Mechanistic insights into EgGST1, a Mu class glutathione S-transferase from the cestode parasite Echinococcus granulosus.

Glutathione transferases (GSTs) comprise a major detoxification system in helminth parasites, displaying both catalytic and non-catalytic activities. The kinetic mechanism of these enzymes is complex and depends on the isoenzyme which is being analyzed. Here, we characterized the kinetic mechanism of rEgGST1, a recombinant form of a cytosolic GST from Echinococcus granulosus (EgGST1), which is related to the Mu-class of mammalian enzymes, using the canonical substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Initial rate and product inhibition studies were consistent with a steady-state random sequential mechanism, where both substrates are bound to the enzyme before the products are released. Kinetic constants were also determined (pH 6.5 and 30 °C). Moreover, rEgGST1 lowered the pKa of GSH from 8.71 ± 0.07 to 6.77 ± 0.08, and enzyme-bound GSH reacted with CDNB 1 × 105 times faster than free GSH at pH 7.4. Finally, the dissociation of the enzyme-GSH complex was studied by means of intrinsic fluorescence, as well as that of the complex with the anthelminth drug mebendazole. This is the first report on mechanistic issues related to a helminth parasitic GST.

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.

Induction of intestinal multidrug resistance-associated protein 2 (Mrp2) by spironolactone in rats.

The effect of spironolactone (SL) pretreatment (200micromol/kg b.w./day, 3 consecutive days) on intestinal multidrug resistance-associated protein 2 (Mrp2) was evaluated in rats. A significant increase in protein levels in upper regions of small intestine, where Mrp2 is mainly present, was detected by western blotting. Real time PCR studies suggest a transcriptional regulation. The administration of ketoconazole, a pregnane X receptor (PXR) antagonist, was able to prevent the increase in Mrp2 mRNA levels induced by SL. The serosal to mucosal transport of dinitrophenyl S-glutathione, a model substrate of Mrp2 was evaluated in jejunal sac model. The data indicate that SL increased Mrp2 activity, well correlating with its up-regulation. We conclude that SL is able to induce intestinal Mrp2 transcriptionally, PXR being a potential mediator. We propose that SL could be of potential therapeutic application particularly in situations of down-regulation of intestinal Mrp2.

Characterization of Taenia solium cysticerci microsomal glutathione S-transferase activity.

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 micromol min(-1) mg(-1) with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an appKm (GSH)=0.161 microM, appKm (CDNB)=14.5 microM, and appVmax of 0.15 and 27.9 micromol min(-1) mg(-1) for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding Ki's for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.

Involvement of Mrp2 in hepatic and intestinal disposition of dinitrophenyl-S-glutathione in partially hepatectomized rats.

The ability of the liver and small intestine for secretion of dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), into bile and lumen, respectively, as well as expression of Mrp2 in both tissues, were assessed in 70-75% hepatectomized rats. An in vivo perfused intestinal model was used. A single i.v. dose of 30 micromol/kg b.w. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered and its glutathione conjugate, DNP-SG, was determined by HPLC in bile and intestinal perfusate. One and seven days after hepatectomy, biliary excretion of DNP-SG was decreased by 90 and 50% with respect to shams, respectively, when expressed per mass unit. In contrast, intestinal excretion was increased by 63% or unchanged one and seven days post-hepatectomy, respectively. Tissue content of DNP-SG 5 min after CDNB administration was substantially decreased in liver and significantly increased in intestine, one day post-hepatectomy. Western and immunofluorescence studies revealed preserved levels and localization of Mrp2 in both tissues from hepatectomized animals, irrespective of the time analyzed. In spite of preserved expression of Mrp2, the higher availability of DNP-SG in intestinal cells, likely as a consequence of increased glutathione-S-transferase-mediated conjugation of CDNB, may explain the in vivo findings. Further experiments in isolated hepatocytes suggested that decreased synthesis of DNP-SG rather than altered canalicular transport is responsible for the substantial impairment in excretion of this compound into bile. Taken together, these results indicate that the intestine may partially compensate for liver DNP-SG disposition, particularly shortly after surgery, while liver capability is recovering.

Effect of acaricides on the activity of a Boophilus microplus glutathione S-transferase.

In the present study, we report the effect of several acaricides on the enzyme activity of a Boophilus microplus recombinant glutathione S-transferase (rGST). GST was expressed in Escherichia coli and was purified with glutathione (GSH) affinity column chromatography. The kinetic constants were determined by reacting GST with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione. We report the effect of several acaricides on the enzyme activity of rGST. Some acaricides (ethion, amitraz, chlorpyrifos, DDT, cypermethrin, diazinon, ivermectin, deltamethrin and flumethrin) inhibited rGST. Contrarily, coumaphos had an activating effect. Although the accurate mechanisms of the B. microplus resistance to acaricides remain elusive, this work helps in understanding how acaricides can interact with GST.

Quantitative and qualitative gender-related differences in jejunal glutathione S-transferase in the rat effect of testosterone administration.

Gender-related differences and the regulation by testosterone of glutathione S-transferase were studied in rat jejunum. We analyzed enzyme activity and the relative content of GST subunits. Four experimental groups of adult rats were studied: normal males, castrated males, castrated males injected with testosterone and normal females. Glutathione S-transferase activity was assayed using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Differences in subunit composition among groups were evaluated by western blot analysis. The results demonstrated that 1-chloro-2,4-dinitrobenzene conjugation rate is higher in normal males than in normal females and castrated males. Testosterone administration to castrated males raised the activity up to the level observed in normal males. No significant difference in glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was observed among groups. Western blot analysis revealed that males and females differ in all subunits tested that is, rGSTA2, rGSTM1, rGSTM2 and rGSTP1, and that testosterone regulates the content of rGSTM1, rGSTM2 and rGSTP1. In conclusion, jejunal GST shows a gender-dependent regulation affecting both enzyme activity and subunit composition, and testosterone appears to be one of the factors involved.

Possible role of blood insulin levels on glutathione S-transferase activities from different tissues of male rats.

The activity of in vitro glutathione S-transferase towards 1-chloro-2,4-dinitrobenzene was examined in liver, renal cortex, and small intestine (duodenum, jejunum, ileum) after the in vivo treatment of male Wistar rats with streptozotocin or alloxan. The studies were performed at 2, 10, 24, and 48 h and 7 and 15 days after streptozotocin treatment or 24 and 48 h after alloxan treatment. The results indicated that while the blood levels of insulin-glucose did not show variations, there were no alterations of the glutathione S-transferase activity in the tissues tested. On the other hand, when the treatments caused modifications on blood insulin-glucose levels, there were changes of glutathione S-transferase activity in all tissues (except in the ileum) in such a way that a direct relationship between plasma insulin levels and glutathione S-transferase activity could be demonstrated. These results were also confirmed through insulin administration to control and diabetic rats. The data demonstrate a possible regulation of glutathione S-transferase activity by blood insulin and (or) glucose levels in the tissues tested.

Effect of aflatoxin B1 treatment in vivo on the in vitro activity of hepatic and extrahepatic glutathione S-transferase.

The effect of aflatoxin B1 (AFB1) on the glutathione S-transferase activity (GST) and on non-protein thiol levels of different tissues was studied in adult male Wistar rats. Animals received a single dose of the toxin (100 or 500 micrograms/kg body wt., p.o.), and were studied 6 or 24 h after administration. GST was determined in liver, renal cortex, duodenum, jejunum-ileum and distal ileum, using 3 substrates: 1-chloro-2,4-dinitrobenzene (CDNB), trans-4-phenyl-3-buten-2-one (PBO) and 1,2-epoxyethylbenzene (STOX). The non-protein thiol content of all tissues tested increased with the lowest dose at 6 h, returning to normal values at 24 h, while the higher dose produced a significant decrease in reduced thiol levels at 6 h, returning to normal values at 24 h. AFB1 administration induced, independently of dose and tissue, total GST (CDNB) and epoxide-transferase activity (STOX) while A--C-type transferases (PBO) were inhibited. Almost all activities returned to normal values at 24 h. In cases of enzyme induction there was in general an increase in Vmax and a decrease in apparent Km. The opposite was seen in cases of inhibition. In conclusion, the results provide evidence that extrahepatic GST could be important in the overall process of detoxification of AFB1. The behavior seen in hepatic and extrahepatic tissues revealed the functions of catalysis (B-type transferases) and covalent bond formation, as well as inactivation by probable AFB1 metabolites (A--C-type transferases).