Modulation of antioxidant defence system in brain of rainbow trout (Oncorhynchus mykiss) after chronic carbamazepine treatment

We investigated the effect of long-term exposure to CBZ on the antioxidant system in brain tissue of rainbow trout. Fish were exposed to sublethal concentrations of CBZ (1.0 μg/L, 0.2 mg/L or 2.0 mg/L) for 7, 21, and 42 days. Oxidative stress indices (LPO and CP) and activities of antioxidant enzymes (SOD, CAT, GPx and GR) in fish brain were measured. In addition, non-enzymatic antioxidant (GSH) was determined after 42 days exposure. Carbamazepine exposure at 0.2 mg/L led to significant increases (p < 0.05) of LPO and CP after 42 days and, at 2.0 mg/L, after 21 days. Activities of the antioxidant enzymes SOD, CAT, and GPx in CBZ-treated groups slightly increased during the first period (7 days). However, activities of all measured antioxidant enzymes were significantly inhibited (p < 0.05) at 0.2 mg/L exposure after 42 days and after 21 days at 2.0 mg/L. After 42 days, the content of GSH in fish brain was significantly lower (p < 0.05) in groups exposed to CBZ at 0.2 mg/L and 2.0 mg/L than in other groups. Prolonged exposure to CBZ resulted in excess reactive oxygen species formation, finally resulting in oxidative damage to lipids and proteins and inhibited antioxidant capacities in fish brain. In short, a low level of oxidative stress could induce the adaptive responses of antioxidant enzymes, but long-term exposure to CBZ could lead to serious oxidative damage in fish brain.     

Goldfish exposure to cobalt enhances hemoglobin level and triggers tissue-specific elevation of antioxidant defenses in gills, heart and spleen

Cobalt ions can enhance the generation of reactive oxygen species (ROS), which may be the reason for cobalt toxicity. This study aimed to determine whether Co(2+) toxicity in goldfish is related to induced oxidative stress in gills, heart and spleen, and to assess responses of antioxidant systems. Exposure of goldfish to 50, 100 and 150 mg L(-1) of Co(2+) for 96 h elevated total hemoglobin in blood by 23, 44 and 78%, respectively. In gills, cobalt exposure enhanced lipid peroxide levels and activities of primary antioxidant enzymes; superoxide dismutase (SOD) rose by 125% and glutathione peroxidase (GPx) increased by 53-296%. Glutathione-S-transferase (GST) activity also increased by 117-157% and glucose-6-phosphate dehydrogenase (G6PDH) enhanced by 46-96%. Heart showed limited effects of fish exposure to 50 or 100 mg L(-1) of Co(2+), but the exposure to 150 mg L(-1) of Co(2+) elevated concentrations of lipid peroxides by 123% and activities of GPx by 98% and SOD by 208%. The most substantial effects of goldfish exposure to Co(2+) were observed in spleen: a decrease in total protein concentration by 44-60% and high molecular mass thiols by 59-82%, reduced activities of catalase by 24-58% and GR by 25-68%, whereas the level of low molecular mass thiols increased by 153-279% and activities of GPx, GST, G6PDH were enhanced by 114-120%, 192-769%, and 256-581%, respectively. The data show that fish exposure to 50-150 mg L(-1) of Co(2+) elevates blood hemoglobin level, mimicking effects of hypoxia, and causes the activation of defense systems against ROS.     

The influence and the mechanism of docosahexaenoic acid on a mouse model of Parkinson's disease

This study aimed to investigate the effects of docosahexaenoic acid (DHA) on the oxidative stress that occurs in an experimental mouse model of Parkinson’s disease (PD). An experimental model of PD was created by four intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (4 × 20 mg/kg, at 12 h intervals). Docosahexaenoic acid was given daily by gavage for 4 weeks (36 mg/kg/day). The motor activity of the mice was evaluated via the pole test, and the dopaminergic lesion was determined by immunohistochemical analysis for tyrosine hydroxylase (TH)-immunopositive cells. The activity of antioxidant enzymes in the brain were determined by spectrophotometric assays and the concentration of thiobarbituric acid-reactive substances (TBARS) were measured as an index of oxidative damage. The number of apoptotic dopaminergic cells significantly increased in MPTP-treated mice compared to controls. Although DHA significantly diminished the number of cell deaths in MPTP-treated mice, it did not improve the decreased motor activity observed in the experimental PD model. Docosahexaenoic acid significantly diminished the amount of cell death in the MPTP + DHA group as compared to the MPTP group. TBARS levels in the brain were significantly increased following MPTP treatment. Glutathione peroxidase (GPx) and catalase (CAT) activities of brain were unaltered in all groups. The activity of brain superoxide dismutase (SOD) was decreased in the MPTP-treated group compared to the control group, but DHA treatment did not have an effect on SOD activity in the MPTP + DHA group. Our current data show that DHA treatment exerts neuroprotective actions on an experimental mouse model of PD. There was a decrease tendency in brain lipid oxidation of MPTP mice but it did not significantly.     

Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats

Homocystinuria is a neurometabolic disease caused by severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction, being that the pathomechanism is not fully understood. In the present study we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely 2′7′dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3–0.6 μmol/g body weight), and/or creatine (50 mg/kg body weight) from their 6th to the 28th days age. Controls and treated rats were decapitated at 12 h after the last injection. Chronic homocysteine administration increased 2′7′dichlorofluorescein (DCFH) oxidation, an index of production of reactive species and TBARS levels, an index of lipoperoxidation. Antioxidant enzyme activities, such as SOD and CAT were also increased, but GPx activity was not altered. The content of GSH, sulfhydril and carbonyl were decreased, as well as levels of nitrite. Creatine concurrent administration prevented some homocysteine effects probably by its antioxidant properties. Our data suggest that the oxidative insult elicited by chronic hyperhomocystenemia may provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function. Creatine prevents some alterations caused by homocysteine.     

Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles

Hexavalent chromium (Cr^6 +) is a common pollutant transient metal with high toxicity in the environment. The toxicological effects partly result from oxidative damage due to the production of excessive reactive oxygen species (ROS) in the reductive process of Cr^6 +. To explore the influence of ROS induced directly by Cr^6 + on the oxidative stress generation and antioxidant system, the full length cDNAs of antioxidant-related genes cat, gpx1 and Cu/Zn-sod were successfully acquired from pengze crucian carp first and analyzed. Furthermore, the mRNA expression of the antioxidant genes encompassing catalase (cat), copper/zinc superoxide dismutase (Cu/Zn-sod) and glutathione peroxidase (gpx1), antioxidant enzyme activities of CAT, SOD, and GPx and total protein content were further studied in the gill, intestine and liver of pengze crucian carp (Carassius auratus var. Pengze) juveniles upon acute exposure to Cr^6 + at concentrations of 0.1, 1.0, 10 and 100 mg/L for 4 days. Differential significant changes of the antioxidant enzymes and gene expression were observed in different tissues. The findings contribute to better understanding the antioxidant mechanisms induced by Cr^6 + and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem.     

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12 h and 24 h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24 mg/L at 12 h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24 mg/L of CuCl₂. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.     

Stability of the anti-oxidative enzymes in aqueous and detergent solution

Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1–5 days for GPx and 7–10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2–3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0 – 3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlatingin vitro catalase or GPx measurements within vivo anti-oxidative protection may underestimate tissue defences.     

Chronic Exposure to Tributyltin Induces Brain Functional Damage in Juvenile Common Carp (Cyprinus carpio)

The aim of the present study was to investigate the effect of Tributyltin (TBT) on brain function and neurotoxicity of freshwater teleost. The effects of long-term exposure to TBT on antioxidant related indices (MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; GPx, glutathione peroxidase), Na⁺-K⁺-ATPase and neurological parameters (AChE, acetylcholinesterase; MAO, monoamine oxidase; NO, nitric oxide) in the brain of common carp were evaluated. Fish were exposed to sublethal concentrations of TBT (75 ng/L, 0.75 μg/L and 7.5 μg/L) for 15, 30, and 60 days. Based on the results, a low level and short-term TBT-induced stress could not induce the notable responses of the fish brain, but long-term exposure (more than 15 days) to TBT could lead to obvious physiological-biochemical responses (based on the measured parameters). The results also strongly indicated that neurotoxicity of TBT to fish. Thus, the measured physiological responses in fish brain could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity.     

Study on a 65‐mer peptide mimetic enzyme with GPx and SOD dual function

Excessive reactive oxygen species (ROS) levels are harmful to the body. The peroxidase, GPx, and the superoxide dismutase, SOD, are important antioxidant enzymes for preventing ROS‐induced damage. Se‐CuZn‐65P is an enzyme mimetic with dual GPx and SOD antioxidant function. However, currently, its production is mainly based on the cysteine auxotrophic expression technique, which is inefficient, expensive, and time consuming. In this study, we combined protein engineering and the chemical mutation method to synthesize Se‐CuZn‐65P. The DNA sequence encoding the 65 amino acid peptide with the desired sequence transformations to incorporate the SOD and the GPx catalytic sites was cloned and expressed in a soluble protein expression vector. The protein yield increased up to 152 mg/L, which is 10 times higher than in previous studies. The SOD and GPx activity of Se‐CuZn‐65P was high (1181 U/mg and 753 U/μmol, respectively). The binding constant of glutathione was 5.6 × 10⁴ L·mol^?1, which shows that Se‐CuZn‐65P efficiently catalyzed hydrogen peroxide reduction by glutathione. Mitochondrial damage experiments confirmed the double protective role of the Se‐CuZn‐65P peptide and demonstrated functional synergy between the SOD and the GPx domains, which indicates its potential to be used in the treatment of ROS‐related diseases. Our research may give a new thought to increase the yield of mimic.     

PPAR’s and Diosgenin a chemico biological insight in NIDDM

Diosgenin a steroidal saponin found widely in nature is reported to contain several biological activities in recent years. The present work elaborates the modulation of the lipid and antioxidant profile by Diosgenin in diabetic condition. Type 2 diabetes was induced in experimental animals by feeding high fat diet (HFD) for 8 weeks followed by streptozotocin (STZ) injection (sub-diabetogenic dose; 35 mg/kg body weight). Diosgenin administered orally at two doses (40 and 80 mg/kg body weight) for 14 days reduced hyperglycemia, hypercholesterolemia and hypertriglyceridemia (p < 0.001). Oxidative stress a crucial marker of diabetes and obesity associated complications was analyzed and noteworthy changes were observed. Improved levels of the antioxidant enzymes SOD and GPx and a minimized level of lipid peroxidation were also observed in Diosgenin treated rats. Further, analyzing the lipid accumulation by Oil Red O staining in 3T3-L1 preadipocytes confirmed its adipogenic activity which was influenced by PPAR γ and PPAR α. This was also substantiated through docking studies of Diosgenin with the PPARs. Altogether, Diosgenin a phytochemical of natural origin is found to mitigate diabetes induced oxidative stress and dyslipidemia which is crucial in cardio-metabolic risks by modulating the PPARs.     

Modulation of glutathione-related antioxidant defense system of fish chronically treated by the fungicide propiconazole

Recently, residual fungicides are generally recognized as relevant sources of aquatic environmental pollutants. However, the toxicological effects of these contaminants have not been adequately researched. In this study, the chronic effect of PCZ, a triazole-containing fungicide commonly present in aquatic environment, on GSH-related antioxidant system and oxidative stress indices of rainbow trout (Oncorhynchus mykiss) were investigated. Fish were exposed at sub-lethal concentrations of PCZ (0.2, 50 and 500 μg/L) for 7, 20 and 30 days. GSH levels and GSH-related enzyme activities, including GPx, GR and GST, were quantified in three tissues—liver, gill and muscle. The levels of LPO and CP were also measured as makers of oxidative damage. In addition, the correlations of the measured parameters in various tissues were evaluated by using PCA. The results of this study indicate that chronic exposure of PCZ has resulted in different responses in various tissues and the gill was the most sensitive tissue; however, before these parameters are used as potential biomarkers for monitoring residual fungicides in aquatic environment, more detailed experiments in laboratory need to be performed in the future.     

A fused selenium-containing protein with both GPx and SOD activities

As a safeguard against oxidative stress, the balance between the main antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) was believed to be more important than any single one, for example, dual-functional SOD/CAT enzyme has been proved to have better antioxidant ability than either single enzyme. By combining traditional fusion protein technology with amino acid auxotrophic expression system, we generated a bifunctional enzyme with both GPx and SOD activities. It displayed better antioxidant ability than GPx or SOD. Such dual-functional enzymes could facilitate further studies of the cooperation of GPx and SOD and generation of better therapeutic agents.     

Antioxidant enzyme activities of human peripheral blood mononuclear cells exposed to trace elements

The trace elements copper, zinc, and selenium are important immune modulators and essential cofactors of the antioxidant enzymes. In the present study, the proliferative effect of human peripheral mononuclear cells (PBMCs) that have been exposed to copper, zinc, and selenium and the corresponding activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, were determined. Zinc and copper stimulated the PBMC proliferation in a dose-dependent manner within the dose range 25-200 micromol/L. SOD and GPx activities in PBMCs exposed to zinc were inhibited, whereas catalase activity was unaffected. All the three antioxidant enzymes in the cells exposed to copper were inhibited. Selenium exerted more potent inhibition of the cell proliferation while causing stimulation of the antioxidant enzymes at the lowest dose (25 micromol/L) than at the highest dose (200 micromol/L) tested. A significant negative correlation was observed between proliferation and antioxidant enzyme (SOD and GPx) activities in trace-element-exposed PBMC. The present findings substantiate the importance of trace elements as immune modulators and the involvement of enzymatic antioxidant system in the immune cell regulation.     

Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity

To elucidate the physiological mechanism of chilling stress mitigated by cinnamic acid (CA) pretreatment, a cucumber variety (Cucumis sativus cv. Jinchun no. 4) was pretreated with 50 μM CA for 2 d and was then cultivated at two temperatures (15/8 and 25/18 °C) for 1 d. We investigated whether exogenous CA could protect cucumber plantlets from chilling stress (15/8 °C) and examined whether the protective effect was associated with the regulation of antioxidant enzymes and lipid peroxidation. At 2 d, exogenous CA did not influence plant growth, but induced the activities of some antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) in cucumber leaves, and it also elevated the contents of reduced glutathione (GSH) and ascorbate (AsA). When CA was rinsed and the CA-pretreated seedlings were exposed to different temperatures, the antioxidant activities in leaves at 3 d had undergone additional change. Chilling increased the activities of CAT, GSH-PX, APX, GSH and AsA in leaves, but the combination of CA pretreatment and chilling enhanced the antioxidant activities even more. Moreover, chilling inhibited plant growth and increased the contents of malonaldehyde (MDA), superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in cucumber leaves, and the stress resulted in 87.5% of the second leaves being withered. When CA pretreatment was combined with the chilling stress, we observed alleviated growth inhibition and decreased contents of MDA, H₂O₂ and O₂⁻ in comparison to non-pretreated stressed plants, and found that the withered leaves occurred at a rate of 25.0%. We propose that CA pretreatment increases antioxidant enzyme activities in chilling-stressed leaves and decreases lipid peroxidation to some extent, enhancing the tolerance of cucumber leaves to chilling stress.     

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n = 14) and healthy age-matched controls (n = 14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Genotoxicity and oxidative stress in fish after a short-term exposure to silver nanoparticles

This study examined the effects of waterborne silver nanoparticles (AgNPs) on juvenile fish Piaractus mesopotamicus (“pacú”), and analyzed toxicological endpoints such as metal burdens, oxidative stress and genotoxicity in a short-term assay. Fish were individually exposed to 0 (control), 2.5, 10, and 25 μg AgNPs/L. After 24 h, silver accumulation was greater in the brain than the liver and gills at all silver concentrations. Fish exposed to higher AgNPs concentrations showed major alterations in oxidative stress markers. An increase in lipid peroxidation (LPO) levels was observed in the liver of fish exposed to 10 μg AgNPs/L with no changes in the antioxidant enzymes activities. In the case of the 25 μg AgNPs/L treatment, a hepatic activation of the enzymatic antioxidant defense occurred, and LPO levels resulted unaltered. On the other hand, the brain presented the highest LPO levels at both 10 and 25 μg AgNPs/L exposures. The AgNPs toxicity was also evidenced by the DNA damage in fish erythrocytes at higher concentrations. Summarizing, a short exposure to sublethal concentrations of AgNPs is enough to generate deleterious effects on fish, including DNA damage.     

Cytotoxic effects of 4-octylphenol on fish hepatocytes

The present study was conducted to determine cytotoxic effects of 4-octylphenol (4-OP) on primary cultured hepatocytes of pearl mullet (Alburnus tarichi). Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) level, antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and glutathione (GSH) content were measured after 24-h exposure to 4-OP. 4-OP caused dose- and time-dependent increases in LDH release. Significant induction of MDA level and decrease in GSH content were found. SOD and GPx activities were decreased while GST activity was increased. These findings suggest that 4-OP leads to cytotoxicity by depressing antioxidant defenses in fish hepatocytes.     

Potential chemoprevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by polyphenolics from Acacia nilotica bark

Chemopreventive potential of Acacia nilotica bark extract (ANBE) against single intraperitoneal injection of N-nitrosodiethylamine (NDEA, 200 mg/kg) followed by weekly subcutaneous injections of carbon tetrachloride (CCl₄, 3 ml/kg) for 6 weeks induced hepatocellular carcinoma (HCC) in rats was studied. At 45 day after administration of NDEA, 100 and 200 mg/kg of ANBE were administered orally once daily for 10 weeks. The levels of liver injury and liver cancer markers such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), total bilirubin level (TBL), α-feto protein (AFP) and carcinoembryonic antigen (CEA) were substantially increased following NDEA treatment. However, ANBE treatment reduced liver injury and restored liver cancer markers. ANBE also significantly prevented hepatic malondialdehyde (MDA) formation and reduced glutathione (GSH) in NDEA-treated rats which was dose dependent. Additionally, ANBE also increased the activities of antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) in the liver of NDEA-administered rats. Eventually, ANBE also significantly improved body weight and prevented increase of relative liver weight due to NDEA treatment. Histological observations of liver tissues too correlated with the biochemical observations. HPLC analysis of ANBE showed the presence of gallic, protocatechuic, caffeic and ellagic acids, and also quercetin in ANBE. The results strongly support that A. nilotica bark prevents lipid peroxidation (LPO) and promote the enzymatic and non-enzymatic antioxidant defense system during NDEA-induced hepatocarcinogenesis which might be due to activities like scavenging of oxy radicals by the phytomolecules in ANBE.     

Co- and Post-Treatment with Lysine Protects Primary Fish Enterocytes against Cu-Induced Oxidative Damage

The aim of the work was primarily to explore the protective activity pathways of lysine against oxidative damage in fish in vivo and in enterocytes in vitro. First, grass carp were fed diets containing six graded levels of lysine (7.1–19.6 g kg^-1 diet) for 56 days. Second, the enterocytes were treated with different concentrations of lysine (0–300 mg/L in media) prior to (pre-treatment), along with (co-treatment) or following (post-treatment) with 6 mg/L of Cu for 24 h. The results indicated that lysine improved grass carp growth performance. Meanwhile, lysine ameliorated lipid and protein oxidation by elevating the gene expression and activity of antioxidant enzymes (superoxide dismutase (SOD), glutathioneperoxidase (GPx), glutathione-S-transferase (GST) and reductase (GR)), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels in fish intestine. The in vitro studies showed that co- and post-treatment with lysine conferred significant protection against Cu-induced oxidative damage in fish primary enterocytes as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) OD values, along with alkaline phosphatase (ALP) and lactate dehydrogenase activities, and the depletion of protein carbonyl (PC), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine contents. Moreover, lysine co-treatment decreased the activities and mRNA level of cellular SOD, GPx, GST and GR compared with the Cu-only exposed group. Gene expression of the signalling molecule Nrf2 showed the same pattern as that of SOD activity, whereas Kelch-like ECH-associated protein 1b (Keap1b) followed the opposite trend, indicating that co-treatment with lysine induced antioxidant enzymes that protected against oxidative stress through Nrf2 pathway. In addition, post-treatment with lysine increased proteasomal activity and blocked the Cu-stimulated increase in mRNA levels of GST and associated catalase (CAT) and GST activities (P<0.01 and P<0.001). GR activity and gene expression, and glutathione (GSH) content followed an opposite trend to GST activity (P<0.05). Thus, post-treatment of lysine elevated protein and DNA repair abilities and ameliorated the cellular redox state of enterocytes. The overall results suggest that lysine plays a significant role in the protection of fish intestine in vivo and in vitro through the induction of key antioxidant protection.