Inhibition Of Glutathione S-transferase Research Articles

Characterization of glutathione S-transferases in the detoxification of metolachlor in two maize cultivars of differing herbicide tolerance

Glutathione S-transferases (GSTs) have been widely studied in relation to their role in herbicide tolerance and detoxification. However, a detailed characterization of GSTs from herbicide tolerant and sensitive maize cultivars is still lacking. In this study, we determined the mechanism of differential tolerance between two maize cultivars which had 4-fold difference tolerance to metolachlor. The metabolism rate of metolachlor was more rapid in the tolerant cultivar (Zea mays L. cv Nongda86) than the susceptible one (Zea mays L. cv Zhengda958). Addition of the GST inhibitor ethacrynic acid reduced the metabolism of metolachlor indicating the involvement of GSTs in the differential detoxification of metolachlor. The expression profiles of 32 GST isozymes were measured using quantitative RT-PCR. The results showed the expression of GST genes were slightly up-regulated in Nongda86, but severely inhibited in Zhengdan958 24h after metolachlor treatment. The genes GSTI, GSTIII, GSTIV, GST5, GST6 and GST7, which can detoxify chloroacetanilide herbicides, were all expressed higher in Nongda86 compared to Zhendgan958. The result of GST activity was consistent with the gene expression profiles. Collectively, higher-level expression of GST genes, leading to higher GST activity and faster herbicide detoxification, appears to be responsible for the difference in tolerance to metolachlor in two maize cultivars.

Developing piperlongumine-directed glutathione S-transferase inhibitors by an electrophilicity-based strategy

We report a case of successful design of glutathione S-transferase (GST) inhibitors via a natural product-inspired and electrophilicity-based strategy. Based on this strategy, a novel piperlongumine analog (PL-13) bearing a para-trifluoromethyl group and an α-chlorine on its aromatic and lactam rings, respectively, surfaced as a promising GST inhibitor, thereby overcoming cisplatin resistance in lung cancer A549 cells.

Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical.

Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chemically-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clinical investigations. The primary route of isothiocyanate metabolism is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymatic activity.

Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells

NRF2 stabilizes redox potential through genes for glutathione and thioredoxin antioxidant systems. Whether blockade of glutathione and thioredoxin is useful in eliminating cancer stem cells remain unknown. We used xenografts derived from colorectal carcinoma patients to investigate the pharmacological inhibition of glutathione and thioredoxin systems. Higher expression of five glutathione S‐transferase isoforms (GSTA1, A2, M4, O2, and P1) was observed in xenograft‐derived spheroids than in fibroblasts. Piperlongumine (2.5–10 μmol/L) and auranofin (0.25–4 μmol/L) were used to inhibit glutathione S‐transferase π and thioredoxin reductase, respectively. Piperlongumine or auranofin alone up‐regulated the expression of NRF2 target genes, but not TP53 targets. While piperlongumine showed modest cancer‐specific cell killing (IC 50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC 50 difference between cancer spheroids and fibroblasts: P = 0.002). The synergism of dual inhibition was evaluated by determining the Combination Index, based on the number of surviving cells with combination treatments. Molar ratios indicated synergism in cancer spheroids, but not in fibroblasts: (auranofin:piperlongumine) = 2:5, 1:5, 1:10, and 1:20. Cancer‐specific cell killing was achieved at the following drug concentrations (auranofin:piperlongumine): 0.25:2.5 μmol/L, 0.5:2.5 μmol/L, or 0.25:5 μmol/L. The dual inhibition successfully decreased CD44v9 surface presentation and delayed tumor emergence in nude mouse. However, a small subpopulation persistently survived and accumulated phosphorylated histone H2A. Such “persisters” still retained lesser but significant tumorigenicity. Thus, dual inhibition of glutathione S‐transferase π and thioredoxin reductase could be a feasible option for decreasing the tumor mass and CD44v9‐positive fraction by disrupting redox regulation.

A novel class of ethacrynic acid derivatives as promising drug-like potent generation of anticancer agents with established mechanism of action

The well-known diuretic Ethacrynic acid (EA, Edecrin), showing low anti-proliferative activities, was chemically modified at different positions. The new EA derivatives have been tested in vitro in anti-proliferative assays on both tumor KB (epidermal carcinoma) and leukemia HL60 (promyelocytic) cells suitable targets for anticancer activity. Reduction of the α-β double bond of EA completely abolished anti-cancer activities, whereas introduction of either 2-(4-substituted phenyl)ethanamine (series A) or 4-(4-substituted phenyl)piperazine (series B) moieties generated compounds showing moderate to strong anti-proliferative activities against human cancer cell lines. Several substitutions on the phenyl of these two moieties are tolerated. The mechanism of action of the EA derivatives prepared in this study is more complex than the inhibition of glutathione S-transferase π ascribed as unique effect to EA and might help to overcome tumor resistances.

Glutathione-S-transferase FosA6 of Klebsiella pneumoniae origin conferring fosfomycin resistance in ESBL-producing Escherichia coli.

The objectives of this study were to elucidate the genetic context of a novel plasmid-mediated fosA variant, fosA6, conferring fosfomycin resistance and to characterize the kinetic properties of FosA6. The genome of fosfomycin-resistant Escherichia coli strain YD786 was sequenced. Homologues of FosA6 were identified through BLAST searches. FosA6 and FosA(ST258) were purified and characterized using a steady-state kinetic approach. Inhibition of FosA activity was examined with sodium phosphonoformate. Plasmid-encoded glutathione-S-transferase (GST) FosA6 conferring high-level fosfomycin resistance was identified in a CTX-M-2-producing E. coli clinical strain at a US hospital. fosA6 was carried on a self-conjugative, 69 kb IncFII plasmid. The ΔlysR-fosA6-ΔyjiR_1 fragment, located between IS10R and ΔIS26, was nearly identical to those on the chromosomes of some Klebsiella pneumoniae strains (MGH78578, PMK1 and KPPR1). FosA6 shared >99% identity with chromosomally encoded FosA(PMK1) in K. pneumoniae of various STs and 98% identity with FosA(ST258), which is commonly found in K. pneumoniae clonal complex (CC) 258 including ST258. FosA6 and FosA(ST258) demonstrated robust GST activities that were comparable to each other. Sodium phosphonoformate, a GST inhibitor, reduced the fosfomycin MICs by 6- to 24-fold for K. pneumoniae and E. coli strains carrying fosA genes on the chromosomes and plasmids, respectively. fosA6, probably captured from the chromosome of K. pneumoniae, conferred high-level fosfomycin resistance in E. coli. FosA6 functioned as a GST and inactivated fosfomycin efficiently. K. pneumoniae may serve as a reservoir of fosfomycin resistance for E. coli.

Multilevel assessment of ivermectin effects using different zebrafish life stages

Several studies have shown high toxicity of the veterinary pharmaceutical ivermectin (a semisynthetic avermectin) for aquatic invertebrates however, few data is found for fish species. The present study evaluated the toxicity of ivermectin, to embryos, juveniles, and adults of zebrafish at different levels of biological organization including developmental, behavioural and biochemical. Toxicity tests were performed based on OECD protocols and mortality and behavioural changes were assed for all stages. Biochemical responses were assessed in adults and embryos and included cholinesterases (ChEs), catalase (CAT) (only in embryos), glutathione-S-Transferase (GST), lactate dehydrogenase (LDH) and vitellogenin (VTG) like proteins (only in embryos). Genotoxicity was evaluated in adults. Results showed a higher sensitivity of juvenile and adults of zebrafish (96h-LC10 values of 14.0 and 55.4μg/L, respectively). For embryos a 96h-LC10 of 147.1μg/L was calculated, moreover developmental anomalies and hatching inhibition were observed only at high concentrations (>400μg/L), whereas biochemical and behavioural responses occurred at lower concentrations (<60μg/L). Behavioural responses (lethargy) occurred in all life stages. Biochemical responses were observed including the inhibition of GST in adults and changes in ChE, CAT, LDH activities and VTG levels in embryos. Ivermectin did not show to be genotoxic for adult fish. The species sensitivity distribution analysis, based on fish and invertebrate species, indicated a Hazardous Concentration for 5% of the population (HC5) value of 0.057μg/L; suggesting high sensitivity of both groups to ivermectin and a high risk of this compound to aquatic ecosystems.

Differential Responses to Preemergence and Postemergence Atrazine in Two Atrazine‐Resistant Waterhemp Populations

Waterhemp [Amaranthus tuberculatus (Moq) Sauer] is a difficult‐to‐control dicot weed in the United States. Atrazine [6‐chloro‐N‐ethyl‐N’‐(1‐methylethyl)‐1,3,5‐triazine‐2,4‐diamine] is commonly used for preemergence (PRE) and postemergence (POST) waterhemp control in maize (Zea mays L.). Previous research reported that atrazine metabolism via glutathione S‐transferase (GST) activity contributes to atrazine POST resistance in two waterhemp populations from Illinois, designated MCR (McLean County, Illinois, resistant) and ACR (Adams County, Illinois, resistant). Objectives were to quantify responses of these populations to atrazine PRE and determine if the combination of a GST inhibitor and atrazine PRE or POST increases their control. Dose‐response analyses indicated MCR was resistant to atrazine PRE relative to ACR or WCS (Wayne County sensitive; herbicide‐sensitive population), despite MCR and ACR exhibiting equivalent levels of atrazine resistance POST. The ACR response to atrazine PRE (LD50) was intermediate compared with MCR and WCS. Seedling survival of ACR was reduced by 4‐chloro‐7‐nitrobenzofurazan (NDB‐Cl; a GST inhibitor) and atrazine PRE more than atrazine PRE alone, but not in MCR. Atrazine following NBD‐Cl applied POST inhibited seedling growth in ACR, but not in MCR. Enhanced atrazine activity with NBD‐Cl further supports rapid metabolism via GSTs as the main atrazine‐resistance mechanism in ACR. GST(s) that metabolize atrazine in MCR may not have been completely inhibited by NBD‐Cl, indicating that similar yet distinct atrazine‐resistance mechanisms exist in MCR compared to ACR. In conclusion, atrazine PRE (with or without NBD‐Cl) still controls ACR when applied at typical field‐use rates in maize, but the length of residual activity may be shorter than in sensitive populations.Core Ideas Two waterhemp populations are resistant to atrazine POST due to increased metabolism by GST enzymes. Since atrazine can also be soil applied, quantifying the levels of resistance in these populations is important for weed management in maize. The MCR population was resistant to atrazine PRE and displayed a higher level of resistance to atrazine PRE than the ACR population. NBD‐Cl, a metabolic inhibitor of GST enzymes, enhanced atrazine activity in the ACR population but not MCR. The length of residual activity of atrazine may be shorter for controlling these populations in maize compared with sensitive populations.

Arene Osmium Complexes with Ethacrynic Acid-Modified Ligands: Synthesis, Characterization, and Evaluation of Intracellular Glutathione S-Transferase Inhibition and Antiproliferative Activity

Four arene osmium complexes were prepared containing derivatives of ethacrynic acid, a potent inhibitor of glutathione S-transferases, either by direct coordination or via N- or P-donor ligands. The complexes were characterized by spectroscopic and analytical methods and, for Os(η6-p-cymene)(acetylacetonato)(2-(2,3-dichloro-4-(2-ethylenebutanoyl)phenoxy)acetato) and Os(η6-p-cymene)Cl2(2-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetoxy)ethyl nicotinato), by single-crystal X-ray diffraction. The cytotoxicity of the complexes toward human ovarian cancer cells and nontumorous human embryonic kidney cells was investigated, and two of the complexes, for which ruthenium analogues are known, helped to delineate the influence of the metal ion. Inhibition studies of intracellular glutathione S-transferases (GSTs, detoxification enzymes implicated in drug resistance) indicate that the observed cytotoxicity of the complexes involves GST inhibition, as well as other targets following dissociation of the ethacry...

Glutathione-Associated Enzymes In Anticancer Drug Resistance.

The importance of thiol-mediated detoxification of anticancer drugs that produce toxic electrophiles has been of considerable interest to many investigators. Glutathione and glutathione S-transferases (GST) are the focus of much attention in characterizing drug resistant cells. However, ambiguous and sometimes conflicting data have complicated the field. This article attempts to clarify some of the confusion. The following observations are well established: (a) tumors express high levels of GST, especially GST psi, although the isozyme components vary quite markedly between tissues and the isozymes are inducible; (b) nitrogen mustards are good substrates for the GST alpha family of isozymes which are frequently overexpressed in cells with acquired resistance to these drugs; (c) most drugs of the multidrug-resistant phenotype have not been shown to be GST substrates and although GST psi is frequently overexpressed in multidrug-resistant cells, most indications are that this is an accompaniment to, rather than a cause of, the resistant phenotype; (d) transfection of GST complementary DNAs has produced some lines with increased resistance to alkylating agents. Most studies of the relationships between GST and resistance have overlooked the potential importance of other enzymes involved in the maintenance of cellular glutathione homeostasis, and this has complicated data interpretation. Translational research aimed at applying our knowledge of glutathione pathways has produced preclinical and clinical testing of some glutathione and GST inhibitors, with some encouraging preliminary results. In brief, GSTs are important determinants of drug response for some, not all, anticancer drugs. Caution should be encouraged in assessing cause/effect relationships between GST overexpression and resistance mechanisms.

Effects of Ethacrynic Acid Addition to Diet on Fitness and Development in the Psocid Liposcelis bostrychophila Badonnel.

Fertility life table provides a comprehensive description of arthropod population dynamics by the estimation of parameters about arthropod population growth potential. It can also clarify the sublethal effects of chemicals on insects. Ethacrynic acid (EA), an inhibitor of glutathione S-transferases, is a diuretic compound that has been confirmed to modulate drug resistance in organisms. In this study, the effects of EA on growth and development of Liposcelis bostrychophila Badonnel were investigated in the laboratory to explore the potential possibilities of EA as an active agent to manage insecticide-resistant psocids. The treatment of psocids was obtained by feeding on the routine diet containing 3% EA for three successive generations, and psocids on routine diet served as control. The results indicated that EA possessed some negative effects on the life-table parameters of the psocid in F1 and F2 generations. The addition of EA to diet stunted psocids growth by lengthening development time and increasing mortality with a greater effect in the F2 generation. In the third generation of psocids on EA diet, it seemed there was a return to normal. Psocid fitness was influenced by addition of EA to standard diet. Using rm values, the fitness for EA diet in F1, F2, and F3 compared with the counterpart of routine diet was calculated as 0.80, 0.74, and 0.87, respectively.

Preparation of GST Inhibitor Nanoparticle Drug Delivery System and Its Reversal Effect on the Multidrug Resistance in Oral Carcinoma.

During the chemotherapy of cancer, drug resistance is the first issue that chemotherapeutic drugs cannot be effectively used for the treatment of cancers repeatedly for a long term, and the main reason for this is that tumor cell detoxification is mediated by GSH (glutathione) catalyzed by GST (glutathione-S-transferase). In this study, a GST inhibitor, ethacrynic acid (ECA), was designed to be coupled with methoxy poly(ethylene glycol)-poly(lactide) (MPEG–PLA) by disulfide bonds to prepare methoxy poly(ethylene glycol)-poly(lactide)-disulphide bond-mthacrynic acid (MPEG–PLA–SS–ECA) as a carrier material of the nanoparticles. Nanoparticles of pingyangmycin (PYM) and carboplatin (CBP) were prepared, respectively, and their physicochemical properties were investigated. The ECA at the disulfide could be released in the presence of GSH, the pingyangmycin, carboplatin and ECA were all uniformly released, and the nanoparticles could release all the drugs completely within 10 days. The half maximal inhibitory concentration (IC50) of the prepared MPEG–PLA–SS–ECA/CBP and MPEG–PLA–SS–ECA/PYM nanoparticles in drug-resistant oral squamous cell carcinoma cell lines SCC15/CBP and SCC15/PYM cells was 12.68 μg·mL−1 and 12.76 μg·mL−1, respectively; the resistant factor RF of them in the drug-resistant cells were 1.51 and 1.24, respectively, indicating that MPEG–PLA–SS–ECA nanoparticles can reverse the drug resistance of these two drug-resistant cells.

Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1st instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB.

Bioactivation of luteolin by tyrosinase selectively inhibits glutathione S-transferase

Glutathione S-transferase (GST) plays a significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in drug efficacy. Tyrosinase is an abundant enzyme found in melanoma. In this study, we used a tyrosinase targeted approach to selectively inhibit GST. In the presence of tyrosinase, luteolin (10 μM) showed 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. With respect to GSH, both luteolin-SG conjugate and luteolin-quinone inhibited ≥90% of GST activity via competitive reversible and irreversible mixed mechanisms with Ki of 0.74 μM and 0.02 μM, respectively. With respect to CDNB, the luteolin-SG conjugate inhibited GST activity via competitive reversible mechanism and competitively with Ki of 0.58 μM, whereas luteolin-quinone showed irreversible mixed inhibition of GST activity with Ki of 0.039 μM. Luteolin (100 μM) inhibited GST in mixed manner with Ki of 53 μM with respect to GSH and non-competitively with respect to CDNB with Ki of 38 μM. Luteolin, at a concentration range of 5–80 μM, exhibited 78–99% GST inhibition in human SK-MEL-28 cell homogenate. Among the 3 species of intact luteolin, luteolin-SG conjugate, and luteoline-quinone, only the latter two have potential as drugs with Ki < 1 μM, which is potentially achievable in-vivo as therapeutic agents. The order of GST inhibition was luteolin-quinone >> luteolin-SG conjugate >>> luteolin. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a luteolin-quinone and luteolin-glutathione conjugate, which inhibited GST. For the first time, in addition to intracellular GSH depletion, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase. Such strategy could potentially be used to selectively inhibit GST, a drug detoxifying enzyme, in melanoma cells.

Growth Inhibition and Cytotoxicity in Human Lung and Cervical Cancer Cell Lines and Glutathione S-Transferase Inhibitory Activity of Selected Sri Lankan Traditional Red Rice (Oryza Sativa L.) Brans

Extracts, fractions and gastrointestinal-resistant protein hydrolysates (GRPH) from rice bran (RB) of four Sri Lankan traditional varieties were studied for growth inhibition (GI) and cytotoxicity against human lung cancer (NCI-H460), cervical cancer (HeLa) cell lines and effect on glutathione S-transferase (GST) in vitro. RB extracts showed significantly high (P < 0.05) GI (GI50 μg/mL: HeLa: 19.24 ± 3.47 to 112.58 ± 0.05; NCI-H460: 54.63 ± 4.89 to 170.31 ± 4.71) and cytotoxicity (LC50 μg/mL: HeLa: 240.12 ± 9.23 to 476.22 ± 0.05; NCI-H460: 255.36 ± 1.81 to 412.07 ± 17.73) compared with GRPH. Highest GI (GI50: 30.63 ± 9.19 to 44.40 ± 5.34 μg/mL) and cytotoxicity (LC50: 141.31 ± 14.47 to 169.51 ± 2.04 μg/mL) was evident in nonpolar fractions (dichloromethane: Goda Heeneti and Sudu Heeneti; hexane: Dik Wee and Sudu Heeneti). No cytotoxicity was evident to normal cells. All RB extracts had high GST inhibition. Properties observed indicate its usefulness in managing lung and cervical cancers. Practical Applications Lung and cervical cancers are two of the most commonly diagnosed cancers in men and women, respectively, and leading causes of mortality. Although numerous potent chemotherapeutic drugs are available for management, most of these drugs are very expensive and are associated with numerous side effects. Further, anticancer drugs containing electrophilic centers can easily form a conjugate with glutathione, which is facilitated by glutathione S-transferase (GST) and excreted from the body resulting in lower efficacy. Therefore, GST inhibitors are used as adjuvants during cancer chemotherapy. The selected rice varieties in this study claimed to possess health-enhancing properties according to Sri Lankan ethnomedicine. The findings of this study clearly showed that bran extracts, fractions and gastrointestinal-resistant protein hydrolysates of selected rice varieties had growth inhibition and cytotoxicity against both lung and cervical cancers. Further, all of these selected varieties had high GST inhibition. Therefore, selected rice brans may be useful in the management of lung and cervical cancers.

Insecticides resistance in the Culex quinquefasciatus populations from northern Thailand and possible resistance mechanisms

The mosquito vector Culex quinquefasciatus is known to be resistant to insecticides worldwide, including Thailand. This study was the first investigation of the insecticide resistance mechanisms, involving metabolic detoxification and target site insensitivity in C. quinquefasciatus from Thailand. Adult females reared from field-caught larvae from six provinces of northern Thailand were determined for resistant status by exposing to 0.05% deltamethrin, 0.75% permethrin and 5% malathion papers using the standard WHO susceptibility test. The overall mortality rates were 45.8%, 11.4% and 80.2%, respectively. A fragment of voltage-gated sodium channel gene was amplified and sequenced to identify the knock down resistance (kdr) mutation. The ace-1 gene mutation was determined by using PCR-RFLP. The L1014F kdr mutation was observed in all populations, but the homozygous mutant F/F1014 genotype was found only in two of the six provinces where the kdr mutation was significantly correlated with deltamethrin resistance. However, none of mosquitoes had the G119S mutation in the ace-1 gene. A laboratory deltamethrin resistant strain, Cq_CM_R, has been established showing a highly resistant level after selection for a few generations. The mutant F1014 allele frequency was significantly increased after one generation of selection. A synergist assay was performed to assess the metabolic detoxifying enzymes. Addition of bis(4-nitrophenyl)-phosphate (BNPP) and diethyl maleate (DEM), inhibitors of esterases and glutathione S-transferases (GST), respectively, into the larval bioassay of the Cq_CM strain with deltamethrin showed no significant reduction. By contrast, addition of piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenases, showed a 9-fold reduction of resistance. Resistance to pyrethroids in C. quinquefasciatus is widely distributed in northern Thailand. This study reports for the first time for the detection of the L1014F kdr mutation in wild populations of C. quinquefasciatus in Thailand. At least two major mechanisms, kdr and cytochrome P450 monooxygenases, confer resistance to deltamethrin in Thai C. quinquefasciatus populations.

Influence of glutathione-S-transferase (GST) inhibition on lung epithelial cell injury: role of oxidative stress and metabolism.

Oxidant-mediated tissue injury is key to the pathogenesis of acute lung injury. Glutathione-S-transferases (GSTs) are important detoxifying enzymes that catalyze the conjugation of glutathione with toxic oxidant compounds and are associated with acute and chronic inflammatory lung diseases. We hypothesized that attenuation of cellular GST enzymes would augment intracellular oxidative and metabolic stress and induce lung cell injury. Treatment of murine lung epithelial cells with GST inhibitors, ethacrynic acid (EA), and caffeic acid compromised lung epithelial cell viability in a concentration-dependent manner. These inhibitors also potentiated cell injury induced by hydrogen peroxide (H2O2), tert-butyl-hydroperoxide, and hypoxia and reoxygenation (HR). SiRNA-mediated attenuation of GST-π but not GST-μ expression reduced cell viability and significantly enhanced stress (H2O2/HR)-induced injury. GST inhibitors also induced intracellular oxidative stress (measured by dihydrorhodamine 123 and dichlorofluorescein fluorescence), caused alterations in overall intracellular redox status (as evidenced by NAD(+)/NADH ratios), and increased protein carbonyl formation. Furthermore, the antioxidant N-acetylcysteine completely prevented EA-induced oxidative stress and cytotoxicity. Whereas EA had no effect on mitochondrial energetics, it significantly altered cellular metabolic profile. To explore the physiological impact of these cellular events, we used an ex vivo mouse-isolated perfused lung model. Supplementation of perfusate with EA markedly affected lung mechanics and significantly increased lung permeability. The results of our combined genetic, pharmacological, and metabolic studies on multiple platforms suggest the importance of GST enzymes, specifically GST-π, in the cellular and whole lung response to acute oxidative and metabolic stress. These may have important clinical implications.

Putative Role of Glutathione S‐transferases as Mediators of Cell Growth, Development, and Differentiation in the Eukaryotic Model Organism, Dictyostelium discoideum

In addition to their role in detoxification, Glutathione S‐transferase (GST) enzymes influence signal transduction, development, and differentiation in many eukaryotic cells. Transcriptome analysis of the cellular slime mold, Dictyostelium discoideum (strain AX4) expresses five GST (α1‐α5 class) transcripts whose function(s) remain unknown. This study characterized the activity and expression of GST isozymes in D. discoideum at various stages of growth and development. A GSH‐agarose pull down assay of the lysates from vegetative amoebae identified two protein species of ~24kDa; mass spectrophotometric analysis identified GSTα3&gt; GSTα1&gt;GSTα2 as predominant species. Measurements of GST activity in amoeba using the substrate 1‐chloro‐2‐4‐dinitrobenzene (CDNB) revealed functional enzyme activity, which was significantly reduced by as much as 50% in the presence of 500 nM ethacrynic acid (EA, a GSTs inhibitor). Ethacrynic acid also delayed the formation of aggregates and multicellular development. Levels of GSTs activity differed significantly between vegetative amoebae and multicellular stages of development (aggregate, slug, culmination, fruiting body), indicating the importance of GSTs to influence cell signaling and gene expression during morphogenesis and differentiation. Current studies aim to determine: a) the levels of each isozyme and their substrate specificities; b) the impact of GST null mutants on growth and development of the organism. These studies will contribute to elucidating the expanding the role and function of GST enzymes in eukaryotic signal transduction that regulates cell growth, chemotaxis, and differentiation.

Acute toxicity and sublethal effects of gallic and pelargonic acids on the zebrafish Danio rerio.

Gallic and pelargonic acids are naturally found in a variety of plants and food products. Despite their extensive use in man-made applications, little is known regarding their potential risks to aquatic vertebrates. The aim of this work was to assess the acute toxicity of these polyphenolic and fatty acid compounds to the zebrafish. In order to get insights into sublethal effects, the enzyme activity of usual biomarkers related to oxidative stress and biotransformation were also assessed in fish. These latter included total superoxide dismutase, catalase as well as total glutathione peroxidase for antioxidant defence mechanisms and glutathione S-transferase for biotransformation related enzyme. Gallic acid was practically non-toxic (96-h lethal concentration (LC50) > 100mg/L) whereas pelargonic acid was slightly toxic (96-h LC50 of 81.2mg/L). Moreover, biomarker analyses indicated enhanced superoxide dismutase activity in fish exposed to 20, 40 and 100mg/L of gallic acid compared to control. A dose-dependent induction of glutathione peroxidase and glutathione S-transferase was reported following gallic acid exposure at the tested concentrations of 10, 20 and 40mg/L, with the exception of 100mg/L of substance where basal activity levels were reported. In the case of pelargonic acid, there was no change in antioxidant enzyme activity while an inhibition of glutathione S-transferase was observed from organisms exposed to 45, 58 and 76mg/L of test solution. The results concerning sublethal effects on biological parameters of zebrafish highlighted thereby the need for further investigations following chronic exposure to both organic acids.

Evaluation of Active Hexose Correlated Compound (Ahcc) on Phase Ii Drug Metabolism Pathways and the Implications for Supplement-Drug Interactions

Background: The evaluation of active hexose correlated compound (AHCC) on hepatic metabolism mediateddrug interaction is critical in current clinical setting as there is little published information on the potential effect on drug efficacy and safety. The primary objective of this study was to evaluate the potential phase II hepatic metabolism pathways associated with the metabolism of AHCC and to determine potential drug/AHCC interactions. Methods: Four primary hepatic metabolism phase II pathways were evaluated: glutathione S-transferase (GST), quinone oxidoreductase (QOR), catechol-O-methyltransferases (COMT) and uridine diphosphate (UDP)- glucuronosyltransferase (UGT). Pooled human liver microsomes and human liver S9 fractions were utilized to evaluate QOR and UGT metabolism inhibition assays. The pool human liver S9 fractions were used to assess GST activity. Cryopreserved inducible human liver hepatocytes were used to evaluate potential induction of UGT and COMT metabolism. All experiments were carried out in triplicate. Results: Data demonstrated that AHCC is not an inhibitor of GST or UGT pathways, but may be a potential inhibitor of QOR pathway. Evaluation of induction of the phase II pathways demonstrated that AHCC showed potential induction of the UGT 1A3 and 1A6 pathways. There was no induction of the COMT pathway. Conclusion: Historically, drug interaction studies have only focused on Phase I metabolism pathways, so currently there is very limited information regarding the phase II metabolism of most commonly used medications. In conclusion, additional studies are warranted to determine potential of any phase II hepatic interactions with AHCC when administered with other medications or supplement that are substrates of these pathways.