Delineating involvement of MAPK/NF-κB pathway during mitigation of permethrin-induced oxidative damage in fish gills by melatonin.

Present study evaluated involvement of transcription factors during permethrin-induced gill toxicity and its amelioration by melatonin. First, adult Notoptertus notopterus females were exposed to permethrin at nominal concentrations [C: 0.0, P1: 0.34, P2: 0.68 µg/L] for 15 days followed by intramuscular melatonin administration (100 µg/kg body weight) for 7 days. Gill MDA, XO, LDH levels increased, while Na+-K+-ATPase, SDH, cytochrome C oxidase levels decreased with increasing permethrin concentrations. Glutathione, SOD, CAT, GST, GRd levels increased in P1 than C, but decreased in P2 than P1, C. Melatonin administration restored gill enzyme and antioxidant levels in P1, P2. Next, isolated gill tissues were exposed to permethrin at 25, 50 µM doses along with melatonin administration (100 µg/mL). NF-κB, NRF2, Keap1, ERK, Akt, caspases protein expression changed significantly during permethrin-induced gill damage. Melatonin administration amended permethrin-induced molecular imbalance through modulation of caspase proteins and MAPK/NF-κB signal transduction pathway via melatonin receptor 1.

Bioactive compounds from four Indian medicinal plants have different potency to induce sex reversal in Nile tilapia: A chromatographic, molecular docking and in silico analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal plants such as Basella alba (Family: Basellaceae), Tribulus terrestris (Family: Zygophyllaceae), Asparagus racemosus (Family: Asparagaceae) and Mucuna pruriens (Family: Fabaceae) are mentioned in Indian traditional system of medicine Ayurveda to possess androgenic activity and increase male virility. The plants have been reported to improve testosterone level and sperm production in experimental male rodents as well. AIM OF THE STUDY: Male Nile tilapias grow more quickly than females and hence are preferred for monosex Nile tilapia culture. Ethanol extracts of B. alba leaves (EB) and T. terrestris seeds (ET), and methanol extract of A. racemosus roots (MA) and M. pruriens seeds (MM) were found effective to induce masculinization in Nile tilapia. The present study intends to evaluate the anti-aromatase activity of EB, ET, MA and MM, to identify the androgenic bioactive compounds in the extracts, and to determine their pharmacokinetics. The study may validate the use of those plant extracts and their major bioactive phytoconstituents in the field of aquaculture and pharmaceuticals. MATERIALS AND METHODS: The four crude plant extracts were first fractioned through column and thin layer chromatography (TLC). Three days old Nile tilapia juveniles (mean weight 0.025 ± 0.009g; mean length 12.50 ± 0.12 mm; n = 50 fish/replicate, 3 replicates/treatment) were then fed diets fortified with the obtained fractions for 30 days. After 30 days, fish were sacrificed and gonad aromatase mRNA expression, and 11-ketotestosterone (11-KT) and estradiol (E2) levels were measured. Fractions yielding the highest male percentage for each plant were subject to gas chromatography-mass spectrometry (GC-MS) analysis. The in silico docking and SwissADME study were conducted with the components showing higher peak percentage in chromatogram. RESULTS: After column chromatography and TLC analysis, EB, ET, MM and MA yielded 6 (EB1 - EB6), 8 (ET1- ET8), 14 (MM1-MM14) and 5 (MA1- MA5) fractions, respectively. Fish fed EB2, ET2, MA2 and MM13 fraction fortified diets showed significantly (p < 0.05) higher male percentage (92.32%-98.39%) compared to other treatment groups. EB2, ET2, MA2 and MM13 fed fish showed significantly (p < 0.05) higher 11-KT level compared to control male (+247.52 - +397.76%) and lower E2 level compared to control female (-95.92% to -90.65%). Aromatase mRNA expression was significantly (p < 0.05) down-regulated by all these four fractions (-1.32 to -5.65 fold) with respect to control female. GC-MS analysis revealed the presence of 1-Octadecene (OD) in EB2, Phenol, 2,4-bis(1,1-dimethylethyl) (PD) in ET2 and MA2, 9,12-Octadecadienoic acid (Z,Z)- (ODDA) in MM13. In silico molecular docking indicated that PD is more effective than ODDA and OD to inhibit aromatase. In addition, PD showed better pharmacokinetics and more drug-likeness compared to OD and ODDA in SwissADME analysis. CONCLUSION: The present results indicate that ET and MA are more potent to produce all-male tilapia by means of aromatase inhibition. PD can be an ideal compound to achieve masculinization in Nile tilapia through dietary administration, but further investigation is required.

Acute exposure to a neem based biopesticide and mahua oil cake changes haemocyte parameters in freshwater crab, Varuna litterata (Decapoda, Crustacea).

Present study aims to evaluate the immunotoxic effects of two biopesticides, Nimbecidine Plus (a neem biopesticide) and mahua oil cake (MOC) on the haemocyte populations of a freshwater crab, Varuna litterata after acute exposure. Four-day static renewal bioassay test was performed where sixteen healthy adult male crabs were exposed to 96-h LC50 values of Nimbecidine Plus (0.006284 ppt) and MOC aqueous extract (7.631 ppt) separately in the laboratory condition. Control groups were maintained throughout the experimental period without any biopesticide exposure. Various haemocyte parameters such as total count (THC), differential count (DHC), haemocyte density, cytomorphological anomalies and reactive oxygen species (ROS) were measured in the biopesticides-exposed and control crabs after 24, 48, 72, and 96 h of exposure. After treatment with Nimbecidine Plus and MOC, several cytomorphological deformities (cytoplasmic and nuclear membrane disintegration, chromatin condensation, pyknosis, karyorrhexis, karyolysis, nuclear vacuolation, altered cell shape, cellular coagulation, cytoplasmic discharge, vacuolation) were observed in hyalinocytes, small granule haemocytes and large granule haemocytes with modulation of their relative percentages at different exposure times. THC, DHC, haemocyte density and ROS levels were significantly altered (p < 0.05) in biopesticides-exposed crabs at different exposure periods. The toxicity of both biopesticides did not persist throughout the entire exposure time. Nimbecidine Plus exhibited nonlinear toxic impacts on different haemocyte parameters at initial, mid and higher exposure periods whereas MOC showed linear toxic effects mostly at initial exposure time. In comparison to MOC, Nimbecidine Plus showed higher immunotoxic effects in V. litterata. Outcome of this experiment might provide useful information to understand the immune responses of V. litterata against biopesticide toxicity.

Melatonin ameliorates lipopolysaccharide induced brain inflammation through modulation of oxidative status and diminution of cytokine rush in Danio rerio.

Lipopolysaccharide (LPS) is known to induce inflammation and immunonomodulation in a piscine model of Danio rerio. Present study aimed to explore the ability of melatonin in attenuating LPS-induced oxidative damages using this model. In LPS-exposed fish, activation of stress marker MDA was observed in brain with corresponding augmentation of multiple pro-inflammatory cytokines (IL1ß, IL6, IL10 and TNFα). In addition, it also showed marked increase in the levels of heat shock factor (HSF) and heat shock proteins (HSPs) in association with transcription factors (NF-kB and NRF2) and mitogen-activated protein kinases (MAPKs). The changes in the levels of these mediators are highly correlated with the induction of pro-inflammatory cytokines. In melatonin-treated fishes, significant amelioration of oxidative stress was observed with reduced levels of MDA and pro-inflammatory cytokines. Melatonin also modulated expression of HSPs that facilitated the brain to overcome inflammation-induced stress by directly initiating NFkB/NRF2 translocation. In summary, melatonin effectively functions to reduce stress induced inflammatory signalling through modulation of oxidative stress and protects the brain from the neuropathological insult.

Effects of salinity stress on antioxidant status and inflammatory responses in females of a "Near Threatened" economically important fish species Notopterus chitala: a mechanistic approach.

In the present study, acute stress responses of adult female Notopterus chitala were scrutinized by antioxidant status and inflammation reaction in the gill and liver at five different salinity exposures (0, 3, 6, 9, 12 ppt). Oxidative defense was assessed by determining superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase, and glutathione reductase activities, while malondialdehyde (MDA), glutathione, and xanthine oxidase levels were determined as indicators of oxidative load. Pro-inflammatory cytokines (IL-1ß, IL-6, IL-10, and TNFα) and caspase 1 levels were also analyzed. Expression levels of transcription factors (NRF2 and NF-κB) and molecular chaperons (HSF, HSP70, and HSP90) were estimated to evaluate their relative contribution to overcome salinity stress. MDA showed a significant (P < 0.05) increase (gill, + 25.35-90.14%; liver, + 23.88-80.59%) with salinity; SOD (+ 13.72-45.09%) and CAT (+ 12.73-33.96%) exhibited a sharp increase until 9 ppt, followed by a decrease at the highest salinity (12 ppt) (gill, - 3.92%; liver, - 2.18%). Levels of cytokines were observed to increase (+ 52.8-127.42%) in a parallel pattern with increased salinity. HSP70 and HSP90 expressions were higher in gill tissues than those in liver tissues. NRF2 played pivotal role in reducing salinity-induced oxidative load in both the liver and gills. Serum cortisol and carbonic anhydrase were measured and noted to be significantly (P < 0.05) upregulated in salinity stressed groups. Gill Na+-K+-ATPase activity decreased significantly (P < 0.05) in fish exposed to 6, 9, and 12 ppt compared to control. Present study suggests that a hyperosmotic environment induces acute oxidative stress and inflammation, which in turn causes cellular death and impairs tissue functions in freshwater fish species such as Notopterus chitala.

Evaluating the role of dietary plant extracts to allow adaptation to thermal stress in a cold stream ornamental fish, Botia rostrata (Günther, 1868).

Increases in ambient temperature affect the biochemical status of fish, and dietary supplementation with bioactive phytoconstituents may promote resilience against environmental stress. This study evaluated the impact of three plant extracts on the biochemical status of a cold stream fish Botia rostrata (Günther, 1868) under high temperatures. After 1 month dietary supplementation separately with Mucuna pruriens methanol extract (0.25 g/kg feed), Tribulus terrestris ethanol extract (0.5 g/kg feed) and Basella alba ethanol extract (1.0 g/kg feed), juvenile fish (Wt. 4.3 ± 0.5g) were exposed to different sublethal heat stress [28 ± 0.5 °C (T1), 32 ± 0.5 °C (T2), 36 ± 0.5 °C (T3)]. Control fish were fed a diet without any plant extract and maintained at 24 ± 0.5 °C. Serum and muscle tissues were collected to measure different biochemical parameters, muscle metabolic enzymes and molecular chaperons before and after heat stress. Before stress, the group fed the Mucuna diet showed significant (P < 0.05) increases in serum glucose [+10.92%], protein [+18.93%], muscle heat shock protein (HSP) 90 [+8.6%] compared to the control group. No significant change (P > 0.05) of stress parameters was observed between control, Tribulus and Basella fed fish. The control group exposed to T3 showed significant differences (P < 0.05) in protein [-26.19%], lactate dehydrogenase [+93.69%], fructose 1,6 bisphosphate [-35.19%], phosphorylase 'a' [+35.72%], HSP60 [+69.54%], HSP70 [+84.85%], HSP90 [+92.07%], heat shock factor (HSF) 1 [+88.48%] suggesting susceptibility of Botia to this temperature. Among the three plant extracts, Mucuna methanol extract was effective to enhance resistance against temperature-induced biochemical alterations. After exposure to T3, only the fish fed Mucuna diet showed no mortality. Fish fed Mucuna diet exposed to 36 ± 0.5 °C showed significantly higher (P < 0.05) glucose [+42.82%], protein [+11.98%], citrate synthase [+59.81%], phosphorylase 'a' [+14.96%], Glucose 6 phosphate dehydrogenase [+60.87%], HSP60 [+34.13%], HSP70 [+41.42%], HSP90 [+65.91%], HSF1 [+61.32%] compared to those in Mucuna fed fish maintained at 24 ± 0.5 °C. These results highlight temperature-induced biochemical alterations in Botia and point towards the potential use of Mucuna in overcoming such adverse high thermal stress.

Organometallic Folate Gold Nanoparticles Ameliorate Lipopolysaccharide-Induced Oxidative Damage and Inflammation in Zebrafish Brain.

Synthesized organometallic gold-based folate nanoparticles (FAuNPs) were characterized, and its defense against lipopolysaccharide (LPS)-induced brain inflammation in Zebra fish was proven. Vitamin entrapment efficiency of these particles was found to be nearly 70%. The in vitro pH-dependent drug release dialysis study of FAuNPs confirmed a slow, sustained, and gradual release of folate for a period of 24 h. Both AuNPs and FAuNPs did not cause any marked changes in food intake, body weight, color, behavioral pattern, blood parameters, and hepatotoxicity. Histology of liver showed no changes between treated and control groups of fishes. The ex vivo study showed significant uptake of FAuNPs to free folate in folate receptor negative Hek293 cells, confirming a strategy to overcome folate deficiency in the brain. Antioxidant status and activities of few crucial brain enzymes were also measured to assess the brain function and found to be returned to the basal level, following FAuNP treatment. The transcription factor NRF2-Keap 1 expression pattern was also noted, and a prominent modulation was observed in the LPS-treated and FAuNP-administered group. Decisive brain enzymes like AChE and Na+K+ATPase were decreased significantly after LPS treatment, which is restored with FAuNP treatment. Caspases increased sharply after LPS treatment and diminished following FAuNP treatment. We conclude that FAuNP due to its high physical stability and uptake could be utilized against severe brain inflammation, leading to brain injury and neurodegeneration.

Crosstalk between heat shock proteins, NRF2, NF-κB and different endogenous antioxidants during lead-induced hepatotoxicity in Puntius ticto.

Present study aims to investigate interaction of molecular chaperons (heat shock protein 70, heat shock protein 90) with transcriptional factors (nuclear factor kappa B/nuclear factor E2-related factor 2/Kelch-like ECH-associated protein 1) to evaluate their role during metal induced stress in fish hepatocytes. Adult Puntius ticto were exposed to lead nitrate at 0 mg/l (control), 1/50th (0.04 mg/l) and 1/20th (0.12 mg/l) of LC50 for 30 days and sacrificed to collect liver tissues. Activity of selected liver enzymes, antioxidants and metallothionein were analyzed. Levels of heat shock protein 70, heat shock protein 90, nuclear factor kappa B, nuclear factor E2-related factor 2 and Kelch-like ECH-associated protein 1 were also measured. Liver enzymes showed a significant increase (p < 0.05) in both Pb exposed groups indicating that the liver might be at risk of damage. Increased level of lipid peroxidation due to metal stress was marked by significant increase (p < 0.05) in malondialdehyde level in fish exposed to the higher Pb concentration compared to control (+ 13.7 %). Significant increase (p < 0.05) in gluthathione reductase (+ 35 %, + 39.2 %), glutathione s-transferase (+ 22.4 %, + 50.4 %) activities and decrease in reduced glutathione level (- 6.75 %, - 12.25 %) in fish exposed to both lower and higher Pb concentration compared to control also indicated metal induced oxidative damage in fish liver. Super oxide dismutase and catalase activities increased significantly (p < 0.05) during exposure to lower Pb concentration, while decreased significantly (p < 0.05) during exposure to higher Pb concentration compared to those in control. Significant (P < 0.05) increase (+ 52.63 %, + 89.47 %) in metallothionein in Pb exposed groups confirmed its role in detoxification process of the metal. Heat shock protein 70 and heat shock protein 90 expression levels increased significantly (p < 0.05) during metal exposure indicating their role as modulator of stress-induced antioxidant protein remodelling. A positive correlation between nuclear factor kappa B/nuclear factor E2-related factor 2/Kelch-like ECH-associated protein 1 with gluthathione regulatory enzymes (gluthathione reductase and glutathione s-transferase) was noted. Current study effectively illuminates the critical role of different factors (heat shock proteins/nuclear factor kappa B/nuclear factor E2-related factor 2/Kelch-like ECH-associated protein 1) to influence the expression and synthesis of antioxidants and other functional enzymes in lead-exposed fish liver.

Enzymatic, non enzymatic antioxidants and glucose metabolism enzymes response differently against metal stress in muscles of three fish species depending on different feeding niche.

Current study aims to determine difference in metal accumulation pattern in muscle of Liza parsia (pelagic, omnivore), Amblypharyngodon mola (surface feeder, herbivore) and Mystus gulio (benthic, carnivore) depending on their niche and feeding habit and how it affects the endogenous antioxidants and glucose metabolism in fish muscle. Fishes were collected from Malancha, Diamond Harbour and Chandanpiri, West Bengal, India. Concentrations of lead, zinc, cadmium, chromium were measured in water, sediment and fish muscle. Metal pollution index (MPI) and bioconcentration factor (BCF) was calculated to evaluate the ability of fish to accumulate specific metals in muscle tissue from the aquatic environment. Metal concentrations were found significantly higher (P < 0.05) in water, sediment, fish muscles from Malancha than Chandanpiri and Diamond Harbour. L. parsia (MPI: 0.4-1.6) showed highest metal deposition in their muscle followed by A. mola (MPI: 0.37-1.38) and M. gulio (MPI: 0.2-1.2). Malondealdehyde, superoxide dismutase, catalase, glutathione S transferase, glutathione reductase and cortisol levels increased in case of L. parsia from Malancha and Chandanpiri. Succinate dehydrogenase, lactate dehydrogenase, Ca+2 ATPase and cytochrome C oxidase levels were significantly (P < 0.05) lower at Malancha and Chandanpiri than Diamond Harbour. Heat shock protein (HSP70) expression was significantly (P < 0.05) higher in all fish species at Malancha followed by Chandanpiri and Diamond Harbour. Glucose, glycogen, hexokinase, phosphofructokinase and glycogen phosphorylase levels varied between sites and selected fish species. Serum cortisol level was measured and found to be the highest in L. parsia from Malancha (2.94 ± 0.12 ng/ml) and the lowest in M. gulio from Diamond Harbour (0.7 ± 0.05 ng/ml). The results indicate that metal toxicity alters antioxidant levels, oxidative status and energy production in fish in species specific manner. Our results also indicate that Mystus has the highest degree of adaptability in response to metal toxicity possibly due to its specific food habit and niche position. Therefore, it can be concluded that maintenance of oxidative and metabolic status to combat metal-induced oxidative load will be helpful for the fishes to acquire better resistance under such eco-physiological stress. Alteration of niche and interactive segregation in aquatic organism may be one of the key modulator of resistance against such stress.

Effectiveness of melatonin to restore fish brain activity in face of permethrin induced toxicity.

Present study demonstrates permethrin induced oxidative damage in fish brain and explores effectiveness of melatonin to ameliorate brain function. Adult female Notopterus notopterus were exposed to nominal permethrin concentrations at 1/20th (0.34 µg/l) and 1/10th (0.68 µg/l) of LC50 for 15 days. The measured permethrin concentrations using gas chromatography (GC-ECD) were 0.28 µg/l and 0.57 µg/l, respectively. Some fish were sacrificed to collect brain tissue after 15 days of exposure. Remaining fish from both groups were administered exogenous melatonin (50 µg/kg, 100 µg/kg body weight) for 7 days and brain tissues were collected. Brain enzymes, ntioxidant factors, HSP70, HSP90, nuclear factor-kappa binding (NFkB), melatonin receptor (MT1R) proteins were measured. Permethrin treatment significantly (P < 0.05) decreased the levels of glutathione and brain enzymes. Malondialdehyde (MDA), xanthine oxidase (XO), HSPs increased at each concentration of permethrin. However, superoxide dismutase, glutathione s-transferase levels increased at low permethrin concentration followed by sharp decrease at higher concentration. Expression of NFkB and MT1R increased significantly (P < 0.05). Melatonin administration reinstated activity of brain enzymes, reduced MDA, XO levels and modulated HSPs. Melatonin also increased expression of NFkB and MT1R. Exogenous melatonin improves oxidative status in permethrin stressed fish brain. Melatonin modulates expression of HSPs that enables brain to become stress tolerant and survive by initiating NFkB translocation. Melatonin could act through melatonin receptor protein to induce synthesis of antioxidant proteins. Therefore the study successfully evaluates the potential of melatonin application for better culture and management of fish against pesticide toxicity.

Arecoline inhibits pineal-testis function in experimentally induced hypothyroid rats.

Arecoline is known to cause endocrine dysfunction. In the current article role of arecoline on pineal-testis activity was investigated in hypothyroid rats induced by propylthiouracil (PTU). PTU treatment caused thyroid dysfunction ultrastructurally with a fall in T3 and T4 levels followed by a rise of thyroid stimulating hormone (TSH) level. Pineal activity was impaired by PTU treatment, as evident from degenerated synaptic ribbons and mitochondria of the pinealocytes with depletion of pineal and serum N-acetyl serotonin and melatonin levels. Leydig cell function was suppressed, evident from reduced smooth endoplasmic reticulum and depletion of testosterone level. Sex accessories function was impaired by showing scanty rough endoplasmic reticulum with depletion of fructose and sialic acid levels. Arecoline treatment that caused pineal dysfunction and testicular stimulation in control rats, suppressed both pineal and testis functions after PTU treatment. The findings suggest that arecoline inhibits pineal-testis function in experimentally induced hypothyroid rats.

Enzymatic, Non-enzymatatic Antioxidant Levels and Heat Shock Protein Expression as Indicators of Metal Induced Toxicity and Reproductive Modulation in Female Indian Major Carp Cirrhinus cirrhosus.

Effects of zinc (Zn) and lead (Pb) exposure on oxidative biomarkers and heat shock protein (HSP) expression, and their possible relation to ovarian steroidogenesis in fish were investigated. Female Cirrhinus cirrhosus were exposed to 1/10th of LC50 of metals for 30 days, and kept for another 30 days without metal exposure. Metal concentrations, antioxidants, HSPs, 17ß-estradiol and steroidogenic enzymes were analysed in brain and ovary after 15 and 30 days of exposure and a 15 and 30 day recovery period. Activities of enzymatic and non-enzymatic antioxidants showed duration dependent variation in both exposure and recovery period. HSP70 and HSP90 expressions increased following metal exposure, with the expression being higher in brain than ovary. 17ß-Estradiol, steroidogenic enzymes decreased significantly (p < 0.05) after metal exposure. The present study suggests that metals have differential and tissue specific influence on oxidative status and manipulate ovarian steroidogenesis probably through the modulation of HSPs.

Evaluation of the acute toxicity of mahua oil cake aqueous extract and its effect on the behavioral responses of the freshwater grapsid crab, Varuna litterata (Fabricius, 1798).

Mortality and behavioral alterations are monitored as the sensitive endpoints in toxicological studies and may be applied as useful biomarkers to assess piscicidal pollution in aquatic environment. Present study assesses acute toxicity of the piscicide, mahua oil cake (MOC), and its effect on the behavioral responses of the freshwater grapsid crab, Varuna litterata, under laboratory conditions. To determine the LC50 values, a 4-day acute static renewal toxicity test was done where 10 adult male crabs (mean length 2.870 ± 0.379 cm; mean weight 9.891 ± 3.951 g) were exposed to different concentrations (1, 5, 10, 15, 20, 25, 30, 35 ppt) of MOC aqueous extract with a control at different exposure periods. The LC50 values are 19.109 ppt for 24 h, 16.052 ppt for 48 h, 11.827 ppt for 72 h, and 7.631 ppt for 96 h. The high LC50 values indicate less sensitivity of this crab to the MOC extract than other aquatic animals. MOC extract has toxic effect on the mouthparts activity, whirling motion of water current producing activity, froth releasing activity, aggregation, balance and coordination actions, medium escaping behavior, locomotor activity, and fecal matter excretion of this crab in different exposure periods. Behavioral responses such as froth releasing activity, aggregation, and medium escaping behavior can be used as biomarkers of MOC pollution in aquatic environment.

Physiological and behavioural responses to acid and osmotic stress and effects of Mucuna extract in Guppies.

Variation in pH (acidification) and salinity conditions have severe impact at different levels of biological organization in fish. Present study focused to assess the effects of acidification and salinity changes on physiological stress responses at three different levels of function: i) hormonal and oxidative response, ii) osmoregulation and iii) reproduction, in order to identify relevant biomarkers. Second objective of the study was to evaluate the efficacy of plant (Mucuna pruriens) extract for alleviating pH and salinity related stress. Guppies (Poecilia reticulata) were exposed to different pH (6.0, 5.5, 5.0) and salinity (1.5, 3.0, 4.5 ppt) for 7, 14 and 21 days. Following exposure to stress for respective duration, fish were fed diet containing methanol extract of Mucuna seeds (dose 0.80 gm/kg feed) for 7, 14 and 21 days to measure their possible recovery response. Stress hormone (cortisol), hepatic oxidative stress parameters [superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRd), glutathione peroxidise (GPx), glutathione S-transferase (GST), malondialdehyde (MDA), glutathione (GSH)], gill osmoregulatory response (Na+-K+ATPase activity), sex steroid profiles and mating behaviours (gonopodial thrust and gestation period) were estimated. Cortisol and MDA levels increased with dose and duration of acid and salinity stress, and cortisol levels were higher in males than in females. Effect on Na+-K+ATPase activity was more intense by salinity stress rather than pH induced stress. Both acid and salinity stress reduced sex steroid levels, and mating response was highly affected by both stresses in a dose- and duration-dependent manner. Mucuna treatment reduced stress-induced alteration of cortisol, MDA, Na+-K+ATPase activity and reproductive parameters. Dietary administration of Mucuna seed extract decreased the intensity of environmental stressors at all three functional levels. Mucuna treatment was more effective against salinity stress than acid stress. Thus, cortisol, oxidative stress marker MDA and Na+-K+ATPase could be effective indicators for acid and salinity stress in wild and domestic fish populations. Dietary administration of Mucuna extract may limit the detrimental effects of acidification and salinity variations that are the inevitable outcomes expected under global climate change conditions.

Melatonin ameliorates H2O2-induced oxidative stress through modulation of Erk/Akt/NFkB pathway.

BACKGROUND: Improper control on reactive oxygen species (ROS) elimination process and formation of free radicals causes tissue dysfunction. Pineal hormone melatonin is considered a potent regulator of such oxidative damage in different vertebrates. Aim of the current communication is to evaluate the levels of oxidative stress and ROS induced damage, and amelioration of oxidative status through melatonin induced activation of signaling pathways. Hepatocytes were isolated from adult Labeo rohita and exposed to H2O2 at three different doses (12.5, 25 and 50 µM) to observe peroxide induced damage in fish hepatocytes. Melatonin (25, 50 and 100 µg/ml) was administered against the highest dose of H2O2. Enzymatic and non-enzymatic antioxidants such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) was measured spectrophotometrically. Expression level of heat shock proteins (HSP70 and HSP90), HSPs-associated signaling molecules (Akt, ERK, cytosolic and nuclear NFkB), and melatonin receptor was also measured by western blotting analysis. RESULTS: H2O2 induced oxidative stress significantly altered (P < 0.05) MDA and GSH level, SOD and CAT activity, and up regulated HSP70 and HSP90 expression in carp hepatocytes. Signaling proteins exhibited differential modulation as revealed from their expression patterns in H2O2-exposed fish hepatocytes, in comparison with control hepatocytes. Melatonin treatment of H2O2-stressed fish hepatocytes restored basal cellular oxidative status in a dose dependent manner. Melatonin was observed to be inducer of signaling process by modulation of signaling molecules and melatonin receptor. CONCLUSIONS: The results suggest that exogenous melatonin at the concentration of 100 µg/ml is required to improve oxidative status of the H2O2-stressed fish hepatocytes. In H2O2 exposed hepatocytes, melatonin modulates expression of HSP70 and HSP90 that enable the hepatocytes to become stress tolerant and survive by altering the actions of ERK, Akt, cytosolic and nuclear NFkB in the signal transduction pathways. Study also confirms that melatonin could act through melatonin receptor coupled to ERK/Akt signaling pathways. This understanding of the mechanism by which melatonin regulates oxidative status in the stressed hepatocytes may initiate the development of novel strategies for hepatic disease therapy in future.

Consequences of oxidative damage and mitochondrial dysfunction on the fatty acid profile of muscle of Indian Major Carps considering metal toxicity.

Current study aims to find interrelation between mitochondrial enzyme function and fatty acid profile in fish muscle and role of antioxidant agents to maintain their balance in response to metal accumulation. Fishes (Labeo rohita, Catla catla, Cirrhinus cirrhosus) were collected from two sites (Nalban Bheri and Diamond Harbour, India). Concentrations of metals (lead, cadmium, copper, nickel, zinc), enzymatic and non-enzymatic antioxidant activity (malondialdehyde, superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione S-transferase), muscle enzyme activity (acetylcholinesterase, succinate dehydrogenase, lactate dehydrogenase, Ca2+ATPase, AMP-deaminase, lipoamide reductase, cytochrome C oxidase, aldolase) and fatty acid composition in muscle tissues were analyzed. Metal concentrations were significantly higher (P < 0.05) in fish muscles from Nalban compared to those in Diamond Harbour. Increased activity of antioxidant enzymes was noted with diminished mitochondrial enzymes activity and altered fatty acid composition in response to higher metal accumulation. Higher metal concentration in fish muscle of Nalban seems to significantly (P < 0.05) affect poly and monounsaturated fatty acid content, possibly due to oxidative damage and accumulation of hazardous reactive oxygen species (ROS) molecules. Changes in fatty acid contents following metal accumulation were observed to be species specific. Current study is the first correlative study to illuminate the level of oxidative damage and possible consequences on muscle cellular integrity, mitochondrial functionality and flesh quality against bioaccumulation of different metals in carps. Future studies are needed to quantify the relative contributions of enzymatic and low-molecular-mass antioxidants in protecting mitochondrial function and maintenance of proper fatty acid oxidation during acclimation to long term metal exposure.

Impact of Metal Toxicity on Oxidative Balance and Mitochondrial Enzyme Function in Muscle of Tilapia.

Present study investigates the effect of metal accumulation on antioxidant level and mitochondrial enzymes function in muscle of Oreochromis mossambicus. Metal accumulation in muscle upregulated stress marker malondialdehyde and the activity of different antioxidant enzymes with no significant alteration in glutathione system. Metal exposure to fish muscle decreased the activity of mitochondrial enzymes. AMP deaminase, aldolase, cytochrome C oxidase and lipoamide reductase showed positive correlation with acetylcholinesterase, glutathione reductase, reduced glutathione and glutathione peroxidase, but negative correlation with superoxide dismutase, catalase, glutathione S-transferase and thiobarbituric acid reactive substance. Analysis of these biomarkers clearly indicates the change in oxidative load in muscle tissues and provides insight to muscle response to the metal exposure. Therefore, the study outlines the potential use of biomarkers in context of muscle mitochondrial enzymes relating to oxidative processes that take place in the fish muscle following metal exposure and toxicity.

Melatonin ameliorates H2O2-induced oxidative stress through modulation of Erk/Akt/NFkB pathway

BACKGROUND: Improper control on reactive oxygen species (ROS) elimination process and formation of free radicals causes tissue dysfunction. Pineal hormone melatonin is considered a potent regulator of such oxidative damage in different vertebrates. Aim of the current communication is to evaluate the levels of oxidative stress and ROS induced damage, and amelioration of oxidative status through melatonin induced activation of signaling pathways. Hepatocytes were isolated from adult Labeo rohita and exposed to H2O2 at three different doses (12.5, 25 and 50 µM) to observe peroxide induced damage in fish hepatocytes. Melatonin (25, 50 and 100 µg/ml) was administered against the highest dose of H2O2. Enzymatic and non-enzymatic antioxidants such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) was measured spectrophotometrically. Expression level of heat shock proteins (HSP70 and HSP90), HSPs-associated signaling molecules (Akt, ERK, cytosolic and nuclear NFkB), and melatonin receptor was also measured by western blotting analysis. RESULTS: H2O2 induced oxidative stress significantly altered (P < 0.05) MDA and GSH level, SOD and CAT activity, and up regulated HSP70 and HSP90 expression in carp hepatocytes. Signaling proteins exhibited differential modulation as revealed from their expression patterns in H2O2-exposed fish hepatocytes, in comparison with control hepatocytes. Melatonin treatment of H2O2-stressed fish hepatocytes restored basal cellular oxidative status in a dose dependent manner. Melatonin was observed to be inducer of signaling process by modulation of signaling molecules and melatonin receptor. CONCLUSIONS: The results suggest that exogenous melatonin at the concentration of 100 µg/ml is required to improve oxidative status of the H2O2-stressed fish hepatocytes. In H2O2 exposed hepatocytes, melatonin modulates expression of HSP70 and HSP90 that enable the hepatocytes to become stress tolerant and survive by altering the actions of ERK, Akt, cytosolic and nuclear NFkB in the signal transduction pathways. Study also confirms that melatonin could act through melatonin receptor coupled to ERK/Akt signaling pathways. This understanding of the mechanism by which melatonin regulates oxidative status in the stressed hepatocytes may initiate the development of novel strategies for hepatic disease therapy in future.

Change in redox state and heat shock protein expression in an Indian major carp Cirrhinus cirrhosus exposed to zinc and lead.

Fish are exposed to different heavy metals that may induce numerous physiological changes. In the present study, we examined the redox state in response to a severe stress resulting from two heavy metals (Zinc and Lead) contamination in carp Cirrhinus cirrhosus. Fish were exposed to 1/10th of LC50 of the respective metals [zinc chloride (2.72 mg/L) and lead acetate (2.53 mg/L)] for 30 days and allowed to recover for another 30 days without any metal exposure. Concentration of metals, different enzymatic and non-enzymatic antioxidant agents and expression levels of heat shock protein (HSP) 70 and 90 were measured in the liver and the kidney of fish. The lipid peroxide levels in fish tissues gradually increased with duration of treatment for both metals. After 15 days of treatment, glutathione (GSH) levels had increased, but decreased as the treatment continued for 30 days and returned to basal levels after a 30-day recovery period. Activities of all the anti-oxidant enzymes, except glutathione peroxidase, in stressed fish were significantly increased compared to those in the control at 15 days and continued till the 30th day of treatment, showing a tendency to return to basal levels after the recovery period. Expression levels of HSP70 and HSP90 gradually increased after zinc and lead treatment, respectively. The expression of HSP was higher in the liver. The results suggest that different heavy metals may have differential effects on the redox state and induction of oxidative stress in carp, in vivo.

Effect of heavy metals on tissue-specific antioxidant response in Indian major carps.

Concentrations of heavy metals (Cu, Ni, Zn, Cd and Pb) were measured in sediments, water and liver and kidney tissues of three Indian major carps (Labeo rohita, Catla catla and Cirrhinus cirrhosus), belonging to two different weight groups (250 and 500 g), collected from ponds at two different sites (Nalban bheri and Diamond Harbour). The tissues were analysed for the levels of different antioxidant defence systems such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRd), glutathione S-transferase (GST), glutathione (GSH) and malondialdehyde (MDA). Concentrations of all the metals were significantly higher (P < 0.05) in sediment, water and the tissues from Nalban bheri compared to those in Diamond Harbour. Metal concentrations were the lowest in C. cirrhosus, which increased with an increase in fish weight, and the liver accumulated higher amount of metals than the kidney. Activities of all enzymatic and non-enzymatic antioxidant parameters except GPx and GRd were significantly higher (P < 0.05) in the tissues from Nalban bheri than those in Diamond Harbour. Significant multicollinearity was found in the values of SOD, CAT, GST, GRd, GPx and MDA with Pb, Cu and Ni in all three fish species at Nalban and with Cd in L. rohita and C. catla. Principal component analysis results revealed that stress response in a polluted site was directly regulated by an amalgamation of GSH profile and the levels of MDA in a synchronized manner. The study indicated a tissue-specific and species-specific difference for heavy metal-induced oxidative stress response in fish and a correlation between different heavy metals and individual oxidative stress markers.