Effect of arsenic (As) and lead (Pb) on glycogen content and on the activities of selected enzymes involved in carbohydrate metabolism in freshwater catfish, Heteropneustes fossilis

Heavy metals show a wide range of effect on fishes, out of which arsenic (As) and lead (Pb) are among the leading heavy metal toxicants. These heavy metals are known to alter different biochemical parameters, including glycogen level, in different tissues of fishes. Glycogen level in fish serves as the main source of energy; hence, in this study, the acute toxicity test of As and Pb and their effect on the glycogen content and the enzymes involved therein (glycogen phosphorylase, glycogen synthase, hexokinase, phosphofructokinase and pyruvate kinase) were studied in the liver and muscle tissues of Heteropneustes fossilis. The 96 h LC50 values of As2O3 and PbCl2 on H. fossilis were found to be 35.09 ppm and 66.20 ppm, respectively. On acute exposure to 96 h LC50 values of As2O3 and PbCl2, the glycogen concentration showed a gradual decrease in both liver and muscle tissues of H. fossilis. However, on chronic exposure (LC50/20th ppm), the glycogen content in liver and muscle of H. fossilis was depleted till 20 days; whereas after 30 days, the glycogen level was recovered in both the tissues. The activities of glycogen metabolic enzymes (glycogen phosphorylase and glycogen synthase) and few selected glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) were also altered in H. fossilis when exposed to acute and chronic concentration of As2O3 and PbCl2. Our present results showed that As and Pb induced toxicity stress on the catfish, H. fossilis, which might have caused to alter the carbohydrate metabolism in the fish.


Introduction
Freshwater gets contaminated with different pollutants and is a matter of concern (Vinodhini and Narayanan 2009). Among the various water pollutants, heavy metals pose a great threat to fishes. The natural aquatic systems are contaminated with heavy metals released from domestic, industrial, and other man-made activities (Velez and Montoro 1998). These heavy metals cause the greatest threat to the health of the aquatic ecosystem (Ohe et al. 2004;Govind and Madhuri 2014), as well as human health (Wennberg 1994). Arsenic (As) and lead (Pb) are recognized as the leading toxicants worldwide (Akhtar et al. 2013), and these heavy metals are accumulated in different tissues, and the rate of their accumulation is in the order of muscle [ liver [ gill for As, and of gill [ liver [ muscle for Pb (Thang et al. 2017). These heavy metals cause serious damage to aquatic life, including fishes (Karbassi et al. 2006).
Glycogen is the main source of energy for animals and its content in the liver and muscle indicates the health of the fish. The rate of glycogen synthesis is a function of the relative activities of glycogen synthase (GSase) and glycogen phosphorylase (GPase). Both GPase and GSase exist in two interconvertible forms, active (a) and less active (b). GPase a catalyzes the rate-limiting step of glycogenolysis, and control glycogenesis (Connett and Sahlin 1996). Both As and Pb decrease the level of glycogen in Oreochromis mossambicus, and Labeo rohita (James et al. 1996;Aruljothi et al. 2013), and a higher concentration of As induces glycogenolysis in the liver of Channa punctatus (Haque et al. 2009). Glycogenolysis is catalysed by As in rabbit muscle by producing unstable glucose-1-arsenate (Klein et al. 1982); whereas, Pb decreases the activity of GSase in rat liver, thus decreases the glycogen content (Hacker et al. 1990).
Glucose is oxidized via the glycolytic pathway for the production of ATPs. Glycolytic pathway is inhibited by As and Pb by inhibiting hexokinase (Lagunas 1980;Yun and Hoyer 2000) and pyruvate kinase activity (Lepper et al. 2010) in a rat model. The sub-lethal exposure of Pb decreases the activity of phosphofructokinase activity in the diencephalon, cerebrum, medulla oblongata, and cerebellum of L. rohita, Clarias batrachus and C. punctatus (Raibole et al. 2013).
The Indian catfish, Heteropneustes fossilis (Bloch, 1794), is one of the most economically important food fishes in the Indian subcontinent and Southeast Asian region. This is primarily a fish in ponds, ditches, beels, swamps, and marshes, but sometimes found in muddy rivers (Jha and Rayamajhi 2010;Froese and Pauly 2018). It is hardy and amenable to a high stocking rate and utilizes atmospheric oxygen for respiration (Vijaykumar et al. 1998;Haniffa and Sridhar 2002). H. fossilis is an exceptionally sturdy and long-lived species; therefore, it served as the best model of study. Hence, in the present study, the effect of As and Pb (both acute and chronic concentrations) on glycogen content and the activities of selected enzymes involved in glycogen metabolism (GPase and GSase) and glucose oxidation (hexokinase, phosphofructokinase, and pyruvate kinase) in both liver and muscle tissues of H. fossilis were investigated.

Fishes
The experimental fish, H. fossilis, with an average weight of 25 ± 2 g and length of 15 ± 2 cm, were procured from commercial sources and acclimatized to laboratory conditions in dechlorinated water for 15 days in a plastic tank. The acclimatized fishes were then used for subsequent experiments.

LC 50 determination
Toxicity tests were performed in accordance to the standard methods mentioned in the Organization for Economic Co-operation and Development (OECD 1992). These tests were carried out in plastic tanks containing 8 L of water and 10 fishes for each group. Following the preliminary screening, different concentrations of As 2 O 3 (25 ppm to 50 ppm) and PbCl 2 (50 ppm to 100 ppm) were used for bioassay with an independent simultaneous maintenance of control. Feeding was stopped 24 h prior to the treatment and during the experiment. The fishes were exposed to different concentrations of As 2 O 3 and PbCl 2 for 96 h, and the mortality percentage was recorded at 24 h, 48 h, 72 h and 96 h post exposure. LC 50 values for As and Pb were calculated by Probit analysis method using Statistical Package for the Social Sciences (SPSS) 17.0 software.
The fishes were then treated with both acute (35.09 ppm As 2 O 3 and 66.20 ppm PbCl 2 ) and chronic (LC 50 / 20th ppm of both As 2 O 3 and PbCl 2 ) concentrations of As and Pb for the estimation of glycogen content and to study the activities of the selected enzymes (glycogen phosphorylase, glycogen synthase, hexokinase, phosphofructokinase and pyruvate kinase) of H. fossilis.

Estimation of glycogen content
The glycogen content in different tissues of H. fossilis was estimated following the method as described by Seifter et al. (1949). In brief, 1 g of fish tissues was taken in 3 ml of 30% KOH solution and boiled for 20 min. After adding 2 ml of saturated Na 2 SO 4 and 3 ml of 95% ethanol, the homogenate was boiled for 20 min and centrifuged at 3000 g for 5 min. The pellet was dissolved in 2 ml of water and 6 ml of 90% ethanol and boiled for 10 min. The precipitate was redissolved in 3 ml water and 3 ml of 2 N HCl and was boiled for 30 min. Prior to the estimation of glycogen, the solution was neutralized by adding 3 ml of 1 N NaOH and used for the estimation of glycogen. For the estimation of glycogen, 1 ml of the neutralized solution was taken and mixed with 3 ml of 0.2% anthrone reagent (prepared in concentrated H 2 SO 4 ) and boiled for 15 min. After cooling down, the OD was taken at 620 nm using a spectrophotometer (Carry 60, Agilent), and the glycogen content was estimated against a standard glycogen graph.

Transmission electron microscopy (TEM) study
Glycogen content in the liver and muscle tissues of H. fossilis was visualized by TEM study. In brief, after treatment, the liver and muscle tissues were cut into small pieces (1 mm 9 1 mm) and were fixed in Karnovsky's fixative for 4 h (David et al. 1973). The samples were then washed thoroughly in 0.1 M cacodylate buffer and post-fixation was carried out in 1% osmium tetraoxide for 1 h at 4°C. The samples were dehydrated in ascending grades of acetone (30%, 50%, 70%, 80%, 90%, 95%, and dry acetone). The acetone was cleared off from the samples by using propylene oxide for 30 min. Embedding of the tissues was carried out in Araldite embedding medium using beam capsule. The embedded blocks were kept at 50°C in an embedding oven for 24 h at 60°C. Ultra-thin sections (600-800 Å ) were cut in a RMC Ultra-Microtome and the sections were then stained using 50% alcoholic solution of uranyl acetate for 10 min at room temperature in the dark, followed by lead nitrate for 5 min (Reynolds 1963). The stained sections were examined using JOEL JEM 2100 Transmission Electron Microscope at an accelerating voltage of 80 kV.

Tissue processing
For assaying the activities of GPase (EC 2.4.1.1) and GSase (EC 2.4.1.11), a 10% tissue homogenate was prepared in a homogenizing buffer containing 20 mM Imidazole-HCl buffer (pH 7.2), 100 mM NaF, 10 mM EDTA, 10 mM EGTA, 15 mM b-mercaptoethanol, and 0.1 mM PMSF (Russell and Storey 1995). The homogenate was sonicated for 3 cycles of 30 s ON and 30 s OFF using Q500 sonicator (Qsonica) for proper breakage of mitochondria. The homogenate was then centrifuged at 10,000 g for 10 min followed by 14,000 g for 30 min at 4°C, and the resultant supernatant was used for the enzyme assays.

Assay of enzyme activities
The activities of enzymes were assayed spectrophotometrically at 340 nm. In brief, 1 ml of reaction mixture was preincubated at 25°C for 5 min. The tissue extract was added to the preincubated reaction mixture in order to start the reaction. OD was recorded at 340 nm at 30 s interval for 10 min and the change in OD values (DE) was taken for calculating the enzyme activity using 6.22 9 10 6 as molar extinction coefficient value for NADH/NADPH. One unit of enzyme activity was expressed as that amount of enzyme, which catalyzed the oxidation or reduction of 1 lmol of coenzymes (NADP ? , NADH and NADPH) per min at 25°C. The specific activity of the enzymes was expressed as the units of enzyme activity per mg protein.

Statistical analysis
The data were obtained from at least three different experiments and statistically analyzed and presented as mean ± SEM. Mean values of the control and the treated samples were then compared using one way ANOVA using Statistical Package for the Social Sciences 17.0 software by Turkey post-hoc analyses with p \ 0.05 regarded as statistically significant.

Acute toxicity test
The acute toxicity test was performed in order to evaluate the toxicity of As and Pb on H. fossilis. The percentage mortality of H. fossilis during the exposure time at 48 h, 72 h and 96 h, respectively, was determined and indicated in Fig. 1 (A, B), which showed that the mortality of the fish was increased with increasing As 2 O 3 and PbCl 2 concentration. The 96 h LC 50 values of As 2 O 3 were calculated and was found to be 35.097 ppm; whereas, that of PbCl 2 was 66.205 ppm. Based on these findings, the 96 h LC 50 values of As 2 O 3 and PbCl 2 were taken for acute study; whereas, the LC 50 /20th ppm values were taken for chronic study for estimating the glycogen content and the activities of selected enzymes as mentioned before involved in glycogen metabolism and glycolysis.

Estimation of glycogen
The glycogen content in liver and muscle tissues of H. fossilis was estimated for both acute and chronic treatment of As 2 O 3 and PbCl 2 and presented in Table 1. Following the acute treatment of both As 2 O 3 and PbCl 2 , the glycogen content showed a gradual decrease in both liver and muscle tissues of H. fossilis. There was a significant decrease in the glycogen level (approximately 50-60%) within 96 h of As 2 O 3 and PbCl 2 acute treatment as compared to the control. However, upon chronic treatment of As 2 O 3 and PbCl 2 , the glycogen content decreased up to 30-50% till 20 days, then there was a sudden increase in the glycogen level on 30 days. In corroboration to biochemical analysis of glycogen content, TEM studies were performed to find out the changes in the glycogen content in both liver and muscle tissues of H. fossilis exposed to 96 h LC 50 values of As 2 O 3 and PbCl 2 . TEM studies of liver and muscle tissues of H. fossilis showed that the glycogen granules were decreased when exposed to an acute concentration of As 2 O 3 and PbCl 2 . Along with the decrease in glycogen level in the liver and muscle tissues of the catfish, the liver cells also showed a degenerated cell membrane, broken endoplasmic reticulum, disoriented nucleus, and dense mitochondria (Fig. 2a-c). Similarly, broken myofibrils were also observed in the muscle cells of the treated catfish exposed to heavy metals (Figs. 2d-f).

Effect of As and Pb on the activities of glycogen metabolic enzymes
The specific activity of GPase a ? b in the liver of H. fossilis did not show any significant changes on acute and chronic treatment of As 2 O 3 and PbCl 2 (Table 2). However, the specific activity of GPase a in liver was increased by 61.54% and 53.85% upon acute treatment with As 2 O 3 and PbCl 2 , respectively; whereas, upon chronic treatment with As 2 O 3 and PbCl 2 , the specific activity of GPase a in liver of H. fossilis was increased by 56.25% and 50.00%, respectively, after 20 days. But, after 30 days of treatment, the specific activity of GPase a in liver of H. fossilis did not show any significant changes ( Table 2). The specific activity of GSase a ? b in liver of H. fossilis also did not show any significant changes upon acute and chronic treatment with As 2 O 3 and PbCl (Table 2). However, upon acute treatment of As 2 O 3 and PbCl 2 , the specific activity of GSase a was decreased by 70.00% and 57.50%, respectively; however, upon chronic treatment of As 2 O 3 and PbCl 2 , the specific activity of GSase a was decreased by 47.27% and 50.91%, respectively, after 20 days, but it showed slightly recovery after 30 days ( Table 2).
The specific activity of GPase a ? b in the muscle of H. fossilis also did not show any significant changes (Table 3). Upon acute treatment with As 2 O 3 and PbCl 2 , the specific activity of GPase a in the muscle of H. fossilis was increased by 66.67% and 75.00%, respectively. However, upon chronic treatment of As 2 O 3 and PbCl 2 , the specific activity of GPase a in the muscle of H. fossilis was also increased by 81.82% and 72.73%, respectively, on 20 days; however, after 30 days of treatment, the specific activity GPase a was recovered and  Values are expressed as mean ± S.E.M. (n =3) Percentage of decrease (-) or increase (?) in glycogen content of the fish exposed to As 2 O 3 and PbCl 2 with respect to their controls is given in parentheses a P value significant at \ 0.05 in As and Pb treated fish with respect to their controls showed no significant changes when compared to the control (Table 3). In the muscle of H. fossilis, the specific activity of GSase a ? b did not show any significant changes upon acute and chronic treatment with As 2 O 3 and PbCl 2 ( Table 3). The specific activity of GSase a in the muscle of H. fossilis was decreased by 57.58% and 53.03% upon acute treatment of As 2 O 3 and PbCl 2 , respectively; however, upon chronic treatment with As 2 O 3 and PbCl 2 , the specific activity of GSase a in the muscle of H. fossilis was decreased by 47.76% and 52.24%, respectively, up to 20 days (Table 3).

Effect of As and Pb on activities of selected glycolytic enzymes
The effect of As and Pb was also tested on the activities of selected glycolytic enzymes. The specific activity of hexokinase in the liver of H. fossilis was decreased by 69.70% and 59.09% upon acute exposure with As 2 O 3 and PbCl 2 , respectively; however, upon chronic treatment, the specific activity of hexokinase in the liver of H. fossilis was decreased gradually up to 62.07% and 60.34%, respectively (Table 4). The specific activity of phosphofructokinase in the liver of H. fossilis did not show any significant changes when treated with both acute and chronic concentration of As 2 O 3 and PbCl 2 ( Table 4). The specific activity of pyruvate kinase in the liver of H. fossilis was decreased by 51.12% and 65.59% upon acute treatment with As 2 O 3 and PbCl 2 , respectively. On chronic treatment with As 2 O 3 and PbCl 2 , the specific activity of pyruvate kinase in the liver of H. fossilis was also found to be decreased gradually up to 49.81% and 46.41%, respectively (Table 4). The specific activity of hexokinase in the muscle of H. fossilis was decreased by 50.00% and 55.26% on acute treatment of As 2 O 3 and PbCl 2 , respectively. However, upon chronic treatment of As 2 O 3 and PbCl 2 , the specific activity of hexokinase was decreased gradually up to 44.83% and 41.38%, respectively (Table 5). The specific activity of phosphofructokinase in the muscle of H. fossilis, showed no significant changes on both acute and chronic treatment of As 2 O 3 and PbCl 2 (Table 5). However, in the muscle of H. fossilis, the specific Fig. 2 Effect of As 2 O 3 and PbCl 2 on glycogen particles in liver (a-c) and muscle (d-f) tissues of H. fossilis. The catfish was treated with LC 50 values of As 2 O 3 and PbCl 2 and glycogen particles were observed using transmission electron microscopy. a Control; b exposed to 35.09 ppm As 2 O 3 for * 96 h; c exposed to 66.20 PbCl 2 for * 96 h; d control; e exposed to 35.09 ppm As 2 O 3 for * 96 h; f exposed to 66.20 PbCl 2 for * 96 h. CM cell membrane, ER endoplasmic reticulum, G glycogen granules, M mitochondria, My myofibrils, N nucleus  (-5.13) 0.041 ± 0.002 (-21.15) 0.029 ± 0.003 a (-47.27) 0.048 ± 0.002 (-11.11) PbCl 2 (3.31) 0.034 ± 0.001 (-10.53) 0.032 ± 0.004 (-13.51) 0.031 ± 0.002 (-20.51) 0.037 ± 0.002 (-28.85) 0.027 ± 0.002 a (-50.91) 0.049 ± 0.005 (-9.26) Values are expressed as mean ± S.E.M. (n =3) Percentage of decrease (-) or increase (?) in specific activity of the fish exposed to As 2 O 3 and PbCl 2 with respect to their controls is given in parentheses a P value significant at \ 0.05 in As and Pb treated fish with respect to their controls. One unit of enzyme activity is defined as 1 lmol of NADP ? reduced in case of GPase or NADH oxidized in case of GSase per minute at 25°C. Specific activity of the enzymes is expressed as the units of enzyme activity per mg protein  Percentage of decrease (-) or increase (?) in specific activity of the fish exposed to As 2 O 3 and PbCl 2 with respect to their controls is given in parentheses a P value significant at \ 0.05 in As and Pb treated fish with respect to their controls. One unit of enzyme activity is defined as 1 lmol of NADP ? reduced in case of GPase or NADH oxidized in case of GSase per minute at 25°C. Specific activity of the enzymes is expressed as the units of enzyme activity per mg protein  (-46.41) Values are expressed as mean ± S.E.M. (n =3) Percentage of decrease (-) or increase (?) in specific activity of the fish exposed to As 2 O 3 and PbCl 2 with respect to their controls is given in parentheses a P value significant at \ 0.05 in As and Pb treated fish with respect to their controls. One unit of enzyme activity is defined as 1 lmol of NADP ? reduced in case of hexokinase or NADH oxidized in case of phosphofructokinase and pyruvate kinase per minute at 25°C. Specific activity of the enzymes is expressed as the units of enzyme activity per mg protein  (-77.91) Values are expressed as mean ± S.E.M. (n =3) Percentage of decrease (-) or increase (?) in specific activity of the fish exposed to As 2 O 3 and PbCl 2 with respect to their controls is given in parentheses a P value significant at \ 0.05 in As and Pb treated fish with respect to their controls. One unit of enzyme activity is defined as 1 lmol of NADP ? reduced in case of hexokinase or NADH oxidized in case of phosphofructokinase and pyruvate kinase per minute at 25°C. Specific activity of the enzymes is expressed as the units of enzyme activity per mg protein activity of pyruvate kinase was decreased by 31.62% and 48.93% upon acute treatment of As 2 O 3 and PbCl 2 , respectively; whereas, upon chronic treatment of As 2 O 3 and PbCl 2 , the specific activity of pyruvate kinase was observed to be decreased gradually up to 68.71% and 77.91%, respectively (Table 5).

Discussion
Sensitivity of fish to As and Pb differs among different species of fishes. A possible explanation for these differences to trace metals might be explained as a function of metallothionein synthesis, which is believed to provide a protective role against toxic effect of metals in aquatic animals including fishes (Roesijadi 1992;Hollis et al. 2001 (Sadeghi and Imanpoor 2015).
In the present study, acute and chronic exposure of H. fossilis to As and Pb exhibited significant alterations in the glycogen content in the liver and muscle tissues of H. fossilis. Glycogen granules were shown to be depleted in the fish exposed to an acute concentration of heavy metals (As and Pb) as evidenced from the TEM studies. Depletion in glycogen granules in liver of zebra fish is also reported by Macirella et al. (2016) when treated with mercury chloride. The depletion of glycogen may be due to rapid turnover of the glycogen. Ananth et al. (2014) and Kavidha and Muthulingam (2014) have also reported the decline of glycogen content in grass carps, Ctenopharyngodon idella and Cyprinus carpio when exposed to As and Pb. Exposure to chronic concentrations of As and Pb showed that the glycogen content was decreased up to 20 days, but after 20 days there was an increase in glycogen content in both liver and muscle tissues of the fish. Similar trend has also been reported by Sastry and Subhadra (1982) in H. fossilis when exposed to cadmium. This may be due to the reduced rates of glycogenolysis resulting from the cumulative toxic effects of As and Pb.
The decreasing glycogen content in both liver and muscle tissues of the fish was resulted by the increasing specific activity of GPase a. Upon acute treatment of As and Pb, GPase a specific activity increases, which correspond with the breakdown of glycogen granules in both liver and muscle tissues of Clarias batrachus (Begum 2004). However, upon chronic exposure to As and Pb the specific activity of GPase a was increased after 20 days of treatment, and recovered after 30 days, which may be correlated with the rapid restoration of glycogen content in both the tissues. In the present study, the decrease in the specific activities of GSase a in liver and muscle tissues of H. fossilis may be correlated with the increase in glycogenolysis. Similar results are also observed when Salmo salar is exposed to cadmium (Soengas et al. 1996).
In this study, the specific activity of hexokinase was decreased when exposed to an acute concentrations of As and Pb. Carvalho and Fernandes (2008) show that hexokinase activity in Prochilodus lineatus decreases when exposed to copper. The specific activity of phosphofructokinase showed no significant changes when treated with As and Pb, which is similar to the effect of copper in Salmo trutta (Beaumont et al. 2000). Pyruvate kinase specific activity was found to decrease when exposed to acute and chronic concentrations of As and Pb. Previous studies also indicate that heavy metals such as copper inhibits pyruvate kinase specific activity in tropical fish, Prochilodus lineatus (Carvalho and Fernandes 2008).

Conclusions
The results of the present study indicated that As and Pb caused significant changes in carbohydrate metabolism in the fish and these alterations could be attributed to the toxic stress induced by these two heavy metals on the model fish.
fellowship under the scheme National Fellowship for Higher Education (NFHE) of ST students. The authors also gratefully acknowledge the facilities and assistance provided by Transmission Electron Microscope Unit, SAIF, NEHU, Shillong.

Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of interest.
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