Air-breathing magur catfish (Clarias magur) regularly face the problem of exposure to high environmental ammonia (HEA) as one of the major pollutants in their natural habitats that causes considerable toxic effects at the cellular level, including that of oxidative stress. The major objective of the present study was to demonstrate the antioxidant activity of endogenously produced nitric oxide (NO) to defend against ammonia-induced oxidative stress in primary hepatocytes of magur catfish during exposure to HEA. Exposure to NH4Cl (5 mmol l−1) led to a significant increase in intracellular ammonia concentration with a sharp rise of hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations within 3 h in primary hepatocytes, which decreased gradually at later stages of treatment. This phenomenon was accompanied by a significant increase in superoxide dismutase (SOD) and catalase (CAT) activity as a consequence of induction of corresponding genes. HEA exposure also led to the stimulation of NO production due to induction of inducible nitric oxide synthase (iNOS) activity, as a consequence of up-regulation of the nos2 gene. Most interestingly, when NO production by hepatocytes under ammonia stress was blocked by adding certain inhibitors [aminoguanidine and 3-(4-methylphenylsulfonyl)-2-propenenitrile] to the culture medium, there was a further rise of H2O2 and MDA concentrations in hepatocytes. These were accompanied by the lowering of SOD and CAT activity with less expression of corresponding genes. Thus, it can be contemplated that magur catfish use the strategy of stimulation of NO production, which ultimately induces the SOD–CAT enzyme system to defend against ammonia-induced oxidative stress.
The level of toxic ammonia may rapidly rise in confined water bodies and intensive fish farming ponds as a consequence of nitrogenous waste excretion by fish and decomposition of biological waste, including that of macro-vegetation, which is present in ponds and stagnant water bodies. Additionally, the toxic ammonia level can also be elevated in these water bodies by the influx of ammonia from various anthropogenic activities such as the discharge of sewage effluents, industrial waste and industrial run-off of chemical fertilizers (for reviews, see Ip et al., 2001; Saha and Ratha, 2007). The elevated ammonia levels in the aquatic environment may impair ammonia excretion and/or result in a net uptake of ammonia from the environment, leading to a rise of ammonia concentration in the blood and body tissues of teleosts (Saha and Ratha, 2007). Ammonia in water exists in two forms, unionized ammonia (NH3) and the ionized form (NH4+), and the sum of both comprise the total ammonia concentration. Although NH3 is much more toxic than NH4+ as it is highly permeable to the cell membrane (Emerson et al., 1975), NH4+ is probably the primary toxic element for animals as more than 95% of total ammonia in the body exists as NH4+ at physiological pH values.
The build-up of high environmental ammonia (HEA) may lead to a severe threat to aquatic animals, including fish, in several ways. HEA induces a range of ecotoxicological effects in fish, including a decrease in growth rate (Dosdat, 2003; Sinha et al., 2012), alteration in energy metabolism (Arillo et al., 1981; Sinha et al., 2015), disruption of ionic balance (Wilkie, 1997; Diricx et al., 2013; Sinha et al., 2015), alteration in hormone regulation (Knoph and Olsen, 1994; Dosdat, 2003), neurotoxicity-like astrocyte swelling (Brusilow, 2002; Albrecht and Norenberg, 2006), overactivation of N-methyl-d-aspartate type glutamate (NMDA) receptors (Marcaida et al., 1992; Fan and Szerb, 1993), increased gill ventilation, loss of equilibrium, hyperexcitability, convulsion and ultimately may lead to death (Pan et al., 2011; Roumieh et al., 2013). Additionally, a few studies suggest that ammonia exerts cellular toxicity by overproduction of reactive oxygen species (ROS), thereby leading to oxidative stress (Murthy et al., 2001). Some recent reports have elucidated ammonia-induced oxidative stress in fishes while living in HEA, such as in pufferfish (Takifugu obscurus) (Cheng et al., 2015), rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), goldfish (Carassius auratus) (Sinha et al., 2014), mudskipper (Boleophthalmus boddarti) (Ching et al., 2009) and Nile tilapia (Oreochromis niloticus) (Benli et al., 2008). Induction of oxidative stress is known to cause extensive damages to biomolecules like DNA, proteins, lipids, and also the cellular membrane (Halliwell, 1999). Nonetheless, in order to convert ROS to harmless metabolites, animals use various enzymatic and non-enzymatic antioxidant defensive strategies including the participation of superoxide dismutase (SOD) and catalase (CAT) in protection and restoration of normal cellular homeostasis under oxidative stress (Birben et al., 2012). Furthermore, the involvement of nitric oxide (NO), generally produced by nitric oxide synthase (NOS), is well documented in cellular homeostasis (Gong et al., 2004), neurotransmission (Vincent, 2010), neuromodulation of the central nervous system (Giraldi-Guimarães et al., 2007), immune response (Bogdan, 2001), signal transduction (Demple, 2002), cell proliferation (Napoli et al., 2013) and apoptosis (Brüne et al., 1998), and even provides some protection to the cellular system against oxidative stress in goldfish (Hansen and Jensen, 2010). However, the mode of action of NO to defend against oxidative stress in fish is not yet fully understood.
The facultative air-breathing magur catfish (Clarias magur, known previously as Clarias batrachus) is found predominantly in tropical Southeast Asia. They usually inhabit stagnant, slow-flowing swampy water bodies of ponds and lakes or wetlands that are often covered with macrovegetation, such as water hyacinth, and are characterized by low dissolved oxygen, and high bicarbonate and ammonia levels in the water bodies (for review, see Saha and Ratha, 2007). Magur catfish occasionally face the problem of exposure to HEA, especially when they are trapped in puddles of water or while burrowing inside the mud peat during summer, where continual excretion of endogenous ammonia into a small volume of the aquatic environment may lead to HEA concentration. The situation is further aggravated for those fish living in paddy fields, where agricultural fertilization can lead to HEA in the aquatic environment (Rao et al., 1994). The magur catfish is known to tolerate a very HEA (up to 75 mmol l−1 NH4Cl) for several months (Saha et al., 2003). The occurrence of various adaptational strategies related to nitrogen metabolism has already been reported in magur catfish, mainly to ameliorate the ammonia toxicity under hyper-ammonia stress (for reviews, see Saha and Ratha, 1998, 2007). Additionally, the enhanced synthesis and accumulation of NO in different tissues of magur catfish due to induction of the inducible nitric oxide synthase gene (nos2) during exposure to HEA have recently been reported (Kumari et al., 2019). Furthermore, the nuclear factor kappa-B (NFκB)-mediated induction of the nos2 gene under hyper-ammonia stress was suggested in another closely related air-breathing catfish (Heteropneustes fossilis) (Choudhury and Saha, 2012). Hence, it is logical to consider that the enhanced production and accumulation of NO might play a significant role in magur catfish in defending against possible ammonia-induced oxidative stress while exposing to HEA. As the liver is the main organ responsible for accumulation and detoxification of various toxic metabolites, it was felt that studies on alterations of certain metabolic processes under ammonia-induced oxidative stress in hepatic cells would be ideal to investigate the adaptational strategies that are commonly being adopted by the magur catfish. Therefore, the present study clearly demonstrates the antioxidant activity of NO that is produced endogenously under ammonia-induced oxidative stress in primary hepatocytes of C. magur by using certain biochemical, enzymatic, molecular and immunological techniques in the presence and absence of certain inhibitors for NO production.
MATERIALS AND METHODS
Antibodies and reagents
A polyclonal antibody specific to inducible nitric oxide synthase (iNOS) was produced against the peptide from the epitope EIGARDFCDPQRYNILEKVGR. The peptide was conjugated to the KLH (Keyhole Limpet Hemocyanin) peptide to immunize the rabbit for obtaining the polyclonal antibody (Imgenex, Odisha, India). Rabbit polyclonal NFκB p65 (sc-109), goat polyclonal CAT (sc-34285) antibody, goat polyclonal glyceraldehyde 3-phosphate dehydrogenase GAPDH (sc-48166), the HRP-conjugated anti-rabbit (sc-2357), HRP-conjugated anti-goat (sc-2020) immunoglobulin G, Bay 11-7085 (BAY) and aminoguanidine (AG) were obtained from Santa Cruz Biotechnology (USA). Rabbit polyclonal SOD (S4946) was obtained from Merck (USA). Anti-goat conjugated with Alexa Fluor 568 (A11057), anti-rabbit conjugated with Alexa Fluor 568 (A10042) was obtained from Thermo Fisher Scientific, USA. Oligonucleotide primers were obtained from GCC Biotech (India). Enzymes, coenzymes and substrates were obtained from Sigma Chemicals (USA). SYBR Premix Ex Taq II was obtained from Takara (Japan). Other chemicals were of analytical grade and obtained from local sources. Milli-Q water was used in all preparations.
Magur catfish, Clarias magur (Hamilton 1822) (175–200 g body mass) aged 18–26 months, were purchased from a single source that was bred and cultured in selected commercial ponds of the Nilbagan Fish Seed Farm situated in Barpeta, Assam, India. Fish were acclimatized in the laboratory approximately for 1 month at 27±2°C with 12 h light:12 h dark photoperiod before isolation of hepatocytes. Only the male fish were used for hepatocyte isolation. Minced dry fish and rice bran (5% of body mass) were given as food every day, and the water, collected from a natural stream, was changed on alternate days. The study was approved by the Institutional Animal Ethics Committee (IAEC) of North-Eastern Hill University, Shillong, India (NEC/IEC/2018/016).
Hepatocyte isolation, culture and experimental set-up
Hepatocytes were isolated and cultured following the method of Banerjee et al. (2017). In brief, fishes were anesthetized in ethyl 3-aminobenzoate methanesulfonic acid (MS-222, 0.2 g l−1) and disinfected with 70% alcohol before the operation. Isolated livers were perfused via the hepatic portal vein for 10 min in a perfusion medium containing 142 mmol l−1 NaCl, 5 mmol l−1 NaHCO3, 6.7 mmol l−1 KCl, 1.3 mmol l−1 MgSO4 and 10 mmol l−1 Hepes. Perfused livers were cut into pieces and transferred into 50 ml centrifuge tubes containing 10 ml Medium III (142 mmol l−1 NaCl, 6.7 mmol l−1 KCl, 2.5 mmol l−1 CaCl2, 3 mmol l−1 Na2HPO4, 10 mmol l−1 Hepes, 5.5 mmol l−1 glucose and 0.5 mg ml−1 collagenase) and incubated for 2 h at 27°C in a shaker inside a CO2 incubator. The whole solution with liver pieces was filtered through a 70 µm cell strainer and the cell suspension filtrate was centrifuged at 40 g for 3 min. The supernatant was discarded and the pelleted cells were resuspended in 10 ml Dulbecco's phosphate-buffered saline (DPBS) and centrifuged once again. The cell pellet was resuspended in 10 ml of Medium 199 (M199, Gibco, USA) supplemented with 1% streptomycin penicillin and 10% fetal bovine serum (FBS), and cells were counted using a Countess Automated Cell Counter (Thermo Fisher Scientific). Cells were seeded at an approximate concentration of 1×106 viable cells per milliliter in 12-well sterile culture plates containing 2 ml M199 and incubated inside a CO2 incubator at 27°C with 2% CO2 for 24 h. Hepatocyte viability of 80–90% in the presence of 5 mmol l−1 NH4Cl until 60 h was confirmed by MTT assay following the method of Mosmann (1983). A total of three sets of experiments with five replicates of hepatocytes, isolated from five different fish, were performed in the culture media. In the first set, hepatocytes were treated with 5 mmol l−1 NH4Cl alone; in set 2, NH4Cl (5 mmol l−1) plus AG (10 μmol l−1); in set 3, hepatocytes were treated with NH4Cl (5 mmol l−1) plus BAY (3 μmol l−1) for a period of 48 h. In the third set of experiments, BAY was added to the culture medium to inhibit NFκB activity as reported by García et al. (2005), and also by Lee et al. (2012) as a broad-spectrum inhibitor against inflammatory signaling pathways including NFκB.
Determination of ammonia, hydrogen peroxide, malondialdehyde and NO concentrations
The intracellular total ammonia (TA: NH4+ plus NH3) concentration in isolated hepatocytes was determined enzymatically following Kun and Kearney (1974) after processing the hepatocytes as described by Saha and Ratha (1998). The intracellular hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations in isolated hepatocytes were determined using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific) and TBARS assay kit (DTBA-100; BioAssay Systems, USA), respectively, following the manufacturers’ instructions. The NO concentration in the culture medium and cell lysates were measured by treating both the culture medium and cell lysates with 5% perchloric acid (PCA) in 1:0.5 ratio to precipitate out the proteins, followed by centrifugation at 10,000 g for 5 min. The concentration of NO in the supernatant was determined spectrophotometrically following the Griess reaction, as described by Sessa et al. (1994).
The protein concentration in cell lysates was determined using Bradford reagent (Bradford, 1976).
The hepatocytes were ultrasonically lysed for 10×4 s in a lysis buffer containing 50 mmol l−1 Tris–HCl (pH 7.4), 150 mmol l−1 NaCl and protease inhibitor on ice, and centrifuged at 10,000 g for 10 min at 4°C. The supernatants were used for assaying the activity of different enzymes.
SOD activity was assayed following the method of Paoletti et al. (1986). The reaction mixture at a final volume of 1 ml contained 50 mmol l−1 potassium phosphate buffer (pH 7.5), 0.5 mmol l−1 riboflavin and 0.4 mmol l−1 nitroblue tetrazolium (NBT), which was pre-incubated at 27°C for 5 min in a quartz cuvette, followed by the addition of 10 µl of cell lysate to start the reaction. The formation of formazan blue was continuously monitored in an ultraviolet (UV)-visible spectrophotometer (Cary 60; Agilent, Santa Clara, CA, USA) at 560 nm for 3 min at 27°C. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT photoreduction.
CAT activity was assayed following the method of Beers and Sizer (1952). The reaction mixture at a final volume of 3 ml contained 50 mmol l−1 potassium phosphate buffer (pH 7.5) and 25 mmol l−1 H2O2, which was pre-incubated at 27°C for 5 min in a quartz cuvette, followed by the addition of 10 µl of cell lysate to the reaction mixture to start the reaction. The H2O2 decomposition was continuously monitored in a UV-visible spectrophotometer (Cary 60, Agilent) at 240 nm for 3 min at 27°C. One unit of CAT activity was defined as the amount of enzyme that catalysed 1 µmol of H2O2 per minute at 27°C. Both the SOD and CAT activity were expressed as units per milligram of protein.
iNOS activity was assayed following the method of Knowles and Salter (1998) with certain modifications (Choudhury and Saha, 2012). The reaction mixture at a final volume of 1 ml contained 100 mmol l−1 sodium phosphate buffer (pH 7.2), 100 mmol l−1 l-arginine, 1.5 mmol l−1 MgCl2, 0.25 mmol l−1 CaCl2, 0.12 mmol l−1 NADPH, 50 mmol l−1 l-valine, 10 units of urease and 0.1 ml of cell lysate. The second set of reaction mixture contained all the above reagents except CaCl2, and also contained 0.5 mmol l−1 AG (an inhibitor of iNOS activity) and 1 mmol l−1 EGTA. Both sets of reaction mixtures were incubated at 27°C for 20 min, and the reaction was stopped by adding 1 ml 10% PCA to precipitate out the protein, followed by centrifugation at 10,000 g for 5 min. Citrulline, formed as the reaction product, was estimated in the supernatant spectrophotometrically at 490 nm in a UV-visible spectrophotometer (Cary 60, Agilent) following the method of Moore and Kauffman (1970) and expressed as enzyme activity. Part of the activity, which was inhibited in the second set of the reaction mixture, was taken as iNOS activity. One unit of enzyme activity was defined as that amount of enzyme which catalysed the formation of 1 μmol of citrulline per hour at 27°C and expressed as units per milligram of protein.
Lactate dehydrogenase (LDH) activity in the culture medium was assayed spectrophotometrically following the method of Bergmeyer (1974). One unit of LDH activity was defined as that amount of enzyme which oxidized 1 μmol of NADH to NAD+ per hour at 27°C and expressed as units per liter.
Total RNA extraction, cDNA synthesis and quantitative real-time PCR analysis
Total RNA was isolated from hepatocytes using TRI Reagent (Sigma Aldrich), following the method of Rio et al. (2010), and quantified spectrophotometrically at 260 nm with the help of QIAxpert (Qiagen, Germany). First-strand cDNA was synthesized from 400 ng of total RNA in a total volume of 20 μl with iScript cDNA synthesis kit (Bio-Rad, USA) as per the standard protocol.
The quantitative real-time polymerase chain reaction (qPCR) analysis was performed in a StepOne plus real-time PCR system (Thermo Fisher Scientific) using Premix Ex Taq II SYBR Green (Takara, Japan). The reaction mixture of 10 μl contained 5 μl of SYBR Green Mix, 0.5 μl of cDNA, 400 nmol l−1 of each primer and Milli-Q H2O. The assay conditions included an initial denaturation step at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s. The qPCR was performed in triplicate for each sample and negative controls using no cDNA were run for each gene. Melting curve analysis was used to confirm the amplification of only a single PCR product. Furthermore, to re-confirm the amplification product, the sequencing of the PCR product was performed in an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific) using the Sanger dideoxy method. Two reference genes (β-actin and α-tubulin) were used to normalize the qPCR data. Relative mRNA expression of each gene was calculated using the modified ΔΔCT method (Livak and Schmittgen, 2001). The used primer pairs were designed, and the specificity of each primer pair was checked by using the Primer-BLAST tool (Ye et al., 2012) (Table 1).
Western blot analysis
Hepatocytes were homogenized in a lysis buffer [50 mmol l−1 Tris–HCl, pH 7.5, 1 mmol l−1 EDTA, 0.1% sodium dodecyl sulphate (SDS), 1% Triton X, 1 mmol l−1 phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail (Roche)] and sonicated for 30×4 s. Lysates were centrifuged at 10,000 g for 10 min at 4°C. Fifty micrograms of cellular protein present in the supernatant for each sample were resolved in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to the polyvinylidene fluoride membrane. Blots were probed using a specific antibody against SOD, CAT, iNOS and NFκB p65 proteins (1:5000 dilution), followed by incubation with HRP-conjugated secondary antibodies, and the chemiluminescence was detected using Clarity Western ECL substrate (Bio-Rad) in an Image Quant LAS 500 system (GE Healthcare Life Sciences, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein loading control.
Immunocytochemistry and confocal laser scanning microscopy
In another set of experiments to analyse the expression SOD, CAT and iNOS enzyme proteins, the isolated hepatocytes were grown over coverslips in multi-well sterile culture plates for 48 h following the method as described above with a CO2 incubator. After treatment, hepatocytes were fixed in 4% paraformaldehyde and washed in PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 1% bovine serum albumin (BSA in PBS) for 1 h. The coverslips were incubated separately with primary antibodies for SOD, CAT and iNOS (1:50,000 dilution) for 48 h at 4°C. After washing for three times in PBS, the coverslips were incubated for 2 h in Alexa Fluor-conjugated secondary antibodies (1:10,000 dilution) in a dark wet chamber. Subsequently, after the final washing, coverslips were mounted in ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Another set of slides were processed in the same way except incubating with primary antibodies, which served as negative controls. Immunostained slides were analysed in a confocal laser scanning microscope (TCS SP5; Leica, Germany). Cross-talk of fluorochromes was excluded by the use of an acousto-optical tunable filter.
The data, collected from different experiments, were statistically analysed by two-way ANOVA test to evaluate the significant differences of values obtained with relation to respective controls, and also the values obtained due to treatment with NH4Cl plus inhibitors with relation to the values obtained due to treatment with NH4Cl alone over time of exposure using GraphPad Prism software. Data are presented as means±s.e.m. (n=5 in each set of the experiment). Levene's test was also performed to verify the homogeneity of variance of different parameters between the control and treated groups of each set of experiments.
The intracellular concentration of ammonia in hepatocytes
The changes in ammonia concentration in primary hepatocytes of magur catfish due to treatment with 5 mmol l−1 NH4Cl in the absence and presence of different inhibitors are presented in Fig. 1A. The average concentration of ammonia in hepatocytes was recorded to be about 45 nmol mg−1 protein throughout the experimental period. However, when the hepatocytes were treated with NH4Cl, a significant 1.4-fold increase in ammonia concentration was observed within 3 h, which rose further at later stages to a maximum 2.3-fold increase after 48 h of treatment. Similarly, treatment of hepatocytes with NH4Cl in the presence of AG (10 µmol l−1) and BAY (3 µmol l−1) separately also led to a significant rise in intracellular ammonia concentration (maximum 2.3- and 2.6-fold increase, respectively, after 48 h of treatment).
Intracellular concentrations of H2O2 and MDA in hepatocytes
Exposure of primary hepatocytes of magur catfish to 5 mmol l−1 NH4Cl in the culture medium led to a sharp 2.3-fold rise in intracellular H2O2 concentration compared with untreated controls within 3 h, followed later by a gradual decrease almost to basal level after 48 h (Fig. 1B). However, when the hepatocytes were treated with NH4Cl in the presence of AG and BAY separately, the H2O2 concentration in hepatocytes increased continuously with the increasing time of incubation, and maximum 3.2- and 3.1-fold increases, respectively, were recorded after 48 h compared with untreated controls (Fig. 1B).
Similarly, the intracellular concentration of MDA also increased sharply (5.9-fold) in hepatocytes due to treatment with NH4Cl alone within 3 h compared with untreated controls, followed later by a gradual decrease reaching almost basal level after 48 h of treatment (Fig. 1C). However, the treatment of hepatocytes with AG and BAY separately in the presence of NH4Cl led to a continuous increase of intracellular MDA concentration in hepatocytes (maximum 13.4- and 12.7-fold rise, respectively) after 48 h compared with untreated controls (Fig. 1C).
LDH leakage in the culture medium
LDH leakage from the primary hepatocytes of magur catfish into the culture medium was assayed as a measure of cellular damage caused by treatment with NH4Cl in the absence and presence of different inhibitors (Fig. 1D). LDH leakage by the control hepatocytes in the culture medium was negligible throughout the experimental periods. Treatment of hepatocytes with NH4Cl led to a marginal increase of LDH leakage from the hepatocytes, evidenced by an increase of LDH activity in the culture medium from 0.8 to 3.75 units l−1 after 48 h of treatment. However, when the hepatocytes were treated with NH4Cl in the presence of AG and BAY separately, a manyfold increase of LDH leakage from hepatocytes was recorded compared with the level that was found during treatment with NH4Cl alone, evidenced by an increase of LDH activity in the culture medium. In the presence of AG and BAY separately along with NH4Cl, the level of LDH activity in the culture medium was found to increase maximally to 37.5 units l−1 (68.0-fold) and 35.6 units l−1 (54.0-fold), respectively, after 48 h compared with untreated controls.
SOD and CAT activity in hepatocytes
SOD activity in control hepatocytes of magur catfish was recorded to be about 35 units mg−1 protein throughout the experimental period (Fig. 2). However, the treatment of hepatocytes with 5 mmol l−1 NH4Cl led to a significant 2.0-fold increase in SOD activity compared with untreated controls within 3 h, which later increased further 3.4-fold after 48 h of treatment (Fig. 2A). Subsequently, when the hepatocytes were treated with NH4Cl in the presence of AG and BAY separately, no significant change of SOD activity could be seen until 6 h. Instead, a significant decrease in activity was observed at later stages of treatment (Fig. 2A).
Similarly, the CAT activity, which was recorded to be about 210 units mg−1 protein in control hepatocytes throughout the experimental periods, increased significantly within 3 h of NH4Cl treatment, followed by a further increase at later stages of treatment, having a maximum 2.4-fold increase of activity after 24 h (Fig. 2B). However, when the hepatocytes were treated with NH4Cl in the presence of AG and BAY separately, a significant decrease of CAT activity could be seen from 3 h onwards compared with untreated controls (Fig. 2B).
Expression of mRNAs for sod1, sod2 and cat in hepatocytes
The effect of NH4Cl on the expression of some antioxidant genes such as sod1, sod2 and cat was also studied in primary hepatocytes of magur catfish (Fig. 2C–E). Significant up-regulation in the expression of all three genes was recorded in the hepatocytes within 3 h of treatment with NH4Cl, evidenced by the increase in mRNA concentrations, which was followed by a further increase at later stages of treatment. The levels of expression of mRNAs for sod1 and cat genes increased maximally 2.7-fold after 48 h, and for sod2, it increased maximally 2.8-fold after 24 h of treatment with NH4Cl. However, when the hepatocytes were treated with NH4Cl in the presence of AG and BAY separately, the levels of expression of mRNAs for all three genes were found to decrease significantly within 3 h, and remained lower than the control levels until 48 h of treatment.
SOD and CAT enzyme protein expression in hepatocytes
Treatment of isolated hepatocytes of magur catfish with NH4Cl also led to a significant rise in SOD and CAT enzyme protein expression within 3 h as determined by western blotting, which later increased further with a maximum 1.8- and 1.9-fold rise, respectively, after 24 h of treatment (Fig. 3). However, when the hepatocytes were treated with NH4Cl in the presence of AG and BAY separately, the enzyme protein concentrations of both SOD and CAT remained significantly lower compared with untreated controls throughout the experimental periods (Fig. 3).
Immunocytochemical analysis of SOD and CAT enzyme proteins in hepatocytes
Western blotting analysis of SOD and CAT enzyme protein expression in primary hepatocytes under different experimental conditions was further supplemented by immunocytochemical analysis (Fig. 4). For both enzymes, the changing patterns of protein expression in hepatocytes are presented only after 24 and 48 h of treatment along with their respective controls, as maximum changes were noticed either after 24 or 48 h of treatment under different experimental conditions. In control hepatocytes, prominent signals for both SOD and CAT could be seen throughout the experimental periods. However, after treatment with NH4Cl, a remarkable increase of signals for both SOD and CAT enzyme proteins were observed after 3 h (data not shown), having a maximum rise of signal intensities after either 24 or 48 h. However, when the hepatocytes were treated with AG and BAY separately along with NH4Cl, a decreasing trend of signal intensity for both enzymes could be seen within 3 h compared with untreated controls (data not shown), which decreased further at later stages of treatment.
NO concentration in the culture medium and in hepatocytes
Treatment of primary hepatocytes of magur catfish with 5 mmol l−1 NH4Cl led to a gradual and significant increase of NO concentration in the culture medium with a maximum 6.5-fold rise after 48 h. However, when AG and BAY were added separately to the culture medium along with NH4Cl, the NO concentrations increased only 1.96- and 1.7-fold, respectively (Fig. 5A).
Similarly, the intracellular concentration of NO in primary hepatocytes was also found to increase gradually with increasing time of exposure to 5 mmol l−1 NH4Cl with a maximum 4.6-fold rise after 48 h compared with the level found in control hepatocytes (Fig. 5B). However, when AG and BAY were added separately to the culture medium along with NH4Cl, a marginal (1.7-fold) increase of intracellular NO concentration was seen to occur in both the cases after 48 h of treatment (Fig. 5B).
iNOS activity, expression of nos2 and iNOS protein in hepatocytes
In control hepatocytes, a negligible amount of iNOS activity (0.005 units mg−1 protein) could be detected throughout the experimental periods (Fig. 6A). However, treatment of hepatocytes with 5 mmol l−1 NH4Cl led to a significant rise of iNOS activity (0.035 units mg−1 protein) within 3 h of treatment, which later increased further, having a maximum 6.0-fold rise of activity after 48 h of treatment compared with the activity recorded after 3 h of NH4Cl treatment (Fig. 6A).
Similarly, treatment of primary hepatocytes with NH4Cl also led to an up-regulation of nos2 gene expression, evidenced by a significant increase of nos2 mRNA concentration within 3 h of treatment, followed by a further increase at later stages of treatment with a maximum 6.9-fold rise of mRNA concentration after 24 h of treatment (Fig. 6B). However, when BAY was added to the culture medium along with NH4Cl, a decreasing trend in the level of expression of mRNA in hepatocytes was observed with increasing time of incubation (Fig. 6B).
The effect of NH4Cl treatment on iNOS enzyme protein expression in primary hepatocytes was also studied by western blotting in the absence and presence of BAY (Fig. 6C). A very faint band of iNOS protein could be seen in control hepatocytes throughout the experimental periods. However, when the hepatocytes were treated with 5 mmol l−1 NH4Cl, a prominent iNOS protein band could be seen within 3 h with a maximum 5.7-fold rise of band intensity after 24 h of treatment. However, in the presence of BAY, the band intensity of iNOS protein increased only 2.0-fold after 24 h of treatment (Fig. 6C,D).
Western blotting analysis of iNOS protein expression in primary hepatocytes under different experimental conditions was further supplemented by immunocytochemical analysis (Fig. 6E). In control hepatocytes, no visible signal for iNOS protein could be seen. However, when the hepatocytes were treated with NH4Cl, a very prominent signal for iNOS protein could be seen after 3 h (data are not shown), followed by a further rise of signal intensity after 24 and 48 h of treatment (Fig. 6E). When BAY was added along with NH4Cl, a lower increase of iNOS signal was seen throughout the experimental periods.
Expression of the rela gene and its translated protein in hepatocytes
Treatment of primary hepatocytes with 5 mmol l−1 NH4Cl also led to the up-regulation of rela gene expression, evidenced by the increase in mRNA concentration (Fig. 7A). It increased maximally (2.8-fold) after 24 h, followed by a gradual decrease at later stages, but remained significantly higher than the control values until 48 h of treatment.
Similarly, the western blotting analysis demonstrated that NH4Cl treatment caused a significant rise in the expression of NFκB (p65) protein in primary hepatocytes, evidenced by the increase of protein band intensity (Fig. 7B). It increased maximally (2.0-fold) after 6 h of treatment, followed by a gradual decrease at later stages, but remained significantly higher than the control level until 48 h of treatment (Fig. 7C).
Ammonia is one of the most commonly available toxicants in the aquatic ecosystem. In general, fishes are known to be more resistant to ammonia toxicity than all other vertebrates (for reviews, see Ip et al., 2001; Saha and Ratha, 2007). Interestingly, air-breathing magur catfish have a unique capacity to tolerate a very high concentration of ammonia in their aquatic habitats (Saha et al., 2003). Development of various adaptational strategies mainly to avoid the in situ accumulation of ammonia to a toxic level during exposure to HEA have already been well documented in magur catfish, such as the conversion of accumulated ammonia to urea via the induced ornithine–urea cycle (OUC), to glutamine via induced glutamine synthetase, and also to some non-essential amino acids (Saha et al., 2002, 2003, 2007; Banerjee et al., 2018). However, reports on the ammonia-induced oxidative stress and possible molecular mechanisms to defend against such stress have not yet been reported in magur catfish. The results of the present study clearly demonstrate the occurrence of some of the mechanisms that are involved in defending against the ammonia-induced oxidative stress response, and the involvement of NO in such adaptive mechanisms in primary hepatocyte culture of magur catfish. Treatment of primary hepatocytes with 5 mmol l−1 NH4Cl led to a significant rise in ammonia concentration in hepatocytes within 3 h, and the high level of intracellular ammonia concentration was maintained throughout the experimental period. This was similar to the observation made previously, where a high build-up of ammonia concentration in different body tissues of magur catfish, including the liver during exposure to HEA in situ, was demonstrated (Saha et al., 2003, 2007). Interestingly, an increase in intracellular ammonia concentration was associated with an increase of ROS production by the hepatocytes, as evidenced by a sharp rise in intracellular concentrations of H2O2 and MDA levels within 3 h, thereby causing oxidative stress. However, this increase of both H2O2 and MDA levels decreased gradually at later stages and returned almost to basal levels after 48 h of treatment with NH4Cl. Subsequently, the activity of two antioxidant enzymes SOD and CAT increased significantly within 3 h, with a further rise of activity at later stages of treatment as a consequence of up-regulation of different related genes such as sod1, sod2 and cat, along with an increase in enzyme protein concentrations in primary hepatocytes. Thus, it is evident that the hepatic cells of magur catfish, as a first line of defence, have the ability to defend against ammonia-induced oxidative stress by up-regulating the expression of sod and cat genes under HEA exposure. However, the activity of SOD and CAT increased considerably within 3 h of treatment with NH4Cl, but a sizeable increase in both mRNAs and enzyme proteins for both genes were seen only from 6 h onwards, suggesting that there could be some other means of regulation of these two enzymes such as cofactor-mediated activation (Harris, 1992) and/or activation by covalent modification (Ma et al., 2017). Such ammonia-induced oxidative stress and subsequent induction of activity of certain anti-oxidant enzymes during exposure to HEA has also been reported in grass carp (Ctenopharyngodon idellus) (Jin et al., 2017), Nile tilapia (O. niloticus) (Hegazi et al., 2010), common carp (C. carpio), goldfish (C. auratus), rainbow trout (O. mykiss) (Sinha et al., 2014) and European seabass (Dicentrarchus labrax) (Sinha et al., 2015). However, the mechanism by which high intracellular ammonia concentration induces free radical production leading to oxidative stress, and the subsequent induction of antioxidant machinery, are not yet fully understood in fish.
The most interesting observation made in the present study is the stimulation of NOS/NO synthetic machinery in primary hepatocytes of magur catfish during exposure to HEA, resulting in a continuous increase of intracellular NO concentrations in both hepatocytes and culture medium. These data were further supplemented by the observations made on the stimulation of iNOS activity as a consequence of up-regulation of the nos2 gene and more expression of iNOS enzyme proteins. Similar up-regulation of the nos2 gene and more production of NO in different tissues of magur catfish were also reported while exposing the fish to 25 mmol l−1 NH4Cl in situ for 14 days (Kumari et al., 2019). Furthermore, in both the cases the enhanced synthesis of NO was accompanied by higher intracellular accumulation of ammonia in hepatocytes (present study), and also in other tissues as well (Kumari et al., 2019). Thus, it appears that the enhanced intracellular ammonia concentration during HEA exposure served as a positive modulator to stimulate NO synthesis in primary hepatocytes by inducing the nos2 gene. However, it is difficult to explain with our present data if it is the high ammonia or the ROS load, generated in hepatocytes under ammonia stress, which serves as a primary signalling molecule to stimulate NO synthesis. Nonetheless, it is apparent that in response to ammonia-induced oxidative stress, the increased synthesis and accumulation of NO in hepatocytes was accompanied by a gradual decrease of H2O2 and MDA levels, as measures of decreasing the ROS load, in primary hepatocytes after an initial sharp rise of intracellular concentrations of both.
When the primary hepatocytes were treated with AG (an inhibitor for iNOS) in the presence of NH4Cl, a gradual increase in intracellular concentrations of both H2O2 and MDA along with a subsequent decrease in NO concentration was observed in hepatocytes. This was accompanied by a significant decrease in SOD and CAT activity along with the downregulation of sod1, sod2 and cat genes expression, as evidenced by a decrease in mRNAs and protein concentrations of both enzymes. These observations are in contrast to the situation observed in hepatocytes treated only with NH4Cl. Similar observations were made when the hepatocytes were treated with BAY (an inhibitor for NFκB) along with NH4Cl in the culture medium. Furthermore, a sharp rise of LDH activity in the culture medium was observed when the hepatocytes were treated with these two inhibitors separately along with NH4Cl, thus suggesting that the inhibition of NO synthesis caused more cellular damage under ammonia-induced oxidative stress. When only NH4Cl was added to the culture medium, a negligible amount of LDH leakage from the hepatocytes was seen in the culture medium, which is similar to the observation made in control hepatocytes.
Thus, it may be that the stimulation of NO synthesis plays a vital role in defending against ammonia-induced oxidative stress in the hepatocytes of magur catfish as an antioxidant strategy. Such NO-mediated antioxidant strategies were also suggested in yeast (Saccharomyces cerevisiae) (Nasuno et al., 2014) and in fission yeast (Scizosaccharomyces pombe) (Astuti et al., 2016). Nasuno et al. (2014) also suggested the NO-mediated antioxidant mechanism in yeast through the activation of transcription factor MAC1, which is ultimately responsible for the induction of the ctr1 gene. Other studies also indicated that higher NO concentration contributes to greater tolerance to H2O2 by suppressing the conversion of Fe3+ to Fe2+, and by up-regulating H2O2 detoxifying enzymes in the bacterial system (Gusarov and Nudler, 2005; Astuti et al., 2016). Furthermore, Husain et al. (2008) suggested the NO-mediated inhibition of the electron transport chain in Salmonella as a novel antioxidant strategy. Similarly, Robb and Connor (2002) also demonstrated the protective role of NO against the oxidative stress-induced mitochondrial damage of astrocytes. However, in all the above-mentioned studies, either the substrate for NO or NO itself was added directly to the experimental system to check the antioxidant activity of NO, but in the present study, neither the substrate for NO production nor NO was added to the culture medium to investigate its antioxidant activity under ammonia-induced oxidative stress. Indeed, stimulation of endogenous NO production and its accumulation, as a consequence of induction of the nos2 gene under hyper-ammonia stress, was observed in the hepatocytes of magur catfish. This ultimately provided protection against ammonia-induced oxidative stress and also from the related cellular damage mainly by activating certain antioxidant genes.
In another set of experiments, we observed that treatment of primary hepatocytes with 5 mmol l−1 NH4Cl led to a significant induction of the rela gene and also greater expression of NFκB (p65) protein. Similar to our observation, the NFκB-mediated induction of nos2 gene with greater expression of iNOS protein was also reported in hepatocytes of another closely related air-breathing catfish (Heteropneustes fossilis) under hyper-ammonia stress during exposure to 25 mmol l−1 NH4Cl (Choudhury and Saha, 2012). The activation of NFκB by H2O2 and the NFκB-mediated activation of the nos2 gene are known to occur in many cell types (Gupta et al., 2010). Thus, it is logical to think that induction of the nos2 gene and iNOS protein expression in primary hepatocytes of magur catfish under ammonia-induced oxidative stress is also mediated through the activation of NFκB as an anti-oxidant strategy.
In conclusion, it may be considered that the hepatocytes of magur catfish have a unique capacity to handle ammonia-induced oxidative stress. They have the ability to protect themselves from oxidative stress and related cellular damage by inducing the SOD–CAT enzyme system through the NO signaling pathway, where the synthesis of NO is stimulated as a consequence of NFκB-mediated induction of nos2 gene under ammonia stress (Fig. 8). It is the first report of such NO-mediated induction of the SOD–CAT enzyme system in any teleost fish to defend against environmental ammonia-induced oxidative stress, and also from associated cellular damage.
Conceptualization: N.S.; Methodology: R.H., D.K.; Validation: N.S.; Formal analysis: N.S.; Investigation: R.H., D.K., E.K., N.S.; Resources: N.S.; Data curation: N.S.; Writing - original draft: R.H., D.K.; Writing - review & editing: N.S.; Supervision: N.S.; Project administration: N.S.; Funding acquisition: N.S.
This study was supported by a project sanctioned to N.S. by the National Agricultural Science Fund, Indian Council of Agricultural Research, New Delhi (ABA-7011/ 1018-19/240).
The authors declare no competing or financial interests.