Glutathione Peroxidase Isoenzymes Research Articles

Serum trace element levels and activity of enzymes associated with oxidative stress in endometriosis and endometrial cancer.

Endometriosis and endometrial cancer are closely related to oxidative stress. However, the direct relationship between copper and zinc levels and oxidative stress in the extracellular and intracellular space remains unclear. The presented study is focused on the determination of serum Zn and Cu levels, glutathione concentration and enzyme activity in three groups: patients diagnosed with endometrial cancer (EC), patients diagnosed with endometriosis (EM), and a healthy control group. Spectrophotometric determination of trace elements revealed that levels of zinc and copper were lower in blood plasma of patients with endometriosis as compared with the other groups; however, there were no significant differences in the Cu/Zn ratio. Furthermore, significantly increased blood serum glutathione levels were detected in both EM and EC groups compared with the control group. While the activity of superoxide dismutase (SOD) was similar across the studied groups, we observed differences in the activity of other enzymes associated with oxidative stress, including glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST), between the control group and the EM and EC patients. Additionally, analysis of gene expression based on free circulating mRNA indicated significant differences in the expression of SOD isoenzymes between the patient groups and the control group; expression of GPx isoenzymes was also altered. Obtained results may have potential application in diagnostics as well as monitoring of endometriosis and endometrial cancer.

Purification and characterization of two glutathione peroxidases from embryo of the camel tick Hyalomma dromedarii

Two glutathione peroxidase isoenzymes were purified from 24-day old embryos of the camel tick Hyalomma dromedarii and designated tick embryo glutathione peroxidase 1 and 2 (TEGPx1 and TEGPx2). The purification procedure involved ammonium sulfate precipitation, as well as ion exchange and gel filtration column chromatography. Glutathione peroxidase isoenzymes subunit molecular mass was determined by SDS-PAGE to be 36 ± 2 kDa and 59 ± 1.5 kDa for TEGPx1 and TEGPx2, respectively. TEGPx1 isoenzyme exhibited a dimeric structure with native molecular mass of 72 kDa while TEGPx2 was a monomeric protein. TEGPx1 and TEGPx2 displayed their pH optima at 7.6 and 8.2. Both isoenzymes cleaved preferentially H2O2 with K m values of 24 and 49 μM. Iodoacetamide competitively inhibited TEGPx1 with K i value of 0.45 mM and 1.10; phenanthroline competitively inhibited TEGPx2 with K i value of 0.12 mM. These results indicate the presence of two different forms of glutathione peroxidase in the developing camel tick embryos. This finding enhances our knowledge and understanding of the physiology of these ectoparasites and will encourage the development of new and untraditional control methods.

Hydroperoxide reduction by thioredoxin‐specific glutathione peroxidase isoenzymes of Arabidopsis thaliana

Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs (AtGPX1-8). Four mature GPX isoenzymes with different subcellular distributions, AtGPX1, -2, -5 and -6, were overexpressed in Escherichia coli and characterized. Interestingly, these recombinant proteins were able to reduce H2O2, cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using thioredoxin but not glutathione or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2O2 were 2-7 times higher than those with cumene hydroperoxide. Km values for thioredoxin and H2O2 were 2.2-4.0 and 14.0-25.4 microM, respectively. These finding suggest that GPX isoenzymes may function to detoxify H2O2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance.

In vitro effect of low concentrations of oltipraz on the antioxidant defence of mouse hepatocytes and Schistosoma mansoni worms

The direct effects of oltipraz (OPZ) on mouse hepatocytes and Schistosoma mansoni worms are studied in vitro at a concentration range of 5–25 µmol/L following one- and three-hour incubations. Oxidative stress is reflected by increases in malondialdehyde (MDA), representing the end products of lipid peroxidation, and depletion of glutathione (GSH), representing protective thiol groups. Activities of glutathione peroxidase isoenzymes, GST and GR as components of antioxidative defence are also determined. The opposite effects of low concentrations of OPZ on mammalian hepatocytes and S. mansoni worms were confirmed. In incubation with S. mansoni, addition of OPZ resulted in significantly increased production of MDA, together with depletion of GSH, both of which were time- and OPZ concentration-dependent. In incubation with mouse hepatocytes, however, there was little change in MDA concentrations, and a gradual increase in GSH levels, both of which were time- and concentration-dependent. Addition of OPZ to the incubation media also affected the activities of antioxidant enzymes. Although total GPx activity increased in both mammalian hepatocyte and S. mansoni experiments, the opposite was noted with the selenium-dependent isoenzyme. While there was a gradual increase in sGPx in hepatocytes, there was a timeand concentration-dependent inhibition in the worm isoenzyme. Contrasting results were also obtained with GR. While increased activity was obtained with the enzyme from mouse hepatocytes, the worm enzyme was inhibited, especially at the upper end of the OPZ concentration range and also following longer periods of incubation. The increase in GST activity followed the same qualitative pattern in both hepatocytes and schistosomes. Therefore, OPZ given in doses that maintain a serum concentration in the range 5–25 µmol/L induces biochemical changes in mouse hepatocytes that could be utilised for chemo-preventive purposes and prevention of oxidative damage. However, progressive oxidative damage to S. mansoni worms occurred despite some protective biochemical changes.

Cellular and subcellular localization of gastrointestinal glutathione peroxidase in normal and malignant human intestinal tissue

The gastrointestinal glutathione peroxidase (GI-GPx) is believed to prevent absorption of hydroperoxides. GI-GPx is expressed in the intestine together with the other three glutathione peroxidase isoenzymes, raising the question of the physiological role of the different GPx types. We therefore studied the cellular and subcellular distribution of GI-GPx in normal and malignant tissue obtained from patients with colorectal cancer or familial polyposis by immunohistochemistry. In healthy ileum epithelium GI-GPx was preferentially enriched in Paneth cells. In unaffected crypts of colon and rectum, it decreased gradually from the ground to the luminal surface. In crypt ground, GI-GPx was uniformly distributed, whereas in cells at the luminal surface it was concentrated in structures capping the nuclei at the apical pole. In colorectal cancer, GI-GPx expression depended on the stage of malignant transformation. In early stages, GI-GPx was increased and pronouncedly associated with the vesicular structures. In progressed stages of malignancy, structures disintegrated and GI-GPx distribution became more diffuse. These observations support the hypothesis that GI-GPx, apart from being a barrier against hydroperoxide absorption, might be involved in cell growth and differentiation.

Cytoprotection against lipid hydroperoxides correlates with increased glutathione peroxidase activities, but not selenium uptake from different selenocompounds.

Cells cultivated under standard conditions were highly deficient in tocopherol, selenium, and glutathione peroxidase (GPx) activities. We investigated whether and to what extent the addition of different selenocompounds to growth media would alter biochemical, physiological, and pathophysiological parameters of cultured liver cells. Cellular uptake of selenium, GPx activities, and cytoprotection were measured and compared in human hepatoma cells (HepG2). Selenite and selenocystine were Se donors of high bioavailability (i.e., with these culture supplements, the increased Se uptake, induction of GPx isoenzymes, and protection of treated cells from lipid hydroperoxides were well correlated). In contrast, selenium from selenomethionine was incorporated into cellular proteins but had no effect on GPx activities or cytoprotection. The data show that not all selenium donors provide selenium, which is bioactivated to act as antioxidant. Thus, cellular selenium content, in general, did not correlate with cytoprotective activity of this trace element. However, cellular GPx activities at different times, with different concentrations, and with different Se donors always correlated with protection from lipid hydroperoxides and may, thus, represent a more reliable parameter to define adequate Se supply.