Induction of nitric oxide synthesis: a strategy to defend against high environmental ammonia-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur.

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.

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

Transcriptome analysis reveals novel insights in air-breathing magur catfish (Clarias magur) in response to high environmental ammonia.

The facultative air-breathing magur catfish (Clarias magur) frequently face different environmental challenges, such as hyper-ammonia, and desiccation stresses in their natural habitats. All these stresses lead to higher accumulation of body ammonia, thereby causing various harmful effects to the fish due to its toxicity. Nonetheless, the mechanisms underlying ammonia-induced toxicity is yet not clear. In the present study, we used RNA sequencing and utilized a modified method for de novo assembly of the transcriptome to provide an exhaustive study on the transcriptomic alterations of magur catfish in response to high environmental ammonia (HEA; 25 mM NH4Cl). The final contig assembly produced a total of 311,076 unique transcripts (termed as unigenes) with a GC content of 48.3% and the average length of 599 bp. A considerable number of SSR marker associated with these unigenes were also detected. A total of 279,156 transcripts were successfully annotated by using various databases. Comparative transcriptomic analysis revealed a total of 3453 and 19,455 genes were differentially expressed in the liver and brain tissues, respectively, in ammonia-treated fish compared to the control. Enrichment analysis of the differentially expressed genes (DEGs) showed that several GO and KEGG pathway terms were significantly over-represented. Functional analysis of significantly elevated DEGs demonstrated that ammonia stress tolerance of the magur catfish was associated with quite a few pathways related to immune response, oxidative stress, and apoptosis, as well as few transporter proteins involved with ammonia and urea transport. Both liver and brain tissues showed HEA-mediated oxidative damage with consequent activation of antioxidant machinery. However, elevated ROS levels led to an activation of inflammatory cytokines and thus innate immune response in the liver. Conversely, in the brain ROS-mediated irreversible cell damages activated apoptosis via both p53-Bax-Bcl2 and caspase-mediated pathways. The present study provides a novel understanding of the molecular responses of this air-breathing catfish against the ammonia-induced stressors, which could elucidate the underlying mechanisms of adaptation of this facultative air-breather living under various environmental constraints.

Antioxidant activity of endogenously produced nitric oxide against the zinc oxide nanoparticle-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur.

The facultative air-breathing magur catfish (Clarias magur) regularly encounter various environmental challenges including the exposure to nanomaterials discarded as industrial wastes in water bodies. The present investigation aimed at determining the possible ZnO NP-induced oxidative stress and also the antioxidant strategy of nitric oxide (NO), generated endogenously, in primary hepatocytes of magur catfish. Exposure of primary hepatocytes to different concentrations of ZnO NPs (5 and 10 µg/mL) led to a sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) within 6 h, which decreased gradually at later stages. This phenomenon was accompanied by an initial decrease of superoxide dismutase (SOD) and catalase (CAT) activities, the expression of their corresponding genes and the enzyme protein levels, with a subsequent significant increase of all these parameters at later stages. Most interestingly, exposure to ZnO NPs also stimulated the NO production by the primary hepatocytes as a consequence of induction of inducible nitric oxide synthase (iNOS) activity, higher expression of nos2 gene and iNOS protein. Furthermore, when the NO production by the hepatocytes was inhibited by either aminoguanidine (inhibitor for iNOS) or BAY (inhibitor for NFκB) in the presence of ZnO NPs, the intracellular concentrations of H2O2 and MDA was significantly elevated. This elevation was accompanied by a subsequent decrease of sod and cat genes expression, thereby suggesting that the inhibition of NO production leads to oxidative stress. Thus, it is believed that the magur catfish uses the strategy of stimulation of endogenous NO production by inducing the nos2 gene and simultaneous NO-mediated induction of sod and cat genes to defend against the NP-induced oxidative stress. It is the first report of such NO-mediated antioxidant strategy in any teleost fish to defend against the NP-induced oxidative stress and corresponding cellular damages.