Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas.

Heterologous expression and functional characterization of avian mu-class glutathione S-transferases.

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.

Effects of depletion of glutathione on abscisic acid- and methyl jasmonate-induced stomatal closure in Arabidopsis thaliana.

Glutathione (GSH) is involved in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we examined the effects of GSH-decreasing chemicals, p-nitrobenzyl chloride (PNBC), iodomethane (IDM), and ethacrynic acid (EA), on ABA- and MeJA-induced stomatal closure in Arabidopsis. Treatments with PNBC, IDM, and EA decreased GSH contents in guard cells. Depletion of GSH by PNBC and IDM enhanced ABA- and MeJA-induced stomatal closure and inhibition of light-induced stomatal opening by ABA, whereas EA did not enhance either ABA- and MeJA-induced stomatal closure or inhibition of light-induced stomatal opening by ABA. Depletion of GSH did not significantly increase the production of the reactive oxygen species (ROS), cytosolic alkalization, or cytosolic Ca(2+) oscillation induced by ABA and MeJA. These results indicate that depletion of GSH enhances ABA- and MeJA-induced stomatal closure without affecting ROS production, cytosolic alkalization, or cytosolic Ca(2+) oscillation in guard cells of Arabidopsis.

Characterization of new potential anticancer drugs designed to overcome glutathione transferase mediated resistance.

Resistance against anticancer drugs remains a serious obstacle in cancer treatment. Here we used novel strategies to target microsomal glutathione transferase 1 (MGST1) and glutathione transferase pi (GSTP) that are often overexpressed in tumors and confer resistance against a number of cytostatic drugs, including cisplatin and doxorubicin (DOX). By synthetically combining cisplatin with a GST inhibitor, ethacrynic acid, to form ethacraplatin, it was previously shown that cytosolic GST inhibition was improved and that cells became more sensitive to cisplatin. Here we show that ethacraplatin is easily taken up by the cells and can reverse cisplatin resistance in MGST1 overexpressing MCF7 cells. A second and novel strategy to overcome GST mediated resistance involves using GST releasable cytostatic drugs. Here we synthesized two derivatives of DOX, 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) and 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) and showed that they are substrates for MGST1 and GSTP (releasing DOX). MGST1 overexpressing cells are resistant to DOX. The resistance is partially reversed by DNS-DOX. Interestingly, the less reactive MNS-DOX was more cytotoxic to cells overexpressing MGST1 than control cells. It would appear that, by controlling the reactivity of the prodrug, and thereby the DOX release rate, selective toxicity to MGST1 overexpressing cells can be achieved. In the case of V79 cells, DOX resistance proportional to GSTP expression levels was noted. In this case, not only was drug resistance eliminated by DNS-DOX but a striking GSTP-dependent increase in toxicity was observed in the clonogenic assay. In summary, MGST1 and GSTP resistance to cytostatic drugs can be overcome and cytotoxicity can be enhanced in GST overexpressing cells.

Studies of glutathione transferase P1-1 bound to a platinum(IV)-based anticancer compound reveal the molecular basis of its activation.

Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.

Identification of glutathione-methacrylates adducts in gingival fibroblasts and erythrocytes by HPLC-MS and capillary electrophoresis.

OBJECTIVES: Methacrylic monomers are released, from dental composite resins, either into the oral cavity or in pulpal tissues, where they can cause local or systemic adverse effects. The mechanisms of these effects are not well understood, probably because such molecules can act at different levels also inducing a depletion of intracellular glutathione (GSH). GSH can detoxify methacrylates by conjugating their α,ß-unsaturated carbon-carbon moiety to the thiol group, with the catalysis of glutathione S-transferases (GST). This reaction determines a GSH cellular depletion and belongs to the metabolism of α,ß-unsaturated esters, protecting the body against the toxic effects of electrophiles. On the basis of the above considerations, this work aim is to set up a method for the detection of the adducts formed by methacrylic monomers with GSH in cells using HPLC coupled to mass spectrometry (HPLC-MS) and micellar electrokinetic capillary chromatography (MECK) techniques. METHODS AND RESULTS: Adducts of glutathione with triethylene glycol dimethacrylate (TEGDMA) and hydroxyethyl methacrylate (HEMA) were incontrovertibly identified by HPLC-MS and MECK in human gingival fibroblasts and erythrocytes, both outside and inside cells. Molecular docking simulations of HEMA and TEGDMA in the experimental structure of glutathione S-transferase, are also reported to rationalize the effectiveness of such enzyme in the catalysis of the above described reaction. SIGNIFICANCE: The setup of a method for the identification of GSH-methacrylate adducts allows to determine when the metabolic pathway involving such compounds is employed by cells for the detoxification of monomers leached from composite resins.

Distribution and mechanism of α-amanitin tolerance in mycophagous Drosophila (Diptera: Drosophilidae).

Many mycophagous species of Drosophila can tolerate the mushroom poison α-amanitin in wild mushrooms and in artificial diet. We conducted feeding assays with sixteen Drosophila species and α-amanitin in artificial diet to better determine the phylogenetic distribution of this tolerance. For eight tolerant and one related susceptible species, we sequenced the gene encoding the large subunit of RNA Polymerase II, which is the target site of α-amanitin. We found no differences in the gene that could account for differences in susceptibility to the toxin. We also conducted feeding assays in which α-amanitin was combined with chemical inhibitors of cytochrome P450s or glutathione S-transferases (GSTs) in artificial diet to determine if either of these enzyme families is involved in tolerance to α-amanitin. We found that an inhibitor of GSTs did not reduce tolerance to α-amanitin, but that an inhibitor of cytochrome P450s reduced tolerance in several species. It is possible that the same cytochrome P450 activity that produces tolerance of α-amanitin might produce tolerance of other mushroom toxins as well. If so, a general detoxification mechanism based on cytochrome P450s might answer the question of how tolerance to α-amanitin arose in mycophagous Drosophila when this toxin is found in relatively few mushrooms.

Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V.

The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S-transferase (GST P1-1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr-108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys-47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X-ray diffraction. Surprisingly, in the absence of Cys-47, Cys-101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys-47. The Cys-47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G-site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H-site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H-site and in the water network at the dimer interface.

Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus.

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 micromol min(-1)mg(-1)), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.

Application of diffusion-edited NMR spectroscopy for the structural characterization of drug metabolites in mixtures.

Diffusion-edited NMR spectroscopy is used to enable the structural characterization of low level metabolites in the presence of endogenous compounds, and organic solvents. We compared data from standard one-dimensional (1D) (1)H, 1D NOESY-presaturation, and 1D diffusion-edited experiments run on 20 microg and 100 microg samples of ethacrynic acid glutathione thioether (EASG) and a previously unreported metabolite of mefenamic acid, mefenamic acid glutathione thioester (MSG). The 1D NOESY-presaturation technique gave spectra with the best signal-to-noise (S/N) ratio, approximately three times that observed with the standard (1)H experiment, with respect to the metabolite signals. However, it was not selective for solvent signals as overlapping metabolite signals were also suppressed by this technique. In some cases, these signals were key to determining the site of glutathione attachment on the parent molecule. 1D NOESY-presaturation spectra also produced baseline distortions and inconsistent integration values. By comparison, 1D diffusion-edited experiments were found to selectively and simultaneously remove multiple solvent signals, resolve overlapping metabolite signals, and provide more uniform integration for metabolite signals overlapping with or proximal to solvent peaks, without producing baseline distortions. However, the diffusion-edited experiments caused significant signal attenuation of the metabolite signals when compared with a standard (1)H spectrum. Partially purified metabolites isolated from biological matrices were also characterized by using two-dimensional diffusion-ordered spectroscopy (DOSY). DOSY spectra acquired on a sample of EASG purified from rat bile proved useful in 'separating' the signals of EASG, from those of a co-eluting bile acid and parent drug ethacrynic acid (EA) in the diffusion-dimension in regions where there was no spectral overlap. In the low-field regions of high overlap, the DOSY experiment did not effectively separate the signals from the individual components. Diffusion based experiments provide a way to determine the total number of components that are present in a metabolite sample as well as an ability to identify them based on the chemical shift information, without the need for laborious chromatography on small samples.

The characterization of cytosolic glutathione transferase from four species of sea turtles: loggerhead (Caretta caretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), and hawksbill (Eretmochelys imbricata).

Glutathione s-transferases (GST) play a critical role in the detoxification of exogenous and endogenous electrophiles, as well as the products of oxidative stress. As compared to mammals, GST activity has not been extensively characterized in reptiles. Throughout the globe, most sea turtle populations face the risk of extinction. Of the natural and anthropogenic threats to sea turtles, the effects of environmental chemicals and related biochemical mechanisms, such as GST catalyzed detoxification, are probably the least understood. In the present study, GST activity was characterized in four species of sea turtles with varied life histories and feeding strategies: loggerhead (Caretta caretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), and hawksbill (Eretmochelys imbricata). Although similar GST kinetics was observed between species, rates of catalytic activities using class-specific substrates show inter- and intra-species variation. GST from the spongivorous hawksbill sea turtle shows 3-4.5 fold higher activity with the substrate 4-nitrobenzylchloride than the other 3 species. GST from the herbivorous green sea turtle shows 3 fold higher activity with the substrate ethacrynic acid than the carnivorous olive ridley sea turtle. The results of this study may provide insight into differences in biotransformation potential in the four species of sea turtles and the possible health impacts of contaminant biotransformation by sea turtles.

Hepatic and branchial glutathione S-transferases of two fish species: substrate specificity and biotransformation of microcystin-LR.

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min(-1) mL(-1) hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.

Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica.

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione. The pi-class GST cDNA (leGSTp) was cloned from the cold-adapted Antarctic bivalve Laternula elliptica. We used degenerated primers designed based on highly conserved regions of known mollusk GSTs to amplify the corresponding L. elliptica mRNA. Full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full sequence of the GST cDNA was 1189 bp in length, with a 5' untranslated region (UTR) of 74 bp, a 3' UTR of 485 bp, and an open reading frame of 630 bp encoding 209 amino acid residues with an estimated molecular mass of 23.9 kDa and an estimated isoelectric point of 8.3. Quantitative RT-PCR confirmed basal expression of leGSTp, which was up-regulated upon heat treatment (10 degrees C for different time periods) by a factor of 2.3 (at 24 h) and 2.7 (at 48 h) in the digestive gland and gill tissues, respectively. The recombinant leGSTp expressed in Escherichia coli was purified by affinity chromatography and characterized. The purified leGSTp exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). The recombinant leGSTp had a maximum activity at approximately pH 8.0, and its optimum temperature was 35 degrees C.

Transscleral diffusion of ethacrynic acid and sodium fluorescein.

PURPOSE: One of the current limitations in developing novel glaucoma drugs that target the trabecular meshwork (TM) is the induced corneal toxicity from eyedrop formulations. To avoid the corneal toxicity, an alternative approach would be to deliver TM drugs through the sclera. To this end, we quantified ex vivo diffusion coefficient of a potential TM drug, ethacrynic acid (ECA), and investigated mechanisms of ECA transport in the sclera. METHODS: An Ussing-type diffusion apparatus was built to measure the apparent diffusion coefficient of ECA in fresh porcine sclera at 4 degrees C. To understand mechanisms of ECA transport, we quantified the transscleral transport of a fluorescent tracer, sodium fluorescein (NaF), that has a similar molecular weight but is more hydrophilic compared to ECA. Furthermore, we developed a mathematical model to simulate the transport processes and used it to analyze the experimental data. The model was also used to investigate the dependence of diffusion coefficients on volume fraction of viable cells and the binding of NaF and ECA to scleral tissues. RESULTS: The diffusion coefficients of ECA and NaF in the sclera were 48.5+/-15.1 x 10-7 cm(2)/s (n=9) and 5.23+/-1.93 x 10(-7) cm(2)/s (n=8), respectively. Both diffusion coefficients were insensitive to cell shrinkage caused by ECA during the diffusion experiments and cell damage caused by the storage of tissues ex vivo before the experiments. Binding of ECA to scleral tissues could not be detected. The apparent maximum binding capacity and the apparent equilibrium dissociation constant for NaF were 80+/-5 mM and 2.5+/-0.5 mM (n=3), respectively. CONCLUSIONS: These data demonstrated that ECA diffusion was minimally hindered by structures in the sclera, presumably due to the lack of cells and binding sites for ECA in the sclera.

Effects of metals and detergents on biotransformation and detoxification enzymes of leaping mullet (Liza saliens).

In this study, feral leaping mullet (Liza saliens) liver microsomal 7-ethoxyresorufin O-deethylase (EROD), and cytosolic glutathione S-transferases (GSTs) activities were investigated using 7-ethoxyresorufin, 1-chloro-2,4-dinitrobenzene (CDNB), and ethacrynic acid (EA) as substrates, respectively. The average EROD activity was found as 1139+/-175 pmol resorufin/min/mg protein. The average GST activities towards CDNB and EA were found as 1364+/-41 and 140+/-19 nmol/min/mg protein, respectively. We have, then, investigated the in vitro effects of some metals and detergents on CYP1A and GST activities in leaping mullet liver. Leaping mullet liver microsomal EROD activity was significantly inhibited by Hg (0.1 mM), Ni (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Sb (0.1 mM), Fe2+ (1 mM), Co (1 mM), Al (1 mM), and Fe3+ (1 mM), with the percent inhibition of 80, 80, 77, 75, 70, 69, 56, 53, 46, and 44, respectively. Similarly, conjugation of CDNB catalyzed by GST was inhibited significantly to lesser extend by Hg (0.1mM), Sb (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Fe3+ (1 mM), Co (1 mM), and Fe2+ (1 mM), with the percent inhibition of 70, 69, 65, 61, 54, 51, 47, and 43, respectively. The degrees of inhibition observed on GST catalyzed EA conjugation by Hg (0.1 mM), Cd (0.1 mM), Sb (0.1 mM), Cu (0.1 mM), and Zn (0.1 mM) were 86, 78, 69, 51, and 42, respectively. In addition to metals, the effect of various detergents on leaping mullet liver EROD, GST-CDNB, and GST-EA activities were studied. It was found that ionic detergents strongly inhibited the EROD activity, whereas much less inhibitions were observed with GST catalyzed activities. Therefore, the CYP1A inhibition potencies of metals and detergents suggest that their contribution to the overall CYP1A induction in polycyclic aromatic hydrocarbons contaminated environmental samples has to be taken into account for better interpretation of environmental studies.

Evolution of highly active enzymes by homology-independent recombination.

The theta-class GST enzymes hGSTT1-1 (human GSTTheta-1-1) and rGSTT2-2 (rat GSTTheta-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C/Os) at positions not accessible by DNA family shuffling. High-throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C/Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k(cat) with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low-homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme "humanization."

Characterization of cytosolic glutathione S-transferases in juvenile Chinook salmon (Oncorhynchus tshawytscha).

Four cytosolic glutathione S-transferase (GST) classes were isolated and characterized from juvenile winter run Chinook salmon (Oncorhynchus tshawytscha) liver. Two techniques were used: (1) gel electrophoresis/immunoblotting against a polyclonal striped bass GST antibody and (2) high-pressure liquid chromatography (HPLC). Nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to elucidate peptide sequences and the proteins were identified as pi, theta, mu and alpha, by searching against the NCBI non-redundant database (nrDB). Catalytic activity of the cytosolic GSTs towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA) were determined to be 0.3+/-0.05 U/mg cytosolic protein and 0.06+/-0.02 U/mg cytosolic protein, respectively.

Ethacrynic acid inhibits multiple steps in the NF-kappaB signaling pathway.

Ethacrynic acid has been used as a safe and effective diuretic for more than 30 years. In this study, we tested the hypothesis that ethacrynic acid is also an anti-inflammatory agent that inhibits signaling by the proinflammatory transcription factor NF-kappaB. We showed that ethacrynic acid inhibited luciferase expression in lipopolysaccharide-stimulated macrophage-like RAW 264.7 cells transfected with an NF-kappaB-dependent luciferase reporter vector and also inhibited NF-kappaB DNA binding in lipopolysaccharide-stimulated RAW 264.7 cells (electrophoretic mobility shift assay). Ethacrynic acid inhibited degradation of IkappaBalpha and IkappaBbeta in lipopolysaccharide-stimulated RAW 264.7 cells. Ethacrynic acid impaired DNA binding of wild-type p65 subunits of NF-kappaB in cells. However, DNA binding of a Cys--> Ser p65 mutant was not inhibited by ethacrynic acid, suggesting that ethacrynic acid inhibits DNA binding by alkylating p65 at Cys. In a cell-free system, binding of p50 homodimers to an NF-kappaB consensus sequence was inhibited by ethacrynic acid at concentrations from 10 to 100 microM, indicating that ethacrynic acid probably also covalently modifies the p50 subunit. These data indicate that ethacrynic acid inhibits activation of the NF-kappaB pathway at multiple points and suggest that this well-studied drug warrants further investigation as a potential therapeutic for various conditions that are associated with excessive inflammation.

Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis.

The gene coding for glutathione S-transferase (GST) has been isolated from the Mytilus edulis hepatopancreas. Open reading frame analysis indicated that the M. edulis GST (meGST) gene encodes a protein of 206 amino acid residues with a calculated molecular mass of 23.68 kDa. The deduced amino acid sequence showed high sequence similarity with the sequence of the pi class GST. The meGST was expressed in Escherichia coli, and the recombinant meGST was purified by affinity chromatography and characterized. The recombinant meGST exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis with respect to CDNB as substrate gave a K(m) of 0.68 mM and a V(max) of 0.10 mmol/min per mg protein. The recombinant meGST had a maximum activity at approximately pH 8.5, and its optimum temperature was 39 degrees C. The predicted three-dimensional structure of the meGST revealed the N-terminal domain possesses a thioredoxin fold and the six helices of the C-terminal domain make a alpha-helical bundle. These features indicate that the meGST belongs to pi class GST.

Interplay between MRP inhibition and metabolism of MRP inhibitors: the case of curcumin.

The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between curcumin-dependent MRP inhibition and its glutathione-dependent metabolism were investigated using two transport model systems. In isolated membrane vesicles of MRP1- and MRP2-expressing Sf9 cells, curcumin clearly inhibited both MRP1- and MRP2-mediated transport with IC(50) values of 15 and 5 microM, respectively. In intact monolayers of MRP1 overexpressing Madin-Darby canine kidney (MDCKII-MRP1) cells, curcumin also inhibited MRP1-mediated activity, although with a 3-fold higher IC(50) value than the one observed in the vesicle model. Interestingly, MRP2-mediated activity was hardly inhibited in intact monolayers of MRP2-overexpressing MDCKII (MDCKII-MRP2) cells upon exposure to curcumin, whereas the IC(50) value in the vesicle incubations was 5 microM. The difference in extent of inhibition of the MRPs by curcumin in isolated vesicles as compared to intact cells, observed especially for MRP2, was shown to be due to a swift metabolism of curcumin to two glutathione conjugates in the MDCKII cells. Formation of both glutathione conjugates was about six times higher in the MDCKII-MRP2 cells as compared with the MDCKII-MRP1 cells, a phenomenon that could be ascribed to the significantly lower glutathione levels in the cell line. The efflux of both conjugates, identified in the present study as monoglutathionyl curcumin conjugates, was demonstrated to be mediated by both MRP1 and MRP2. From dose-response curves with Sf9 membrane vesicles, glutathionylcurcumin conjugates appeared to be less potent inhibitors of MRP1 and MRP2 than their parent compound curcumin. In conclusion, curcumin clearly inhibits both MRP1- and MRP2-mediated transport, but the glutathione-dependent metabolism of curcumin plays a crucial role in the ultimate level of inhibition of MRP-mediated transport that can be achieved in a cellular system. This complex interplay between MRP inhibition and metabolism of MRP inhibitors, the latter affecting the ultimate potential of a compound for cellular MRP inhibition, may exist not only for a compound like curcumin but also for many other MRP inhibitors presently or previously developed on the basis of vesicle studies.