Assessment of antioxidative and selenium status by seleno-dependent glutathione peroxidase activity in different blood fractions using a pig model: issues for clinical nutrition and research.

Blood seleno-dependent glutathione peroxidase (SeGPX) activity is widely used as a metabolic indicator of systemic antioxidative status despite inconsistent responses in the literature. This study aimed to compare SeGPX activity profiles in different blood fractions, expressed with different reference units, and assess their impact on interpretation of results. Two studies on selenium (Se) metabolism in gilts, including long-term and peri-oestrus SeGPX activity profiles, were submitted to analysis of variance with double repeated measures, after data set standardization. Differences between studies were experimental period (three post-pubertal oestrus or five post-pubertal oestrus +30 days of gestation) and sample type (whole blood or blood plasma). No difference was observed between whole-blood long-term profiles (three oestrus) for SeGPX activity/mg haemoglobin (SeGPXhb) vs. SeGPX activity/ml whole blood (SeGPXwb; p = 0.29). No long-term difference was observed in whole blood between profiles according to dietary Se provision (basal and dietary Se-supplemented groups; p ≥ 0.12). Blood plasma long-term profiles (five oestrus + 30 days gestation) for SeGPX/mg blood plasma protein (SeGPXpro) were different from SeGPX/ml blood plasma (SeGPXpla) according or not to Se provision (p ≤ 0.007 and p < 0.001 respectively). However, regardless of Se provision (p ≥ 0.80), when excluding gestation from the model, blood plasma profiles were similar. During the peri-oestrus period (day -4 to +3), regardless of Se provision, SeGPX activity profiles differed according to reference units in both studies (p < 0.001). However, considering Se provision, similar profiles were observed in whole blood and blood plasma (p ≥ 0.27) for basal Se groups, whereas in Se-supplemented groups they differed for both sample types (p ≤ 0.02). In conclusion, reference units influence interpretation of SeGPX activity according to physiological state. During oxidative stress periods, this effect depends upon dietary Se provision.

Expression and characterization of a phospholipid hydroperoxide glutathione peroxidase gene in Schistosoma japonicum.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a major antioxidant enzyme, which plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We isolated and characterized a full-length cDNA sequence encoding GPx gene from a blood fluke, Schistosoma japonicum (designated SjGPx), which contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Semi-quantitative reverse transcription PCR and Western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference approach was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. Significantly reduction in GPx enzyme activities, as well as obvious changes in morphology of intrauterine eggs followed the reduction in SjGPx transcript level. We observed a 63·04% reduction in GPx activity and the eggs severely deformed. Our results revealed that SjGPx protein might be involved in the provision of enzyme activity during egg production.

The glutathione peroxidase gene family in Thellungiella salsuginea: genome-wide identification, classification, and gene and protein expression analysis under stress conditions.

Glutathione peroxidases (GPX) catalyze the reduction of H2O2 or organic hydroperoxides to water or corresponding alcohols using reduced glutathione, which plays an essential role in ROS (reactive oxygen species) homeostasis and stress signaling. Thellungiella salsuginea (Eutrema salsugineum), a relative of Arabidopsis thaliana, displays an extremely high level of tolerance to salt, drought, cold and oxidative stresses. The enzymatic antioxidant systems may contribute to the stress tolerance of T. salsuginea. In the present study, we aimed at understanding the roles of the antioxidant enzymes in T. salsuginea by focusing on the GPX family. We identified the eight GPX genes in T. salsuginea, and the structure of the N-terminal domains indicated their putative chloroplastic, mitochondrial and cytoplasmic location. The exon-intron organization of these genes exhibited a conserved pattern among plant GPX genes. Multiple environmental stresses and hormone response related cis-acting elements were predicted in the promoters of TsGPX genes. The gene and protein expression profiles of TsGPXs in response to high level of salinity and osmotic stresses, in leaves and roots of T. salsuginea were investigated using real-time RT-PCR and western blotting analysis. Our result showed that different members of the GPX gene family were coordinately regulated under specific environmental stress conditions, and supported the important roles of TsGPXs in salt and drought stress response in T. salsuginea.

Glutathione peroxidase family - an evolutionary overview.

Glutathione peroxidases (EC 1.11.1.9 and EC 1.11.1.12) catalyze the reduction of H(2)O(2) or organic hydroperoxides to water or corresponding alcohols using reduced glutathione. Some glutathione peroxidase isozymes have a selenium-dependent glutathione peroxidase activity and present a selenocysteine encoded by the opal TGA codon. In the present study, insights into the evolution of the whole glutathione peroxidase gene family were obtained after a comprehensive phylogenetic analysis using the improved number of glutathione peroxidase sequences recorded in the PeroxiBase database (http://peroxidase.isb-sib.ch/index.php). The identification of a common ancestral origin for the diverse glutathione peroxidase clusters was not possible. The complex relationships and evolutionary rates of this gene family suggest that basal glutathione peroxidase classes, present in all kingdoms, have originated from independent evolutionary events such as gene duplication, gene losses, lateral gene transfer among invertebrates and vertebrates or plants. In addition, the present study also emphasizes the possibility of some members being submitted to strong selective forces that probably dictated functional convergences of taxonomically distant groups.

Characterization of glutathione peroxidase diversity in the symbiotic sea anemone Anemonia viridis.

Cnidarians living in symbiosis with photosynthetic dinoflagellates (commonly named zooxanthellae) are exposed to high concentrations of reactive oxygen species (ROS) upon illumination. To quench ROS production, both the cnidarian host and zooxanthellae express a full suite of antioxidant enzymes. Studying antioxidative balance is therefore crucial to understanding how symbiotic cnidarians cope with ROS production. We characterized glutathione peroxidases (GPx) in the symbiotic cnidarian Anemonia viridis by analysis of their isoform diversity, their activity distribution in the three cellular compartments (ectoderm, endoderm and zooxanthellae) and their involvement in the response to thermal stress. We identified a GPx repertoire through a phylogenetic analysis showing 7 GPx transcripts belonging to the A. viridis host and 4 GPx transcripts strongly related to Symbiodinium sp. The biochemical approach, used for the first time with a cnidarian species, allowed the identification of GPx activity in the three cellular compartments and in the animal mitochondrial fraction, and revealed a high GPx electrophoretic diversity. The symbiotic lifestyle of zooxanthellae requires more GPx activity and diversity than that of free-living species. Heat stress induced no modification of GPx activities. We highlight a high GPx diversity in A. viridis tissues by genomic and biochemical approaches. GPx activities represent an overall constitutive enzymatic pattern inherent to symbiotic lifestyle adaptation. This work allows the characterization of the GPx family in a symbiotic cnidarian and establishes a foundation for future studies of GPx in symbiotic cnidarians.

The Glutathione Peroxidase Gene Family in Gossypium hirsutum: Genome-Wide Identification, Classification, Gene Expression and Functional Analysis.

The plant glutathione peroxidase (GPX) family consists of multiple isoenzymes with distinct subcellular locations, tissue-specific expression patterns and environmental stress responses. In this study, 13 putative GPXs from the genome of Gossypium hirsutum (GhGPXs) were identified and a conserved pattern among plant GPXs were exhibited, besides this they also responded to multiple environmental stresses and we predicted that they had hormone responsive cis-elements in their promoter regions. Most of the GhGPXs on expression in yeast can scavenge H2O2. Our results showed that different members of the GhGPX gene family were co-ordinately regulated under specific environmental stress conditions, and suggested the importance of GhGPXs in hormone treatments and abiotic stress responses.

Temporal leakage of Cu,Zn superoxide dismutase and loss of two low-molecular-weight forms of glutathione peroxidase-1 from buffalo (Bubalus bubalis) sperm after freezing and thawing.

The postthaw motility and fertility of frozen-thawed buffalo spermatozoa are substantially low as compared with those of cattle sperm. The sperm motility and fertility have been positively correlated with the antioxidant enzyme activities of human and canine sperm. However, the extent of antioxidant enzyme loss during cryopreservation, although reported for human and cattle sperm, is still not clear for buffalo sperm. Thus, in the present study, an attempt was made to determine the activities of various antioxidant enzymes in buffalo spermatozoa cryopreserved for various durations (0, 30, and 60 days) and the mechanism of antioxidant enzyme loss, if any, during the process. Total superoxide dismutase (SOD) activity of cryopreserved sperm decreased and that of extended seminal plasma increased progressively with the increase in duration of cryopreservation indicating the possible time-dependent leakage of these enzymes from cryopreserved sperm into the extended seminal plasmas. The catalase and glutathione peroxidase (GPx) enzyme activities could not be detected in buffalo sperm but could be detected in fresh and extended seminal plasmas. Total GPx activities of extended seminal plasma decreased progressively with the increase in duration of cryopreservation. To confirm the presence of these enzymes at protein levels, specific antioxidant enzymes such as Cu,Zn SOD of 16 kDa and three molecular weight forms (57.7, 40.9, and 26.05 kDa) of GPx-1 were detected in buffalo sperm by Western blot. Furthermore, the intensities of 16-kDa Cu,Zn SOD in 60-day cryopreserved sperm and those of two low-molecular-weight forms of GPx-1 (40.9 and 26.05 kDa) in 30-day cryopreserved sperm decreased significantly (P < 0.05) as compared with those of noncryopreserved (0-day cryopreserved) sperm indicating selective and temporal leakage of only low-molecular-weight antioxidant proteins in the initial phase. However, all the mentioned GPx-1 forms disappeared in 60-day-old cryopreserved sperm. Immunocytochemistry experiment also revealed that Cu,Zn SOD proteins are distributed over the acrosomal region of noncryopreserved buffalo spermatozoa, and the fluorescence signal decreased substantially in 60-day cryopreserved sperm. Thus, the present study reported that there is temporal leakage of Cu,Zn SOD and loss of two low-molecular-weight forms of GPx-1 from the cryopreserved buffalo spermatozoa after freezing and thawing.

Molecular cloning and characterization of a phospholipid hydroperoxide glutathione peroxidase gene from a blood fluke Schistosoma japonicum.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and plays critical roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. A full-length cDNA sequence corresponding to GPx gene from Schistosoma japonicum (designated SjGPx) was isolated and characterized. SjGPx contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Phylogenetic analysis revealed that the SjGPx was highly related to the other PHGPx-related members, and clustered into the trematode subclade II. Semi-quantitative reverse transcription PCR and western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. We observed a 53.86% reduction in total GPx activity and the eggs severely deformed. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.6- to 2.1-fold induction of the GPx activity. Our results revealed that the SjGPx protein is selenium-dependent PHGPx, which might actively participate in the detoxification of oxidative damage during egg production.

Tissue-specific functions of individual glutathione peroxidases.

The family of glutathione peroxidases comprises four distinct mammalian selenoproteins. The classical enzyme (cGPx) is ubiquitously distributed. According to animal, cell culture and inverse genetic studies, its primary function is to counteract oxidative attack. It is dispensible in unstressed animals, and accordingly ranks low in the hierarchy of glutathione peroxidases. The gastrointestinal isoenzyme (GI-GPx) is most related to cGPx and is exclusively expressed in the gastrointestinal tract. It might provide a barrier against hydroperoxides derived from the diet or from metabolism of ingested xenobiotics. The extreme stability in selenium deficiency ranks this glutathione peroxidase highest in the hierarchy of selenoproteins and points to a more vital function than that of cGPx. Plasma GPx (pGPx) behaves similar to cGPx in selenium deficiency. It is directed to extracellular compartments and is expressed in various tissues in contact with body fluids, e.g., kidney, ciliary body, and maternal/fetal interfaces. It has to be rated as an efficient extracellular antioxidant device, though with low capacity because of the limited extracellular content of potential thiol substrates. Phospholipid hydroperoxide glutathione peroxidase (PHGPx), originally presumed to be a universal antioxidant enzyme protecting membrane lipids, appears to have adopted a variety of specific roles like silencing lipoxygenases and becoming an enzymatically inactive structural component of the mitochondrial capsule during sperm maturation. Thus, all individual isoenzymes are efficient peroxidases in principle, but beyond their mere antioxidant potential may exert cell- and tissue-specific roles in metabolic regulation, as is evident for PHGPx and may be expected for others.

Characterization and expression analysis of phospholipid hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to cadmium.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is an antioxidant enzyme in the glutathione peroxidases (GPx) family that reduces hydroperoxides of phospholipids and maintains the integrity of biomembranes. Here, we report the identification and characterization of a full length cDNA of PHGPx from the ecotoxicologically important aquatic midge Chironomus riparius (CrPHGPx1) from the Expressed Sequence Tags (ESTs) database generated through pyrosequencing. The 837 base pair (bp) cDNA contained an open reading frame of 597 bp, and a 75 bp 5' and a 159 bp 3'untranslated region. The theoretical molecular mass of the deduced amino acid (aa) sequence (197 aa) was 22.40 kDa with an estimated pI of 8.77. The Cys-codon was present at residue 74 and also the active site residues Gln(91) and Trp(164). The active-site motifs and GPx family signature motifs LAFPCNQF(101-108) and WNFTK(163-168) were also found. Phylogenetic analysis showed that CrPHGPx1 is grouped with PHGPx1 from other species and is more closely related to insects belonging to the dipteran order. The mRNA of CrPHGPx1 was detected in larvae, pupae and adults. The expression of CrPHGPx1 is induced by cadmium exposure indicating that the mRNA expression of CrPHGPx1 is differently regulated in response to oxidative stress caused by environmental stressors.

Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium.

Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7 ppb) and high (347 ppb) environmental Cd concentrations for 24-48 h significantly decreased gpx4a expression in Coho olfactory rosettes, whereas olfactory gpx4b mRNA expression was not modulated by exposures at these concentrations. In summary, Coho salmon express two paralogs of gpx4, a key enzyme in the maintenance of signal transduction processes that protect against cellular oxidative damage. The Cd-associated downregulation of salmon olfactory gpx4a expression in particular, may be associated with the loss of olfactory signal transduction that accompanies metal-associated loss of olfaction in salmonids.

Characterization of an ovary-specific glutathione peroxidase from the shrimp Metapenaeus ensis and its role in crustacean reproduction.

In vertebrates, both reactive oxygen species (ROS) and the corresponding scavenging system components especially glutathione peroxidase (GPx) are indispensible for normal development of the gonads. To investigate the function of GPx in crustaceans, we cloned and characterized a full length GPx (MeGPx) transcript in the penaeid shrimp Metapenaeus ensis. Phylogenetic analysis showed that MeGPx clustered with the GPx from mollusks and nematodes and shared much higher similarity with vertebrate GPx3 and GPx5 than with GPx1 or GPx2. Multiple sequence alignment further demonstrated that MeGPx is evolutionarily conserved among invertebrates, with common functionally important motifs. MeGPx was specifically expressed in shrimp ovaries, but not in other tissues studied, including testis. In situ hybridization showed that MeGPx was highly expressed in pre-vitellogenic and mid-vitellogenic oocytes, while no expression was detected in late-vitellogenic oocytes. Moreover, real time PCR showed high level expression of MeGPx in the early ovaries. Since active protein synthesis and deposition occurred in mid-vitellogenic oocytes, MeGPx might play a pivotal role in preventing oocytes from oxidative damage and balancing ROS production. The present findings on shrimp GPx provide insights on the regulation of ROS in the ovarian maturation process and the role of GPx in crustacean reproductive biology.

Evolutionary and structural insights into the multifaceted glutathione peroxidase (Gpx) superfamily.

Glutathione peroxidase (GPx) is a widespread protein superfamily found in many organisms throughout all kingdoms of life. Although it was initially thought to use only glutathione as reductant, recent evidence suggests that the majority of GPxs have specificity for thioredoxin. We present a thorough in silico analysis performed on 724 sequences and 12 structures aimed to clarify the evolutionary, structural, and sequence determinants of GPx specificity. Structural variability was found to be limited to only two regions, termed oligomerization loop and functional helix, which modulate both reduced substrate specificity and oligomerization state. We show that mammalian GPx-1, the canonic selenocysteine-based tetrameric glutathione peroxidase, is a recent "invention" during evolution. Contrary to common belief, cysteine-based thioredoxin-specific GPx, which we propose the TGPx, are both more common and more ancient. This raises interesting evolutionary considerations regarding oligomerization and the use of active-site selenocysteine residue. In addition, phylogenetic analysis has revealed the presence of a novel member belonging to the GPx superfamily in Mammalia and Amphibia, for which we propose the name GPx-8, following the present numeric order of the mammalian GPxs.

Glutathione peroxidases and redox-regulated transcription factors.

Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides. cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-kappaB activation by interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis. GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells. GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways. In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs.

Attempt to differentiate between individual glutathione peroxidases in biological samples.

Routine determinations of glutathione peroxidases (GPx) still suffer from poor standardization and usually from lack of specification. The different types of glutathione peroxidases present in cellular homogenates may be differentially estimated by their distinct substrate specificities. Complex lipid hydroperoxides required for this approach, however, are not generally available nor easily standardized due to their molecular heterogeneity and tendency to form micelles. We therefore developed a simple procedure for the differential estimation of the major cellular types of GPx, the cytosolic GPx (cGPx) and the phospholipid hydroperoxide glutathione peroxidase (PHGPx) taking advantage of the peculiar susceptibility of PHGPx to deoxycholate. It proved to reliably determine the activities of both purified cGPx and PHGPx, in mixtures thereof, and in homogenates of tissue samples (e.g., testes), and some (e.g. ECV 304) but not all (e.g. THP-1) cultured cell lines. The method allows the differential estimation of cGPx and PHGPx, if the samples do not contain further types of GPx.

mRNA stability and selenocysteine insertion sequence efficiency rank gastrointestinal glutathione peroxidase high in the hierarchy of selenoproteins.

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.