Melanin decolorization by lysosome-related extract in Saccharomyces cerevisiae modified to overproduce glutathione peroxidase.

All eukaryotes have lysosomes that contain hydrolytic enzymes, such as protease, that degrade waste materials and cellular fragments. As a cellular organelle, lysosomes function as the digestive system of the cell, serving both to degrade material taken up from outside the cell and to digest obsolete components of the cell itself. In a previous study, melanin compounds were bleached using lysosome-related organelle extract (LOE) in which glutathione peroxidase (GPX) contributed decisively to melanin decolorization. In this study, Saccharomyces cerevisiae was engineered to overproduce GPX, which increases the melanin color reduction activity of LOE. In addition, the peroxidase activity of the recombinant yeast was measured for each compartment. In spite of the modification to overexpress the GPX protein, with the peroxidase activity of the lysosome fraction specifically higher, the overall peroxidase activity of the cells remained constant. The overexpression of GPX2 among the GPX present in S. cerevisiae increased both the melanin-decolorization activity and the peroxidase activity of LOE. These results indicate that the peroxidase activity is related to the melanin decomposition and antioxidant enzymes such as GPX. In an artificial skin tissue test, the LOE extracted from the recombinant yeast was efficient in reducing the melanin. These results confirmed the enzyme's ability to penetrate corneous tissue, and they suggest the possibility of further development as a new whitening cosmetic. KEY POINTS: • Modification of Saccharomyces cerevisiae to overexpress glutathione peroxidase (GPX). • The lysosome fraction of the recombinant strain enhanced the decolorizing function. • The LOE penetrates the skin barrier and works effectively on artificial skin tissue.

Protective Effects of a Lutein Ester Prodrug, Lutein Diglutaric Acid, against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells.

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.

Expression of COX-2 and Nrf2/GPx3 in the anterior vaginal wall tissues of women with pelvic organ prolapse.

PURPOSE: To evaluate COX-2 and Nrf2/GPx3 expressions in the lamina propria of the anterior vaginal wall tissues of women with and without pelvic organ prolapse (POP). METHODS: Tissue samples of anterior vaginal wall were examined using HE staining, immuohistochemical staining and Western blot for the expressions of COX-2/PGE2, Nrf2/GPx3, MMP2, TIMP1, collagen I and collagen III (n = 35, per group). RESULTS: Compared with control group, collagen fibers of the anterior vaginal wall were disorganized and discontinuous. Expressions of Nrf2, GPx3, TIMP1, collagen I and collagen III were found significantly lower in POP group (P < 0.05); while, expressions of COX-2, PGE2, and MMP2 were found significantly higher in POP group (P < 0.05). Statistically significant correlations of COX-2 and Nrf2/GPx3 were showed (P < 0.01). CONCLUSION: We found that the interaction between inflammation and oxidative stress was closely related to the development of POP. This study demonstrates that COX-2 and Nrf2 pathways may be involved in pathogenesis of POP, as promising potential therapeutic targets and agents.

An adenoviral vector encoded with the GPx-1 gene attenuates memory impairments induced by ß-amyloid (1-42) in GPx-1 KO mice via activation of M1 mAChR-mediated signalling.

In the present study, we examined whether glutathione peroxidase-1 (GPx-1), a major H2O2 scavenger in the brain, affects memory deficits induced by Aß (1-42) in mice. Treatment with 400 pmol/5 µl Aß (1-42) (i.c.v.) resulted in a reduction of GPx-1 expression in wild-type (WT) mice. An Aß (1-42)-induced reduction in acetylcholine (ACh) level was observed in the hippocampus. Treatment with Aß (1-42) consistently resulted in reduced expression and activity of choline acetyltransferase (ChAT) and in an increase in expression and activity of acetylcholinesterase (AChE). Upon examining each of the muscarinic acetylcholine receptors (mAChRs) and nicotinic AChRs, we noted that Aß (1-42) treatment selectively reduced the levels of M1 mAChR. In addition, Aß (1-42) induced a significant reduction in phospho-cAMP response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) expression. The cholinergic impairments induced by Aß (1-42) were more pronounced in GPx-1 knockout mice than in WT mice. Importantly, an adenoviral vector encoded with the GPx-1 gene (Ad-GPx-1) significantly rescued Aß (1-42)-induced cholinergic impairments in GPx-1 knockout mice. In addition, M1 mAChR antagonist dicyclomine significantly counteracted Ad-GPx-1-mediated increases in p-CREB and BDNF expression, as well as memory-enhancing effects in GPx-1 knockout mice, thus indicating that M1 mAChR might be a critical mediator for the rescue effects of Ad-GPx-1. Combined, our results suggest that GPx-1 gene protected against Aß (1-42)-induced memory impairments via activation of M1 mAChR-dependent CREB/BDNF signalling.

Involvement of GPx-3 in the Reciprocal Control of Redox Metabolism in the Leukemic Niche.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell-cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

Seleno-l-methionine suppresses copper-enhanced zinc-induced neuronal cell death via induction of glutathione peroxidase.

Excessive zinc ion (Zn2+) release is induced in pathological situations and causes neuronal cell death. Previously, we have reported that copper ions (Cu2+) markedly exacerbated Zn2+-induced neuronal cell death by potentiating oxidative stress, the endoplasmic reticulum (ER) stress response, and the activation of the c-Jun amino-terminal kinase (JNK) signaling pathway. In contrast, selenium (Se), an essential trace element, and amino acids containing selenium (such as seleno-l-methionine) have been reported to inhibit stress-induced neuronal cell death and oxidative stress. Thus, we investigated the effect of seleno-l-methionine on Cu2+/Zn2+-induced neuronal cell death in GT1-7 cells. Seleno-l-methionine treatment clearly restored the Cu2+/Zn2+-induced decrease in the viable cell number and attenuated the Cu2+/Zn2+-induced cytotoxicity. Accordingly, the levels of ER stress-related factors (especially, CHOP and GADD34) and of phosphorylated JNK increased upon CuCl2 and ZnCl2 co-treatment, whereas pre-treatment with seleno-l-methionine significantly suppressed these upregulations. Analysis of reactive oxygen species (ROS) as upstream factors of these pathways revealed that Cu2+/Zn2+-induced ROS production was clearly suppressed by seleno-l-methionine treatment. Finally, we found that seleno-l-methionine induced the antioxidative protein, glutathione peroxidase. Taken together, our findings suggest that seleno-l-methionine suppresses Cu2+/Zn2+-induced neuronal cell death and oxidative stress via induction of glutathione peroxidase. Thus, we think that seleno-l-methionine may help prevent refractory neurological diseases.

Glutathione peroxidase-1 overexpression reduces oxidative stress, and improves pathology and proteome remodeling in the kidneys of old mice.

This study investigated the direct roles of hydrogen peroxide (H2 O2 ) in kidney aging using transgenic mice overexpressing glutathione peroxidase-1 (GPX1 TG). We demonstrated that kidneys in old mice recapitulated kidneys in elderly humans and were characterized by glomerulosclerosis, tubular atrophy, interstitial fibrosis, and loss of cortical mass. Scavenging H2 O2 by GPX1 TG significantly reduced mitochondrial and total cellular reactive oxygen species (ROS) and mitigated oxidative damage, thus improving these pathologies. The potential mechanisms by which ROS are increased in the aged kidney include a decreased abundance of an anti-aging hormone, Klotho, in kidney tissue, and decreased expression of nuclear respiratory factor 2 (Nrf2), a master regulator of the stress response. Decreased Klotho or Nrf2 was not improved in the kidneys of old GPX1 TG mice, even though mitochondrial morphology was better preserved. Using laser capture microdissection followed by label-free shotgun proteomics analysis, we show that the glomerular proteome in old mice was characterized by decreased abundance of cytoskeletal proteins (critical for maintaining normal glomerular function) and heat shock proteins, leading to increased accumulation of apolipoprotein E and inflammatory molecules. Targeted proteomic analysis of kidney tubules from old mice showed decreased abundance of fatty acid oxidation enzymes and antioxidant proteins, as well as increased abundance of glycolytic enzymes and molecular chaperones. GPX1 TG partially attenuated the remodeling of glomerular and tubule proteomes in aged kidneys. In summary, mitochondria from GPX1 TG mice are protected and kidney aging is ameliorated via its antioxidant activities, independent and downstream of Nrf2 or Klotho signaling.

GPx3 dysregulation impacts adipose tissue insulin receptor expression and sensitivity.

Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of patients with diabetes are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of IR with a reduction of mRNA expression of selenoproteins Txnrd3, Sephs2, and Gpx3 in gonadal adipose tissue. Consistently, GPX3 is also decreased in adipose tissue of insulin-resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation, and elevated IR expression in WAT. Again, IR expression correlated positively with Gpx3 expression, a phenotype that is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduced IR expression and insulin sensitivity in 3T3-L1 preadipocytes. Overall, our data identify GPx3 as a potentially novel regulator of IR expression and insulin sensitivity in adipose tissue.

Isoliquiritigenin attenuates lipopolysaccharide-induced cognitive impairment through antioxidant and anti-inflammatory activity.

BACKGROUND: Oxidative stress and neuroinflammation are central pathogenic mechanisms common to many neurological diseases. Isoliquiritigenin (ISL) is a flavonoid in licorice with multiple pharmacological properties, including anti-inflammatory activity, and has demonstrated protective efficacy against acute neural injury. However, potential actions against cognitive impairments have not been examined extensively. We established a rat model of cognitive impairment by intracerebroventricular injection of lipopolysaccharide (LPS), and examined the effects of ISL pretreatment on cognitive function, hippocampal injury, and hippocampal expression of various synaptic proteins, antioxidant enzymes, pro-inflammatory cytokines, and signaling factors controlling anti-oxidant and pro-inflammatory responses. RESULTS: Rats receiving LPS alone demonstrated spatial learning deficits in the Morris water maze test as evidenced by longer average escape latency, fewer platform crossings, and shorter average time in the target quadrant than untreated controls. ISL pretreatment reversed these deficits as well as LPS-induced decreases in the hippocampal expression levels of synaptophysin, postsynaptic density-95, brain-derived neurotrophic factor, superoxide dismutase, glutathione peroxidase, and BCL-2. ISL pretreatment also reversed LPS-induced increases in TUNEL-positive (apoptotic) cells, BAX/BCL-2 ratio, and expression levels of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and C-C motif chemokine ligand 3. Pretreatment with ISL increased the expression levels of phosphorylated (p)-GSK-3ß, nuclear NRF2, HO-1 mRNA, and NQO1 mRNA, and reversed LPS-induced nuclear translocation of nuclear factor (NF)-κB. CONCLUSIONS: ISL protects against LPS-induced cognitive impairment and neuronal injury by promoting or maintaining antioxidant capacity and suppressing neuroinflammation, likely through phosphorylation-dependent inactivation of GSK-3ß, enhanced expression of NRF2-responsive antioxidant genes, and suppression of NF-κB-responsive pro-inflammatory genes.

Oxidative Stress Produced by Hyperthyroidism Status Induces the Antioxidant Enzyme Transcription through the Activation of the Nrf-2 Factor in Lymphoid Tissues of Balb/c Mice.

Hyperthyroidism is an endocrine disorder characterized by excessive secretion of thyroid hormones T3 and T4. Thyroid hormones exert pleiotropic actions on numerous tissues and induce an overall increase in metabolism, with an increase in energy demand and oxygen consumption. Therefore, the purpose of this study was to investigate the effects of hyperthyroidism on the production of reactive oxygen species (ROS) in lymph node and spleen cells of euthyroid and hyperthyroid mice, analyzing antioxidant mechanisms involved in the restitution of the cellular redox state. For this, thirty female Balb/c (H-2d) mice were randomly divided into two groups: euthyroid (by treatment with placebo) and hyperthyroid (by treatment with 12 mg/l of T4 in drinking water for 30 days). We found a significant increase in ROS and an increase in the genomic and protein expression of the antioxidant enzymes catalase (CAT) and glutathione peroxidase-1 (GPx-1) in lymph node and spleen cells of hyperthyroid mice. In vitro treatment with H2O2 (250 µM) of the lymphoid cells of euthyroid mice increased the expression levels of CAT and GPx-1. The hyperthyroidism increased the phosphorylation levels of Nrf2 (nuclear factor erythroid 2-related factor) and the kinase activity of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK). Additionally, we found an increase in the expression of the classic isoenzymes of PKCα, ß and γ. In conclusion, these results indicated that the increase in ROS found in the hyperthyroid state induces the antioxidant enzyme transcription through the activation of the Nrf-2 factor in lymphoid tissues. This shows the influence of hyperthyroidism on the regulation of the cellular antioxidant system.

Differential alteration in reproductive toxicity of medaka fish on exposure to nanoscale zerovalent iron and its oxidation products.

Nanoscale zerovalent iron (nZVI) is a redox-active nanomaterial commonly used in remediation of soil and groundwater pollution and wastewater treatment processes. A large quantity of nZVI (e.g., >100 mg/L) accidentally released from in situ sites to nearby oxygenized aquifers could be rapidly oxidized to iron oxides (e.g., Fe3O4 or Fe2O3) and ions (e.g., Fe2+), for acute hypoxia effects to aquatic life. However, we do not know the ecotoxicological fate of nZVI and its oxidation products at lower, environmentally concentrations in surface water receiving waterborne transportation or effluent discharge in terms of exposure to aquatic vertebrate species. This study assessed the causal effect on reproductive toxicity in medaka adults (Oryzias latipes) of carboxymethyl cellulose-stabilized nZVI (CMC-nZVI), Fe2+ and iron oxide nanoparticles (nFe3O4) with 21-day aqueous exposure at 5 and 20 mg/L (Fe-equivalent). Such concentrations did not significantly change the dissolved oxygen, oxidation-reduction potential or pH values in the 3 iron solutions during the fish exposure period. Neither CMC-nZVI nor Fe2+ treated adults showed altered daily egg production (fecundity) and oxidative stress responses in observed tissues, as compared to controls. However, the fecundity in nFe3O4 (20 mg/L)-treated pairs was significantly decreased, with increased incidence of abnormal immature oocytes in the ovary. As well, nFe3O4 treatment suppressed activities of the antioxidants superoxide dismutase and expression of glutathione peroxidase (gpx) in the brain and ovary. Although nFe3O4 or Fe2+ treatments inhibited mRNA expression of hepatic estrogen receptor (er-α) in females, plasma levels of sex hormones and (Na, K)-ATPase activity in gills of treated fish did not differ from controls for both sexes. Hence, oxidation products (e.g., nFe3O4) from nZVI at lower milligram-per-liter levels may be potent in inducing nanoparticle-specific reproductive toxicity in medaka fish by inducing oxidative stress in female gonads. MAIN FINDING: nZVI oxidation product nFe3O4 at lower mg/L induces nanoparticle-specific reproductive toxicity in medaka fish.

Effects of Acute Ethanol Administration on Brain Oxidative Status: The Role of Acetaldehyde.

BACKGROUND: Ethanol (EtOH), one of the most widely consumed substances of abuse, can induce brain damage and neurodegeneration. EtOH is centrally metabolized into acetaldehyde, which has been shown to be responsible for some of the neurophysiological and cellular effects of EtOH. Although some of the consequences of chronic EtOH administration on cell oxidative status have been described, the mechanisms by which acute EtOH administration affects the brain's cellular oxidative status and the role of acetaldehyde remain to be elucidated in detail. METHODS: Swiss CD-I mice were pretreated with the acetaldehyde-sequestering agent d-penicillamine (DP; 75 mg/kg, i.p.) or the antioxidant lipoic acid (LA; 50 mg/kg, i.p.) 30 minutes before EtOH (2.5 g/kg, i.p.) administration. Animals were sacrificed 30 minutes after EtOH injection. Glutathione peroxidase (GPx) mRNA levels; GPx and glutathione reductase (GR) enzymatic activities; reduced glutathione (GSH), glutathione disulfide (GSSG), glutamate, g-L-glutamyl-L-cysteine (Glut-Cys), and malondialdehyde (MDA) concentrations; and protein carbonyl group (CG) content were determined in whole-brain samples. RESULTS: Acute EtOH administration enhanced GPx activity and the GSH/GSSG ratio, while it decreased GR activity and GSSG concentration. Pretreatment with DP or LA only prevented GPx activity changes induced by EtOH. CONCLUSIONS: Altogether, these results show the capacity of a single dose of EtOH to unbalance cellular oxidative homeostasis.

Glutathione peroxidase-1 overexpressing transgenic mice are protected from cocaine-induced drug dependence.

Converging evidence has demonstrated that oxidative burdens are associated with drug dependence induced by psychostimulants. Here, we investigated whether oxidative stress directly mediates conditioned place preference and behavioral sensitization (drug dependence) induced by cocaine and whether glutathione peroxidase-1 (GPx-1), a major GPx, modulates cocaine-induced psychotoxic changes in mice. Cocaine-induced drug dependence was followed by increases in c-Fos-immunoreactivity (c-Fos-IR) in the nucleus accumbens. Simultaneously, cocaine significantly increased oxidative parameters and nuclear factor κB (NFκB) activity (i.e. nuclear translocation and DNA binding activity) in the striatum (including nucleus accumbens). Genetic depletion of GPx-1 made mice susceptible to drug dependence induced by cocaine in mice, while genetic overexpression of GPx-1 protected the mice from drug dependence. Pyrrolidine dithiocarbamate (PDTC), a NFκB inhibitor, significantly attenuated the sensitivity induced by the genetic depletion of GPx-1 in mice. However, PDTC did not exhibit any additive effects against the protection afforded by the genetic overexpression of GPx-1. Our results suggest that drug dependence induced by cocaine requires oxidative stress and NFκB activation, and that the GPx-1 gene is a potential protective factor against cocaine-induced drug dependence through positive modulation of NFκB.

DHA supplementation during prenatal ethanol exposure alters the expression of fetal rat liver genes involved in oxidative stress regulation.

Prenatal ethanol (EtOH) exposure is known to induce adverse effects on fetal brain development. Docosahexaenoic acid (DHA) has been shown to alleviate these effects by up-regulating antioxidant mechanisms in the brain. The liver is the first organ to receive enriched blood after placental transport. Therefore, it could be negatively affected by EtOH, but no studies have assessed the effects of DHA on fetal liver. This study examined the effects of maternal DHA intake on DHA status and gene expression of key enzymes of the glutathione antioxidant system in the fetal liver after prenatal EtOH exposure. Pregnant Sprague-Dawley dams were intubated with EtOH for the first 10 days of pregnancy, while being fed a control or DHA-supplemented diet. Fetal livers were collected at gestational day 20, and free fatty acids and phospholipid profile, as well as glutathione reductase (GR) and glutathione peroxidase-1 (GPx1) gene expressions, were assessed. Prenatal EtOH exposure increased fetal liver weight, whereas maternal DHA supplementation decreased fetal liver weight. DHA supplementation increased fetal liver free fatty acid and phospholipid DHA independently of EtOH. GR and GPx1 messenger RNA (mRNA) expressions were significantly increased and decreased, respectively, in the EtOH-exposed group compared with all other groups. Providing DHA normalized GR and GPx1 mRNA expression to control levels. This study shows that maternal DHA supplementation alters the expression of fetal liver genes involved in the glutathione antioxidative system during prenatal EtOH exposure. The fetal liver may play an important role in mitigating the signs and symptoms of fetal alcohol spectrum disorders in affected offspring.

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft-bodied tick vector.

The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft-bodied tick vector, Ornithodoros turicata. Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata. A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase (gpx), thioredoxin peroxidase (tpx), manganese superoxide dismutase (sod-1), copper-zinc superoxide dismutase (sod-2), and catalase (cat) by reverse-transcriptase droplet digital polymerase chain reaction (RT-ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi, suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.

Estrogen and progesterone dependent expression of visfatin/NAMPT regulates proliferation and apoptosis in mice uterus during estrous cycle.

Visfatin is an adipokine which has an endocrine effect on reproductive functions and regulates ovarian steroidogenesis. There is scant information about the expression, regulation, and functions of visfatin in the mammalian uterus. The present study examined expression and localization of visfatin in the mouse uterus at various stages of the natural estrous cycle, effects of estrogen and progesterone on localization and expression of visfatin in the ovariectomised mouse uterus and effect of visfatin inhibition by a specific inhibitor, FK866 on proliferation and apoptosis in the uterus. Western blot analysis of visfatin showed high expression in proestrus and metestrus while it declined in estrus and diestrus. Immulocalization study also showed strong immunostaining in the cells of endometrium, myometrium, luminal and glandular epithelium during proestrus and metestrus that estrus and diestrus. The uterine visfatin expression closely related to the increased estrogen levels in proestrus and suppressed when progesterone rose to a high level in diestrus. The treatment with estrogen to ovariectomised mice up-regulates visfatin, PCNA, and active caspase3 whereas progesterone up-regulates PCNA and down-regulates visfatin and active caspase3 expression in mouse uterus. The co-treatment with estrogen and progesterone up-regulates visfatin and down-regulates PCNA and active caspase3. In vitro study showed endogenous visfatin inhibition by FK866 increased expression of PCNA and BCL2 increased catalase activity while FK866 treatment decreased expression of active caspase3 and BAX with decreased SOD and GPx activity. BrdU labeling showed that inhibition of visfatin modulates the uterine proliferation. This study showed that expression of visfatin protein is steroid dependent in mouse uterus which is involved in the regulation of proliferation and apoptosis via modulating antioxidant system in the uterus of mice during the reproductive cycle.

Expression and characterization of glutathione peroxidase of the liver fluke, Fasciola gigantica.

Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as an anti-oxidant that helps to protect parasites from damage by free radicals released from the host immune cells, it has both diagnostic as well as vaccine potential against fasciolosis. In this study, we have cloned, characterized, and detected the expression of the GPx protein in Fasciola gigantica (Fg). FgGPx (582 bp) was cloned by polymerase chain reaction (PCR) from complementary DNA (cDNA) from an adult fluke. Its putative peptide has no signal sequence and is composed of 168 amino acids, with a molecular weight of 19.1 kDa, and conserved sequences at NVACKUG, FPCNQFGGQ, and WNF. Phylogenetic analysis showed that GPx is present from protozoa to mammals and FgGPx was closely related to Fasciola hepatica GPx. A recombinant FgGPx (rFgGPx) was expressed in Escherichia coli BL21 (DE3) and used for immunizing mice to obtain polyclonal antibodies (anti-rFgGPx) for immunoblotting and immunolocalization. In immunoblotting analysis, the FgGPx was expressed in all stages of F. gigantica (eggs, metacercariae, newly excysted juveniles (NEJ), 4-week-old juveniles, and adults). This mouse anti-rFgGPx reacted with the native FgGPx at a molecular weight of 19.1 kDa in adult whole body (WB) and tegumental antigens (TA) as detected by immunoblotting. The FgGPx protein was expressed at a high level in the tegument, vitelline glands, and eggs of the parasite. Anti-rFgGPx exhibited no cross-reactivity with the other parasite antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. The possibility of using rFgGPx for immunodiagnosis and/or as a vaccine for fasciolosis in animals of economic importance will be explored in the future.

In-office tooth bleaching with 38% hydrogen peroxide promotes moderate/severe pulp inflammation and production of ll-1ß, TNF-ß, GPX, FGF-2 and osteocalcin in rats.

OBJECTIVES: To study the intensity of inflammatory infiltrate and production of interleukin-1ß (ll-1ß), tumor necrosis factor-ß (TNF-ß), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats. MATERIAL AND METHODS: Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1ß, TNF-ß, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05). RESULTS: The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1ß, TNF-ß, and GPX in bleached groups at 24 h and strong staining for ll-1ß, TNF-ß, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01). CONCLUSIONS: Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.

Genetic overexpression of glutathione peroxidase-1 attenuates microcystin-leucine-arginine-induced memory impairment in mice.

Microcystin-leucine-arginine (MCLR) is the most common form of microcystins, which are environmental toxins produced by cyanobacteria, and its hepatotoxicity has been well-documented. However, the neurotoxic potential of MCLR remains to be further elucidated. In the present study, we investigated whether intracerebroventricular (i.c.v.) infusion of MCLR induces mortality and neuronal loss in the hippocampus of mice. Because we found that MCLR impairs memory function in the hippocampus at a low dose (4 ng/µl/mouse, i.c.v.) without a significant neuronal loss, we focused on this dose for further analyses. Results showed that MCLR (4 ng/µl/mouse, i.c.v.) significantly increased oxidative stress (i.e., malondialdehyde, protein carbonyl, and synaptosomal ROS) in the hippocampus. In addition, MCLR significantly increased superoxide dismutase (SOD) activity without corresponding induction of glutathione peroxidase (GPx) activity, and thus led to significant decrease in the ratio of GPx/SODs activity. The GSH/GSSG ratio was also significantly reduced after MCLR treatment. GPx-1 overexpressing transgenic mice (GPx-1 Tg) were significantly protected from MCLR-induced memory impairment and oxidative stress. The DNA binding activity of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in these mice was significantly enhanced, and the ratios of GPx/SODs activity and GSH/GSSG returned to near control levels in the hippocampus. Importantly, memory function exhibited a significant positive correlation with the ratios of GPx/SODs activity and GSH/GSSG in the hippocampus of MCLR-treated non-transgenic (non-Tg)- and GPx-1 Tg-mice. Combined, our results suggest that MCLR induces oxidative stress and memory impairment without significant neuronal loss, and that GPx-1 gene constitutes an important protectant against MCLR-induced memory impairment and oxidative stress via maintaining antioxidant defense system homeostasis, possibly through the induction of Nrf2 transcription factor.

Association between Antioxidant Enzyme Activities and Enterovirus-Infected Type 1 Diabetic Children.

OBJECTIVE: To examine the effect of infection with Enterovirus (EV) in children with type 1 diabetes (T1D) on the activities of serum antioxidant enzymes in diabetic and nondiabetic controls. SUBJECTS AND METHODS: Three hundred and eighty-two diabetic and 100 nondiabetic children were tested for EV RNA using reverse transcriptase (RT)-PCR. The activities of serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were also estimated in diabetic patients infected with EV (T1D-EV+), those not infected with EV (T1D-EV-), and in nondiabetic controls. RESULTS: The frequency of EV was higher in diabetic children (100/382; 26.2%) than in healthy controls (0/100). Levels of fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c) and C-reactive protein (CRP) were significantly higher but C-peptide was significantly lower in diabetic children than in controls. CRP levels were higher in the T1D-EV+ group than in the T1D-EV- group, and higher in all diabetic children than in nondiabetic controls. The activities of the antioxidant enzymes GPx, SOD, and CAT decreased significantly in diabetic children compared to in controls. Moreover, the activities of the enzymes tested were significantly reduced in the T1D-EV+ group compared to in the T1D-EV- group. CONCLUSION: Our data indicate that EV infection correlated with a decrease in the activity of antioxidant enzymes in the T1D-EV+ group compared to in the T1D-EV- group; this may contribute to ß cell damage and increased inflammation.