Increased GST Activity Research Articles

Increased carbonylation of the lipid phosphatase PTEN contributes to Akt2 activation in a murine model of early alcohol-induced steatosis

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is a key event in the pathogenesis of alcoholic liver disease (ALD), which ranges from simple steatosis to fibrosis. The lipid phosphatase PTEN plays a central role in the regulation of lipid metabolism in the liver. In this study, the effects of chronic ethanol feeding and carbonylation on the PTEN signaling pathway were examined in a 9-week mouse feeding model for ALD. Chronic ethanol consumption resulted in altered redox homeostasis as evidenced by decreased GSH, decreased Trx1, and increased GST activity. Both PTEN expression and PTEN phosphorylation were significantly increased in the livers of ethanol-fed mice. Carbonylation of PTEN increased significantly in the ethanol-fed mice compared to pair-fed control animals, corresponding to decreased PTEN 3-phosphatase activity. Concomitantly, increased expression of Akt2 along with increased Akt phosphorylation at residues Thr308, Thr450, and Ser473 was observed resulting in increased Akt2 activity in the ethanol-fed animals. Akt2 activation corresponded to a decrease in cytosolic SREBP and ChREBP. Subsequent LC/MS/MS analysis of 4-HNE-modified recombinant human PTEN identified Michael addition adducts of 4-HNE on Cys71, Cys136, Lys147, Lys223, Cys250, Lys254, Lys313, Lys327, and Lys344. Computational-based molecular modeling analysis of 4-HNE adducted to Cys71 near the active site and Lys327 in the C2 domain of PTEN suggested inhibition of enzyme catalysis via either stearic hindrance of the active-site pocket or prevention of C2 domain-dependent PTEN function. We hypothesize that 4-HNE-mediated PTEN inhibition contributes to the observed activation of Akt2, suggesting a possible novel mechanism of lipid accumulation in response to increased reactive aldehyde production during chronic ethanol administration in mice.

Effect of blueberry pretreatment on diethylnitrosamine-induced oxidative stress and liver injury in rats

Diethylnitrosamine (DEN) treatment increases the generation of reactive oxygen species (ROS), apoptosis, necrosis and proliferation in the liver. Blueberries (BB; Vaccinium corymbosum L.) contain polyphenols and other active components and have high antioxidant capacities. We investigated the effect of BB pretreatment on DEN-induced liver injury and oxidative and nitrosative stress in male rats. Rats were fed with 5% and 10% BB containing diet for six weeks and DEN (200mg/kg; i.p.) was applied two days before the end of this period. Liver function tests were determined in serum and histopathological evaluation was performed in the liver tissue. Apoptosis-related proteins, Bax and B cell lymphoma-2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) expressions were also examined. Oxidative and nitrosative stress were evaluated in the liver by measuring thiobarbituric acid reactive substances, diene conjugate, protein carbonyl and nitrotyrosine levels, and glutathione levels and glutathione peroxidase, superoxide dismutase and glutathione transferase (GST) activities. Pretreatment with high dose of BB reduced apoptotic, necrotic and proliferative changes in the liver induced by DEN. Dietary BB also decreased hepatic lipid peroxidation, protein oxidation and nitrotyrosine levels together with increased GST activity. In conclusion, BB may have an inhibiting effect on acute liver injury by reducing apoptosis, necrosis, proliferation, oxidative and nitrosative stress in DEN-treated rats.

Auxin signalling is involved in cadmium-induced glutathione-S-transferase activity in barley root

Transient exposure of barley roots to Cd, IAA or H2O2 for 30 min resulted in a significant root growth inhibition. Cd significantly increased the GST activity of roots 6 h after the end of short-term treatment. This increase was more relevant in root segment containing differentiation zone than in root segment just immediately behind the root apex. In contrast to Cd treatment, the short-term exposure of barley roots to IAA resulted in a significant increase of GST activity along the whole root tip and this increase was detectable already 3 h after the treatment with 10 μM IAA. Similarly to IAA, exogenously applied 10 mM H2O2 for 30 min caused significant increase of GST activity along the whole root tip 6 h after the treatment. This increase was already detectable 3 h after the exposure, but only in the differentiation zone of root tip. Auxin influx or signalling inhibitor considerable decreased the Cd- or IAA-induced GST activity in barley root tips. The strong activation of GST even after a brief exposure of barley roots to Cd support the crucial role of GST in the Cd-induced stress response in which presumably IAA and H2O2 play an important signalling role including the activation of GST.

Linking Hematological, Biochemical, Genotoxic, and Behavioral Responses to Crude Oil in the Amazon Fish Colossoma macropomum (Cuvier, 1816)

Despite safety protocols, crude oil extraction and transportation in the Amazon basin has a potential for inadvertent oil spills, which can impact aquatic organisms in local rivers. The objective of this study was to assess the effects of crude oil on juvenile Amazonian fish tambaqui, Colossoma macropomum, at various biological levels. Furthermore, the effect of crude oil on response to alarm substance, an important communication system in fish, was reported for the first time. Fish exposed to crude oil showed a 90 % decrease in their response to alarm substance and a 60 % decrease in swimming activity relative to control fish. Basic hematology was not affected, although an increase of 200 % of DNA damage and an increase of GST activity were observed in animals exposed to crude oil. Inverse correlations were found between genotoxicity end points and behavioral parameters, suggesting that genotoxic end points can also reflect behavioral changes.

Modulation of the cellular redox status by the Alternaria toxins alternariol and alternariol monomethyl ether

The mycotoxin alternariol (AOH) has been reported to possess genotoxic properties, inducing enhanced levels of DNA damage after only 1h of incubation. In the present study we addressed the question whether the induction of oxidative stress might contribute to the genotoxic effects of AOH or its naturally occurring monomethylether (AME). In the dichlorofluorescein (DCF) assay, treatment of HT29 cells for 1h enhanced the formation of dichlorofluorescein, indicative for ROS formation. The total glutathione (tGSH) was transiently decreased. In accordance with the results of the DCF assay, AOH and AME enhanced the proportion of the transcription factor Nrf2 in the nucleus. Concomitantly, the Nrf2/ARE-dependent genes γ-glutamylcysteine ligase (γ-GCL) and glutathione-S-transferase (GSTA1/2) showed enhanced transcript levels. After 24h of incubation this effect was also reflected on the protein level by an increase of GST activity. However, in spite of the positive DCF assay and the activation of the redox-sensitive Nrf2/ARE-pathway, the level of oxidative DNA damage, measured in the comet assay by the addition of formamidopyrimidine-DNA-glycosylase (fpg) remained unaffected. Of note, after 3h of incubation no significant DNA damaging potential of AOH and AME was detectable, indicating either inactivation of the compounds or enhanced DNA repair. In summary, the mycotoxins AOH and AME were found to modulate the redox balance of HT29 cells but without apparent negative effect on DNA integrity.

Inhibition and recovery of molecular biomarkers of earthworm Eisenia andrei after exposure to organophosphate dimethoate

Dimethoate is a broad-spectrum organophosphorous insecticide produced and used in many countries against a wide range of pests. Like other pesticides, dimethoate also affects many non-target organisms, including soil organisms. In this study for the first time effects of dimethoate on molecular biomarkers (AChE, CES, CAT and GST activity, concentration of GSH and efflux pump activity) of Eisenia andrei earthworms were evaluated using the artificial OECD soil. Earthworms were exposed to environmentally relevant doses of dimethoate (0.03, 0.1, 0.3, 1 and 3 mg/kg) for 1, 3, 6, 10, 15, 21 and 28 days and besides the time-related responses of biomarkers, the recovery of AChE, CES and CAT activity was also investigated. Results showed that dimethoate caused dose dependent significant changes in measured biomarkers – inhibition of AChE, CES and CAT activity; increase in GST activity and concentration of GSH; and both induction and inhibition of efflux pump activity. Additionally, the selected molecular biomarkers showed slow recovery rate and at the highest applied concentration of dimethoate, the inhibition of enzymes was evident even after 28 days of recovery. The obtained results indicate that application of dimethoate in agriculture could have harmful effects on earthworms since application of environmentally relevant doses, i.e. doses that are applied in agriculture, caused significant changes in measured biomarkers.

Oxidative Response and Antioxidative Mechanism in Germinating Soybean Seeds Exposed to Cadmium

Seeds of soybean (Glycine max L.) exposed to 50 mg/L (Cd50), 100 mg/L (Cd100) and 200 mg/L (Cd200) cadmium solution for 24, 48, 72 and 96 h were examined with reference to Cd accumulation, oxidative stress and antioxidative responses. Soybean seeds accumulated Cd in an exposure time-and dosage-dependent manner. FRAP (ferric reducing ability of plasma) concentration, GSH/hGSH content, and GST activity showed a pronounced exposure time-dependent response. Cd100 enhanced FRAP concentration in germinating soybean seeds as compared to Cd50 treatment after 24 h exposure. Cd200 however increased statistically GST activities after 72 and 96 h exposure. Under all Cd dosages, GSH/hGSH concentrations were depressed with increasing exposure time. Reduction of GSH/hGSH content and concomitant increase of GST activity suggested a possible participation of GSH into GSH-Cd conjugates synthesis. MDA content is a potential biomarker for monitoring Cd phytotoxicity because it responds significantly to treatment dosage, exposure time and dosage × exposure time interaction. Increase of proline content may be a response to acute heavy metal toxicity in soybean seeds.

Responses of juvenile sea bass, Dicentrarchus labrax, exposed to acute concentrations of crude oil, as assessed by molecular and physiological biomarkers

In the present study, juvenile sea bass were exposed for 48 and 96h to an Arabian light crude oil and their responses were assessed at the molecular and physiological levels. The aim of the study was therefore to assess (i) the short term effects of crude oil exposure by the measurement of several molecular biomarkers, (ii) the consequences of this short term exposure on fish health by using growth and condition indices measured after a decontamination period of 28 and 26d in seawater. Hydrocarbon petroleum concentrations was monitored during the 96h experiments and an increase of PAH concentrations were found in fish following both exposure times. An 7-ethoxyresorufin-O-deethylase (EROD) induction was observed after 48h of exposure, while a significant decrease in the sea bass specific growth rate in length and for the RNA:DNA ratio was observed 28d after that exposure ceased. The EROD induction doubled after the 96h exposure, and a significant increase in GST activities was observed. A significant decrease in the specific growth rates, the otolith recent growth, the RNA:DNA ratio and the Fulton’s K condition index were then observed in sea bass 26d after the 96h exposure to mechanically dispersed crude oil compared to the control. The present study shows that growth and condition indices can prove useful in assessing fish health status following an oil spill. Their complementary analysis with sensitive molecular biomarkers as EROD could improve the determination of oil spill impact on fish populations.

The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae)

The aim of this study was to evaluate the anesthesia induction and recovery times of sub-adult and post-larvae white shrimp (Litopenaeus vannamei) that were treated with eugenol and the essential oils (EOs) from Lippia alba and Aloysia triphylla. Oxidative stress parameters in the hemolymph of this species were also analyzed. The concentrations of eugenol, A. triphylla EO and L. alba EO recommended for anesthesia were 200, 300 and 750μLL−1 for sub-adults and 175, 300 and 500μLL−1 for post-larvae, respectively. The concentrations studied during the transport of sub-adults were between 20 and 50μLL−1 eugenol, 20–30μLL−1A. triphylla EO and 50μLL−1L. alba EO. For post-larvae, the optimal concentrations for transport were 20μLL−1 eugenol and between 20 and 50μLL−1A. triphylla EO. The white shrimp sub-adults that were exposed to A. triphylla EO (20μLL−1) showed increases in their total antioxidant capacities (150%), catalase (70%) and glutathione-S-transferase (615%) activity after 6h. L. alba EO (50μLL−1) and eugenol (20μLL−1) also increased GST activity (1292 and 1315%) after 6h, and eugenol (20μLL−1) decreased the total antioxidant capacity (100%). Moreover, concentrations above 30μLL−1 for the EOs of A. triphylla and L. alba and 20μLL−1 eugenol were effective at inducing anesthesia and improving the antioxidant system against reactive oxygen species (ROS) after 6h.

Susceptibility to oxidative stress and modulated expression of antioxidant genes in the copper-exposed polychaete Perinereis nuntia

To identify and evaluate potentially useful biomarkers for oxidative stress as early warning indices in the polychaete, Perinereis nuntia, we exposed P. nuntia to copper (Cu) and measured several biomarker enzymes (glutathione S-transferase; GST, glutathione peroxidase; GPx, Metallothionein-like protein; MTLPs, and catalase; CAT) and genes (Pn-GSTs, Pn-CAT, and Pn-MT) with a cellular oxidative index, reactive oxygen species (ROS) level. Accumulated Cu concentrations in P. nuntia increased in a time-dependent manner. Intracellular ROS reached high levels 6h after exposure in P. nuntia with an increase of GST activity and glutathione (GSH) content. Particularly, GSH in polychaetes showed a positive correlation with Cu contents accumulated in P. nuntia. Messenger RNA expressions of GST sigma and GST omega showed relatively high expressions at 50μg/L of Cu exposure, even though the moderate increase of rest of GST isoforms was also observed. Also regarding long-term exposure, we reared P. nuntia in sediments for 15days, and found that there was an obvious increase of Pn-GSTs, Pn-CAT, and Pn-MT genes with elevated concentrations of Cu and Cd in polychaete body, compared to initial levels, suggesting that P. nuntia in sediment was affected by metals as well as by other organic pollutants to induce oxidative stress genes and enzymes. These findings suggest that oxidative stress is a potential modulator of defense system of P. nuntia. Several potential biomarker genes are available as early warning signals for environmental biomonitoring.

Impact of toxicant exposure on the proteomic response to intertidal condition in Mytilus edulis

Intertidal blue mussels display physiological adaptations to emersion-submersion cycle that can be impacted by response to chemicals. In order to study the interference of cellular response to pollutants on intertidal physiology, we analysed proteomic (2-DE) responses in gills of mussels exposed for 14 days to regular emersion (intertidal condition) or continuous submersion (subtidal condition) and to a mixture (B[a]P/phenantrene) of polycyclic aromatic hydrocarbons (PAHs). Antioxidant activities were measured as general stress markers. In clean context, emersion generated several over-expressions of proteins mainly involved in cytoskeleton, chaperoning, energetic metabolism and transcription regulation. Mussels exposed to PAHs showed equivalent accumulation levels of contaminants in both physiological conditions but an increased GST activity specifically in intertidal context, highlighting the high degree of stress underwent in this group, as well as over-expressions of Cu/Zn SOD and stress proteins in subtidal context. Presence of contaminants partly impacted the response to emersion: cytoskeletal rearrangements and energetic adjustments were mostly maintained whereas stress response was dramatically altered. These findings highlight the potential adverse effects of toxicants on physiological adjustments linked to air-exposure, thus suggesting to take into account in the evaluation of environmental risk the multiplicity of stresses that wild animals are likely to encounter.

Short-term physiological effects of a xenobiotic mixture on the freshwater mussel Elliptio complanata exposed to municipal effluents

The aim of this study was to investigate the short-term effects of tertiary-treated municipal effluents on the freshwater mussel Elliptio complanata. Caged mussels were immersed during 2 weeks in a river located North of Montreal Island, upstream/downstream the outfall and in one reference site located at the beginning of the Rivière des Prairies. A selection of biomarkers was analyzed to depict changes on various physiological systems: general physiology (mussel viability, condition index and gonado-somatic index), immune status (hemocyte viability, cellularity, phagocytosis efficiency, NK-like cytotoxic activity and lysozyme activity), inflammation (cyclo-oxygenase activity), detoxification (glutathione-S-transferases activity) and vitellogenesis (alkali-labile phosphate level). The analysis of total and fecal coliform counts in water and of heterotrophic bacteria levels in mussel tissues showed that the bacteriological quality of the water strongly decreased from the reference site to the downstream site. This was correlated with a significant loss of weight and an increase of mussel mortality. Cellularity and phagocytosis efficiency were significantly increased in the downstream site compared to the reference site. Though not statistically significant, lysozyme activity was also increased. NK-like cytotoxicity, activity of the pro-inflammatory enzyme COX and the levels of ALP and MT were not significantly changed. Conversely, the municipal effluents induced a significant increase of GST activity in downstream site, indicating a stimulation of detoxification metabolism. Altogether, these results confirm that a short-term exposure to a mixture of bacterial and chemical compounds released by the wastewater treatment plant La Pinière induces adverse physiological effects in E. complanata, as observed with the modulation of immune response and induction of detoxification metabolism.

Antimicrobial Peptides Increase Tolerance to Oxidant Stress in Drosophila melanogaster

It is well appreciated that reactive oxygen species (ROS) are deleterious to mammals, including humans, especially when generated in abnormally large quantities from cellular metabolism. Whereas the mechanisms leading to the production of ROS are rather well delineated, the mechanisms underlying tissue susceptibility or tolerance to oxidant stress remain elusive. Through an experimental selection over many generations, we have previously generated Drosophila melanogaster flies that tolerate tremendous oxidant stress and have shown that the family of antimicrobial peptides (AMPs) is over-represented in these tolerant flies. Furthermore, we have also demonstrated that overexpression of even one AMP at a time (e.g. Diptericin) allows wild-type flies to survive much better in hyperoxia. In this study, we used a number of experimental approaches to investigate the potential mechanisms underlying hyperoxia tolerance in flies with AMP overexpression. We demonstrate that flies with Diptericin overexpression resist oxidative stress by increasing antioxidant enzyme activities and preventing an increase in ROS levels after hyperoxia. Depleting the GSH pool using buthionine sulfoximine limits fly survival, thus confirming that enhanced survival observed in these flies is related to improved redox homeostasis. We conclude that 1) AMPs play an important role in tolerance to oxidant stress, 2) overexpression of Diptericin changes the cellular redox balance between oxidant and antioxidant, and 3) this change in redox balance plays an important role in survival in hyperoxia.

Overexpression of a specific soybean GmGSTU4 isoenzyme improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants

Plant glutathione transferases (GSTs) superfamily consists of multifunctional enzymes and forms a major part of the plants herbicide detoxification enzyme network. The tau class GST isoenzyme GmGSTU4 from soybean, exhibits catalytic activity towards the diphenyl ether herbicide fluorodifen and is active as glutathione-dependent peroxidase (GPOX). Transgenic tobacco plants of Basmas cultivar were generated via Agrobacterium transformation. The aim was to evaluate in planta, GmGSTU4's role in detoxifying the diphenyl ether herbicides fluorodifen and oxyfluorfen and the chloroacetanilides alachlor and metolachlor. Transgenic tobacco plants were verified by PCR and Southern blot hybridization and expression of GmGSTU4 was determined by RT-PCR. Leaf extracts from transgenic plants showed moderate increase in GST activity towards CDNB and a significant increase towards fluorodifen and alachlor, and at the same time an increased GPOX activity towards cumene hydroperoxide. GmGSTU4 overexpressing plants when treated with 200 μM fluorodifen or oxyfluorfen exhibited reduced relative electrolyte leakage compared to wild type plants. Moreover all GmGSTU4 overexpressing lines exhibited significantly increased tolerance towards alachlor when grown in vitro at 7.5 mg/L alachlor compared to wild type plants. No significant increased tolerance was observed to metolachlor. These results confirm the contribution of this particular GmGSTU4 isoenzyme from soybean in the detoxification of fluorodifen and alachlor, and provide the basis towards the development of transgenic plants with improved phytoremediation capabilities for future use in environmental cleanup of herbicides.

THE ANTIOXIDATIVE PROPERTIES OF WHITE CABBAGE (BRASSICA OLERACEA VAR. CAPITATA F. ALBA) FRESH AND SUBMITTED TO CULINARY PROCESSING

ABSTRACT White cabbage, fresh or fermented belongs to major dietary ingredients in Central Europe. Here we concentrated on antioxidative properties of cabbage since such activities are believed to prevent undesirable effects in human organism. These properties were assessed for juices in cell-free system by antioxidant capacity, in HT29 cells by protection against DNA oxidation, and stimulation of GSTs and DNA repair. Although cabbage does not possess high antioxidative potential compared with other plantborne foods, it may provide a very effective antioxidative barrier especially if culinary processing caused the release of antioxidants. All juices increased GST activities to various degree regardless of cabbage origin or year of cultivation and stimulated DNA repair enzymes. Cabbage juices also prevented DNA damage when applied to HT29 cells concomitantly with H2O2; however, no protective effect was seen after prolonged (24 h) incubation with these juices prior to ROS exposure. Interestingly, in this tumor cell line, both cabbage juices displayed moderate inhibitory effect on cell growth and induced DNA fragmentation readily detectable by comet assay. PRACTICAL APPLICATIONS The presented research demonstrated different antioxidative activities for cabbage juices, that is, the actual food component, at dietarily relevant concentrations, which allows one to draw practical conclusions. Technologically, the direct ability to scavenge ROS, especially its increase upon heat processing, suggests that cabbage may protect other food components against undesirable oxidative changes. In another study, we confirmed that animal fats low in natural antioxidants, therefore very liable to thermooxidative spoilage, are effectively protected by cabbage phytocomplex. Biologically, the ability to induce in human cells a variety of protective mechanisms at low concentrations implies that the consumption of this vegetable should be recommended. However, our results also demonstrated dangers of overdosing. The exposure to cabbage juices prior to ROS strongly sensitized cells to oxidative stress and in this context, the use of concentrated extracts from cruciferous vegetables, offered by several producers as dietary supplements, seems to require more careful consideration.

Effects of grape pomace on the antioxidant defense system in diet-induced hypercholesterolemic rabbits.

The effects of grape seeds extract and grape peels extract prepared from grape pomace on the activity of antioxidant enzymes, degree of lipid peroxidation in serum and liver tissue were investigated in rabbits fed on high cholesterol diet. New Zealand white rabbits were divided as follows ; 1) NOR (normal group); 2) CHOL (cholesterol group); 3) GSH (cholesterol + grape seed extract group); 4) GPE (cholesterol + grape peel extract); 5) GSP (cholesterol + grape seed powder); 6) GPP (cholesterol + grape peel powder); 7) GE (cholesterol + grape seed and peel extract); 8) GP (cholesterol + grape seed and peel powder). Eight groups of rabbits were studied for 8 weeks. At the end of the experimental period, rabbits were sacrificed and the liver tissue were removed. Then, GSH, GPx, GST, CAT and MDA in the liver were measured. In liver tissues, total glutathione contents (GSH), glutathione peroxidase (GPx) and catalase (CAT) activity, which was significantly higher by grape seed extract supplementation. The level of malondialdehyde (MDA) was lower in the serum of rabbits fed grape seed extract or grape peel powder plus cholesterol than in the serum of rabbits fed cholesterol alone. It is therefore likely that grape seed extract prepared from grape pomace functioned as antioxidants in vivo, negating the effects of the oxidative stress induced by 1% cholesterol diet. The grape seed extract was found effective in converting the oxidized glutathione into reduced glutathione, and in removing H2O2 that is created by oxidative stress. The grape peel powder was found to have small influence on reduced glutathione content, CAT and GPX activity, but it increased GST activity in liver tissues, resulting in promoting the combination of lipid peroxide and glutathione (GSH), and further, lowering the formation of lipid peroxide in the serum. Therefore, grape pomace (grape seed extract and grape peel powder) supplementation is considered to activate the antioxidant enzyme system and prevent damage with hypercholesterolemia.

Effects of salvianolic acid B on lipid peroxidation and metalloproteinase-2activity in fibrotic liver in rat

To investigate the mechanism of salvianolic acid B (Sal B) action against liver fibrosis through preventing lipid peroxidation and regulating MMP-2 activity in liver. The liver fibrotic model was induced through intraperitoneally injection of DMN at a dose of 10 microg x kg(-1) for every other day and lasting for 4 weeks. Sal B was administered (10 mg x kg(-1)), and perindopril (5 mg x kg(-1)) was used as positive control. Hepatic inflammation and collagen were observed with HE and sirius red staining. The liver function including serum ALT, AST activity, Alb and total bilirubin (T. Bil) level were determined. The hepatic lipid peroxidation including SOD and GST activities and GSH content were measured. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The activity of metalloproteinase was assayed by gelatin zymography. The expressions of alpha-SMA, Col I in liver tissue were analyzed by Western blot. The model rats had higher serum T. Bil content, ALT and AST activities but lower Alb content than the normal rats, also had remarkable inflammatory necrosis and collagen deposition in liver, with much higher Hyp content, protein expression of alpha-SMA and collagen I and MMP-2 activity in liver, but had a decreased GSH content, SOD and GST activities. Both Sal B and perindopril attenuated hepatic injury and collagen deposition in model rats, decreased serum ALT activity and hepatic Hyp content, down-regulated alpha-SMA and collagen I protein expressions and metalloproteinase-2 activity than those in the model group, but increased SOD activity and GSH content, and Sal B decreased serum T. Bil content and increased GST activity. Sal B had a much better comprehensive actions than perindopril. Sal B has a good preventive action against liver fibrosis, the action mechanism is related to the prevention from lipid peroxidation and down-regulation of metalloproteinase-2 activity in fibrotic liver.

Oxidative Stress Biomarkers in the Digestive Gland of Theba pisana Exposed to Heavy Metals

The in vivo toxic effects of sublethal treatment of 40 and 80% of 48-h LD(50) of topically applied trace metals [copper (Cu), lead (Pb), and zinc (Zn)] on oxidative stress biomarkers in the digestive gland of Theba pisana were examined. Oxidative individual perturbations were assessed by measuring nonenzymatic (glutathione; GSH) and enzymatic (catalase, CAT; glutathione peroxidase, GPx; and glutathione-S-transferase, GST) antioxidants in digestive gland of the snails. Lipid peroxidation (LPO) was also evaluated as a marker of cell damage. The results indicated that the copper ion was the most potent metal against this snail, followed by zinc and lead, for which the corresponding LD(50) values were 37.88, 261.72, and 652.55 microg/snail, respectively. The no-observed effect concentration (NOEC) values for Cu, Zn, and Pb were 10, 50, and 500 microg/snail, respectively, and the corresponding lowest-observed effect concentration (LOEC) values were 50, 100, and 1000 microg/snail. All trace metals resulted in a significant increase in the level of LPO, whereas a significant decline in the content of GSH was observed when compared with untreated controls. Treatment with both sublethal doses of the metals caused significant increase in CAT activity, except in the case of 40% LD(50) Zn and 80% LD(50) Cu, which exhibited no alteration in CAT when compared to control animals. GPx was significantly increased in snails exposed to 40% LD(50) Cu and Pb as well as 80% LD(50) Cu. However, an opposite effect was observed in snails exposed to 80% LD(50) Pb and in either 40 or 80% LD(50) of Zn-intoxicated animals. Treatment with Pb at two sublethal doses significantly increased GST activity, whereas treatment the animal with Cu caused significant inhibition in this enzyme. Snails exposed to 40% LD(50) Zn showed significant enhancement of GST, whereas snails exposed to 80% LD(50) showed significantly reduced GST activity. Biphasic responses were observed for CAT, GPx, and GST activities in snails exposed to Cu, Pb, and Zn, respectively. This study suggests that upregulation of the antioxidant enzyme activities, elevation of LPO, and the reduction in GSH content is related to oxidative stress in this species that could be useful as biomarkers for the evaluation of contaminated terrestrial ecosystems.

A diet rich in cocoa attenuates N-nitrosodiethylamine-induced liver injury in rats

The effects of cocoa feeding against N-nitrosodiethylamine (DEN)-induced liver injury were studied in rats. Animals were divided into five groups. Groups 1 and 2 were fed with standard and cocoa-diet, respectively. Groups 3 and 4 were injected with DEN at 2 and 4 weeks, and fed with standard and cocoa-diet, respectively. Group 5 was treated with DEN, received the standard diet for 4 weeks and then it was replaced by the cocoa-diet. DEN-induced hepatic damage caused a significant increase in damage markers, as well as a decrease in the hepatic glutathione, diminished levels of p-ERK and enhanced protein carbonyl content, caspase-3 activity and values of p-AKT and p-JNK. The cocoa-rich diet prevented the reduction of hepatic glutathione concentration and catalase and GPx activities in DEN-injected rats, as well as diminished protein carbonyl content, caspase-3 activity, p-AKT and p-JNK levels, and increased GST activity. However, cocoa administration did not abrogate the DEN-induced body weight loss and the increased levels of hepatic-specific enzymes and LDH. These results suggested that cocoa-rich diet attenuates the DEN-induced liver injury.

Bioinformatic analysis and in vitro site-directed mutagenesis of conserved amino acids in BphK LB400, a specific bacterial glutathione transferase

Glutathione transferases [GSTs: EC 2.5.1.18)] are ubiquitous multifunctional prokaryotic and eukaryotic enzymes involved in the cellular detoxification and excretion of a large variety of compounds. However, our understanding of the role of bacterial GSTs in metabolism is still in its infancy. The association of bacterial GST DNA with other genes involved in degradation of toxic pollutants, including polychlorinated biphenyls (PCBs), indirectly suggests a role for bacterial GSTs in biodegradation. Previously, in this laboratory, a specific bacterial GST, BphK LB400 isolated from Burkholderia xenovorans LB400, was shown to be capable of dehalogenating chlorinated organic substrates rendering them less toxic. However, little is known about the specific amino acids in BphK LB400 involved in catalysis in vitro. In this study, bioinformatic analysis of BphK LB400 and other bacterial GSTs, including PCB degraders, identified a number of amino acids that were identical in all bacterial GST sequences analysed. Two amino acids, Cys10 and His106, were selected for in vitro site-directed mutagenesis studies. In vitro GST activity assay results suggest that these two amino acids play a role in determining the catalytic activity of BphK LB400. Studies of bacterial cell extracts expressing BphK LB400 (wildtype and mutant) identified a specific mutant, Cys10Phe, with increased GST activity towards 1-chloro-2,4-dinitrobenzene (the model substrate for GSTs). BphK LB400 (mutant) with increased activity towards toxic chlorinated organic compounds could have potential for bioremediation of contaminated soil in the environment.