Dietary glutathione supplementation attenuates oxidative stress and improves intestinal barrier in diquat-treated weaned piglets.

The aim of this study was to investigate the protective effects of glutathione (GSH) against oxidative stress and intestinal barrier disruption caused by diquat (an oxidative stress inducer) in weaned piglets. Twenty-four piglets were randomly assigned to four treatments with six pigs per treatment for an 18-d trial. Treatments were basal diet, basal diet + diquat challenge, 50 mg/kg GSH diets + diquat challenge and 100 mg/kg GSH diets + diquat challenge. On day 15, piglets in basal diet group and diquat-challenged groups were intraperitoneally injected with sterile saline and diquat at 10 mg/kg body weight, respectively. The results showed that GSH supplementation improved growth performance of diquat-injected piglets from days 15 to 18 (p < 0.05), especially at a dose of 100 mg/kg GSH. Meanwhile, diquat also caused oxidative stress and intestinal barrier damage in piglets. However, GSH supplementation enhanced the antioxidant capacity of serum and jejunum, as evidenced by the increase in GSH content and total superoxide dismutase activities and the decrease in 8-hydroxy-2'-deoxyguanosine concentrations (p < 0.05). GSH also up-regulated the mRNA expressions of intestinal tight junction protein (zonula occludens 1, ZO1; occludin, OCLN; claudin-1, CLDN1) and mitochondrial biogenesis and function (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, PGC1α; mitochondrial transcription factor A, TFAM; cytochrome c, CYCS), compared with diquat-challenged piglets in basal diet (p < 0.05). Thus, the study demonstrates that GSH protects piglets from oxidative stress caused by diquat and 100 mg/kg GSH has a better protective role.

Resveratrol protects intestinal integrity, alleviates intestinal inflammation and oxidative stress by modulating AhR/Nrf2 pathways in weaned piglets challenged with diquat.

This study investigated the effects of resveratrol (RES) on intestinal morphology, antioxidant capacity, intestinal inflammation, and barrier function in weaned piglets challenged with diquat (DIQ). Thirty weaned piglets were randomly assigned to 5 treatments: non-challenged group (CON), DIQ-challenged group (DIQ), and DIQ-challenged group with 10, 30, or 90 mg/kg of RES, respectively. The trail lasted 21 days, and piglets were intraperitoneally injected with DIQ or the same amount of saline on day 15. The results showed that supplementation with 90 mg/kg RES increased (P < 0.05) jejunal villus height and villus height: crypt depth ratio, and decreased (P < 0.05) crypt depth, plasma D-lactate and diamine oxidase (DAO) compared with the DIQ group. Piglets fed with 30 or 90 mg/kg RES prevented the diquat-induced decrease (P < 0.05) of mRNA expression of occludin, claudin-1, ZO-1, and IL-10, and increase (P < 0.05) of TNF-α mRNA expression. Moreover, addition of 90 mg/kg RES increased (P < 0.05) the activities of SOD, GSH-Px, and CAT and decreased (P < 0.05) the MDA levels in jejunal mucosa compared with the DIQ group. Finally, addition of 90 mg/kg RES enhanced (P < 0.05) the mRNA expression of SOD1, SOD2, CAT, GPx1, and HO-1, and increased (P < 0.05) mRNA and protein expression of Nrf2, NQO1, aryl hydrocarbon receptor (AhR), and cytochrome P450 family 1 member A1 (CYP1A1). These data indicated that supplementation with 90 mg/kg RES was effective in protecting the intestinal integrity, alleviating intestinal inflammation and oxidative stress by activating AhR/Nrf2 pathways in diquat-challenged piglets.

Dietary canthaxanthin reduces xanthophyll uptake and red coloration in adult red-legged partridges.

Carotenoids give color to conspicuous animal signals that are often the product of sexual selection. Knowledge of the mechanisms involved in carotenoid-based signaling is critical to understanding how these traits evolve. However, these mechanisms remain only partially understood. Carotenoids are usually viewed as scarce dietary antioxidants whose allocation to ornaments may trade off against health. This trade-off would ensure its reliability as a signal of individual quality. In the case of red (keto)carotenoids, the literature suggests that some species may show constraints in their uptake. Canthaxanthin is one of the most common ketocarotenoids in red ornaments of animals. It is often commercially used as a dietary supplement to obtain redder birds (e.g. poultry). We increased the dietary canthaxanthin levels in captive red-legged partridges (Alectoris rufa). This species shows red non-feathered parts mostly pigmented by another common ketocarotenoid: astaxanthin. We studied the impact on the uptake of carotenoids and vitamins and, finally, on coloration. We also tested the potential protective effect of canthaxanthin when exposing birds to a free radical generator (diquat). Canthaxanthin did not apparently protect birds from oxidative stress, but interfered with the absorption of yellow carotenoids (lutein and zeaxanthin). Zeaxanthin is a precursor of astaxanthin in enzymatic pathways, and their levels in tissues and eggs were lower in canthaxanthin-supplied birds. This led to lower astaxanthin levels in ornaments and paler coloration. As far as we know, this is the first report of a carotenoid supplementation decreasing animal coloration. The results have implications for understanding carotenoid-based signaling evolution, but also for improving husbandry/experimental procedures.

The Evaluation of Antioxidant and Anti-Inflammatory Effects of Eucommia ulmoides Flavones Using Diquat-Challenged Piglet Models.

This study was designed to evaluate the antioxidant and anti-inflammatory effects of Eucommia ulmoides flavones (EUF) using diquat-challenged piglet models. A total of 96 weaned piglets were randomly allotted to 1 of 3 treatments with 8 replication pens per treatment and 4 piglets per pen. The treatments were basal diet, basal diet + diquat, and 100 mg/kg EUF diet + diquat. On day 7 after the initiation of treatment, the piglets were injected intraperitoneally with diquat at 8 mg/kg BW or the same amount of sterilized saline. The experiment was conducted for 21 days. EUF supplementation improved the growth performance of diquat-treated piglets from day 14 to 21. Diquat also induced oxidative stress and inflammatory responses and then impaired intestinal morphology. But EUF addition alleviated these negative effects induced by diquat that showed decreasing serum concentrations of proinflammatory cytokines but increasing antioxidant indexes and anti-inflammatory cytokines on day 14. Supplementation of EUF also increased villi height and villous height, crypt depth, but decreased the histopathological score and MPO activity compared with those of diquat-challenged pigs fed with the basal diet on day 14. Results indicated that EUF attenuated the inflammation and oxidative stress of piglets caused by diquat injection.

Critical Role of FoxO1 in Granulosa Cell Apoptosis Caused by Oxidative Stress and Protective Effects of Grape Seed Procyanidin B2.

Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.

Knockout of cellular glutathione peroxidase gene renders mice susceptible to diquat-induced oxidative stress.

Two experiments were conducted to determine the protection and the underlying mechanisms of cellular glutathione peroxidase (GPX1) against lethal, acute oxidative stress induced by an intraperitoneal injection of 24 mg diquat/kg body weight. In experiment 1, mortality and survival times were compared among selenium (Se)-adequate or deficient GPX1 knockout mice [GPX1(-/-)] and wild-type mice (WT). In experiment 2, mice from these four groups were euthanized at 0, 1, 2, and 3 h after the injection of diquat to elucidate the time course of oxidative events. The stress produced 100% mortality in all of the groups except for the Se-adequate WT, which were euthanized on day 7 for analysis. The Se-deficient WT and the Se-adequate GPX1(-/-) had similar survival times (4.1 and 3.9 h), which were longer (p < .05) than that of the Se-deficient GPX1(-/-) (2.4 h). However, these three GPX1-deficient groups had higher levels (p < .05) of hepatic F2-isoprostanes and carbonyl contents and/or plasma alanine aminotransferase activities than those of the Se-adequate WT. The diquat-induced formations of hepatic F2-isoprostanes in these animals peaked at 1 h and preceded the rise of plasma alanine aminotransferase in the Se-adequate GPX1(-/-). Responses of hepatic superoxide dismutase activities to the diquat treatment were affected by the GPX1 level. In conclusion, GPX1 is the major selenoprotein to protect mice against the lethal oxidative stress induced by diquat.

Excretion of F2-isoprostanes in bile: a novel index of hepatic lipid peroxidation.

Lipid peroxidation is believed to be an important mechanism of liver injury caused by some xenobiotics. However, it has been difficult to demonstrate and quantify this process in vivo. Moreover, little is known about the disposition of lipids oxidized in the liver. F2-isoprostanes are prostanoids produced by nonenzymatic free radical-catalyzed peroxidation of arachidonic acid esterified to phospholipids. Hydrolysis of F2-isoprostanes from phospholipids by phospholipases yields free F2-isoprostanes. Excretion of F2-isoprostanes, both free and esterified to phospholipids, was measured in bile after administration of CCl4. The concentration of lipid-esterified F2-isoprostanes in bile exceeded that of free F2-isoprostanes. CCl4 caused a dose-dependent increase in biliary F2-isoprostane excretion that correlated better with the increase in liver F2-isoprostanes than it did with the increase in plasma F2-isoprostanes. Pretreatment with colchicine ameliorated CCl4-liver injury but did not affect baseline or CCl4-induced biliary F2-isoprostane excretion. Administration of diquat to selenium-deficient rats, which causes hepatic and renal necrosis, was associated with a 13-fold elevation of plasma F2-isoprostanes. However, both hepatic F2-isoprostane concentrations and biliary F2-isoprostane excretion were increased only threefold. These data suggest that quantification of F2-isoprostane excretion in bile may provide a sensitive and quantitative index of hepatic lipid peroxidation.

A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism.

Increasing attention has focused on the role of free radicals derived from oxygen in the pathophysiology of a wide variety of disorders. One of the well-recognized targets of free radical-induced injury is peroxidation of lipids. Using a variety of approaches, we have found that a series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase mechanism involving free radical-catalyzed peroxidation of arachidonic acid. Levels of these compounds in normal human plasma and urine range from 5 to 40 pg/ml and 500 to 4000 pg/mg of creatinine, respectively. In rats, their formation was found to increase as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of CCl4 and diquat. To explore whether these prostanoids can exert biological activity, the effects of one of the compounds formed by this mechanism, 8-epi-prostaglandin F2 alpha, was examined in the kidney in the rat. Infusion of 8-epi-prostaglandin F2 alpha into a peripheral vein (5 micrograms/kg per min) or intrarenally (0.5-2.0 micrograms/kg per min) resulted in marked parallel reductions in renal blood flow and glomerular filtration rate. That the formation of these prostanoids is catalyzed by free radicals and that they can exert potent biological activity suggest that these prostanoids may participate as pathophysiological mediators in oxidant injury. Quantification of these compounds may also provide a noninvasive approach to assess oxidant status in humans. That the formation of these prostanoids occurs independent of the catalytic activity of the cyclooxygenase enzyme suggests that there may be limitations at times regarding the reliability of the use of cyclooxygenase inhibitors to assess the role of prostaglandins in certain pathophysiological processes.

gamma-Glutamyltranspeptidase-positive rat hepatocytes are protected from GSH depletion, oxidative stress and reversible alterations of collagen receptors.

The aim of this study has been to define cytotoxic mechanisms that may cause clonal expansion in the liver of pre-carcinogenic cells. An in vitro model, which has been described previously, was used. Hepatocytes were isolated from carcinogen-treated rats and a high proportion of the cells were gamma-glutamyltranspeptidase (GGT)-positive. The cells were incubated in suspension and exposed to toxic agents in concentrations that induced a moderate increase in cellular leakage within 3 h. Samples were withdrawn and sampled cells were then allowed to attach to collagen-coated plates. Attached cells were stained and the ratio of GGT-positive/GGT-negative cells (GGT-ratio) was determined. The initial GGT-ratio was 10.4 +/- 4.7% and an increased ratio was taken as a sign of toxicity that resulted in a selection of GGT-positive cells. In a first series of experiments it was shown that hydroquinone and menadione increase the GGT-ratio, while diquat, sodium selenite, diethyl maleate or phorone do not. However, diethyl maleate in combination with diquat increased the GGT-ratio. Hydrogen peroxide (5 mM) increased the GGT-ratio as effectively as hydroquinone (0.3 mM). Lower concentrations of H2O2 (0.05 mM) increased the GGT-ratio in GSH-depleted cells. The changes induced by hydroquinone and H2O2 in low concentration were reversible. In another series of experiments, plates coated with antibodies against beta 1-integrin were used. An increase in the GGT-ratio was obtained with anti beta 1-integrin, but not with broad spectrum anti-rat hepatocyte or anti-rat beta 2-microglobulin antibodies as substrata. These data suggested an involvement of the beta 1-integrin in the selection. Taken together, these data indicate that GGT-positive hepatocytes are protected against GSH depletion and oxidative stress that may result in reversible receptor alterations.

N-acetylcysteine and glutathione-dependent protective effect of PZ51 (Ebselen) against diquat-induced cytotoxicity in isolated hepatocytes.

The glutathione peroxidase (GSH-Px)-like reduction of H2O2 by the selenoorganic compound 2-phenyl-1,2-benzoisoselenazol-3(H)-one (PZ51: Ebselen) was studied using glutathione (GSH) and the therapeutic agent N-acetylcysteine (NAC) to provide reducing equivalents. In a purely chemical system containing H2O2 and in an enzymatic system of glucose/glucose oxidase-generated H2O2 Ebselen alone did not reduce H2O2. Ebselen in combination with either GSH (1 mM) or NAC (1 mM) was capable of reducing H2O2 in both systems. In these non-cellular systems GSH was a more effective source of reducing equivalents than NAC. The GSH-Px-like activity of Ebselen was further investigated in a cellular system. The redox-cycling bipyridylium compound diquat generates active oxygen species, depletes intracellular glutathione, and is cytotoxic in isolated hepatocytes pretreated with the glutathione reductase inhibitor 1,3-bis(Z-chloro-ethyl)-1-nitrosourea (BCNU). Ebselen alone did not ameliorate diquat cytotoxicity, but in combination with either GSH (1 mM) or NAC (1 mM) it produced a significant delay in diquat-induced cytotoxicity. Further additions of either GSH (0.5 mM) or NAC (0.5 mM) at 30 min intervals provided significantly more protection against diquat-induced cytotoxicity and intracellular GSH depletion than the single 1 mM addition. Thus, the combination of Ebselen and NAC may provide an effective antidote in cases of overexposure to bipyridylium herbicides, such as diquat and paraquat.