xanthine oxidase (XOD) is an essential enzyme that converts hypoxanthine to xanthine, subsequent to uric acid. This enzyme contains FAD, molybdenum and Iron are exclusively found in liver, intestine and little amount in other tissues of animals (Sathyanarayana, 2005) also stated XOD played a vital role in transformation of toxic ammonia into nontoxic uric acid. Xanthine oxidase produces hydrogen peroxide which is very dangerous to the animal, and then it converts into HO and O 2 . Further, the uric acid may act as an antioxidant and free radical scavenger protects the cells from oxidative damage (Sheehan et al., 2001; Guskovet al., 2002). Naveed and Janaiah (2011) reported that the reduction in XOD activity in liver of fish, Channa punctatus exposed to triazophos leads to increase in cellular damage and may be due to nonavailability of Iron to the fish during toxic period.

Hematological parameters
The main hematological parameters in fish including red blood cell counts (RBC), hematocrit (Ht), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) may be influenced by intrinsic and externalfactors. Toxicology studies show that the disruptive action of different insecticides on the erythropoietic tissue such as kidney and spleen may decrease erythrocyte number and hemoglobin content as an anemic sign, and even lead to death of fish. A low red cell or hemoglobin count indicates anemia, or severe bleeding. Low hemoglobin usually means the animal has anemia. Anemia results from conditions that decrease the number or size of red cells, such as excessive bleeding, a dietary deficiency, destruction of cells because of a transfusion reaction or mechanical heart valve, or abnormality formed hemoglobin (Hisa and Connie, 1998). Decreases in the number or size of red cells also decrease the amount of space they occupy, resulting in a lower hematocrit. A low hematocit, combined with abnormal blood tests, confirms the diagnosis. Decreased erythrocyte count and haemoglobin content in freshwater fish Channa punctatus, (Anees, 1978) and A low white blood cell count may mean dysfunction in hematological tissues (spleen and kidney) or certain infectious diseases. Lower than normal levels of lymphocytes (lymphopenia) can be an indicator of immune system deficiency.
Poisonous substances treatments can also deplete the body's supply of lymphocytes, as can exposure to diazinon. Lymphopenia as a consequence of methy-parathion based pesticide was reported by Nath and Banerjee (1996) in Heteropneustes fossilis and also by Siwicki et al.,

Blood biochemical parameters
Measurement of blood biochemical parameters are used as important diagnostic tool for the detection of abnormalities in the liver and other tissues . Liver serves to protect the body from potentially harmful ingredients absorbed from the intestinal tract, gills, and skin as well as toxic by-products of metabolism by various mechanisms that previously described.
The significant differences in glucose concentrations in plasma between the control and treatment fish, following the action of different insecticides, which may be considered to be the manifestation of stress . In agreement with our results, Ceron, et al., Glucose increase is a general response of fish to acute pollutant effects, including organophosphates insecticides (Banaee, 2012). Plasma glucose was elevated in treated silver catfish Rhamdia quelen after all periods of clomazone exposure (Lazzari, et al., 2006).
Albumin and globulin make up most of the protein within the body and are measured in the total protein of the plasma. Total protein, albumin and globulin tests are used to monitor the course of diseases in immune disorders, liver dysfunction and impaired kidney activity . According to the test results, levels of total protein, albumin and globulin were decreased in fish exposed to diazinon. Decreased total protein levels may be due to starvation, malnutrition and chronic liver diseases . Other authors also found that the levels of total protein and albumin are decreased in the fish exposed to different pollutants and insecticides (Vijayan et  Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are found in the liver, heart, skeletal muscle, kidney, pancreas, spleen, erythrocyte, brain and gills . When diseases or injuries affect these tissues, the cells are destroyed and these enzymes are released into plasma. Keizer et al (1995) showed that diazinon in fish liver can be metabolized to diazoxon and other metabolites including pyrimidinol by cytochrome P450 monoxygenase and during this process, reactive oxygen species (ROS) are generated. The increase in intracellular levels of ROS may lead to lipid peroxidation resulting in an increased permeability of liver cell membrane. As a result, liver enzymes including AST and ALT are released into plasma. Similarly, ROS produced from the metabolism of diazinon may damage other tissues such as gills, muscle, heart, kidney and spleen causing the leakage of enzymes into plasma. In this sense, if the cellular injury is chronic AST and ALT levels will remain elevated ( Lactate dehydrogenase (LDH) is an enzyme found in almost all body tissues, such as heart, kidneys, liver, skeletal muscle, brain, erythrocyte and gills . LDH measurement is used to detect tissue disorders and as an aid in the diagnosis of tissue damage . Although a significant decrease in LDH content in plasma was observed in fish exposed to diazinon on 7th day, LDH activity was not significantly reduced on 14th and 28th days. Our data on 7 days after initial experiment are in accordance with several reports that revealed decreased LDH activity in tissues under various pesticide toxicity conditions in different fishes such as O. mossambicus (Saha and Kaviraj, 2009)  Creatine kinase (CK) is an enzyme found in muscle, heart, gills and brain . Although, fish exposed to higher dose of diazinon exhibited higher CK activities in plasma on 7th day, plasma CK concentrations resumed to its normal levels or even a reduced activity on Creatinine is a breakdown product of CK in muscle. The creatinine test has been usually used to diagnose impaired kidney function and to detect renal damage (Toffaletti and McDonnell, 2008; Banaee et al., 2011) However, the lack of changes in creatinine levels are in accordance with the previous results obtained by Gilbert et al (1989) and  which reported that plasma creatinine was usually not an accurate biomarker to distinguish dysfunction in kidney tissue. The creatinine test has been usually used to diagnose impaired kidney function and to detect renal damage (Toffaletti and McDonnell, 2008).
The alkaline phosphatase (ALP) plays a significant role in phosphate hydrolysis and in membrane transport as well as is a good bio-indicator of stress in biological systems. The importance of measuring alkaline phosphatase is to check the liver dysfunction (Banaee et al., 2011) and the cellular membrane health. Alkaline phosphatase (ALP) is an enzyme found in different body tissues; in liver, is produced by the cells lining the small bile ducts (Agrahari et al., 2007). Increased activity of ALP in blood plasma on 14th day may be related to hepatic tissue damage and dysfunction due to pesticide toxicity; the elevation in ALP activity on may be due to an increase in transphosphorylation activity (Sharma, 1990). Agrahari et al. (2007) found increased levels of ALP in plasma of Channa punctatus exposed to monocrotophos. These changes in ALP activities in plasma were earlier reported in tilapia (Rao, 2006) and in common carp . The return of ALP activity to basal values could indicate a fish adaptation to the diazinon treatment at sub-lethal concentrations.
Acid phosphatase plays an important role in carbohydrate metabolism. This enzyme can be found inside the membrane of lysosomes. So, any damage to the membrane of lysosomes can cause the release of this enzyme into muscle and increase its levels.

Immune system
The immune system of fish is important for defense against a variety of pathogens. The system is very sensitive to homeostatic adjustments via endocrine regulation and is influenced by the biochemical status of the nervous system. Thus, any impairment in the nervous system and disturbance in the biochemical homeostasis can weaken the immune system of fish.
Different insecticides at sub-lethal levels have been recognized as stressors causing immune-suppression in fish (Werner and Oram, 2008). In addition, some insecticides may exert immunotoxic effects by altering the transcription of important mediators of the

Metabolism and growth
The change from anabolic to catabolic processes in fish exposed to various insecticides involves important alterations in intermediary metabolism and has important consequences for somatic growth. Changing levels of stress hormones -catecholamine and corticosteroids -, alterations in the ability of digestive enzyme biosynthesis and behavioral changes -hunter and forage behavior -are main factors influencing on the metabolism and growth of fish. For example, histopathological damage to liver, pancreas or hepato-pancreas and intestine can also reduce efficiency of food digestion in fish. Because, these tissues have critical role in regulation biochemical parameters, especially proteins, lipids and carbohydrates, hormones as well as in synthesis and secretion of digestive enzymes, are a target organs for toxicity of insecticides. So, the effect of insecticides on these organs can change metabolism of proteins, lipids and carbohydrates. The purpose of this section of the chapter is to describe briefly the metabolic activity known to be sensitive to various forms of insecticides toxicity. However, it has refused to more descriptions, because of breadth of the topic.
The most important factors decreasing fish growth consist of disorder in feeding behaviors, decrease in feeding rate, dysfunction in metabolism process and waste of energy to overcome the stress caused by insecticide exposure (Tripathi et al., 2003). For example, disorder in the metabolism of carbohydrates, proteins and lipids in various tissues, particularly liver of fish exposed to insecticides, may reduce their growth rates. Begum (2004) found out that protein and carbohydrate metabolism in the liver and muscle tissue is disrupted on the exposure to a carbofuran insecticide. In addition, exposure during embryonic or larval stage can result in behavioral abnormalities, such as decreased ability to capture prey after hatching, functional deficiencies or slowing of growth and finally death (

Carbohydrate metabolism
Most researchers have reported that the increased blood glucose is usually observed in fish under undesirable conditions and it helps the animal by providing energy substrates to vital organs to cope with the increased energy demand . Elevation of blood glucose levels was widely used as a secondary marker of a stress response (Toal et al., 2004). On the basis of our literature review it is clear that insecticides can be acted as a stressor in fish. Because, hyperglycemia has been reported in many fish exposed to different insecticides. For example, increases in blood glucose levels have been reported in Heteropneustes fossilis (Saha and Kaviraj, 2009)  Glycogenesis, glycogenolysis, glycolysis and gluconeogenesis are processes that play important roles in regulating blood glucose and carbohydrate metabolism. Glycogenesis is the process of glycogen synthesis from glucose molecules, in which various enzymes such as involved. Hexokinase (HK), glucokinase, and glycogen synthase are key enzyme in the glycogenesis process. Studies show that interfere with activity of these enzymes associated with insecticides exposure of organisms can inhibit glycogen stored in liver (Rezg et al., 2006). Reduced glycogen contents in live and muscle of fish have been reported by some authors.
Glycogenolysis is the catabolism of glycogen which leads to breakdown of glycogen to glucose and catalyzed by enzymes glycogen phosphorylase (GP), phosphoglucomutase (PGM), and glucose 6-phosphotase. Depletion of glycogen content in liver and muscle of fish during exposure to organophosphate insecticides was reported by many researchers (Ghosh, 1987;Nemcsok et al., 1987;Husain and Ansari, 1988).
In fish, catecholamine and corticosteroid hormones have an important role to promote gluconeogenesis. Cortisol is possible through increased levels activity of liver enzyme such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and tyrosine aminotransferase (TAT) have a stimulatory effect on gluconeogenic mechanism.Since in the gluconeogenesis process, glucose is produced from otherorganic molecules like pyruvate, lactate, glycerol, and aminoacids; elevated liver enzyme activity have an important role in the progress of the gluconeogenesis process. Although, pyruvate carboxylase, phosphoenolpyruvatecarboxykinase (PEPCK), fructose 1,6bis-phosphatase, andglucose-6-phosphatase are key enzymes of this process, there are a limit information about their activities in the gluconeogenesis process in fish exposed to insecticides.
Glycolysis is a cascade of biochemical reactions by which a molecule of glucose is oxidized to two molecules of pyruvic acid and two high energy electron carrying molecules of NADH. Glycolysis can occur with or without oxygen. In the presence of oxygen, glycolysis is the first stage of cellular respiration. Without oxygen, glycolysis allows cells to make small amounts of ATP. Glycolysis is catalyzed by enzymes such as HK, phosphor-fructokinase (PFK), and lactate dehydrogenase (LDH).
LDH is an enzyme participated in anaerobic pathway of carbohydrate metabolism. The increase of LDH activity is a diagnostic index widely used to recognize increases of anaerobic metabolism resulting from depletion of energy under anaerobic and environmental stress conditions.The increase of LDH activity can be attributed to the conversion of accumulated pyruvate into lactate which is transported through muscle to hepatopancreas and regenerated glucose and glycogen to supply energy fish exposed to insecticides. In other words, the increase of LDH activity in liver and muscle reflects a possible improvement in tissue glycolytic capacity.

Protein metabolism
Proteins are vital ingredient involved in the architecture of the cell, which is the main source of amino acids for building up of new tissues and for the synthesis of biologically important molecules such as enzymes, hormones, etc as well as the source of energy for fish. Alterations in protein content of various tissues of fish exposed to different concentrations of insecticides are linked through a biochemical metabolic pathway. For example, Bose et al. (2011) reported the increase in the protein level in liver of freshwater fish (Oreochromis niloticus) was maybe due to check the influence of thiamethoxam and effort to recover from the stress of insecticide at lower doses. They found when the concentration of thiamethoxam was increased; there was decrease in liver protein level. Thus, reduce a significant portion of protein in different tissues, especially the liver, may have been due to their degradation and possible utilization for metabolic purposes. Increases in free amino acid levels were the result of breakdown of protein for energy and impaired incorporation of amino acids in protein synthesis. Since, free amino acids are used in gluconeogentic pathway to glucose production, reduced levels of protein synthesis in fish exposed to insecticides.
Transaminase play an important role in breakdown of protein to free amino acids which may be used an energy source for glyconegenic pathways or used to synthesis new proteins to repair damaged tissues. So, change in plasma free amino acid levels indicates either an increase or a decrease in protein catabolism or biosynthesis.
Aspartate transaminase (AST), or serum glutamic oxaloacetic transaminase (SGOT) is a pyridoxal phosphate (PLP)-dependent transaminase enzyme. AST catalyzes the reversible transfer of α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. Alanine transaminase or ALT is a transaminaseenzyme. It is also called serum glutamic pyruvic transaminase (SGPT). It catalyzes the transfer of an amino group from alanine to α-ketoglutarate, the products of this reversible transamination reaction being pyruvate and glutamate. Aspartate aminotransferase and alanine aminotransferase are found in the liver, heart, skeletal muscle, kidney, pancreas, spleen, erythrocyte, brain and gills . Tyrosine aminotransferase (or tyrosine transaminase) is an enzyme present in the liver and catalyzes the conversion of tyrosine to 4-hydroxyphenylpyruvate. Alteration in deamination and transamination of amino acid are associated with changes in nitrogen metabolism, which can be detected in terms of plasma nitrogenous metabolite levels. Free amino acid is also contributed in the formation of excretory product by the process of conjugation.
The increase in intracellular levels of ROS can lead to lipid peroxidation resulting in an increased permeability of liver cell membrane. As a result, liver enzymes including AST and ALT are released into plasma. In this sense, if the cellular injury is chronic AST and ALT levels will remain elevated (Srivastava et al., 2004;Rao, 2006). Increased activities of AST and ALT were observed in plasma of Channa punctatus (Agrahari et al., 2007) exposed to organophosphorus pesticides. Banaee et al., (2008) have reported increased levels of AST and ALT followed by the exposure of common carp to diazinon. Similarly, ROS produced from the metabolism of organophosphate insecticide could be damaged other tissues such as gills, muscle, heart, kidney and spleen causing the leakage of enzymes into plasma .
Impact of different insecticides on the hormones involved in the process of protein synthesis can also affect the tissue protein levels. This was in conformity with the effects of dimethoate on C. punctatus (Tripathi et al., 2003). Similar changes in protein content was observed in Labeo rohitha (Ramani, 2001), Etroplus maculatus (Sulekha and Mercy, 2011), Anabas testudineus (Sulekha, 2002; Sulekha and Mercy, 2009) exposed to monocrotophos and phosphamidon. Histological damage to various tissues, especially the liver tissue, caused by exposure to insecticides can also decrease the tissue's ability to synthesis protein. For example, Murray et al., (2003) announced that organophosphate insecticides through methylation and phosphorylation of cellular proteins may lead to a reduction in the reconstruction of necrotic tissues.
In case, due to the presence of unsaturated and sulphuric molecules in the biochemical structure of some amino acids such as phenylalanine, mitonin, cysteine, histidine, and tryptophan that they are sensitive and vulnerable to free radicals and specifically reactive oxygen species (Sureda et al., 2006;Tejada et al., 2007) and it helps breaking the sequence of amino acids, aggregation of amino acid chains and even changing the biochemical structure of amino acids and it leads to proteolytic changes in protein compounds (Stadmann, 1992(Stadmann, , 1993; Asada and Barba, 2004).
Albumin and globulin make up most of the protein within the body and are measured in the total protein of the plasma. Total protein, albumin and globulin tests are used to monitor the course of diseases in immune disorders, liver dysfunction and impaired kidney activity .  showed that the levels of total protein, albumin and globulin were decreased in fish exposed to diazinon. Decreased total protein levels may be due to starvation, malnutrition and chronic liver diseases (Kirby et al., 1995;Martin et al., 2010). Other authors also found that the levels of total protein and albumin are decreased in the fish exposed to different pollutants and pesticides (Vijayan et al., 1997;Velisek et al., 2009).

Lipid metabolism
Lipids play an important role as source of energy for fish. Since, most insecticides are lipophilic compounds, they can easy pass through biological barriers which content lipids and accumulate in fat tissue.Lipids molecules are highly susceptible to oxidative reactions. Due to cell membrane lipid peroxidation of unsaturated fatty acids, short chain fatty acids with R-COOH, R-OOH, R-CHO, and R-OH bases are created which seriously affect the cellular membrane functions such as the activity of hormone receptors and neural mediators, ion transport channels and the activity of membrane enzymes and the transportation of specific molecules. On the other hand, the formation of malondialdehyde (MAD) during peroxidation process of fatty acids having double bonds can create covalent bonds and polymerize cellular membrane components (Sureda et al., 2006;Tejada et al., 2007). In addition, accumulation of fatty acids in the cytosol increased peroxidation of fatty acids in peroxisomes and the endothelial reticulum, resulting in overproduction of ROS and further damage.
Increased levels of stress hormones such as cortisol in blood of fish exposed to various insecticides, stimulates lipid breakdown in adipose tissue. Both elevated and reduced free fatty acids levels in plasma have been observed in different fish species exposed to insecticides. Changes in cholesterol and triglycerides levels in the blood and other tissues such as muscles and liver of fish treated insecticides indicated that effect of these compounds on lipid metabolism.

Growth
From a biochemical point view, ratio of the RNA/DNA can be used as a bio-indicator measure of body growth. Recently, many researchers have focused on the impact of different insecticides on the metabolism of nucleic acid in various tissues of fish (Rathod and Kshirsagar, 2010). Insecticides toxicity indicates change in nucleic acid biosynthesis. Disturbance in the metabolism of nucleic acid can lead to reduction in the RNA content. Also, the deterrent effect of different insecticides such as organophosphate compounds on acid phosphatase and alkaline phosphatase activity in different tissues of fish can also adversely effect on nucleic acid synthesis (Das and Mukherjee, 2000).
Apparently, some insecticides have the potential to inhibit DNA synthesis. For example, the toxicity of dichlorvos has also been related to alterations in DNA replication and chromosome aberration, which causes mutations and cellular hyper-proliferation as a result of local irritation.In fact, insecticides and their metabolites may interfere with the process of DNA synthesis and gene expression by different mechanism. On the other hand, the propagation of malondialdehyde into cells can make the ground for its reaction with nitrogen alkalis of DNA strands (Sureda et al., 2006;Tejada et al., 2007).For example, reduced synthesis of nucleic acids andimpair in the process of proliferation DNA strands as well as inhibition of enzyme activities involved in DNA replication and repair mutations can affect the final product of gene expression. In the other hand, damage to DNA strands caused by oxidative stress and insecticide's metabolites bind to DNA strands (DNA adduct) can lead to impair in the transcription and genes expression. Furthermore, inhibition of DNA synthesis, thus, might affect both protein as well as amino acid levels by decreasing the level of RNA in protein synthesis machinery.

Histopathology
Histopathological investigations on different tissues of exposed fish are useful tools for toxicological studies and monitoring water pollutions. Tissue alterations in fish exposed to a different concentration of insecticides are a functional response of organisms which provides information on the nature of the toxicant. In histopathology, we can provide information about the health and functionality of organs. Tissues injuries and damages in organs can result in the reduced survival, growth and fitness, the low reproductive success or increase of susceptibility to pathological agents.
Reactive oxygen species (ROS) produced during the insecticides detoxification process in liver tissue may react with vital macromolecules such as lipid, protein, carbohydrate and nucleic acid and result in oxidative damage to aquatic organisms (Üner et al., 2006). ROS derived damage to natural and structure cellular components are generally considered as a serious mechanism involved in the histological disorders (Sepici-Dinçel et al., 2009). On the other hand, organophosphate insecticides through methylation and phosphorylation of cellular proteins (Murray et al., 2003) may lead to a reduction in the reconstruction of necrotic tissues. The deter-rent effect of organophosphate insecticides on acid phosphatase and alkaline phosphatase activity in different tissues of fish can also adversely effect on nucleic acid synthesis (Das and Mukherjee, 2000).Frequency and intensity of tissue lesions depend on the concentrations of insecticides and the length of the period fish are exposed to toxins. Nevertheless, many insecticides cause specific or non-specific histopathological damage ( (Cengiz, 2006;Cengiz&Unlu, 2006), fenitrothion .The pathological changes in the different tissues such as gill, liver, kidney and spleen of fish treated with different insecticides can disturb homeostasis and lead to physiological disorders in these animals.

Gill
Fish gills have many important functions including exchange of gases, transport of many mono and divalent ions, excretion of waste nitrogen, and uptake and excretion of various xenobiotics (Zayed and Mohamed, 2004;Evans et al., 2005). Histopathology of gill is the appropriate bioindicator to pollution monitoring. One of the lesions most frequency found on the gills of rainbow trout exposed to 0.1 mg/L diazinon was epithelial hyperplasia of both primary and secondary epithelium, as can be seen in Figure 1. Our results indicated that the major alterations in the gills of rainbow trout exposed to 0.2 mg/L diazinon were edma and epithelial hyperplasia, mucosa cell hyperplasia and fusion of the secondary lamellae. Damage to gill tissue may interfere with gas exchange performance of gill and cause respiratory disorders, ion-regulation and osmoregulation dysfunction and inefficacy of the excretion of waste nitrogen metabolite in exposed fish (Nero et

Liver
Histopathological analysis reported important alterations in liver, including necrosis, and cytoskeleton disarray, changes in nuclear shape and heterochromatin distribution as well as intense damages in Disse's space between hepatocytes and sinusoid vessels. Increased vacuolization of the endothelial cells, morphological derangement and necrosis in the Disse's space were also evidenced in liver fish exposed to diazinon. These results are in accordance with Cattaneo  ). This disturbance in the morphological structure of liver could be associated with a disruption in tissue function, which could be also related to the decreased antioxidant capability in diazinon treated fishes.

Kidney
The head kidney of teleost fish is composed of a variety of cells, including parenchymal cells, lymphoid and hematopoietic tissues (Mela et al., 2007). The functional unit of the kidney is nephron. Morphologically, the nephron of bony fish includes glomerulus, tubules and collecting ducts. In the fish exposed to 0.1 mg/L diazinon, disorientation in the glomerular structure, dilation in the inter space of urinary tubular, cloudy swelling were observed. Histopathological damage in the kidney tissues of fish exposed to 0.2 mg/L diazinon were characterized by degeneration in the epithelial cells of renal tubule, necrosis in the hematopoietic tissue, degeneration of glomerulus, dilation of glomerular capillaries, appearance of vacuoles in cytoplasm epithelial cells of renal tubules with hypertrophied cells and narrowing of the tubular lumen. Increase of ROS production in the diazinon metabolism

Spleen
The spleen has a fibrous capsule, and small trabeculae extend into the parenchyma, which can be divided into a red and white pulp. In rainbow trout, the aggregations of melanomacrophages are less well-defined and lack a capsule, but the association with blood vessels and lymphocytes is maintained (Press and Evensen, 1999). Expansion of red pulp with vascular congestion and a significant deposition of hemosiderin granules in a melano-macrophage center are important histopathological damages observed in spleen of fish exposed to both concentrations of diazinon. One of the typical macroscopic changes associated with diazinon toxicity is enlargement and roughness of the spleen. These results are similar to the 3,4dichloroaniline and captanin effects that have been described in common goby (Pomatoschis tusmicrops) and rainbow trout, respectively (

Digestive tract (intestine)
The intestine of rainbow trout has a mucosa, submucosa, muscularis and serous membrane. The mucosa epithelium has thin and elongated absorptive cells or enterocytes, goblet cells and lymphocytes. Enterocytes are a single layer of columnar cells with apical brush border and basal elongated nucleus with one nucleolus. There are also often lymphocytes at the basal and apical regions of the epithelium. Atrophy and necrosis of mucosal cell, exfoliate of mucosal epithelium, lymphocyte infiltration to lamina propia, reduction in the elastic properties and capillary bleeding in intestine tissue are important histopathological alterations observed in fish exposed to both concentrations of diazinon. Necrosis, degeneration, and accumulation of lymphocyte in lamina propia were observed in the intestine of mosquitofish, G. affinis, exposed to Thiodan and deltamethrin (Cengiz et al., 2001; and Cirrhinus mrigala treated with lambda-cyhalothrin (Velmurugan et al., 2007). This result is similar to the observations by Glover et al. (2007) in Atlantic salmon (S. salar) to dietary endosulfan exposure.

Gonads
Some  The ovaries of C. carpio have shown significant changes on exposure to sub-lethal concentration of diazinon. The oocyte with de-shaped yolk vesicles, vacuolated follicular epithelium and degenerative cytoplasm were reported in fish exposed to diazinon by Banaee et al., 2008.

Conclusion
In conclusion, the present literature review shows that exposure to insecticides, firstly, caused an induction of antioxidant enzyme activities indicating the activation of the insecticides detoxification pathways and the antioxidant defenses. However, acute to chronic insecticides treatment disrupted the behavioral response, alterations hematological, biochemical and im- munological parameters. In addition, the histopathological analysis performed in gills, liver, spleen, kidney, gonads and intestine of fish exposed to different concentration of insecticides reported significant damage in all tissues indicating that it was a useful methodology for monitoring the effects of insecticides on wild and cultured fish. These changes may be potentially disruptive for the survivability of fish in aquaculture farm and natural resources. This fact should be taken into consideration when this insecticide is used for pest control in agriculture fields surrounding surface water and ground water resources.