Effects of temperature on complexes I and II mediated respiration, ROS generation and oxidative stress status in isolated gill mitochondria of the mud crab Scylla serrata.

Effects of fluctuations in habitat temperature (18-30°) on mitochondrial respiratory behavior and oxidative metabolic responses in the euryhaline ectotherm Scylla serrata are not fully understood. In the present study, effects of different temperatures ranging from 12 to 40°C on glutamate and succinate mediated mitochondrial respiration, respiratory control ratio (RCR), ATP generation rate, ratio for the utilization of phosphate molecules per atomic oxygen consumption (P/O), levels of lipid peroxidation and H2O2 in isolated gill mitochondria of S. serrata are reported. The pattern of variation in the studied parameters was similar for the two substrates at different temperatures. The values recorded for RCR (≥3) and P/O ratio (1.4-2.7) at the temperature range of 15-25°C were within the normal range reported for other animals (3-10 for RCR and 1.5-3 for P/O). Values for P/O ratio, ATP generation rate and RCR were highest at 18°C when compared to the other assay temperatures. However, at low and high extreme temperatures, i.e. at 12 and 40°C, states III and IV respiration rates were not clearly distinguishable from each other indicating that mitochondria were completely uncoupled. Positive correlations were noticed between temperature and the levels of both lipid peroxidation and H2O2. It is inferred that fluctuations on either side of ambient habitat temperature may adversely influence mitochondrial respiration and oxidative metabolism in S. serrata. The results provide baseline data to understand the impacts of acute changes in temperature on ectotherms inhabiting estuarine or marine environments.

Seasonal variability of antioxidant biomarkers in mud crabs (Scylla serrata).

Studies on oxidative stress (OS) in crustacea are widely used as ecotoxicological indices to assess the environment risk produced by the impact of several stressor and pollutants. In the present study, effects of seasonality on OS physiology markers such as antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase), small antioxidant molecules (ascorbic acid and reduced glutathione), oxidative stress indices (lipid peroxidation, protein carbonylation and hydrogen peroxide) and total antioxidant capacity in hepatopancreas, gills and abdominal muscle of adult mud crab Scylla serrata, sampled from Chilika lagoon of India, were determined in winter, summer and rainy seasons. Results indicate that variations in enzymatic and non-enzymatic antioxidants with relation to season were not only tissue specific but also were gender specific. The levels of OS parameters were higher in hepatopancreas in comparison to gills and abdominal muscle of the crabs in all seasons. OS indices in tissues of the crabs were mainly higher in summer season when temperature and salinity of the lagoon were high with low oxygen content. Although OS was lower in winter season and moderate in rainy season in tissues of male crabs, it was higher in gills and hepatopancreas of females in rainy season. Correlation analyses between hydrological parameters of the lagoon (temperature, salinity and dissolved oxygen content) and OS physiology parameters in tissues of crabs suggest that abiotic factors influence the levels of antioxidant enzymes and, thereby the OS status in a tissue and sex specific manner. Collectively, the results of the present work suggest that further investigation is warranted before using OS parameters in S. serrata as biomarkers to monitor estuarine environment as these are influenced by gender, tissue and season.

Curcumin and vitamin E modulate hepatic antioxidant gene expression in PTU-induced hypothyroid rats.

In the present study, regulatory role of vitamin E and curcumin on antioxidant gene (AOG) expression in hypothyroid rat liver is reported. Adult male rats were rendered hypothyroid by administration of 0.05 % 6-propyl-thiouracil in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days. Expression of antioxidant genes (Cu/Zn-superoxide dismutase; SOD1, Mn superoxide dismutase; SOD2, catalase; CAT, glutathione peroxidase; GPx1 and glutathione reductase; GR) was evaluated using RT-PCR and Western blot analyses. The activities of antioxidant enzymes were measured in mitochondrial fraction (MF) and post-mitochondrial fraction (PMF) of rat liver. In addition measurement of glutathione redox status was also carried out in both the fractions. The enhanced transcripts of CAT, GPx1 and GR in hypothyroid rat liver were alleviated by administration of vitamin E and curcumin. Elevated levels of translated product of all AOGs in hypothyroid group were remained unchanged after antioxidant administration. However, enhanced SOD1, GPx1 and decreased GR activities in PMF were normalized by vitamin E and curcumin. Similarly the increased SOD2, GPx1 and decreased CAT activities in MF were also normalized by vitamin E and curcumin supplementation. Administration of vitamin E and curcumin enhanced mitochondrial GSH level; whereas the enhanced GSH level in PMF of hypothyroid rats was alleviated by vitamin E. Thus it can be concluded that besides the antioxidant role of vitamin E and curcumin, they also regulate hepatic antioxidant gene expression in hypothyroid rats.

Constitutional, organopathic and combined homeopathic treatment of benign prostatic hypertrophy: a clinical trial.

BACKGROUND: Benign Prostatic Hypertrophy (BPH) is common in older men. This study compared homeopathic treatment strategies using constitutional medicines (CM) or organopathic medicines (OM) alone or in combination (BCOM) in patients suffering from BPH. METHODS: 220 men aged 30-90 years were recruited in Odisha, India. Patients presenting symptoms of prostatism, with or without evidence of bladder outflow obstruction were included in the study. Patients with serum prostate specific antigen (PSA)> 4 nmol/mL, malignancy, complete urine retention, stone formation and gross bilateral hydronephrosis were excluded. Patients were sequentially allocated to OM, CM or BCOM. The main outcome measure was the International Prostate Symptom Score (IPSS). RESULTS: 73, 70 and 77 patients respectively were sequentially allocated to OM, CM or BCOM. 180 patients (60 per group) completed treatment and were included in the final analysis. Overall 85% of patients showed improvement of subjective symptoms such as frequency, urgency, hesitancy, intermittent flow, unsatisfactory urination, feeble stream, diminution of residual urine volume but there was no reduction in prostate size. Treatment response was highest with BCOM (38.24%) compared to OM (31.62%) and CM (30.15%). Effect sizes were highest for the decrease in IPSS, residual urine volume and urinary flow rate.

Biology and conservation of the genus Scylla in India subcontinent.

Mud crabs usually inhabit brackish water bodies of South-East Asia including India. The biological significance of the species is its survibility in wide range of hydro-biological fluctuating environments including salinity. The life cycle of the species includes two important larval stages at which they are vulnerable to various predators. Pathophysiological status of the species is a challenge for its aquaculture. Inspite of a huge economical and high food values, informations on biochemical, molecular, physiological, ecological and taxonomical aspects of mud crabs (Scylla sp.) in comparison to other important aquaculture candidates are very limited. The present review is an attempt to string together various informations available on mud crabs (S. serrata) so that it will provide a platform to have all the basic informations regarding the species for interested workers. It is believed that understanding the pathology, ecophysiology, genetics and reproduction of the species in its natural environment will not only help in its conservation but also will help in its enhanced production.

Catechin and curcumin interact with S protein of SARS-CoV2 and ACE2 of human cell membrane: insights from computational studies.

The recent outbreak of the coronavirus (SARS-CoV2) is an unprecedented threat to human health and society across the globe. In this context, development of suitable interventions is the need of the hour. The viral spike protein (S Protein) and the cognate host cell receptor ACE2 can be considered as effective and appropriate targets for interventions. It is evident from the present computational study, that catechin and curcumin, not only exhibit strong binding affinity to viral S Protein and host receptor ACE2 but also to their complex (receptor-binding domain (RBD) of the spike protein of SARS-CoV2 and ACE2; RBD/ACE2-complex). The binding affinity values of catechin and curcumin for the S protein, ACE2 and RBD/ACE2-complex are - 10.5 and - 7.9 kcal/mol; - 8.9 and - 7.8 kcal/mol; and - 9.1 and - 7.6 kcal/mol, respectively. Curcumin directly binds to the receptor binding domain (RBD) of viral S Protein. Molecular simulation study over a period of 100 ns further substantiates that such interaction within RBD site of S Protein occurs during 40-100 ns out of 100 ns simulation trajectory. Contrary to this, catechin binds with amino acid residues present near the RBD site of S Protein and causes fluctuation in the amino acid residues of the RBD and its near proximity. Both catechin and curcumin bind the interface of 'RBD/ACE2-complex' and intervene in causing fluctuation of the alpha helices and beta-strands of the protein complex. Protein-protein interaction studies in presence of curcumin or catechin also corroborate the above findings suggesting the efficacy of these two polyphenols in hindering the formation of S Protein-ACE2 complex. In conclusion, this computational study for the first time predicts the possibility of above two polyphenols for therapeutic strategy against SARS-CoV2.

Application of oxidative stress indices in natural populations of Perna viridis as biomarker of environmental pollution.

Oxidative stress indices were measured in gills and digestive glands of Perna viridis collected from three coastal locations in Goa i.e., Bambolim, Marmugao Harbour and Malim. In addition to lipid peroxidation, the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase and two non-enzymatic antioxidants (ascorbic acid and reduced glutathione) were investigated in order to understand their variation with respect to pollution status of the sampling locations. We observed a significant increase in lipid peroxidation and antioxidant enzymes of both the tissues at Mormugao Harbour and Malim, suggesting that the animals at these two locations are at higher level of oxidative stress as compared to those at Bambolim. Conversely, low levels of non-enzymatic antioxidants such as ascorbic acid and reduced glutathione, observed at Mormugao Harbour and Malim indicate that the animals may use these compounds to counteract stress in the tissues. This study shows that changes in lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase and reduced glutathione in tissues of P. viridis can be used as molecular biomarkers in environmental monitoring programs.

Curcumin restores hepatic epigenetic changes in propylthiouracil(PTU)Induced hypothyroid male rats: A study on DNMTs, MBDs, GADD45a, C/EBP-ß and PCNA.

6-n-propyl-2-thiouracil (PTU), a thioamide drug, is used as an effective anti-thyroid agent to treat hyperthyroidism and Graves' disease. However, acute liver oxidative damage is an important side effect of the drug. In the present study, we report that PTU administration to rat induces hepatic epigenetic changes by upregulating expression of DNMT1, DNMT3a, DNMT3b, MBD4, MeCP2, p53 and Gadd45a and down-regulation of PCNA and C/EBP-ß. This is accompanied by decrease in the cell population and augmentation of cellular lipid peroxidation, an index of oxidative stress, in liver. On the other hand, co-administration of curcumin, a polyphenol extract from the rhizome of Curcuma longa L, along with PTU ameliorates PTU- induced oxidative stress and epigenetic parameters except for the expression of MBD4. Also, co-administration of curcumin with PTU resulted in restoration of hepatic cell population and histoarchitecture. The protective effect of curcumin to PTU-induced hepatotoxicity is attributed to its antioxidative properties.

Alleviation of enhanced oxidative stress and oxygen consumption of L-thyroxine induced hyperthyroid rat liver mitochondria by vitamin E and curcumin.

In the present study, the role of vitamin E and curcumin on hyperthyroidism induced mitochondrial oxygen consumption and oxidative damage to lipids and proteins of rat liver are reported. Adult male rats were rendered hyperthyroid by administration of 0.0012% l-thyroxine in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days. Hyperthyroidism induced elevation in serum aspartate aminotransferase and alanine aminotransferase activities were reduced significantly in response to vitamin E and curcumin treatment. On the other hand, effects of vitamin E and curcumin on hyperthyroidism induced hepatic complexes I and II mediated respiration were found to be different. While curcumin administration ameliorates hyperthyroidism induced state 3 and state 4 respiration in complex I, vitamin E treatment was effective only in reducing state 4 respiration of complex I. On the contrary, curcumin administration was ineffective in modulating hyperthyroidism induced complex II respiration, but vitamin E treatment to hyperthyroid rats resulted in augmentation of complex II respiration both at state 3 and state 4 level. Moreover, vitamin E and curcumin treatment resulted in alleviation of hyperthyroidism induced lipid peroxidation. Enhanced protein carbonylation in hyperthyroid rats is decreased only in response to simultaneous supplementation of vitamin E and curcumin. Above findings suggest that both vitamin E and curcumin have differential regulation on complexes I and II mediated mitochondrial respiration and have a protective role against L-thyroxine induced hepatic dysfunction and oxidative stress.

Modulation of antioxidant defences in digestive gland of Perna viridis (L.), on mercury exposures.

Sub-lethal effects of mercury exposure (110th of LC(50), i.e. 0.045 mg l(-1)) for 5, 10 and 15 d was investigated on oxidative stress parameters and antioxidant defences in digestive gland of Perna viridis. In addition to this an in vitro effect of mercury single and supplemented with reduced glutathione on lipid peroxidation was studied. Increased lipid peroxidation (during first 10 days and also during in vitro exposures), protein carbonyl and hydrogen peroxides (from 5th till last day of exposure) indicate the resultant oxidative stress in the mercury exposed specimen. DNA damage (F-value) response although less distinct on 5th and 15th d, its low values on 10th d and significant correlation with hydrogen peroxide suggests the toxic role of free radicals towards DNA integrity. Superoxide dismutase, which remains low initially (5th d) and increases later suggests its immediate response against superoxide radical. Higher activities of catalase, glutathione peroxidase and glutathione reductase on 15th d and glutathione-S-tranferase from 10th d onwards suggests the adaptive behaviour of the tissue against oxyradicals. Increasing levels of non-enzymatic antioxidant molecules, such as reduced glutathione and ascorbic acid indicated its involvement in counteracting oxidative damage. Further role of reduced glutathione in reducing Hg toxicity is evident in in vitro experiments where lipid peroxidation remains low in mercury concentrations supplemented with reduced glutathione. The elevated levels of metallothionein from 5th to 10th d suggest involvement of this protein in detoxification of reactive oxygen species and toxic metal. The above results suggest that both enzymatic and non-enzymatic antioxidants play an important role in protecting cell against Hg toxicity, which can be used as a biomarker of metal contamination in aquatic environment.

Rat testicular mitochondrial antioxidant defence system and its modulation by aging.

Accumulation of oxidative damage caused by reactive oxygen species (ROS) underlies fundamental changes found during aging. In the present study, age related effect on testicular mitochondrial oxidant generation and antioxidant defence profile was investigated in Wistar rats at 3 months (young adults), 12 months (old adults) and 24 months (senescent animals) of age. Mitochondrial oxidative stress parameters viz., lipid peroxidation (LPx), protein carbonylation, hydrogen peroxide (H2O2) generation and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), levels of total, oxidized (GSSG) and reduced glutathione (GSH) were studied to find out their roles in maintenance of mitochondrial glutathione redox pool as a function of age. Increased levels of LPx, H2O2 and decreased GSH content accompanied by a decline in activities of SOD, GPx and GR with advancing age suggest that antioxidant defense profile of testicular mitochondria exhibit age related alterations which might play a critical role in regulating physiological functions of the testis such as steroidogenesis and spermatogenesis.

Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature.

Oxidative damage and antioxidant properties have been studied in Perna viridis subjected to short-term exposure to Hg along with temperature (72h) and long-term temperature exposures (14 days) as pollution biomarkers. The elevated thiobarbituric acid reactive substances (TBA-RS) levels observed in gills and digestive gland under exposure to Hg, individually and combined with temperature, as also long-term temperature stress have been assigned to the oxidative damage resulting in lipid peroxidation (LPX). Increased activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) both in gills and digestive glands under long-term exposures to temperatures are more prominent to heat rather than cold stress suggesting activation of physiological mechanism to scavenge the ROS produced during heat stress. Also decreased values of reduced glutathione (GSH) on long exposures to temperature stress indicate utilisation of this antioxidant, either to scavenge oxiradicals or act in combination with other enzymes, was more than its production capacity under heat stress. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sublethal effects of pollution.

Effect of T3 treatment on glutathione redox pool and its metabolizing enzymes in mitochondrial and post-mitochondrial fractions of adult rat testes.

T3 (3,3', 5-triiodo-L-thyronine; 20 microg/100 g body weight/day in 0.01 N NaOH, i.p for 1, 3 and 5 days) treatment modulated reduced (GSH) and oxidized (GSSG) glutathione contents along with the activities of its metabolizing enzymes (such as glutathione peroxidase, glutathione reductase and glutathione S-transferase) in the testis of Wistar rats. However, the magnitude and nature of changes in the above biochemical parameters in response to T3 treatment were noticed to be different between mitochondrial and post-mitochondrial fractions. This was accompanied with elevated levels of lipid hydroperoxide and ascorbic acid in the crude homogenate of testis. The level of hydrogen peroxide in the post-mitochondrial fractions of testes did not change on first day, decreased on 3rd day and increased on 5th day of the hormone treatment when compared to respective controls. Nevertheless, its content in mitochondria was significantly elevated in response to all the three durations of the hormone treatment having the highest induction on 3rd day. The changes observed in the levels of GSH and GSSG and its metabolizing enzymes in response to T3 treatment reflect an alteration in the redox state of testis, which may be a causative factor for the impairment of testicular physiology as a consequence of oxidative stress.

Kinetics and mechanism of reduction of ferricytochrome c by glutathione and L-cysteine: a comparative study.

The kinetics and mechanism of the reduction of ferricytochrome c [Cyt c(III)] by substrates namely glutathione (GSH) and L-cysteine (L-cys) have been investigated spectrophotometrically employing [substrate]T >> [Cyt c(III)]T. The reaction exhibits first order dependence in [substrate]T and [Cyt c(III)]T. The pseudo-first order rate constant increases with an increase in pH, indicating that the conjugate base form of the HCyt c(III) is a better oxidant than the parent HCyt c(III). The electron transfer rate constants between the oxidants and GSH for both the k1 and k2 paths are found to be greater than that with L-cysteine. Hence, GSH is a better reductant of Cyt c(III) as compared to L-cysteine. A suitable mechanism has been proposed on the basis of experimental findings. The deprotonation constant for HCyt c(III) and the second order rate constants of k1 and k2 paths for the present reaction at 25 degrees C have been determined.

Investigating the Conformational Structure and Potential Site Interactions of SOD Inhibitors on Ec-SOD in Marine Mud Crab Scylla serrata: A Molecular Modeling Approach.

Superoxide dismutases (SODs) act as a first line of the enzymatic antioxidant defense system to control cellular superoxide anion toxicity. Previously, several inhibitors have been widely identified and catalogued for inhibition of SOD activity; however, still the information about the mechanism of interaction and points toward the inhibitor interactions in structures of SODs in general and in extracellular (Ec)-SOD in particular is still in naive. In the present research, we present an insight to elucidate the molecular basis of interactions of SOD inhibitors with Ec-SOD in mud crab Scylla serrata using molecular modeling and docking approaches. Different inhibitors of SOD such as hydrogen peroxide [Formula: see text], potassium cyanide, sodium dodecyl sulfate (SDS), [Formula: see text]-mercaptoethanol and dithiocarbamate were screened to understand the potential sites that may act as sites for cleavage or blocking in the protein. SOD-SDS and [Formula: see text] complex interactions indicate residues Pro72 and Asp102 of the predicted crab Ec-SOD as common targets. The GOLD result indicates that Pro72, Asp102 and Thr103 are commonly acting as the site of interaction in Ec-SOD of S. serrata with SOD inhibitors. For the first time, the results of this study provide an insight into the structural properties of Ec-SOD of S. serrata and define the possible involvements between the amino acids present in its active sites, i.e., in the regions from 70 to 84 and from 101 to 103 and different inhibitors.

Experimental hyperthyroidism-induced oxidative stress and impairment of antioxidant defence system in rat testis.

Short-term hyperthyroidism, induced by daily administration of T3 (20 microg/100g body weight) for one, three, and five days consecutively, modulates oxidative stress and antioxidant defence parameters in mitochondrial and postmitochondrial fractions of testis in adult rats. Alteration in antioxidant defences along with oxidative stress parameters in testis by thyroid hormone was found to be associated with a decline in the number of sperms and disturbances in histoarchitecture of seminiferous tubules in the testes; the results indicated that induced hyperthyroid state altered testicular physiology by influencing antioxidant defence system of testes.

Response of testicular antioxidant enzymes to hexachlorocyclohexane is species specific.

AIM: To find out whether the response of testicular oxidative stress parameters to hexachlorocyclohexane (HCH) is species specific. METHODS: In rats and mice (n=5 in each group), HCH was administered at a dose of 20 mg/kg/day intraperitoneally for 30 days in 0.1 ml of refined groundnut oil. The control groups received equal volume of the vehicle. Animals were sacrificed 24 hours after the last injection and various oxidative stress parameters were measured immediately. RESULTS: The level of both endogenous as well as FeSO4 and ascorbic acid-stimulated lipid peroxidation was increased significantly in the HCH-treated rats, whereas the pattern was just the reverse in case of mice. Although the level of H2O2 content increased in response to HCH in both groups, a totally different trend was observed for the activity of the principal H2O2-metabolising enzyme, catalase. In case of rats, a significant decline in the activity of catalase was recorded in response to HCH whereas a sharp augmentation in the enzyme activity was noticed in mice. Similarly, the decreased activity of superoxide dismutase observed in rats remained unaltered in mice. CONCLUSION: HCH induces oxidative stress in the testis of both rats and mice. However, the pattern of response of testicular oxidative stress parameters seems to be species specific.

Effects of hypothyroidism induced by 6-n-propylthiouracil and its reversal by T3 on rat heart superoxide dismutase, catalase and lipid peroxidation.

The present study critically evaluates the effects of hypothyroid and hyperthyroid states on lipid peroxidation and two enzymes of active oxygen metabolism, namely superoxide dismutase (SOD) and catalase (CAT) in the rat heart mitochondrial and post-mitochondrial fractions. Lipid peroxidation, an index of oxidative stress, was elevated in the heart tissue in hypothyroid state but reduced upon T3 supplementation. Hyperthyroidism registered increased SOD activity in post-mitochondrial fraction. Mitochondrial SOD activity was reduced in hypothyroid state, which was further reduced by T3 administration. In contrast, different thyroid states had no effect on catalase activity in the mitochondrial fraction. The hypothyroid state however, significantly augmented catalase activity in post-mitochondrial fraction. The results suggest that the antioxidant defence status of cardiac tissue is well modulated by thyroid hormone.

Effects of salinity on O2 consumption, ROS generation and oxidative stress status of gill mitochondria of the mud crab Scylla serrata.

Mitochondrial respiration, activities of electron transport chain enzymes and formation of oxidative stress parameters were investigated in mitochondria isolated from gill tissue of mud crabs (Scylla serrata) as a function of salinity (10 ppt, 17 ppt and 35 ppt). Mitochondrial oxygen consumption rate was higher for succinate as substrate compared with those of glutamate, malate and pyruvate. Complex I and complex II mediated respirations were higher at low salinity (10 ppt) than high salinity (17 ppt and 35 ppt). Although activities of electron transport chain enzymes particularly complexes I (EC 1.6.5.3), II (EC 1.3.99.1) and II-III (EC 1.3.2.1) were elevated linearly in response to salinity treatment, activity of complex V (ATPase, EC 3.6.1.34) was decreased at 35 ppt salinity. However, ATPase activity was higher at 17 ppt salinity in comparison to 10 ppt and 17 ppt salinity. Results of the experiment suggest that high salinity (35 ppt) causes hypoxic state in mitochondria of mud crabs. Hypoxic condition induced by high salinity was accompanied with increased hydrogen peroxide production resulting oxidative stress in mitochondria of crabs. A possible mechanism of hypoxia-induced reactive oxygen species generation and OS due to salinity stress in the crabs is discussed.