Total GST Activities Research Articles

A Thiophene Derivative, 2‐Bromo‐5‐(2‐(methylthio)phenyl)thiophene, Has Effective Anticancer Potential with Other Biological Properties

AbstractThiophene derivatives are studied intensively due to their anticancer, antimicrobial, antioxidant and some other clinically important properties. In this study, the biological properties of a new thiophene derivative, so called BMPT, with a moderate drug score of 0.29 were tested. BMPT showed selective cytotoxicity for LnCap, HepG2 and Caco‐2 cancer cell lines with EC50 values of 138.573 μM, 185.931 μM, and 108.657 μM, respectively, but not on the control cell line HEK293. The increased expressions were detected for caspase3, caspase8, caspase9 and Bax; but Bcl‐2 expressions were decreased at cancer cell lines. BMPT decreased the total thiol content and total GST activities at HepG2 47 % and 40 %, respectively, and, molecular docking supported inhibitory models for GSTP1‐1, GSTA1‐1 and GSTM2‐2 were mixed, uncompetitive and uncompetitive, respectively. BMPT also showed potent antibacterial activity against Staphylococcus aureus. Related with its selective inhibition on GST activities, BMPT is proposed to have significant apoptotic effect leaded by changes, especially, in caspase 3, Bcl‐2 and Bax expressions.

Bacterial symbionts influence host susceptibility to fenitrothion and imidacloprid in the obligate hematophagous bed bug, Cimex hemipterus

The use of insecticides remains important in managing pest insects. Over the years, many insects manifested physiological and behavioral modifications resulting in reduced efficacy of insecticides targeted against them. Emerging evidence suggests that bacterial symbionts could modulate susceptibility of host insects against insecticides. Here, we explore the influence of host microbiota in affecting the susceptibility of insect host against different insecticides in the blood-sucking bed bug, Cimex hemipterus. Rifampicin antibiotic treatment resulted in increased susceptibility to fenitrothion and imidacloprid, but not against deltamethrin. Meanwhile, the host fitness parameters measured in the present study were not significantly affected by rifampicin treatment, suggesting the role of bacterial symbionts influencing susceptibility against the insecticides. 16S metagenomics sequencing revealed a drastic shift in the composition of several bacterial taxa following rifampicin treatment. The highly abundant Alphaproteobacteria (Wolbachia > 90%) and Gammaproteobacteria (Yersinia > 6%) in control bed bugs were significantly suppressed and replaced by Actinobacteria, Bacilli, and Betaproteobacteria in the rifampicin treated F1 bed bugs, suggesting possibilities of Wolbachia mediating insecticide susceptibility in C. hemipterus. However, no significant changes in the total esterase, GST, and P450 activities were observed following rifampicin treatment, indicating yet unknown bacterial mechanisms explaining the observed phenomena. Re-inoculation of microbial content from control individuals regained the tolerance of rifampicin treated bed bugs to imidacloprid and fenitrothion. This study provides a foundation for a symbiont-mediated mechanism in influencing insecticide susceptibility that was previously unknown to bed bugs.

Circadian rhythms in insecticide susceptibility, metabolic enzyme activity, and gene expression in Cimex lectularius (Hemiptera: Cimicidae).

Many insect species display daily variation of sensitivity to insecticides when they are exposed to the same concentration at different times during the day. To date, this has not been investigated in bed bugs. To address this, we explored circadian rhythms in insecticide susceptibility, xenobiotic metabolizing (XM) gene expressions, and metabolic detoxification in the common bed bug, Cimex lectularius. An insecticide susceptible Monheim strain of C. lectularius was most tolerant of deltamethrin during the late photophase at ZT9 (i.e. nine hours after light is present in the light-dark cycle (LD) cycle) and similarly repeated at CT9 (i.e. nine hours into the subjective day in constant darkness (DD)) suggesting endogenous circadian involvement in susceptibility to deltamethrin. No diel rhythm was observed against imidacloprid insecticide despite significant daily susceptibility in both LD and DD conditions. Rhythmic expressions of metabolic detoxification genes, GSTs1 and CYP397A1 displayed similar expression patterns with total GST and P450 enzyme activities in LD and DD conditions, respectively. The oscillation of mRNA levels of GSTs1 and CYP397A1 was found consistent with peak phases of deltamethrin susceptibility in C. lectularius. This study demonstrates that circadian patterns of metabolic detoxification gene expression occur within C. lectularius. As a consequence, insecticide efficacy can vary dramatically throughout a 24 hour period.

Radio-protective effect of some new curcumin analogues

In the present study, novel symmetrical curcumin analogues (2–7) have been synthesized by substituting the phenolic OH of curcumin with different linkers providing additional keto-enol tautomerism, very essential for radioprotective activity. The structures of the synthesized compounds (2–7) were elucidated by elemental analysis, IR, 1H-NMR, 13C-NMR and mass spectral data and were found consistent with the assigned structures. The curative effect of these new compounds, against the oxidative stress due to exposure of rats to the whole body γ-irradiation (7Gy) was investigated. Gamma-irradiated rats exhibited elevations of ALT, AST activities, urea, creatinine, triglycerides, total cholesterol, malondialdehyde (MDA), nitric oxide (NO), Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α) and Nuclear Factor-kappa B (NF-κB) levels. Contrariwise, the total protein, albumin, total calcium level, SOD, CAT, GSH-Px, GST activities and GSH content were decreased. Treatment of gamma-irradiated rats with the new curcumin analogues (2–7) showed significant amelioration in the in-vivo antioxidant status, liver and kidney functions, as well as the anti-inflammatory markers (IL-6, TNF-α and NF-κB). Inhibition of NF-κB could be responsible for the improvement of the antioxidant and anti-inflammatory status in gamma-irradiated animals, by down-regulation of IL-1β and TNF-α level. In conclusion, the new curcumin analogues (2–7) exhibited post-protective effect on gamma-irradiation, by NF-κB inhibition.

Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito Anopheles gambiae

Mosquitoes exhibit ∼24h rhythms in physiology and behavior, regulated by the cooperative action of an endogenous circadian clock and the environmental light:dark cycle. Here, we characterize diel (observed under light:dark conditions) time-of-day changes in metabolic detoxification and resistance to insecticide challenge in Anopheles gambiae mosquitoes. A better understanding of mosquito chronobiology will yield insights into developing novel control strategies for this important disease vector. We have previously identified >2000 rhythmically expressed An. gambiae genes. These include metabolic detoxification enzymes peaking at various times throughout the day. Especially interesting was the identification of rhythmic genes encoding enzymes capable of pyrethroid and/or DDT metabolism (CYP6M2, CYP6P3, CYP6Z1, and GSTE2). We hypothesized that these temporal changes in gene expression would confer time-of-day specific changes in metabolic detoxification and responses to insecticide challenge. An. gambiae mosquitoes (adult female Pimperena and Mali-NIH strains) were tested by gene expression analysis for diel rhythms in key genes associated with insecticidal resistance. Biochemical assays for total GST, esterase, and oxidase enzymatic activities were undertaken on time-specific mosquito head and body protein lysates. To determine for rhythmic susceptibility to insecticides by survivorship, mosquitoes were exposed to DDT or deltamethrin across the diel cycle. We report the occurrence of temporal changes in GST activity in samples extracted from the body and head with a single peak at late-night to dawn, but no rhythms were detected in oxidase or esterase activity. The Pimperena strain was found to be resistant to insecticidal challenge, and subsequent genomic analysis revealed the presence of the resistance-conferring kdr mutation. We observed diel rhythmicity in key insecticide detoxification genes in the Mali-NIH strain, with peak phases as previously reported in the Pimperena strain. The insecticide sensitive Mali-NIH strain mosquitoes exhibited a diel rhythm in survivorship to DDT exposure and a bimodal variation to deltamethrin challenge. Our results demonstrate rhythms in detoxification and pesticide susceptibility in An. gambiae mosquitoes; this knowledge could be incorporated into mosquito control and experimental design strategies, and contributes to our basic understanding of mosquito biology.

Elevated antioxidant response and induction of tau-class glutathione S-transferase after glyphosate treatment in Vigna radiata (L.) Wilczek

Glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum herbicide, acting on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products. It has also been reported to generate oxidative stress which influences the antioxidant response of target plants. The effect of glyphosate application on total protein, CAT, POD and GST activities was investigated and elevated expression of the oxidative stress enzymes was obtained after glyphosate treatment. Tau-class GSTs are plant-specific, and are chiefly involved in xenobiotics and oxidative stress metabolisms. Many herbicides and safeners have been known to selectively induce tau-class GSTs in different plant species. Here we also report the induction of tau-class GSTs after glyphosate treatment in the seedling roots of two Vigna radiata varieties (PDM11 and PDM54). GSH-agarose affinity chromatography and mass spectrometry revealed that the tau-class GSTs induced in the two varieties were different; the tau-class GSTs present in the untreated controls were also different in the two varieties. The present study highlights the elevated antioxidant response, the induction of tau-class GST and the genotypic variation in the type of tau-GST in control and glyphosate treated varieties of V. radiata.

Differential expression of glutathione- S-transferase isoenzymes in various types of anemia in Taiwan

Background Published reports concerning the expression of GST in various anemias including aplastic, hemolytic, iron deficiency and thalassemia anemia has been insufficient. We improved the conventional GST assay by incorporating a chloroform treatment to remove the interference of hemoglobin and evaluated the altered expression of GSTs in various anemias in Taiwan. Methods We incorporated a chloroform treatment to eliminate the interference of hemoglobin. Erythrocyte total GST and isoenzymes activities from 35 control subjects and 125 subjects of various anemias, including aplastic, hemolytic, iron deficiency and thalassemia anemias were measured spectrophotometrically. Results Chloroform treatment did not significantly affect GST activities in erythrocytes of control subjects while the activities of erythrocyte total GST and α-GST were significantly increased in all anemic patients ( P < 0.001). The expression of μ-GST was significantly decreased, although at a less extent, in cases of aplastic, iron deficiency and thalassemia anemia ( P < 0.05), but π-GST was not physiologically different in various types of anemia. Conclusion The determination of changes in erythrocyte GST activity is a promising indicator of oxidative stress conditions that occur in various types of anemia. Measurement of GST activity might be useful for the evaluation of prophylactic treatment in trials of antioxidant strategies.

Modest induction of phase 2 enzyme activity in the F-344 rat prostate

BackgroundProstate cancer is the most commonly diagnosed malignancy in men and is thought to arise as a result of endogenous oxidative stress in the face of compromised carcinogen defenses. We tested whether carcinogen defense (phase 2) enzymes could be induced in the prostate tissues of rats after oral feeding of candidate phase 2 enzyme inducing compounds.MethodsMale F344 rats were gavage fed sulforaphane, β-naphthoflavone, curcumin, dimethyl fumarate or vehicle control over five days, and on the sixth day, prostate, liver, kidney and bladder tissues were harvested. Cytosolic enzyme activities of nicotinamide quinone oxidoreductase (NQO1), total glutathione transferase (using DCNB) and mu-class glutathione transferase (using CDNB) were determined in the treated and control animals and compared.ResultsIn prostatic tissues, sulforaphane produced modest but significant increases in the enzymatic activities of NQO1, total GST and GST-mu compared to control animals. β-naphthoflavone significantly increased NQO1 and GST-mu activities and curcumin increased total GST and GST-mu enzymatic activities. Dimethyl fumarate did not significantly increase prostatic phase 2 enzyme activity. Compared to control animals, sulforaphane also significantly induced NQO1 or total GST enzyme activity in the liver, kidney and, most significantly, in the bladder tissues. All compounds were well tolerated over the course of the gavage feedings.ConclusionOrally administered compounds will induce modestly phase 2 enzyme activity in the prostate although the significance of this degree of induction is unknown. The 4 different compounds also altered phase 2 enzyme activity to different degrees in different tissue types. Orally administered sulforaphane potently induces phase 2 enzymes in bladder tissues and should be investigated as a bladder cancer preventive agent.

Effect of supplemental l-ascorbyl-2-polyphosphate (APP) in enriched live food on the immune response of Penaeus vannamei exposed to ammonia-N

The effects of supplemental vitamin C, in the form l-ascorbyl-2-polyphosphate (APP) in enriched live food dietary ( Artemia) on reactive oxygen intermediates (ROIs) and free radical scavenging enzymes (such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) activities in muscle of Penaeus vannamei exposed to ambient ammonia-N were investigated. The results showed ROIs values of shrimps fed the starved and enriched Artemia increased with increased ammonia-N concentration. The ROIs value of shrimps fed the enriched Artemia exposed to increasing ammonia-N concentration were 37.4%, 26.4% and 31.1% lower ( P < 0.05) compared with those of shrimps fed the starved Artemia exposed to the same ammonia-N concentration. Total SOD, CAT, GPX, GST and GR activities of shrimps fed the enriched Artemia exposed to ammonia-N (2.568–3.852 mmol/l), were all higher ( P < 0.05) than that of shrimps fed the starved Artemia exposed to the same ammonia-N concentration. In addition, total SOD, CAT, GPX, GST and GR activities of shrimps of both dietary treatments decreased ( P < 0.05) with increased ammonia-N concentration. The results demonstrated that supplementation of ascorbic acid in enriched live food ( Artemia) enhanced the anti-oxidant capacity of shrimp, increasing its defense system that may fight against environmental stress leading to reduced ammonia toxicity.

Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50-60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (approximately 40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to trans-plancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.

Effect of Extracellular Matrix Composition on the Expression of Glutathione S-transferase Isoenzymes in Organotypical Hepatocyte Cultures

Collagen gel sandwich and immobilization cultures of hepatocytes, using hydrated collagen type I as extracellular matrix (ECM), have been proposed as long-term in vitro models in pharmaco-toxicology. The in vivo ECM composition in the space of Disse is, however, much more complex. As a differentiated hepatocyte phenotype is thought to be highly dependent on ECM composition and biophysical characteristics, we modulated the ECM to mimic the in vivo situation. Moreover, commercially available collagen type I (Boehringer-Ingelheim) was compared to the one prepared in the laboratory from rat tails. ECM composition had no effect on albumin secretion or hepatocyte morphology in both collagen gel sandwich and immobilization cultures. Total, Alpha and Mu class GST activities in organotypical cultures with a complex or a simple collagen type I ECM were similar. The Pi class GST activity increased as a function of culture time in all culture models. Thus, mimicking the in vivo composition of the ECM did not improve the changes in GST expression that were observed in simple collagen gel cultures. The collagen type I matrix is therefore assumed to confer sufficient protection to help the hepatocytes to maintain their differentiated phenotype to a certain extent. Moreover, we hypothesize that the collagen gel matrix may act as a scaffold to keep newly synthesized ECM components in the proximity of the basolateral surfaces of the hepatocytes.

Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 °C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of l-proline (final concentration 60 μg/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. l-Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.

Cell morphology, albumin secretion and glutathione S-Transferase expression in collagen gel sandwich and immobilization cultures of rat hepatocytes

In order to investigate whether collagen gel sandwich and immobilization cultures of rat hepatocytes are suitable in vitro models for long-term pharmaco-toxicological studies, the expression of the key phase II biotransformation enzyme, glutathione S-transferase (GST, EC 2.5.1.18), has been studied in the presence or absence of l-proline (60μg/ml) in the culture medium. Additionally, hepatocytes morphology was followed and albumin secretion into the medium measured. As judged by inverse phase light microscopy and transmission electron microscopy, cells cultured in both organotypical models remained viable and well differentiated for at least 14 days. Albumin secretion increased 2.5-fold after 7 days of culture, in comparison with the values found after 2 days, and remained thereafter relatively constant. When l-proline was added to the medium of sandwich and immobilization gel cultures, steady-state secretion levels of 7.1 and 5.1 μg albumin/hr, respectively, were already obtained after 4 days of culture. Total, Mu, Alpha and Pi class GST activities were determined using a general substrate and isoenzyme specific substrates, respectively. After 7 days of culture, total GST activities were decreased as compared with the values obtained for freshly isolated cells. On the contrary, Mu class GST activities were kept at a constant level. Alpha class GSTs were maintained at a 50% activity level and GST 7-7 activity was shown to be slightly induced. l-proline prevented an initial decline in total and Mu class GST activities in both culture models. The GST subunit pattern, measured after affinity chromatography by reversed phase HPLC, reflected the GST activity results.

Effect of pyruvate on glutathione s-transferase expression in primary cultures of rat hepatocytes

Recently, primary rat hepatocyte cultures supplemented with 30 m m pyruvate and various hormones have been proposed as suitable long-term in vitro models for xenobiotic metabolism studies. In this study, the effect of 30 m m pyruvate on the cytosolic phase II biotransformation enzyme glutathione S-transferase (GST, EC 2.5.1.18) has been investigated. Adult male rat hepatocytes were brought into primary culture in serum free Williams' medium E with and without supplementation of 30 m m pyruvate. Total, Mu and Alpha class GST activities as well as GST subunit patterns were determined on the day of the isolation and as a function of culture time. Cell morphology and albumin secretion were also examined. After 7 days of culture, the morphology of pyruvate-treated cultures was still intact and bile canaliculi were clearly visible, whereas control cells without pyruvate had deteriorated and lost their normal morphology. The albumin secretion rate was higher in pyruvate-supplemented than in non-supplemented cultures. Total, Mu and Alpha class GST activities were well maintained in the presence of pyruvate; conversely, without pyruvate, all GST isoenzyme activities were significantly decreased. These findings were confirmed at the protein level. However, when hepatocytes received medium containing pyruvate for 7 days, the Pi class subunit 7, normally not present in adult liver cells, was expressed at a much higher level than was the case in untreated hepatocyte cultures. This study clearly shows that GST activities and GST proteins, as well as cell morphology and albumin secretion, are better maintained in primary monolayer cultures of adult rat hepatocytes supplemented with 30 m m pyruvate than in control cultures. However, it was also found that pyruvate is a very strong inducer of GST Pi. Consequently, the percentage GST subunit pattern of cultured rat hepatocytes is significantly affected by pyruvate supplementation, which may have consequences for in vitro xenobiotic metabolism studies.

Subunit specificity and organ distribution of glutathione transferase-catalysed S-nitrosoglutathione formation from alkyl nitrites in the rat

Glutathione transferase (GST)-catalysed S-nitrosoglutathione (GSNO) formation from alkyl nitrites was determined with the homodimers 1-1, 2-2, 3-3, and 4-4 isolated from rat liver. The 4-4 isoform showed a high specificity for the alkyl nitrites. Total GST activities were studied in homogenates from different organs. The liver showed highest GST activity both with amyl nitrite and with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, the activity ratio of amyl nitrite over CDNB being 3.8. In lung and heart, these ratios were 6.2 and 5.7, respectively, indicating a selectivity of these organs for alkyl nitrite metabolism and GSNO formation.

6] Purification of soluble human glutathione S-transferases

Publisher Summary This chapter focuses on the purification of soluble human glutathione S-transferases. Soluble glutathione transferases (GSTs) catalyze glutathione-dependent nucleophilic substitution and addition reactions with endogenous and xenobiotic electrophiles as well as the reduction of hydroperoxides, quinones, and quinoneimines. The human kidney is a good source of GSTPI-I but also contains alpha class GSTs together with small amounts of mu class GSTs, including M3-3. The procedure is very similar to that for the liver GSTs with the following modifications. The soluble fraction from 40 g of kidney is applied to the 40-ml affinity column. The concentration of GSTs in skeletal muscle is in the region of 4% that of the liver. However, because skeletal muscle accounts for 40% of body mass, the total hepatic and skeletal muscle GST activities are similar. Skeletal muscle is a very important component of extrahepatic GST activity. Enzyme purity may be assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectrofocusing, and reversedphase high-performance liquid chromatography (HPLC).