Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC₅₀ values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O⁶-benzylguanine (IC₅₀s around 30 μM), GST P1-1 by sulphinpyrazone (IC₅₀ = 66 μM), GST A1-1 by sulphasalazine, and camptothecin (34 and 74 μM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 μM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC₅₀ = 1.4 μM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.     

Activation of rat glutathione transferases in class mu by active oxygen species

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.     

Comparison of glutathione S-transferase and general esterase in two bugs, Eurygaster integriceps Puton (Heteroptera: Scutelleridae) and Brachynema germari Kolenati (Heteroptera: Pentatomidae)

Glutathione S -transferases (GSTs) and general esterases play important roles in the detoxification of many substances including allelochemicals from plants and chemical pesticides. In this study, to determine the feeding status and effects of agronomical practices on GST and general esterase, two bug species, Eurygaster integriceps and Brachynema germari , were selected with differences in these cases. GST and general esterase responses varied when switching from 1-chloro-2,4-dinitrobenzene (CDNB) and α -naphtyl to 1,2-dichloro-4-nitro-benzene (DCNB) and β -naphtyl in E. integriceps and B. germari , respectively. The activities of GST and esterase by using CDNB and α -naphtyl were higher than that of DCNB and β -naphtyl for both insects. It was shown that the optimal pH for GST and general esterase activity varied between pH 5.5 and 6. While the Michaelis constant (K_m) value related to E. integriceps toward CDNB and α -naphtyl was lower than K_m for B. germari , K_m values calculated for E. integriceps toward DCNB and β -naphtyl corresponding to those related to A. hejeri were similar to CDNB and α -naphtyl values. The maximal reaction velocity (V_max) values related to E. integriceps for both substrates (CDNB and α -naphtyl) were always higher than those from B. germari and showed significant differences. At the basement of the native-PAGE electrophoresis, five bands were observed in for GST and three were visualized for B. germari , with a large, darker band for E. integriceps in the case of esterases. Studies on the detoxification enzymes of herbivores should be undertaken to determine accurately the effect of the host plants on the organisms eating them, particularly in terms of biochemical and ecological advantages.     

Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC50 values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O6-benzylguanine (IC50s around 30 microM), GST P1-1 by sulphinpyrazone (IC50 = 66 microM), GST Al-1 by sulphasalazine, and camptothecin (34 and 74 microM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 microM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC50 = 1.4 microM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.     

Combined effects of cadmium and nickel on testicular xenobiotic metabolizing enzymes in rats

When male rats were given a single dose of cadmium (Cd) (3.58 mg CdCl₂·H₂O/kg, ip) 72 hr prior to sacrifice, the testicular 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP), and cumene hydroperoxide (CHPx) decreased significantly as compared to controls. Cd also inhibited reduced glutathione (GSH) level while increasing the lipid peroxidation (LP) level significantly. When the animals were given a single dose of nickel (Ni) (59.5 mg NiCl₂·6H₂O/kg, ip) 16 hr prior to sacrifice, significant decreases were observed in EROD and GST activities toward CDNB, EAA, EPNP, and CHPx, and GSH level. No significant alterations were noted in DCNB GST activity and LP level by Ni. For the combined treatment, rats received the single dose of Ni 56 hr after the single dose of Cd and were killed 16 hr later. In these animals, lesser depressions were observed on EROD activity and LP level than those of Cd alone. The combination of metals significantly inhibited GST activities and GSH level but not to a greater degree than noted by Cd or Ni alone. Plasma testosterone levels of Cd-, Ni-, and combination-treated rats decreased significantly compared to controls. The strongest depression was achieved by Cd alone. Cd, both alone and in combination with Ni, increased the tissue Ni uptake significantly. Ni, however, did not produce such an effect on the tissue uptake of Cd in either case. Cd treatment caused interstitial edema and coagulation necrosis in seminiferous tubules and also caused fibrinoidal necrosis in vascular endothelium. Ni treatment did not produce any pathological testicular alterations compared to controls. Combined treatment produced fewer pathological alterations (i.e., only interstitial edema) than that of Cd treatment. These results reveal that the combination of Cd and Ni does not have a synergistic effect on testicular xenobiotic metabolizing enzymes, and in contrast, Ni has an ameliorating effect on pathological disturbances caused by Cd alone in the rat testis.     

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.     

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 25.8 kDa, 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.     

Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes

GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb₁ and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb₁ after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb₁ was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.Abbreviations ABTS 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) - CDNB I-Chloro-2,4-dinitrobenzene - DCNB 1,2-dichloro-4-nitrobenzene; DEX, dexamethasone - DMSO dimethylsulfoxide - GST glutathione Stransferase - MC methylcholanthrene - N, NIC nicotinamide - -NF -naphthoflavone - PB phenobarbital - PBS phosphate buffered saline     

A study of gender,strain and age differences in mouse liver glutathione-S-transferase

The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-1, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Effects of phenol compounds, glutathione analogues and a diuretic drug on glutathione S-transferase, glutathione reductase and glutathione peroxidase from canine erythrocytes.

1. Phenol compounds (ellagic acid, quercetin and purpurogallin), glutathione analogues (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogallin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.     

Plasmodium vivax: molecular cloning, expression and characterization of glutathione S-transferase

Malaria parasite glutathione S-transferases (GSTs) are postulated to be essential for parasite survival by protecting the parasite against oxidative stress and buffering the detoxification of heme-binding compounds; therefore, GSTs are considered potential targets for drug development. In this study, we identified a Plasmodium vivax gene encoding GST (PvGST) and characterized the biochemical properties of the recombinant enzyme. The PvGST contained 618 bp that encoded 205 amino acids and shared a significant degree of sequence identity with GSTs from other Plasmodium species. The recombinant homodimeric enzyme had an approximate molecular mass of 50kDa and exhibited GSH-conjugating and GSH-peroxidase activities towards various model substrates. The optimal pH for recombinant PvGST (rPvGST) activity was pH 8.0, and the enzyme was moderately unstable at 37 degrees C. The K(m) values of rPvGST with respect to GSH and CDNB were 0.17+/-0.09 and 2.1+/-0.4mM, respectively. The significant sequence homology and similar biochemical properties of PvGST and Plasmodium falciparum GST (PfGST) indicate that they may have similar molecular structures. This information may be useful for the design of specific inhibitors for plasmodial GSTs as potential antimalarial drugs.     

Glutathione S-transferase isoenzymes in the two-spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae)

Isoenzymes of glutathione S-transferase (GST) in adult Adalia bipunctata, an aphidophagous predator, were studied. Cytosolic GST activity was studied in each beetle developmental stage. The highest activities towards both 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dinitro-1-iodobenzene (DNIB) occurred in adults. The enzyme distribution was investigated in adults. While most of the enzymatic activity was found in the abdomen (40-50 and 34-63% respectively) using several concentrations of both CDNB and DNIB, significant differences were observed for the head and the thorax depending on the substrate. Activities were more abundant in the thorax with DNIB (37-47%) compared to the 13-19% obtained with CDNB. Some GST activity was also detected in the elytra. GSTs were purified by epoxy-activated Sepharose 6B affinity chromatography and applied to an HPLC column to determine the native molecular weight (69 kDa). Three isoenzymes were separated by chromatofocusing at pH ranges 7-4. Three bands with molecular mass from 23 to 26 kDa were visualised on SDS-PAGE. Their isoelectric points were 6.66, 6.36, and 6.21. The substrate specificities and the kinetic parameters (Vm and Km) of the isoenzymes showed large differences depending on the isoenzyme. Arch.     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

一个新的产黄青霉谷胱甘肽转移酶基因的克隆和鉴定

以产黄青霉(Penicillium chrysogenum Thom)cDNA为模板,克隆得到一个新的谷胱甘肽转移酶基因PcgstB,其开放阅读框长651bp,编码216个氨基酸的蛋白质。与已知序列进行BLASTp比较显示,该蛋白具有保守的GST结构域,与烟曲霉GstB的序列一致性最高,达65%。将PcgstB与原核表达载体pTrc99A连接得到表达质粒pTrc-gstB,转化大肠杆菌DH5α,经IPTG诱导后获得以可溶形式表达的重组PcGstB蛋白。以1-chloro-2,4-dinitrobenzene(CDNB)为底物检测,确认该蛋白具有GST活性。     

Cooperative kinetics of the recombinant glutathione transferase of Taenia solium and characterization of the enzyme

Glutathione transferases (GSTs) are essential enzymes in many organisms due their diverse functions and, in helminths they are the main detoxification system. For Taenia solium, two cytosolic GSTs with molecular masses of 25.5 and 26.5 kDa (Ts26GST) have been found. Ts26GST was cloned to be studied in its recombinant form (recTs26GST). Although the primary structure is related to the mu class, the kinetic parameters for CDNB (V_max = 51.5 μmol min⁻¹ mg⁻¹; K_m = 1.06 mM; k_cat = 22.2 s⁻¹) are related with some alpha GSTs. The substrate and inhibitor class markers reaffirmed these bimodal characteristics. Inhibition studies with anthelminthics indicate that recTs26GST is sensitive to mebendazole, displaying a non competitive inhibition pattern suggesting that at least two molecules are binding to recTs26GST. On the other hand, the kinetic curves for CDNB and GSH showed a positive cooperativity that was corroborated using fluorometric assays. Those assays indicate that CDNB binding is highly influenced by GSH, probably by modulation of the Ts26GST conformational ensamble.