Impact of ethanol extract of Anamirta cocculus (Linn.) seeds on tissue damage biomarkers of the predatory catfish Heteropneustes fossilis (Bloch.)

ABSTRACT Seeds of the phytopiscicide Anamirta cocculus are used recently to eradicate unwanted fishes from aquaculture ponds during pond preparation. However, the mode of action of A. cocculus and the biochemical responses caused to exposed fishes largely remain unexplained. The present study attempts to assess the impact of 24 h LC50 (18.79 ppm) of the ethanolic extract of A. Cocculus seeds on the tissue damage biomarkers viz., alkaline phosphatase (ALP), acid phosphatase (ACP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of the predatory wild fish Heteropneustes fossilis. The tissues assayed were brain, gills, liver, kidney, muscle, accessory respiratory organs (ARO) and blood. The exposure has caused varying levels of significant reductions in the activities of all these enzymes in various tissues except blood. The activities of serum phosphatases and aminotransferases became significantly higher in the later stages of exposure. The observations clearly indicate the tissue-damaging effects of A. cocculus seed extract.


Introduction
Many civilizations throughout the world have exploited piscicidal plants (ichtyotoxicants) as barbascos for capturing wild fish for human consumption [1,2]. Anamirta cocculus (Linn.) is one such piscicidal plant extensively used by the native tribes and local people in the Indian subcontinent and neighbouring geographical regions for traditional fishing from the wild [3,4]. In addition to their primary use as a fishing agent, reports also indicate the probabilities of utilizing the piscicidal plants in the management of nuisance fishes in aquaculture ponds [5,6]. Eradication of weed fishes and other nuisance organisms such as predatory fishes and insects from culture ponds before stocking the desired species is an important step in aquaculture management. This is significant in view of the high rate of fecundity of weed fishes and voracious carnivorous nature of the predatory ones. While the high fecundity rate of weed fishes leads to severe competition for food, space and dissolved oxygen with the cultured species, predatory species may compete as well as prey upon the desired ones. The difficulty in eliminating air-breathing predatory fishes from even the dewatered culture ponds is mainly attributed to their capacity to survive inside moist cracks, crevices, burrows and even inside the bottom mud [7].
In the absence of safe and effective means to eradicate the unwanted fish fauna in stocking ponds, aqua farmers find it easy to use synthetic chemicals such as malachite green, sodium cyanide and antimycin, and even chemical pesticides to get rid of them and thereby leading to ecological as well as public health concerns [6,[8][9][10]. Use of pesticides and chemicals for the eradication of unwanted fish fauna and insects in fish culture ponds during pond preparation could have long-lasting impacts and may affect non-target organisms as well as human health through food chains and bioaccumulation. Non-target animals including the cultivated fishes are greatly affected by the indiscriminate use of the synthetic piscicidal agents [11]. Therefore, plant origin piscicides are preferred to synthetic ones because of the biodegradation potential and nonbioaccumulative nature of the former [7]. Studies by Jothivel and Paul [7,12] have established the significance and effectiveness of the seeds of A. cocculus as an organic management tool for the eradication of weed and predatory fishes including air-breathing ones.
Since the fish caught by applying A. cocculus seeds are traditionally consumed by humans, it is important to examine the toxic effects of the seed extract on the physiology and biochemistry of fishes as the seeds are reported to contain picrotoxin derivatives [3,13]. The activities of some enzymes like alkaline phosphatase (ALP; E.C., 3.1.3.1), acid phosphatase (ACP; E.C., 3.1.3.2), aspartate aminotransferase (AST; E.C., 2.6.1.1) and alanine aminotransferase (ALT; E.C., 2.6.1.2) are generally used to indicate the physiological impacts of toxicants on biological organisms including fishes. These enzymes are normally found within the cells of many organs including, liver, heart, gills, kidney and muscle but their increased presence in plasma is generally considered as an indication of tissue or organ damage [14][15][16].
ACP is a ubiquitous lysosomal hydrolase that hydrolyses phosphate groups at acidic pH and is widely considered as an indicator of lysosomal integrity and functions as these two parameters are generally altered to a great extent due to the actions of the xenobiotics. As part of the patho-physiological processes, a pronounced change in ACP synthesis occurs during tissue damage or diseases and is usually released by the lysosomes during stress-induced tissue/cell damage [17]. ACP also facilitates transphosphorylation with an important role in the general energetics of the organisms [18] and according to Bull et al. [17], ACP could be diagnostically used as serological and histological biomarkers of tissue damage.
ALP is a homodimeric metabolic enzyme that catalyses the hydrolysis and transphosphorylation of a wide variety of phosphate monoesters in the presence of a phosphate acceptor in alkaline medium. It is an ectoenzyme, attached to the outer face of the plasma membrane through a phosphatidylinositolglycophospholipid anchor covalently attached to the Cterminus of the enzyme [19,20]. ALP is a marker enzyme for the membrane system including endoplasmic reticulum and is usually utilized to assess the integrity of plasma membrane [21,22]. This membrane-bound enzyme is present on all cell membranes where active transport occurs. It is commonly found at bile pole of hepatocytes, in pinocytic vesicle and Golgi complex [23]. Changes in ALP level and activity are reported to be associated with various diseases of liver and bone as well as exposure to xenobiotics [16,24]. Since ALP hydrolyses phosphate monoesters, the enzyme's hyper production could constitute a threat to the life of the cells that are dependent on a variety of phosphate esters for their vital process as it may lead to indiscriminate hydrolysis of phosphate ester metabolites and may affect the transfer of metabolites across the cell membrane [25]. While ACP is a lysosomal enzyme and cellular damage is usually accompanied by an increase in its activity, ALP is generally a brush border enzyme involved in transport and transphosphorylation functions. The serum level of ALP increases under toxic stress due to the collapsing of the membrane-bound enzyme.
ALT and AST are members of the transaminase family of enzymes that are also known as aminotransferases. Aminotransferases or transaminases removes the α-amino groups of most of the L-amino acids and transfer them to the α-carbon atom of α-ketoglutarate once they the reach liver and thereby leaving behind the corresponding α-keto acid along with the amino acid. In short, as the α-amino acid is deaminated, α-ketoglutarate becomes aminated to form L-glutamate. The glutamate then functions as an amino group donor for biosynthetic pathways or excretory pathways that lead to the elimination of nitrogenous waste products [26]. More specifically while ALT catalyzes the transfer of amino groups between L-alanine and glutamate to meet physiological needs, AST catalyzes the transfer of amino and keto groups between alpha-amino acids and alpha-keto acids [27,28]. While ALT is also known as glutamate pyruvate transaminase, AST is also designated as glutamate-oxaloacetate transaminase. ALT as well as AST play critical roles in energy metabolism and are especially important for alternate metabolic pathways in stress situations such as toxicant exposure.
Stress caused by the toxicities of various xenobiotics including plant extracts are expected to cause tissue damages in the vital organs such as liver, kidney, heart and muscle, and the impact can be assessed by biomonitoring the tissue damage biomarkers such as ALP, ACP, ALT and AST [15,29]. Even though reports are available regarding the piscicidal, antimicrobial and biopesticidal status of A. cocculus seeds [7,12,30,31], to the best of our knowledge, studies regarding the impact of this seed extract on the various enzyme biomarkers and thereby an insight into their probable mechanism of action are not available. Therefore efforts have been made in the present study for analysing the impacts of the ethanolic extract of A. cocculus seeds on the activities of the tissue damage biomarkers such as ACP, ALP, AST and ALT in selected tissues of the air-breathing catfish Heteropneustes fossilis.
As energetics of the fish play important roles in mediating the manifestation of piscicidal property of xenobiotics and aminotransferases are key enzymes involved in gluconeogenesis that has vital importance during toxic conditions, the present study may contribute to the database of toxicity of A. cocculus seeds on the metabolism of the exposed fish. Selection of H. fossilis as the test species is based on some distinctive reasons such as (i) it is widely distributed throughout India and in neighbouring geographical areas; (ii) it is a common wild inhabitant in most of the natural fresh water culture ponds; (iii) this species is a sturdy one due to the presence of welldeveloped accessory respiratory organs (ARO) and can easily survive even drought conditions by remaining buried in moist or muddy micro niches available in the ponds and are not easily accessible to the normal methods of mechanical eradication and (iv) even though it is considered as a good table fish, it causes considerable economic loses especially in carp culture systems due to its predatory nature. Therefore it is an unwanted wild fish in many culture systems, which has to be completely eliminated preferably through organic methods before stocking the desired species.

Fish and plant material
Healthy specimens of freshwater catfish H. fossilis (16.5 ± 1.4 cm body length and 40.2 ± 2.1 g body weight) regardless of their sex were collected locally from freshwater ponds in and around Lalpet, Cuddalore District, Tamil Nadu, India. They were immediately transported to the laboratory with oxygen supplementation to reduce stress and acclimated to the laboratory conditions for 30 days in large aquaria bearing well water. The physicochemical parameters of the well water used for acclimation as well as bioassays were determined by following A.P.H.A et al. [32] and are given in Table 1. During the acclimation period, the fish were fed with minced goat liver for a period of 4 h (h) before the renewal of the medium. Water was renewed after every 24 h with routine cleaning of the aquaria leaving no faecal matter, unconsumed food or dead fish (if any). The seeds of A. cocculus were collected from the wild from Kerala state, India and sun-dried. The dried seeds of A. cocculus were broken and the endosperms separated from the shells by a sterilized needle and powdered with the help of an electric grinder. One hundred gram of powdered endosperm was packed in the thimble of Soxhlet apparatus and was extracted with ethanol. The extract was finally concentrated using rotary vacuum evaporator and kept in freezer. The percentage of yield of the ethanolic extract of the powdered endosperm was 35% as already reported by Qadir et al. [31].

Bioassay and tissue sample collection
Before the commencement of the experiment, the 24 h LC 50 of ethanol extract of A. cocculus seeds for H. fossilis was calculated following Finney's [33] method and was found to be 18.79 ppm. In order to study the acute toxic effects of ethanol extract of A. cocculus seeds on H. Fossilis, 4 groups of 10 fish each in triplicate were exposed to the exposure medium containing the ethanol extract (18.79 ppm; 24 h LC 50 ) prepared in well water ( Total hardness 88 ± 1 ppm following 24 h static bioassay system. Each experimental group of 10 fishes were exposed separately to 100 L of the medium. Parallel control groups were also maintained without the addition of the seed extract. Feeding was withheld 24 h prior to the commencement of the experiment in the control as well as experimental groups. Blood and tissue samples were collected from five fish each from the control as well as experimental group after the expiry of 6, 12, 18 and 24 h of the experiment. At each sampling interval, blood sample collections were done from caudal vein following A.F.S. et al. [34]. The fish were sacrificed for collection of tissue samples namely brain, gill, liver, kidney, muscle and ARO. Tissues were quickly dissected out aseptically, rinsed in ice-cold 0.33 M sucrose and blotted dry, weighed, homogenized in ice-cold 0.33 M sucrose solution and 5% cell-free homogenate were prepared and centrifuged at 10,000×g for 20 min at 4°C. The supernatant was stored in a freezer for enzyme assays. Blood samples were collected without any anticoagulant and kept at room temperature for 30 min for separation of serum and were centrifuged (20,000×g for 10 min). The serum was collected and stored in a freezer. Five percent serum samples were used for biochemical assays. Activities of ACP, ALP, ALT and AST were assayed either immediately or within two days.

Estimation of protein and determination of enzyme activities
Protein contents of the samples were determined according to Lowery et al. [35] and the protein contents of the samples were measured in mg/g of tissue.
ALP activity was measured by following the method of Tennis Wood et al. [36]. The reaction medium contained 0.5 mL of 0.4% p-nitro phenyl phosphate substrate together with 0.5 mL glycine buffer (pH 10.5) incubated at 37°C for 5 min. While 0.5 mL of enzyme source (serum or homogenate) was added to the test samples, 0.5 mL of distilled water was added to the blank. The reaction mixture was incubated at 37°C for 30 min and reaction was arrested by adding 10 mL of 0.2 N sodium hydroxide. Controls were also prepared by adding the enzyme source after arresting the reaction. The molar concentration of p-nitrophenol (PNP) was determined by using graded PNP as standard. The reaction product PNP was measured at 415 nm against blank and expressed as µ moles of PNP liberated/minute/mg of protein.
ACP activity was determined by basically following the method of Tennis Wood et al. [36]. The final reaction mixture consisted of 0.5 mL of 0.4% p-nitrophenyl phosphate substrate together with 0.5 mL of 1.0 M citrate buffer (pH 4.85) incubated for 5 min at 37°C. While 0.5 mL of enzyme source (serum or homogenate) was added to the test samples, 0.5 mL of distilled water was added to the blank. The reaction mixture was incubated at 37°C for 30 min and the reaction was arrested by adding 3.8 mL of 0.1 N NaOH. Appropriate controls were also maintained and to them the enzyme source was added after arresting the reaction. A set of graded standards was also prepared in the same manner using PNP. Reaction product PNP was measured at 415 nm against reagent blank and the enzyme activity expressed as µ moles of PNP liberated/minute/mg of protein.
AST was assayed following the method of King [37]. The final reaction mixture consisted of 1 mL of buffered substrate (1.33 g of L-aspartic acid and 15 mg of α-Ketoglutaric acid were dissolved in 20.5 mL of phosphate buffer and 1 N sodium hydroxide to adjust pH 7.5 and made up to 50 mL with phosphate buffer) incubated for 10 min at 37°C to which 0.2 mL of the respective enzyme source was added and incubation continued for 1 h. The reaction was arrested with the addition of 1.0 mL of 2,4-Dinitrophenylhydrazine (DNPH) and kept at room temperature for 20 min. Appropriate controls were also maintained to which enzyme was added after arresting the reaction. Finally, 10 mL of 0.4 N sodium hydroxide solution was added and the colour developed was read at 520 nm against reagent blank. A set of graded standard pyruvate was also treated in the same way. The enzyme activity was expressed as µ moles of pyruvate liberated/minute/mg of protein.
ALT was assayed following the method of King [37]. The reaction mixture consisted of 1 mL of buffered substrate (1.78 g of DL-alanine and 30 mg of α-ketoglutaric acid were dissolved in 20 mL of phosphate buffer at pH 7.5 with 1 N sodium hydroxide and made up to 100 mL with buffer) incubated for 10 min at 37°C to which 0.2 mL of the respective enzyme source was added and incubation continued for 30 min and the reaction was arrested by adding 1.0 mL of DNPH and kept at room temperature for 20 min. To the controls, enzyme was added after arresting the reaction. Colour was developed with the addition of 10 mL of 0.4 N sodium hydroxide. A set of graded standard pyruvate was also prepared in the same way. The colour developed was read at 520 nm against reagent blank. The enzyme activity was expressed as µ moles of pyruvate liberated/minute/mg of protein.

Statistical analysis
Data collected from 5 replicates of enzymatic assays at each sampling interval were subjected to one way analysis of variance followed by Duncan's multiple range test (DMRT). Since there were no significant (p > .05) difference in the activity of these enzymes among the various control groups till the expiry of 24 h, the pooled average of all the control values was taken into account.

Behavioural changes
The immediate responses of the fish in the toxicant medium (24 h LC 50 ) were restlessness and violent swimming movements as if to escape from the xenobiotic. This was followed by respiratory distress with highly increased opercular movement and gulping activity at the water surface. Gradually the exposed fish started to release streaks of mucus from all over the body and exhibited jerky, erratic swimming and lose of balance. Many a time they violently moved in a vertical position with head above the water level. They also showed muscular twitching, tetany and gradually settled down at the bottom and around 50% of them finally died. The dead one turned upside down. No such behavioural changes and mortality were observed in the control groups.
On acute exposure to the seed extract of A. cocculus, H. fossilis also showed marked alterations in the activities of tissue damage biomarker enzymes viz., ALP, ACP, AST and ALT (Tables 2-9).

Effect on ALP and ACP activities
Maximum activities of ALP (2.24 ± 0.02) and ACP (2.36 ± 0.01) were recorded in the liver of the control fish (Tables 3 and 5). Almost all the analysed tissues of the   exposed fish except blood serum showed a decreasing trend of ALP and ACP activities (Tables 2-5 and Figure 1) in comparison to the respective control values. However, the patterns of decrease in the activities of these enzymes were different in different tissues. While the activities of ACP and ALP in gill and liver of the exposed H. fossilis showed significant (p < .05) and continuously decreasing trends, the percentages of their decreases in brain, ARO and muscle were not so prominent in the first half of the experiment (Tables 2-5 and Figure 1(a,b)). However, there were sharp decreases in the activities of ACP and ALP especially in the latter half of the exposure (p < .05) in these tissues of the exposed fish in comparison to the respective controls. Maximum reductions in the activities of ALP (37.60% from control) and ACP (37.75% from control) were observed in kidney and ARO, respectively (Figure 1(a,b)). The sera of the exposed fish showed remarkable resemblance in the activities of both ALP and ACP (Tables 2-5 and Figure 1(a,b)). In both the cases, there were continuous increases in their activities in comparison to the control ones. While in both the cases the increases were not significant (p > .05) in the earlier stages of exposure, the trend became significant (p < .05) after 18 h onwards (Tables 2-5 and Figure 1(a,b)). Another prominent observation is the significant decrease in the activities of these enzymes  (p < .05) in the serum after 24 h when compared to the previous stage (i.e. 18 h), but still remain significantly higher than the respective control levels (Figure 1(a,b)).

Effect on AST and ALT activities
The percentage of activities of AST and ALT in the various tissues of the exposed fish showed a gradual decreasing trend and reaches the minimum activity after 24 h except in the case of blood in comparison to the respective control values (Tables 6-9 and Figure 2(a,b)). Among the tissues, the rates of decreases in the activities of AST and ALT in brain and ARO, were prominently different from other tissues especially in the first half of the experiment (Tables 6-9 and Figure 2(a,b)). However, the enzyme activities were drastically decreased (p < .05) after the expiry of 18 h onwards (Figure 2(a,b)) in comparison to the earlier stages of exposure as well as control tissues. Like ALP and ACP, liver of the control fish recorded the maximum activities of AST (2.48 ± 0.01) as well as ALT (1.80 ± 0.01) also (Tables 2-9). Similarly, maximum percentages of reductions in the activities of these enzymes were also observed in the liver of the exposed fish (54.84 ± 0.01% for AST and 55.56 ± 0.01% for ALT) after 24 h (Figure 2(a,b)).   In the case of the serum of the exposed fishes, the activities of both the enzymes were minimum in the control fish and remained less pronounced in the first half of the experiment (Tables 6-9 and Figure 2(a,b)). However, during the second half, especially after 18 h of exposure, their activities in the sera of the exposed fish increased prominently reaching a maximum of about 126% (p < .05) for AST and 88% (p < .05) for ALT above the respective control values (Figure 2(a,b)).

Discussion
The seeds of A. cocculus reportedly contain sesquiterpene lactones viz., picrotoxin (formed of picrotoxinin and picrotin), methyl picrotoxate, dihydroxy picrotoxinin and picrotoxic acid. Among them, picrotoxin is a neurotoxicant inhibiting gamma-aminobutyric acid (GABA) actions leading to convulsions [13]. The marked variations as observed in the behavioural patterns of the exposed fish clearly indicate the extent and intensity of physiological stress experienced by them. The initial restlessness and increased swimming outbursts are indicators of common avoidance responses by the organisms towards xenobiotics. However, the highly increased intensity of these behavioural alterations culminating in muscular twitching, tetany loss of balance and death are clear indications of neurotoxicity and interferences in the central nervous system (CNS) activity as the picrotoxin is reported to act as a noncompetitive inhibitor of GABA receptor coupled chloride ionophores that mediate postsynaptic inhibition in the CNS of many organisms [13].
The highly pronounced opercular movement and gulping activity could be efforts put forward by the exposed fish to overcome the respiratory distress caused by the seed extract as well as to meet the increased oxygen demand due to the highly overburdened energy requirements. Acute energy crisis could be resulted due to the violent behavioural changes, physiological emergencies as well as the profused secretion of mucus, which is largely composed of glycoproteins and could ultimately lead to depletion in the immediately available carbohydrate stock. The significant alterations in the activities of transaminases as observed in this study also clearly indicate their critical role in the energetics of the exposed fish.
The present study has revealed critical alterations in the activities of ACP, ALP, ALT and AST (Tables 2-9 and Figures 1 and 2) under the toxicity of A. cocculus seed extract. Phosphatases are involved in various physiological activities such as metabolism of phospholipids, phosphoproteins, nucleotides and carbohydrates as well as the synthesis of proteins, oxidative phosphorylation, transport of metabolites, growth and differentiation [19,20,23,26]. They are also used as good indicators of stress conditions in the biological system [18,17,20]. Profiling of phosphatases activities is a commonly used diagnostic tool to assess the toxicity stress of xenobiotics in fishes [29,38].
ALP is involved in the membrane transport and as such is a good indicator of stress conditions in the biological system, including various disease conditions [14,29,39]. The ALP activity has been used as a stress marker to evaluate the effects of environmental toxicants including heavy metals and organic chlorides on a variety of small organic molecules as well as large biomolecules such as DNA and protein [40][41][42]. Therefore measurement of ALP activity for studying the toxicity of A. cocculus seeds seems to be appropriate and the results also indicate that the activity of ALP is significantly altered by A. cocculus toxicity. ALP also has an important role in the permeability of membrane sites in the neurons [43], liver functions [44,45], bone formation [24,46], hydrolysis and transphosphorylation of a wide variety of phosphate monoesters [42,47] etc. Therefore variations observed in the activities of ALP of the present study have the potential to disturb the normal functioning of the cells due to the toxic stress of A. cocculus seeds. The variations in the ALP titre observed in the present study could be related to the gradual physiological changes induced by the seed extract as the circulating levels of ALP vary significantly only in the latter half of the exposure. Such variations in ALP activity in catfishes have been reportedly brought about by other piscicidal plants also [38,48].
Even though the main sources of ALP are liver and bone [49], it is also produced by other tissues such as kidneys, intestine, leukocytes and placenta [50]. The present study has also revealed the non-hepatic origin of ALP from various tissues such as muscle, kidney, gills, ARO and brain. The significant decrease in the ALP titre of the various tissues and its increase in the serum along with the other tissue damage biomarkers in the exposed fish clearly suggest the possibility of necrosis and lability of the membrane system including plasma membrane. Its increased presence in the serum along with the reduced titres in the various organs of the exposed fish (Tables 2 and 3) especially in the latter half of the experiment indicates damage to the membrane system and leakage into the circulation. This finding is important in view of the reports that ALP is a membranebound enzyme and is engaged in the act of dephosphorylation of a variety of molecules throughout the body and its alteration is likely to affect the membrane permeability leading to derangements in the transport of metabolites across the membrane system [18,50,51]. Various authors have also reported increased leakage of tissue damage biomarker enzymes including ALP into the blood stream following cellular damage, organ dysfunction and altered membrane permeability after administration of plant extracts [14,15,29,52]. According to them, the increased presence of ALP would also be a threat to the integrity of the cells as it may lead to indiscriminate hydrolysis of phosphate ester containing metabolites or organelles and would adversely affect the facilitation of transfer of metabolites across the cell membrane leading to a chain of biochemical events. In the light of this report, the highly increased presence of ALP in the serum of the exposed fish of the present study (Tables 2 and 3 and Figure 1(a)) is also detrimental to the vital physiological process of the fish as it may trigger a cascade of toxicity induced alterations. The collapse of membrane permeability leading to impaired transport of metabolites in the present study could have also contributed to the overall retardation of the energy production and ultimate death of the exposed fish as the organism is under increased energy demand to withstand the toxic stress.
ACP is considered as an inducible enzyme because its activity usually goes up when there is a toxic impact and the enzyme begins to counteract the toxic effect. Subsequently, the enzyme activity may begin to drop either as a result of having partially or fully encountered the toxicity or as a result of cell damage and the collapse of the system [29,53]. However, the results of the present study are not exactly in agreement with these reports as the enzyme activity never increased above the control level except in the serum throughout the study. This may be attributed to the increased toxicity of the extract as it might not be either giving a chance of counteraction by the enzyme or may be interfering with the enzyme synthesis. However, the second half of the experiment was more or less in agreement with these reports [29,53] indicating cell damage in various tissues and collapse of the organ system leading to leakage of enzymes to circulatory system. According to Lowe et al. [54], alterations in the lysosomal membrane permeability can have severe consequences such as leakage of hydrolytic enzymes including ACP, which could have a detrimental effect on the cell. Such destructive impacts on the membrane systems might be causing the leakage of other intracellular enzymes also.
In the present study, the significantly increased presence of ACP in the serum, especially in the later phases of exposure along with the decreased enzyme titre in the tissues (Tables 4 and 5 and Figure 1(b)), indicates tissue damage. This is because of the fact that the expression of lysosomal enzymes requires membrane damage which is usually referred to as lability [55] and ACP is one such enzyme that requires lysosomal lability for its expression. Lysosomal lability is usually accompanied by autolytic degradation of the tissue causing leakage of the enzymes into circulation [53].
The present study clearly revealed the presence of AST and ALT in all the analysed tissues. However, their quantities varied significantly among the tissues (Tables 6-9). AST and ALT titres in the various tissues except serum also exhibited varying percentages of significant reductions at different intervals of exposure to the extract in comparison to the respective control tissues (Figure 2(a,b)). Therefore it could be accrued that rather than restricting to liver/heart, reductions in ALT-AST titre in the respective tissue could be considered as indicators of damage or trauma caused by A. cocculus seed extract. Aminotransferases, in general, have critical roles in the degradation of amino acids by retaining and transferring the amino groups. This is very important in maintaining the intracellular amino acid pool and initiating the gluconeogenesis. In this context, the highly decreased levels of AST and ALT in the respective tissues upon continued exposure in the present study clearly indicate the adverse impact of A. cocculus seed extract on the amino acid metabolism as well as gluconeogenesis. Under stress conditions, transamination and deamination processes take place at an increasing rate and AST and ALT activities may be elevated to meet the increased energy demands caused by the toxicant by facilitating increased gluconeogenesis [56]. Reports of Tiwari and Singh [21,57] also underscore such a possibility of gluconeogenesis under toxic stress. However, the significantly increased activities of serum ALT and AST in the present study may not be attributed to increased secretion of these enzymes to meet the new physiological need arised due to A. cocculus toxicity. Rather, the increased serum enzyme activities after exposure to A. cocculus extract should be viewed in comparison to the level of enzyme activities in other tissues of the exposed fish and thereby indicating the possibility of severe tissue damage and leakage of enzymes to circulatory system.
Although ALT and AST are grouped under tissue damage biomarkers, ALT is generally regarded as more liver-specific and AST as heart/muscle-specific enzyme [58]. As ALT is produced mainly in hepatocytes, it is present in large quantity in the liver. Even though lesser in quantity, it is present in other tissues also [50]. The present study is also in confirmation with this report.
On the other hand, in the present study AST is found in comparatively more quantities than ALT in many tissues (Tables 6-9). Other reports also indicate the presence of comparatively higher amounts of AST in various normal tissues in addition to the liver [28]. Such an increased presence in various tissues attributes more metabolic roles to AST in those tissues when compared to ALT.
Only low levels of ALT and AST are normally found in the blood. This is mainly because of the integrity of the membrane systems of the cells and cytosolic origin of these enzymes. However, as cell membrane is the first line defence against xenobiotics, it becomes the first site of exposure also. Therefore, due to toxic exposures, the integrity of the membrane systems gets easily damaged and their permeability gets altered drastically leading to the leakage of the cytosolic enzymes into the circulatory system. In the present study, even though there is a simultaneous decrease in the activities of other enzymes such as ALP and ACP in various tissues, a concomitant significant increase in their serum titre could not be seen especially in the first half of the experiment (Tables 2-5 and Figure 1). Therefore, the decreases in the enzyme titres in the various tissues of the present study especially in the earlier stages of exposure could not be primarily attributed to membrane system damage alone. Rather than such a probability, the phenomenon may be attributed to the seed extract mediated inhibition of these enzymes in the exposed fish. Other investigators have also suggested the possibility of extracts of piscicidal plants exposure induced inhibition of enzyme systems [15,45]. However, it is worth mentioning that later stages of exposure witnessed significant decreases in enzyme titres in the various tissues of the exposed fish along with concomitant increases in the serum levels of these enzymes (p < .05; Tables 2-9 and Figures 1 and 2). Such progressive reductions in ALP, ACP, AST and ALT activities in various tissues might have been due to breakdown of the synthetic machinery as well as leakage of these enzymes from cytosol due to the toxicity induced damages to the membrane system of the cells of the tissues. Reports in favour of necrosis, cellular damage and loss of functional integrity of membrane architecture resulting in the increased release of these enzymes from intracellular locations to the blood are also available [29,59]. Even though xenobiotics delivered to the liver are meant to be metabolized and excreted, the process itself frequently leads to liver injury and cell membrane damage leading to the release of a variety of enzymes including AST and ALT into the blood stream [60].
From the percentage of reductions of ALP, ACP, AST and ALT activities of the various tissues (Figures 1 and 2) of the exposed fish, it may be noted that even though there is some uniformity in the pattern of reductions, the extent of reduction in different tissues are not same. This is evident from the fact that the reductions in the enzyme titres in brain and ARO, especially in the first half of the experiment (up to 12 h), are significantly less than those of liver, muscle, kidneys and gills. However in contrast to this, there are drastic reductions in both the tissues after 18 h onwards. Even though the exact reason for such inter tissue variations are not clear, it may be well correlated to the physiological roles of these two tissues especially in the survival of the organism. The brain being the master controlling unit of the nervous system, the organism as a hole and the organ as a unit might be excelling to the maximum possible limit to counter the toxicity and keep its physiological functions intact to the optimum. Given the facts that brain constantly make use of fresh glucose as the energy source [61,62] and AST and ALT are involved in the gluconeogenesis that is important under toxic stress; naturally, explain to some extent the reason for maintaining comparatively near normal activities of these enzymes in the brain up to 12 h of exposure. This would permit the central nervous system to function to the optimum possible. However, due to the continued exposure, the fragile physiological defence might be collapsing, leading to the drastic reductions in the enzyme titres in the brain tissue from 18 h of the experiment onwards.
Similarly in the case of ARO also, the near-normal activities of the enzymes up to 12 h of exposure (Tables 2-9 and Figures 1 and 2) could be attributed to the high physiological significance of the organ. Even though gill and ARO do the same physiological function viz., respiration, it is the ARO, that is involved in aerial respiration and provides the major share of oxygen supply especially during stress conditions [63,64]. Therefore during the earlier periods of exposure, when other organs exhibit marked decreases in the activities of ALP, ACP, AST and ALT; ARO along with brain exhibits near normal activities of these enzymes and thereby may be trying to keep up the delivery of the oxygen supply. On the other hand, the marked decreases in the enzyme profiles of gill in comparison to ARO may be attributed to various reasons such as its proximity to the toxicant, vulnerable histological architecture etc. While the exposed nature of gills to the ambient water medium make it highly vulnerable to the impact of toxicity of the extract, the ARO remain deeply embedded in the body myotomes [65] and are not directly exposed to the toxicant as they are involved only in aerial respiration. Similarly, the highly branched architecture of the gills resulting in the increased surface area and its single layered respiratory epithelium along with its highly vascular nature [66] even though make aquatic respiration more effective, equally make the organ susceptible to the dissolved toxicants also. Therefore the peculiarities in the morphology and physiology of ARO could be at least partially responsible for the manifestation of comparatively less toxicity in the earlier stages of the exposure. However, in the later half, due to the via media (circulatory system) effect, the tissues of ARO might also be coming under severe toxic stress leading to the collapsing of the membrane systems of its cells as evidenced by the sharp decrease in the enzyme profiles (Tables 2-9 and Figures 1 and 2). Therefore release of these enzymes into the extracellular spaces following tissue damages might be resulting in their simultaneous detectable increase in the serum as reported by various workers [59,67].
Under the toxic stress of A. cocculus a variety of toxico-pathological alterations including tissue damage may be taking place and in the ongoing process, the organism needs a higher share of energy to maintain the physiological activities especially in the brain as well as ARO. This may be one of the reasons for the maintenance of AST and ALT activities in the near normal level in these organs especially up to 12 h of exposure as observed in the present study. This view is in accordance with the reports that transaminases play an important role at the junction between the carbohydrate and protein metabolism by interconverting the strategic compounds viz., ketoglutarate, pyruvate and oxaloacetate on one hand and alanine, aspartate and glutamate on the other hand [14,21,57]. Even though the increased presence of ALT and AST in serum is generally attributed to hepatic damages, Giboney [68] has reported that elevation of ALT and AST should not be considered exclusively as an indicator of liver pathology alone. The results of the present study are also in agreement with this report. Significant reductions in the AST and ALT levels of various tissues (Tables 6-9) clearly demonstrate it as an indicator of non-hepatic damages also.
The present study clearly indicates the relationship between the impact of A. cocculus toxicity and the duration of exposure as well as the morpho-physiological status of the concerned organ/tissue. The initial periods of exposure might have either caused the instantaneous utilization of the enzymes to defend the toxicity caused by the seed extract or dysfunction of the cellular machinery resulting in the reduction/inhibition of synthesis of these enzymes and might not be causing extensive tissue damage. As a result, in the initial stages of exposure, the enzyme titres of the various organs show a gradual decreasing trend without a corresponding sharp increase in the serum titres. In other words, tissue damage is not much prominent in the earlier periods of exposure and as a result leakage from the tissues might be minimum (Figures 1 and 2). However during later half of the experiment, the toxicity of the seed extract might have caused serious cellular damages leading to disturbed morpho-physiological integrity of the organs. Therefore it may be assumed that the toxicity of A. cocculus seed is proportional to the period of exposure under given conditions and concentration. On the other hand, the morpho-physiological state of the organ also has a stake in the manifestation of A. cocculus toxicity. This is very clearly indicated by the non-significant decreases in the enzyme titres of brain and ARO especially during the first half of the exposure unlike most of the other tissues (Tables 2-9 and Figures 1 and 2).

Conclusions
The toxicity of Anamirta cocculus seeds has brought about biochemical alterations in various tissues of Heteropneustes fossilis. This biochemical manifestations of the toxicity are time dependant at the given exposure concentration. The ethanol extract of A. cocculus seeds significantly altered the phosphatases and transaminases activities in various tissues of H. fossilis. The present study has witnessed significant reductions in the activities of ALP, ACP, AST and ALT in various tissues such as brain, gills, liver, kidney, muscle and ARO of H. fossilis at various stages of exposure. However the serum levels of all these enzymes showed an increasing trend and became significantly higher especially after 12 h onwards, may be due to the seepage of these enzymes from the damaged tissues. Therefore the study demonstrates the ability of these enzymes to indicate tissue damages caused by the toxic stress of A. cocculus seeds. Further, present study also indicates the impact of A. cocculus seed extract on the metabolism of the exposed fish especially on gluconeogenesis through its actions on aminotransferase activities. It may be concluded that when body tissue is damaged, additional ALP, ACP, AST and ALT are released into the blood stream leading to a surge in their serum titre as observed in the present study. Such an emptying of enzyme due to tissue damage is again indicated by the simultaneous reduction of the enzyme content especially in gills, liver, kidney and muscle in the first half of the exposure itself. The pattern is followed by brain and ARO in the latter half. This may be due to their morpho-physiological status in comparison to the other tissues investigated. The present investigation may be useful to understand some basic mechanism of tissue damage in H. fossilis exposed to the phytopiscicide viz., ethanolic seed extract of A. cocculus that may be used as an aqua cultural management tool to control unwanted fish fauna in the culture ponds before stocking. Further efforts are needed to isolate and characterize the compound responsible for potential piscicidal activity of the ethanol extract of A. cocculus seeds.