Glutathione S-transferase Enzyme Activity Research Articles

Biomarkers of oxidative stress in the post-embryonic characterization of the neotropical annual killifish.

Annual killifish are among the most remarkable extremophile species with the shortest vertebrate life span. Few studies have reported on the oxidative balance throughout their life cycle and its association to the natural aging process of these neotropical animals in a natural environment. We standardized and analyzed physiological markers related to the redox balance of the annual killifish (Cynopoecilus fulgens) throughout the post-embryonic life cycle (enzyme activity of Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione S-transferase, as well as the determination of the levels of Lipoperoxidation, Carbonylated Proteins, and Total Proteins). We tested the influence of environmental variables on these biomarkers. Individuals were collected, including juveniles, adults, and seniles, in three sampling units around the Parque Nacional da Lagoa do Peixe, located in the Coastal Plain of Rio Grande do Sul. We observed that males and females used different physiological strategies of their redox balance during their life cycle, and their oxidative balance was influenced by their reproductive period and environmental variables (water temperature, abundance of predators, abundance of another sympatric annual killifish species, and abundance of C. fulgens). The population of each temporary pond presented different physiological responses to the adaptation of their life cycle, and there was an influence of environmental component as a modulator of this cycle. Our study offers reference values that will be useful for comparison in future research with short-lived organisms.

Toxicological Impacts on Antioxidant Responses, Stress Protein, and Genotoxicity Parameters of Aluminum Oxide Nanoparticles in the Liver of Oreochromis niloticus.

The aim of this study was to determine the toxic effects of aluminum oxide nanoparticles (Al2O3 NPs) on oxidative stress, stress protein, and genotoxicity parameters in Oreochromis niloticus. Ninety-six-hour LC50 value of Al2O3 NPs was found as 52.4 ppm for O. niloticus. The fish were exposed to 2.6 ppm (5% of the 96-h LC50) and 5.2 ppm (10% of the 96-h LC50) for 3 days and 7 days. Various biochemical parameters, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, glutathione (GSH), thiobarbituric acid reactive substance (TBARS), heat shock protein 70 (HSP70; stress protein), and genotoxicity biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, were determined. Results showed that antioxidant enzymes were significantly decreased in SOD, CAT, and GPx enzyme activity, but GST enzyme activity was significantly increased in 7 days. The oxidative stress parameters, GSH levels, were significantly decreased while 8-OHdG and TBARS levels were increased in 3 and 7 days. HSP70 levels were decreased in the concentrations of Al2O3 NPs and exposure times. Our results showed that as a result of changes in oxidative stress parameters, stress protein, and genotoxicity parameters, O. niloticus liver tissue is highly sensitive and toxic to aluminum oxide nanoparticle exposure.

Protective effect of glutathione S-transferase enzyme activity against aflatoxin B1 in poultry species: relationship between glutathione S-transferase enzyme kinetic parameters, and resistance to aflatoxin B1

Comparative studies designed to investigate the role of glutathione S-transferase (GST) activity on the enzyme catalyzed trapping of aflatoxin B1-8,9-epoxide (AFBO) with glutathione, and the relationship with aflatoxin B1 (AFB1) resistance have not been conducted in poultry. Hepatic cytosolic fractions of chickens, quail, turkeys and ducks were used to measure in vitro the enzymatic parameters maximal velocity (Vmax), Michaelis-Menten constant (Km) and intrinsic clearance (CLint) for GST activity. AFB1 used ranged from 2.0 to 157.5 µM and the AFB1-GSH produced was identified and quantitated by HPLC. Significant differences were found in GST Vmax values, being the highest in chickens, followed by quail, ducks and turkeys. The Km values were also significantly different, with chickens < ducks < turkeys < quail. Chickens had the higher CLint value in contrast to ducks. Differences by sex showed that duck females had a higher CLint value than the turkey and quail, whereas duck males had a CLint close to that of turkey. The ratio “AFBO production /AFB1-GSH production” follows the order duck>turkey>quail>chicken, in agreement with the known poultry sensitivity. The extremely high “AFB1 epoxidation activity/ GST activity” ratio observed in ducks might be the explanation for the development of hepatocellular carcinoma in this species.

Comparative transcriptome combined with biochemical and physiological analyses provide new insights toward cadmium accumulation with two contrasting Nicotiana species.

Cadmium (Cd) is known as one of the most hazardous elements in the environment and a persistent soil constraint toxic to all flora and fauna. In this study, we conducted physiological, biochemical, and transcriptomic analyses of Nicotiana rustica (N. rustica) and Nicotiana tabacum (N. tabacum) treated with CdCl2 to know the underlying molecular mechanisms of Cd accumulation. As a result, N. rustica had more dry weight than N. tabacum. Additionally, N. rustica accumulated higher Cd concentration (69.65 times), Cd2+ influx (1.32-fold), glutathione S-transferases (GST) enzyme activity (2.54 times), GSH/GSSG (oxidized form of GSH) ratio, increase of superoxide dismutase and CAT and a lower H2 O2 and superoxide (O2 •- ) accumulation in their roots than N. tabacum. Cd mainly distributed in the cytoplasm of both species and N. rustica had a significant proportion in the cell wall. Furthermore, the transcriptomic analysis revealed 173 and 710 differentially expressed genes (DEGs) between control and Cd-stressed plants in the leaves and roots of N. rustica, while 576 and 1543 DEGs were found in the leaves and roots of N. tabacum, respectively. In N. rustica, phenylpropanoid biosynthesis and phenylalanine metabolism were the most enriched pathways, while GSH metabolism, ATP-binding cassette transporters and phenylpropanoid biosynthesis were the most enriched in N. tabacum. Finally, we found that DEGs related to metal influx, sequestration, remobilization, and chelation were responsible for Cd accumulation. These results indicated that N. rustica accumulated higher Cd content than N. tabacum, suggesting that each species utilized different response mechanism under the same Cd stress conditions. The DEGs identified in this study might lead to the identification of genes or pathways related to Cd regulation. This study identifies important regulators related to Cd accumulation.

Synthesis, Cytotoxicity, Antioxidant and Antimicrobial Activity of Indole Based Novel Small Molecules

Aim: In this study experiments were carried out to explore antioxidant, antimicrobial, cytotoxic properties of novel indole derivative 1-ethyl-2-phenyl-3-phenylethyl-3-thiophen-2-yl-1Hindole (EPI) together with its effect on glutathione S-transferases (GST) activities in human liver carcinoma (HepG2) cells. Background:: Indoles probably represent one of the most important heterocyclic structures that have been attracting the interest of many scientists in drug discovery. Objective: The present study was carried out to explore antioxidant, antimicrobial, cytotoxic properties of novel indole derivative 1-ethyl-2-phenyl-3-phenylethyl-3-thiophen-2-yl-1H-indole (EPI) and its effect on glutathione S-transferases (GST) activities in human liver carcinoma (HepG2) cells. Materials and Methods: Pd-catalyst Sonogashira coupling reactions, MTT Assay, Antioxidant capacity test, Antimicrobial test, GST enzyme activity test. Results: 1-ethyl-2-phenyl-3-(phenylethynyl)-1H-indole had antioxidant and antimicrobial properties. It displayed significant induction in glutathione S-transferases (GST) enzyme activity in human liver cancer cell lines (HepG2), but cytotoxic effect on all tested cancer cell lines could not be observed. Conclusion:: All of these results showed that 1-ethyl-2-phenyl-3-(phenylethynyl)-1H-indole had antioxidant and antimicrobial properties without cytotoxic effect, which could make it a promising active component with further studies.

Effects of Different Ammonia Levels on Tribenuron Methyl Toxicity in Daphnia magna.

The present study investigates the toxicity of the herbicide tribenuron methyl (TBM) as an anthropogenic agent and ammonia as an abiotic factor on Daphnia magna at environmentally relevant concentrations. These stressors may coexist in surface waters in agricultural regions. To achieve this objective, D. magna were exposed to TBM at a nominal concentration of 0.81μg/L in association with a low ammonia (LA) concentration of 0.65mg/L and a high ammonia (HA) concentration of 1.61mg/L in acute toxicity tests of 96-h duration and chronic toxicity tests of 21-day duration. The D. magna also were exposed to TBM, HA, and LA singly. The D. magna were analysed for various biomarkers of sublethal toxicity. Glutathione peroxidase (GPx), glutathione S-transferase (GST), cholinesterase (ChE) enzyme activities, and levels of thiobarbituric acid reactive substances (TBARS) and total protein were determined spectrophotometrically. Mitochondrial membrane potential (MMP) was analysed by microscopy with fluorescence staining. Cytochrome c and 5' AMP-activated protein kinase (AMPK) were analysed by Western blotting. Morphometric properties were examined microscopically. This is the first study in which AMPK, an indicator of intracellular energy, was measured in D. magna. GST and ChE enzyme activities and TBARS and total protein levels did not change during acute exposures (i.e., 96h) in all treatments. GPx activity increased in D. magna from the HA + TBM treatment compared with single-exposure groups. The level of cytochrome c protein was elevated in D. magna from the LA and LA + TBM treatments. AMPK protein levels increased in all treatments with daphnids, except in the LA group. MMP was depolarised in D. magna from all treatments, whereas the most notable change was observed in HA + TBM mixture group in chronic exposures. The results show that GST and ChE may not be sensitive biomarkers for evaluating the sublethal toxic effects to D. magna exposed to environmentally relevant concentrations of ammonia and TBM. Acute and chronic exposure to ammonia and TBM probably caused an energetic crisis in D. magna. Therefore, AMPK and MMP are promising biomarkers for these toxicants.

Protective Effects of 3-benzoyl-7-hydroxy Coumarin on Liver of Adult Rat Exposed to Aluminium Chloride

In this study, the effects of 3-benzoyl-7-hydroxy coumarin molecule on mineral and antioxidant enzymes were investigated in rat liver exposed to oxidative stress with aluminium chloride (AlCl3). Adult male Wistar albino rats were divided into four groups as Control, Coumarin, AlCl3, and Coumarin + AlCl3. Coumarin at the dose of 10 mg / kg and AlCl3 at the dose of 8.3 mg / kg were administered for 30 days every other day. In AlCl3 group, malondialdehyde (MDA), iron (Fe), aluminium (Al) and copper (Cu) levels increased compared to the control group, while glutathione (GSH) level, glutathione S-transferase (GST), and carboxylesterase (Ces) enzyme activity levels decreased. In Coumarin + AlCl3 group, MDA, Fe, Al and Cu levels decreased with the effect of coumarin compared to AlCl3 group, while GSH level, and GST enzyme activity levels increased. According to our results, AlCl3 generates oxidative stress in rat livers, and we believe that 3-benzoyl-7-hydroxy coumarin has an ameliorative effect on antioxidant enzyme system, Al, Fe and Cu levels.

Cell free Microcystis aeruginosa spent medium affects Daphnia magna survival and stress response

Primary consumers in freshwater ecosystems, such as the zooplankton organism Daphnia magna, are highly affected by cyanobacteria, both as they may use it as a food source but also by cyanobacterial metabolites present in the water. Here, we investigate the impacts of cyanobacterial metabolites focussing on the environmental realistic scenario of the naturally released mixture without crushing cyanobacterial cells or their uptake as food. Therefore, D. magna were exposed to two concentrations of cell free cyanobacterial spent medium from Microcystis aeruginosa PCC 7806 to represent higher and lower ecologically-relevant concentrations of cyanobacterial metabolites. Including microcystin-LR, 11 metabolites have been detected of which 5 were quantified. Hypothesising concentration and time dependent negative impact, survival, gene expression marking digestion and metabolism, oxidative stress response, cell cycle and molting as well as activities of detoxification and antioxidant enzymes were followed for 7 days. D. magna suffered from oxidative stress as both catalase and glutathione S-transferase enzyme activities significantly decreased, suggesting enzyme exhaustibility after 3 and 7 days. Moreover, gene-expressions of the 4 stress markers (glutathione S-transferase, glutathione peroxidase, catalase and thioredoxin) were merely downregulated after 7 days of exposure. Energy allocation (expression of glyceraldehyde-3-phosphate dehydrogenase) was increased after 3 days but decreased as well after 7 days exposure. Cell cycle was impacted time dependently but differently by the two concentrations, along with an increasing downregulation of myosin heavy chain responsible for cell arrangement and muscular movements. Deregulation of nuclear hormone receptor genes indicate that D. magna hormonal steering including molting seemed impaired despite no detection of microviridin J in the extracts. As a consequence of all those responses and presumably of more than investigated molecular and physiological changes, D. magna survival was impaired over time, in a concentration dependent manner. Our results confirm that besides microcystin-LR, other secondary metabolites contribute to negative impact on D. magna survival and stress response.

The detoxification enzymes activity profile in susceptible Aedes and Culex mosquitoes

The status of susceptible mosquito is important for monitoring and managing insecticide resistance in field population. The purpose of this study is to profile the differential expression level of enzymatic activities of metabolic enzymes exhibited in the mosquito species of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus from the susceptible strain. The fourth instar larvae of each strain were subjected to biochemical assay. The total protein content and enzymatic activities of Glutathione S-transferase (GST), α-esterase (α-est), β-esterase (β-est) Cytochrome P450 (Cyt P450) and Acetylcholinesterase (AChE) from each mosquito strains were elucidated. Significant difference (P<0.05) was detected between the total protein content between all species of susceptible strain mosquitoes. One-way ANOVA showed that the specific enzymatic activities of GST, α-est and Cyt P450 of all test mosquitoes were significantly different upon comparison with each species (P<0.05). The mean of enzymatic activities of insensitive AChE showed no significant difference upon comparison with each other (P>0.05). The enzymatic activities of β-est shows no significant difference between Ae. aegypti and Cx. quinquefasciatus mosquitoes (P>0.05) but the enzymatic activity of β-est in Ae. albopictus manifested significant difference upon comparison with the enzymatic activities of the other two test species (P<0.05). The results obtained may provide more information about the enzymatic activities of metabolic enzymes in Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus mosquitoes which might be beneficial for public sector for the application of proper vector control measures.

Biochemical and Histological Biomarkers in Crassostrea sp. (Bivalvia, Ostreidae) for Environmental Monitoring of a Neotropical Estuarine Area (São José Bay, Northeastern Brazil).

This study aimed to compare biochemical and histological biomarkers in oysters to identify impacted areas in a Brazilian port region. Oysters belonging to the Crassostrea genus were collected in two points in São José Bay (Brazil): (A1) Curupu Island (control area) and (A2) Braga Port (impacted area). Digestive glands from oysters were used to analyze the enzymatic activity of glutathione S-transferase and Catalase. The gills were used for standard histology analyses. Water samples were collected for metal analyses. Our results indicated that there was a change in the activity of oyster GST and CAT enzymes, especially in A2. Histological gill analysis indicated more frequent changes in A2. The analyzed metals presented higher values in A2. The results of this study suggest that enzymatic alterations, histological changes and higher metal values are indicative of initial stress caused by contaminants in São José Bay, especially in the port region.

Redox Imbalance Associates with Clinical Worsening in Spinocerebellar Ataxia Type 2.

Background Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease presenting with redox imbalance. However, the nature and implications of redox imbalance in SCA2 physiopathology have not been fully understood. Objective The objective of this study is to assess the redox imbalance and its association with disease severity in SCA2 mutation carriers. Methods A case-control study was conducted involving molecularly confirmed SCA2 patients, presymptomatic individuals, and healthy controls. Several antioxidant parameters were assessed, including serum thiol concentration and the superoxide dismutase, catalase, and glutathione S-transferase enzymatic activities. Also, several prooxidant parameters were evaluated, including thiobarbituric acid-reactive species and protein carbonyl concentrations. Damage, protective, and OXY scores were computed. Clinical correlates were established. Results Significant differences were found between comparison groups for redox markers, including protein carbonyl concentration (F = 3.30; p = 0.041), glutathione S-transferase activity (F = 4.88; p = 0.009), and damage (F = 3.20; p = 0.045), protection (F = 12.75; p < 0.001), and OXY (F = 7.29; p = 0.001) scores. Protein carbonyl concentration was positively correlated with CAG repeat length (r = 0.27; p = 0.022), while both protein carbonyl concentration (r = −0.27; p = 0.018) and OXY score (r = −0.25; p = 0.013) were inversely correlated to the disease duration. Increasing levels of antioxidants and decreasing levels of prooxidant parameters were associated with clinical worsening. Conclusions There is a disruption of redox balance in SCA2 mutation carriers which depends on the disease stage. Besides, redox changes associate with markers of disease severity, suggesting a link between disruption of redox balance and SCA2 physiopathology.

Caffeine versus antioxidant combination (Antox) and their role in modifying cadmium-induced testicular injury in adult male albino rats.

The aim of the current work was to compare the roles of caffeine and antioxidants in prevention of cadmium-induced testicular damage when given, in addition to cadmium, in adult male albino rats. Histopathological and ultra-structural examination as well as biochemical and molecular assessments were done. Cadmium chloride (4mg/kg body weight) was administered via oral gavage from day 21 to 28 of the experiment. Caffeine (25mg/kg) via intra-peritoneal injection and antioxidant preparation (Antox) 10mg/kg via oral gavage were given as a pre-treatment for 21days and concomitantly with Cd from day 21 to 28. Real-time PCR was done for determination of 3, 17 β hydroxy steroid dehydrogenase steroidogenic acute regulatory protein, caspase-9 and mitofusin 1,2 gene expression. Testosterone level, glutathione S-transferase enzyme activity, reactive oxygen species, malondialdehyde and superoxide dismutase were measured spectrophotometrically by ELISA. Histological and ultra-structural evaluation revealed disturbance of normal architecture, vacuolisation and necrosis. Vascular dilatation and congestion and collagen fibre deposition were present. A statistically significant difference was seen in all parameters when caffeine and antioxidants were given against cadmium-induced testicular injury. Overall, we conclude that both caffeine and antioxidants have the ability to reverse cadmium-induced testicular injury when given as pre-treatment prior to cadmium exposure.

Ameolirative effects of garlic (Allium sativum L.,) in streptozotocin induced oxidative stress in diabetic brain of rat

The aim of the present study was to evaluate the protective effect of methanol garlic extract on the enzymes related with polyol pathway, advanced glycation end products, markers of oxidative stress and antioxidant status in brain of streptozotocin induced diabetic rats. Antioxidant capability of methanol extract of garlic was evaluated by 2,2-diphenyl-2-picrylhydrazyl hydrate radical and FOX (ferrous ion oxidation-xylenol orange) H2O2 scavenging test. Diabetes was induced by single i.p injection of STZ (32mg/kg per body Wt.,). Blood glucose levels and body wt, were measured on every 7th day over a period of 30 days. The diabetic rats treated with garlic extract at two doses 250mg/kg and 500mg/kg body wt., by oral administration. Diabetic rats showed significant increase in food and water intake, decrease in blood glucose levels, body weights, but could not show any recovery by garlic treatment. Garlic treatment significantly decreased aldose reductase (AR); sorbitol dehydrogenase (SD) and glutathione S-transferase (GST) enzyme activities. A decrease of malndialdehyde (MDA), Protein carbonyls (PC), Pentosidine advance oxidation protein products (AOPP), Advanced glycation end products (AGEs) was also observed. Additionally garlic administration produced a restoration of brain superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) in diabetic rats. Garlic treatment also reduced the free radical formation and progression of hyperglycemia induced diabetic complications by decreasing influx of glucose into the polyol pathway and increased activity of antioxidant enzymes. The current study reveals exerts, efficiently, an attenuating effect of methanol garlic extract exterted antihyperlgycemic, antioxidant and anti-glycating effects in a dose dependent manner in diabetic rats.

Evaluation and possible mechanism of beet armyworm (Spodoptera exigua Hubner) resistance to chlorpyrifos and their sensitivity to neem oil insecticides

Abstract The uncontrolled and excessive use of insecticides on Spodoptera exigua can cause resistance. The aim of this study is to test resistance of S. exigua to chlorpyrifos and determine the possible mechanism of resistance to S. exigua. The resistance assay was carried out on chlorpyrifos by determining the level of resistance by the comparison of LC50 between the field samples and the standard samples. The resistivity of S. exigua was correlated with the activity of acetylcholinesterase (AChE), esterase, and glutathione S-transferase (GST) enzymes. The samples of S. exigua were also tested for their sensitivity to neem oil insecticides. The results showed that S. exigua samples from Brebes and Cipanas had a resistance ratio (RR) of 5.50 and 3.26, respectively. The results of the present study indicate that the insensitivity of the AChE and the high activity of the GST play a significant role in the mechanism of S. exigua resistance to chlorpyrifos. However, the esterase has fewer roles in the S. exigua resistance mechanism for both samples. In addition, the results of neem oil insecticides test showed that S. exigua from Brebes and Cipanas samples is sensitive to the insecticide with the RR value less than 1; therefore, this biopesticide has the opportunity to manage resistant pests. A novel mechanism for insecticide resistance by insect was proposed.

Responses of Aquatic Nontarget Organisms in Experiments Simulating a Scenario of Contamination by Imidacloprid in a Freshwater Environment.

Several studies have indicated the presence of the neonicotinoid insecticide imidacloprid (IMI) in aquatic ecosystems in concentrations up to 320.0µgL-1. In the present study, we evaluated the effects of the highest IMI concentration detected in surface water (320.0µgL-1) on the survival of Chironomus sancticaroli, Daphnia similis, and Danio rerio in three different scenarios of water contamination. The enzymatic activities of glutathione S-transferase (GST), catalase (CAT), and ascorbate peroxidase (APX) in D. rerio also were determined. For this evaluation, we have simulated a lotic environment using an indoor system of artificial channels developed for the present study. In this system, three scenarios of contamination by IMI (320.0µgL-1) were reproduced: one using reconstituted water (RW) and the other two using water samples collected in unpolluted (UW) and polluted (DW) areas of a river. The results indicated that the tested concentration was not able to cause mortality in D. similis and D. rerio in any proposed treatment (RW, UW, and DW). However, C.sancticaroli showed 100% of mortality in the presence of IMI in the three proposed treatments, demonstrating its potential to impact the community of aquatic nontarget insects negatively. Low IMI concentrations did not offer risks to D. rerio survival. However, we observed alterations in GST, CAT, and APX activities in treatments that used IMI and water with no evidence of pollution (i.e., RW and UW). These last results demonstrated that fish are more susceptible to the effects of IMI in unpolluted environments.

Resistance status and detoxification enzyme activity in ten populations of Panonychus citri (Acari: Tetranychidae) from Turkey

Acaricide resistance monitoring in phytophagous mites is crucial for effective pest management. Citrus growers in Cukurova region, a region that comprised more than 75% of total citrus production in Turkey, complained that acaricides have become inefficient in controlling outbreaks of the citrus red mite, Panonychus citri McGregor (Acari: Tetranychidae). To assess whether such control failures were caused by acaricide resistance, we bio-assayed P. citri populations collected from nine commercial orchards and a susceptible laboratory strain. Based on the mode of action (MoA) of acaricides, protonymphs were bio-assayed with etoxazole, spirodiclofen and spirotetramat, whereas abamectin and pyridaben were used for adults. Additionally, we determined kinetic esterase and glutathione S-transferase (GST) enzyme activities using a micro plate reader. Based on LC50 values, the resistance ratios of the orchard populations compared to the laboratory population varied from 2.81 to 34.82-fold, 1.22–18.35-fold, 1.23–40.43-fold, 1.76–27.50-fold and 2.24–75.06-fold for abamectin, etoxazole, spirodiclofen, spirotetramat and pyridaben, respectively. Total esterase and GST activities of the populations were in the ranges of 15.86–43.28 and 2.07–5.29 mOD/min/mg protein, respectively. The highest enzyme activity levels were detected in the Yakapinar population which was among one of the most resistant populations to all tested acaricides. The activities of detoxification enzymes may play an important role in the metabolic breakdown of the acaricides in resistant P. citri populations. Rotation of acaricides with different modes of action is fundamental to prevent potential resistance problems.

Glutathione S-transferase: Purification and Characterization from Quail (Coturnix coturnix japonica) Liver and the Impact of Some Metal Ions on Enzyme Activity

The objective of this study was to examine the inhibitory effect of some heavy metals on the glutathione S-transferase (GST) enzyme, which is one of the major enzymes of glutathione metabolism purified from quail liver tissues. The quail liver GST enzyme was purified with 15.86 EU/mg specific activity, in a yield of 12.36% and 46.1 purification fold by ammonium sulfate precipitation and glutathione-agarose affinity chromatography. The molecular weight of subunits of the enzyme and the purity were determined by SDS-PAGE. In the characterization studies, the optimum pH of the GST enzyme was determined to be pH = 8.0 in Tris/HCl buffer. Optimum ionic strength was determined to be 140 mM in Tris/HCI buffer. Stable pH was found to be pH = 8.5 in Tris/HCl buffer. Optimum temperature was found to be 50 °C. KM and Vmax values for substrates 1-chloro-2,4-dinitrobenzene (CDNB) and GSH of the enzyme were determined to be KM 0.048 mM Vmax 0.479 EU/mL and KM 0.114 mM Vmax 0.672 EU/mL, respectively. In vitro inhibition effects of metal ions, including Ag+, Ni2+, Cd2+, Fe2+, Pb2+, Co2+ Zn2+, and Al3+, were investigated on the GST enzyme activity. The results showed that Ag+, Cd2+, Ni2+ Zn2+, and Al3+ metal ions inhibited GST enzyme (IC50 values 0.239, 0.250, 0.265, 0.320, 0.594 mM, respectively), while Fe2+, Pb2+, and Co2+ metal ions activated the enzyme. Finally, Ki values and inhibition types for these substances were determined by Lineweaver-Burk graphs.

Developmental toxicity and transcriptome analysis of zebrafish (Danio rerio) embryos following exposure to chiral herbicide safener benoxacor

Benoxacor, a chiral herbicide safener for S-metolachlor, has been detected in streams. However, the potential risk this poses to aquatic ecosystems is not clear. This study used zebrafish (Danio rerio) embryos as a model to assess the enantioselective toxicity of benoxacor and its effects on biological activity and development from 2 h to 96 h post-fertilization (hpf). Results showed that benoxacor had negative effects on hatchability, malformations, and mortality. Compared to either individual enantiomer, embryos exposed to Rac-benoxacor had higher acute and developmental toxicities, glutathione S-transferase (GST) and glutathione peroxidase (GPx) enzyme activities, and nrf 2 expression levels. They also had lower superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) enzyme activity and krt 17, tbx 16, osx, cat, bcl 2, bax, and ifn expression levels. High-throughput RNA sequencing revealed that Rac-benoxacor had a greater effect on gene regulation than either enantiomer. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that changes in oxidoreductase activity, cellular lipid metabolic process, and catalytic activity related genes may be due to the enantioselective effects of benoxacor isomers. These results suggest that the ecotoxicology data and safety knowledge about the effects of chiral benoxacor on zebrafish should be considered in future environmental risk evaluation.

Chromium Exposure in Late Gestation Period Caused Increased Levels of Cr in Brain Tissue: Association with Alteration of Activity and Gene Expression of Antioxidant Enzymes of F1 and F2 Generation Mice.

Chromium is a micronutrient which has found frequent use as supplements during pregnancy and could have a role in altering the antioxidant status in the brain. The present study was undertaken to estimate chromium levels in the brain, antioxidant enzyme activity with their gene expression, and learning and memory parameters on F1 and F2 generation mice when the F0 was exposed to chromium. The chromium levels in the brain were estimated using atomic absorption spectrophotometer. The enzyme activity of glutathione-s-transferase (GST) and catalase (CAT) was estimated and their gene expression was evaluated using RT-PCR. The spatial memory was tested using Morris water maze. The learning and recall memory was tested using the step down latency paradigm. The chromium levels were significantly raised in animals treated with Cr per se in F1 generation and quercetin cotreatment reduced the Cr levels in brain significantly. The enzyme activity of GST was significantly less in Cr-treated animals of both generations and this effect was significantly reversed on cotreatment with quercetin. The gene expression of GST matched the enzyme activity. However, catalase activity did not show significant decrease with Cr but cotreatment with quercetin resulted in significant decrease compared with control and this effect was not matched by its gene expression. We observed no significant change in learning and memory parameters in both generations following Cr exposure. Thus, this study demonstrates that chromium exposure in gestation causes changes in enzyme activity especially GST and this change was matched by change in gene expression in GST but not CAT. There was no effect on memory at the given dose.

New insights into toxic effects of arsenate on four Microcystis species under different phosphorus regimes.

Very little information is available on the stressed growth of Microcystis imposed by arsenate (As(V)) under different phosphorus (P) regimes. In this study, we examined the growth characteristics and arsenic transformation of four Microcystis species exposed under As(V) with two P sources involving dissolved inorganic phosphorus (IP) and organophosphate (D-glucose-6-phosphate disodium salt, GP). Results showed that all the four chosen Microcystis species could grow and reproduce with GP as the only P source, and the difference was insignificant when compared with IP. From optical density (OD), chlorophyll a (Chla), and actual quantum yield (Yield), the tolerance to As(V) of the chosen species was following as FACHB 905 > FACHB 1028 > FACHB 1334 > FACHB 912. Specifically, the 96h EC50 of As(V) for FACHB 905 in IP was approx. 4 orders of magnitude higher than that in GP, but for other three algal species, the 96h EC50 values were similar under the two given different P conditions. Furthermore, all antioxidant enzyme activities of superoxide dismutase (SOD), peroxide dismutase (POD), glutathione S-transferases (GSTs), and metalloproteinase (MTs) in algal cells were significantly increased in GP conditions. Moreover, the enzyme activities of AKP, GSTs, and MTs were inhibited with increasing As(V) levels under both IP and GP conditions. In addition, arsenite (As(III)) and methylated As of monomethylarsonic acid (MMA) and dimethylthioarsinic acid (DMA) were found in FACHB 912 and FACHB 1334 media, indicating that these Microcystis could detoxify As(V) by As biotransformation under IP and GP conditions. Specifically, As(V) reduction was elevated in media of FACHB 1334 and FACHB 905, but was decreased in media of FACHB 912 under GP conditions. Our results highlight the different P sources that impact the toxic effects of arsenate exposure on Microcystis and subsequent As biotransformation.