Mechanisms of Action of Vitamin D as Supplemental Therapy for Pneumocystis Pneumonia.

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the most effective regimen for therapy of Pneumocystis pneumonia (PCP). As many patients with PCP are allergic or do not respond to it, efforts have been devoted to develop alternative therapies for PCP. We have found that the combination of vitamin D3 (VitD3) (300 IU/kg/day) and primaquine (PMQ) (5 mg/kg/day) was as effective as TMP-SMX for therapy of PCP. In this study, we investigated the mechanisms by which vitamin D enhances the efficacy of PMQ. C57BL/6 mice were immunosuppressed by CD4+ cell depletion, infected with Pneumocystismurina for 8 weeks, and then treated for 9 days with the combination of VitD3 and PMQ (VitD3-PMQ) or with TMP-SMX or PMQ to serve as controls. The results showed that vitamin D supplementation increased the number of CD11c+ cells, suppressed the production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], and interleukin-6 [IL-6]) and inducible nitric oxide synthase (iNOS), and enhanced the expression of genes related to antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit), antimicrobial peptides (cathelicidin), and autophagy (ATG5 and beclin-1). These results suggest that the main action of vitamin D is enhancing the ability of the host to defend against Pneumocystis infection.

Hepatoprotective effects of curcumin against diethyl nitrosamine induced hepatotoxicity in albino rats.

Curcumin is widely used as a traditional medicine. This work was aimed to investigate its possible protective effect against chemically induced hepatocellular carcinoma (HCC) in rats. Fifty male albino rats were divided into five groups (n=10, each). The control group received a single dose of normal saline, the diethylnitrosamine (DENA) group received a single intra-peritoneal dose at 200mg/kg body weight, and the 3rd, 4th and 5th groups were given DENA and daily administrated curcunine (CUR) via intra-gastric intubation in doses of 300,200 and 100 mg/kg b.wt. respectively for 20 weeks. Serum, and liver samples were used for determination of alpha feto-protein (AFP), interleukin-2 (IL-2), interleukine-6 (IL-6), serum liver enzymes (AST, ALT, ALP and GGT) levels as well the activities and gene expression of glutathione peroxidise (GPx), glutathione reductase (GR), catalase (CAT) and super oxide dismutase (SOD). Curcumin significantly lowered the serum levels of AFP, IL-2 and IL-6, ALT, ALT, and malondialdehyde (MDA) as well gene expression of IL-2 and IL-6. In contrast it increased the gene expression and activities of Gpx, GRD, CAT and SOD. The protective effect of CUR against DEN-induced hepatocarcinogenesis in albino rats was proven.

Effects of different selenium levels on gene expression of a subset of selenoproteins and antioxidative capacity in mice.

This study aimed to evaluate how excess selenium induces oxidative stress by determining antioxidant enzyme activity and changes in expression of selected selenoproteins in mice. BALB/c mice (n = 20 per group) were fed a diet containing 0.045 (Se-marginal), 0.1 (Se-adequate), 0.4 (Se-supernutrition), or 0.8 (Se-excess) mg Se/kg. Gene expression was quantified in RNA samples extracted from the liver, kidney, and testis by real-time quantitative reverse transcription-polymerase chain reaction. We found that glutathione peroxidase (GPx) and catalase activities decreased in livers of mice fed the marginal or excess dose of Se as compared to those in the Se-adequate group. Additionally, superoxide dismutase and glutathione reductase activities were significantly reduced only in mice fed the excess Se diet, compared to animals on the adequate Se diet. Se-supernutrition had no effect on hepatic mRNA levels of GPx isoforms 1 and 4 (GPx1 and GPx4), down-regulated GPx isoform 3 (GPx3), and upregulated selenoprotein W (SelW) mRNA expression. The excess Se diet led to decreased hepatic mRNA levels of GPx1, GPx3 and GPx4 but no change in testicular mRNA levels of GPx1, GPx3 or SelW. Dietary Se had no effect on testicular mRNA levels of GPx4. Thus, our results suggest that Se exposure can reduce hepatic antioxidant capacity and cause liver dysfunction. Dietary Se was found to differentially regulate mRNA levels of the GPx family or SelW, depending on exposure. Therefore, these genes may play a role in the toxicity associated with Se.

Interrelationship between calmodulin (CaM) and H2O2 in abscisic acid-induced antioxidant defense in the seedlings of Panax ginseng.

Calmodulin (CaM), the predominant Ca(2+) receptors, is one of the best-characterized Ca(2+) sensors in all eukaryotes. In this study the role of CaM and the possible interrelationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA) induced antioxidant defense were investigated in the seedling of Panax ginseng. Treatment of ABA (100 µM) and H(2)O(2) (10 mM) increased the expression of Panax ginseng calmodulin gene (PgCaM) and significantly enhanced the expression of the antioxidant marker genes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and the activities of chloroplastic and cytosolic antioxidant enzymes. Pretreatments with two CaM antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W7) and inhibitor or scavenger, diphenyleneiodonium chloride, and dimethylthiourea of reactive oxygen species almost completely suppressed the up-regulation of antioxidant and PgCaM gene. Moreover, H(2)O(2) production and CaM content was almost completely inhibited by pretreatments with two CaM antagonists. In addition, the expressions of PgCaM gene under different biotic stress were analyzed at different time intervals. Thus it may suggests that CaM are involved in ABA-induced increased expression of PgCaM which triggers H(2)O(2) production through activating trans-plasma membrane NADPH oxidase, resulting in up-regulation of defense related antioxidant gene and also plays a pivotal role in defense response against pathogens.

Neuroprotective diarylheptanoids from the leaves and twigs of juglans sinensis against glutamate-induced toxicity in HT22 cells.

A new diarylheptanoid, juglanin C(1), was isolated from the 80 % methanolic extract of the leaves and twigs of JUGLANS SINENSIS with three known diarylheptanoids, juglanin A(2), juglanin B (3), and (5R)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1(4-hydroxyphenyl)-3-heptanone (4), using bioactivity-guided fractionation and chromatographic techniques. Among the isolated diarylheptanoids, compounds 1 and 2 significantly showed neuroprotective activities against glutamate-induced toxicity in HT22 cells. These two diarylheptanoids significantly reduced the overproduction of cellular peroxide in glutamate-injured HT22 cells. Moreover, these two diarylheptanoids significantly maintained antioxidative defense systems, including glutathione, glutathione reductase, and glutathione peroxidase, under glutamate-induced oxidative stress in HT22 cells.

Ethanol toxicity: rehabilitation of hepatic antioxidant defense system with dietary ginger.

The current investigation has been conducted to investigate the influence of ginger on hepatic antioxidant enzymes system in ethanol treated rats. Ethanol significantly decreased the superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione content while an increase of malondialdehyde (MDA) levels were estimated in the hepatic tissue. This effect was reversed by a treatment with 1% dietary ginger for 4 weeks in rats by improved antioxidant status which suggest that treatment of ginger may have protective role against the ethanol induced hepatotoxicity.

Cytosolic NADP-isocitrate dehydrogenase of pea plants: genomic clone characterization and functional analysis under abiotic stress conditions.

NADPH is an essential electron donor in numerous biosynthetic and detoxification reactions. In animal, yeast and bacteria, the NADP-dependent isocitrate dehydrogenase (NADP-ICDH), which catalyzes the production of NADPH, is being recognized as an essential component of the antioxidative defence mechanisms. In plant cells, there is little information on the antioxidant properties of NADP-ICDH. Using a pea cDNA lambdagt11 library, the full-length cDNA of a NADP-ICDH was obtained. In pea leaves, the analyses of activity, protein and transcript expression of NADP-ICDH under six different abiotic stress conditions (CL, continuous light, HLI, high light intensity, D, continuous dark, LT, low-temperature HT, high-temperature and W, mechanical wounding) revealed a differential regulation at transcriptional and post-translational level depending on the abiotic stress. The activity and protein expression of NADP-ICDH and catalase increased only under HLI but the NADP-ICDH transcripts were up-regulated by cold stress (70%) and W (40%). Under the same conditions, the transcript analysis of glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and ascorbate peroxidase (APX), key components of the antioxidative ascorbate-glutathione cycle, showed similar inductions. These data indicate that in pea plants the cytosolic NADP-ICDH shows a differential response, at mRNA and activity level, depending on the type of abiotic stress and suggests that this dehydrogenase could have a protective antioxidant role against certain environmental stresses in plants.

Upregulation of endogenous glutathione system by 3H-1,2-dithiole-3-thione in pancreatic RINm5F beta-cells as a novel strategy for protecting against oxidative beta-cell injury.

This study was undertaken to investigate the inducibility of glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) by 3H-1,2-dithiole-3-thione (D3T) in beta-cells, and the resultant cytoprotection against oxidant injury. Incubation of the insulin-secreting RINm5F cells with D3T led to significant induction of GSH, GR and GPx. D3T-mediated induction of GSH was abolished by buthionine sulfoximine (BSO), suggesting a critical involvement of gamma-glutamylcysteine ligase (gammaGCL). Consistently, incubation of RINm5F cells with D3T resulted in increased expression of gammaGCL protein and mRNA. Pretreatment of RINm5F cells with D3T provided remarkable protection against oxidant-elicited cytotoxicity. On the other hand, depletion of cellular GSH by BSO sensitized RINm5F cells to oxidant injury. Furthermore, cotreatment of RINm5F cells with BSO to reverse D3T-mediated GSH induction abolished the cytoprotective effects of D3T on oxidant injury. Taken together, this study demonstrates that upregulation of glutathione system by D3T is effective for protecting against oxidative beta-cell injury.

[Effects of deltamethrin on gene expression of some antioxidase, gamma glutamylcysteine synthetase and NFE2 related factor 2 (Nrf2) in brain tissue].

OBJECTIVE: To study the effects of deltamethrin (DM) on the mRNA expression of copper-zinc dependent SOD (CuZn-SOD), glutathione reductase (GR) and gamma glutamylcysteine synthetase (gamma-GCS) light subunit (GCSl), as well as on expression of both mRNA and protein of gamma-GCS heavy subunit (GCSh) and NFE2 related factor 2 (Nrf2) in cerebral cortex and hippocampus of rats. METHODS: Eighteen Wistar male rats were randomizedly divided into three groups, six for each group. The low dosage and high dosage DM treated groups were administrated intraperitoneally with DM (the daily dosage was 3.125, 12.500 mg/kg BWT respectively) for five consecutive days while the control group was administered intraperitoneally with olive oil. The relative amount of mRNA expression of these genes was measured by the method of reverse transcription polymerase chain reaction (RT-PCR) (n = 6). The protein level was detected by the method of immunohistochemistry and image analysis system (n = 4). RESULTS: There was no change in mRNA expression level of CuZn-SOD, GR, GCSh and Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administrated with DM. However, the mRNA level of GCSl gene in cerebral cortex of high dosage group as well as in both cerebral cortex and hippocampus of the low dosage group was significantly lower than that in corresponding tissue in the control group, and was decreased to 71.1%, 63.6% and 75.2% of mRNA level of corresponding tissue in the control group (P < 0.01). There was no obvious effect on protein level of both GCSh and Nrf2 in CA1, CA2, CA3 and dentate gyrus (DG) of hippocampus as well as on that in cerebral cortex in rats treated with DM. CONCLUSION: Under the experimental conditions, there is no obvious effect in the mRNA expression level of CuZn-SOD, GR gene, as well as on expression of both mRNA and protein of Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administered with DM. DM depresses the mRNA expression of GCSl gene, but does not affect the mRNA expression of GCSh gene.

Antioxidant enzymes and tissue regeneration in Eurythoe complanata (Polychaeta: Amphinomidae) exposed to used vehicle crankcase oil.

Polychaetes, Eurythoe complanata, from the Gulf of Cariaco,Venezuela, were exposed to 0.3, 1.6, and 3.3% water-soluble fraction (WSF) of used crankcase oil during 15 and 21 days. The antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), catalase (CAT), and glutathione-S-transferase (GST) were assayed in the body wall tissue. Furthermore, after chemical exposure, the polychaetes were cut into equal halves; then wound healing and the number of regenerated body segments were recorded periodically. GST activity was affected by all the experimental treatments, with activity increasing with WSF concentrations. GPx activity was altered for the contamination period. GR and CAT activities rose in response to increasing WSF concentrations, and were higher for long-term than for short-term exposures. The wound healing of the transected body regions was retarded by WSF exposure. WSF affected the tissue regeneration, which was almost abolished at 3.3% WSF. The exposure period did not affect the tissue-repairing responses. Alteration of GST in contaminated organisms suggested equivalent changes in detoxication of bioaccumulated organic contaminants. The variation of GR and CAT suggests induction of oxidative stress that could reduce the ability of WSF-exposed worms to repair damaged tissue.

Effects of dietary selenium on post-ischemic expression of antioxidant mRNA.

Cardiac ischemia reperfusion leads to oxidative stress and poor physiological recovery. Selenium deficiency down-regulates thioredoxin reductase (Txnrd) and glutathione peroxidase (Gpx) activity, impairing recovery from ischemia-reperfusion. Furthermore, selenium supplementation has been shown to be cardioprotective and lessens oxidative stress in reperfused rat hearts. In this study we have investigated the role of selenium in the mRNA expression of these, and related antioxidant proteins, post ischemia-reperfusion. Male rats were fed varying doses of selenium for five weeks. Hearts were isolated and perfused using the Langendorff method with 22.5 min of global ischemia and 45 min reperfusion. RNA was extracted for quantitative real-time PCR analysis of glutathione peroxidase (Gpx)-1 and 4, glutathione reductase (Gsr), thioredoxin peroxidase-2 (Prdx2), thioredoxin (Txn) and thioredoxin reductase (Txnrd)-1 and 2 gene expression. Selenium deficiency produced significant reductions in Gpx-1, Gpx-4, Prdx2, Txnrd-1 and Txnrd-2 expression. Conversely, selenium supplementation of 1000 microg/kg significantly up-regulated Gpx-1, Gpx-4, Txn, Txnrd-1 and Txnrd-2 transcription. Our results show selenium modulates the cardiac mRNA expression of thioredoxin and glutathione related enzymes post ischemia-reperfusion, and impacts on tolerance to ischemia-reperfusion.

Enhanced gamma-glutamyl transpeptidase expression and superoxide production in Mpv17-/- glomerulosclerosis mice.

Recently, gamma-glutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17-/- cells, enzyme activity and mRNA expression (examined by quantitative RT-PCR) of membrane-bound gamma-glutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4-benzoic acid)porphyrin, the activities of gamma-glutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of gamma-glutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

Production of functional human selenocysteine-containing KDRF/thioredoxin reductase in E. coli.

In a previous study, we reported the isolation of a cDNA encoding KDRF (KM-102-derived reductase like factor) from the human bone marrow-derived stromal cell line KM-102. Analysis of the sequence of this cDNA revealed it to be the previously reported human thioredoxin reductase cDNA. Human thioredoxin reductase, which was recently isolated from human lung adenocarcinoma NCI-H441 cells as a selenocysteine-containing selenoprotein, and its substrate thioredoxin are thought to be essential for protecting cells from the damage caused by reactive oxygen species. To obtain the selenocysteine-containing recombinant KDRF/thioredoxin reductase, we introduced a secondary structure, which is identical to the selenocysteine insertion signal of Escherichia coli formate dehydrogenase H mRNA, downstream of the TGA in the KDRF/thioredoxin reductase cDNA and expressed it in E. coli. As a result, a significant amount of selenocysteine was incorporated into the C-terminus of the KDRF/thioredoxin reductase protein. The selenocysteine-containing KDRF/thioredoxin reductase showed reducing activities toward human and E. coli thioredoxin, whereas non-selenocysteine-containing KDRF/thioredoxin reductase showed no enzyme activity. Our results suggest that this strategy will be applicable to the production of other mammalian selenocysteine-containing selenoproteins in E. coli.

Role of antioxidant enzyme expression in the selective cytotoxic response of glioma cells to gamma-linolenic acid supplementation.

We hypothesized that the cytotoxic effect of GLA observed in glioma but not normal glial cells reflects differences in GLA metabolism and/or antioxidant enzyme levels between these cells. The PUFA content of unsupplemented glioma cells was approximately 50% of that seen in unsupplemented astrocytes. Supplementation with 20 microM GLA for 24 h led to a 230 and 22% increase in glioma and astrocyte PUFA content, respectively, such that both supplemented cell types contained similar levels of PUFA. No major differences were seen in terms of GLA metabolites retained in the cells or secreted into the media following incubation with [(3)H]-GLA. No significant differences were observed in activity of MnSOD or CuZn-SOD between the cells. However, CAT and GPx activity in the glioma cells was significantly higher and lower, respectively, than observed in normal astrocytes. GLA supplementation resulted in a significant increase in CAT activity in normal astrocytes; glioma CAT activity was unchanged. No significant change was seen in the other antioxidant enzymes following GLA supplementation. These results suggest that the cytotoxic effect of GLA on glioma cells reflects both increased PUFA content and an inability to upregulate CAT.

Enhancement of antioxidant and phase II enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention.

Following the oral feeding of a polyphenolic fraction isolated from green tea (GTP) in drinking water, an increase in the activities of antioxidant and phase II enzymes in skin, small bowel, liver, and lung of female SKH-1 hairless mice was observed. GTP feeding (0.2%, w/v) to mice for 30 days significantly increased the activities of glutathione peroxidase, catalase, and quinone reductase in small bowel, liver, and lungs, and glutathione S-transferase in small bowel and liver. GTP feeding to mice also resulted in considerable enhancement of glutathione reductase activity in liver. In general, the increase in antioxidant and phase II enzyme activities was more pronounced in lung and small bowel as compared to liver and skin. The significance of these results can be implicated in relation to the cancer chemopreventive effects of GTP against the induction of tumors in various target organs.

Differential induction of glutathione transferase isoenzymes of mice stomach by diallyl sulfide, a naturally occurring anticarcinogen.

Diallyl sulfide (DAS), an organosulfur compound identified as the flavor component in garlic, has been shown to inhibit chemically induced neoplasia of forestomach and lung in mice. Even though the exact mechanism(s) of anti-neoplastic activity of DAS is not known, several independent studies suggest that this effect may, at least in part, be due to the elevation of glutathione-S-transferase (GST) activity. To gain further insight into the mechanism(s) of anti-carcinogenic activity of DAS, we have determined effect of orally administered DAS (25, 50 and 75 mumol) on levels of alpha, mu and pi class GSTs and glutathione (GSH) peroxidase and GSH reductase activities of female A/J mice stomach. Western blotting revealed presence of alpha, mu and pi class GSTs in mice stomach. A significant increase in all the three classes of GSTs was observed in the stomach of mice treated with DAS. Maximum increase in GST alpha and pi was evident by treating the animals with 75 mumol DAS whereas maximum induction of GST mu occurred after treating mice with 50 mumol DAS. GSH peroxidase activity towards t-butyl-hydroperoxide increased in a dose-dependent fashion in the mice stomach treated with DAS. Even though this activity towards hydrogen peroxide was similar in mice treated with 50 or 75 mumol DAS, these values were significantly higher than that of the control. GSH reductase was also elevated in the stomach of mice treated with 75 mumol DAS. These results suggest that DAS may exert anti-neoplastic effect by modulating GSH dependent detoxification enzymes.

Induction of ascorbate peroxidase and glutathione reductase activities by interactions of mixtures of air pollutants.

The response of ascorbate peroxidase and glutathione reductase activities in peas (Pisum sativum var. Waverex) was investigated after three weeks of exposure to mixed fumigations with SO2, NO2 and O3 (0.050 parts per million each) and increasing concentrations of O3 (0-0.150 parts per million). The results show that plants respond similarly to a high concentration (0.150 parts per million) of a single air pollutant (ozone) and to mixtures of air pollutants (SO2, NO2 and O3) when individual concentrations are low (0.050 parts per million each). In both cases, levels of ascorbate peroxidase and glutathione reductase activities were approximately twice those to be found in plants grown in charcoal-filtered air (p less than 0.01).

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

Influence of dietary selenium on the hepatic and pulmonary enzymes in polychlorobiphenyls-treated rats.

One-month-old male Sprague-Dawley rats maintained for 19 weeks on a low selenium diet with or without supplementation of 2.0 ppm selenium were injected intraperitoneally with either 500 mg PCB (Aroclor 1254)/kg body weight or placebo 5 days prior to sacrifice. In addition to aryl hydrocarbon hydroxylase (AHH) activity, PCB treatment also caused a significant increase in hepatic levels of thiobarbituric acid reactants (TBAR), reduced glutathione (GSH), GSH-peroxidase, GSH reductase, glucose-6-phosphate dehydrogenase (G-6-PD), and GSH-S-transferase in rats on the low selenium diet. The non-selenium-dependent form of GSH peroxidase was mainly responsible for the increase of hepatic GSH peroxidase upon PCB treatment. Only the activities of AHH, GSH-S-transferase, and G-6-PD were significantly higher in the liver of PCB-treated rats fed the selenium-supplemented diet. In contrast, except for AHH activity, the lung GSH and related enzymes were not significantly affected by PCB in either of the two dietary groups. The results suggest that dietary selenium deprivation renders the livers of rats more sensitive to PCB effects.