Influence of selenium sources on age-related and mild heat stress-related changes of blood and liver glutathione redox cycle in broiler chickens (Gallus domesticus)

Selenium is an essential trace element that up-regulates a major component of the antioxidant defense mechanism by controlling the body's glutathione (GSH) pool and its major Se-containing antioxidant enzyme, glutathione peroxidase (GPX). Evidence has emerged suggesting that organic selenium, natural seleno-amino acids found in plants, grains and selenized yeast, maintains the antioxidant defense system more efficiently than inorganic selenium. Inorganic selenium is a pro-oxidant, whereas organic selenium possesses antioxidant properties itself. As a pro-oxidant, inorganic selenium is not suitable for animals or humans. Therefore, we examined the GSH–GPX system in broiler chickens and determined that organic selenium was indeed more beneficial than inorganic selenium. Chickens fed the organic selenium as Sel-Plex^®, a selenized yeast, had elevated GPX activity in both blood and liver in a thermoneutral environment and after heat distress. More importantly, the ability to reduce the oxidized glutathione (GSSG to 2 GSH) was enhanced and facilitated by maintenance of glutathione reductase activity. Organic selenium-fed chickens were less affected by mild heat distress than inorganic selenium-fed chickens, and this assessment was based upon less induction of heat shock protein 70 (hsp70) in organic selenium-fed chickens. Our results clearly show that heat distress, a potent inducer of oxidative stress and hsp70, can be partially ameliorated by feeding organic selenium. We attribute this observation to an enhanced GSH–GPX antioxidant system in organic selenium-fed chickens.     

Influence of selenium on heat shock protein 70 expression in heat stressed turkey embryos (Meleagris gallopavo)

Heat shock protein 70 (hsp70) family of proteins, which functions as molecular chaperones, has been associated with tolerance to stressors in avian species. Selenium (Se) is an essential trace mineral incorporated into the seleno-enzymes such as glutathione peroxidase (GSHpx). GSHpx reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the GSH/GSSG antioxidant system and protects cells from oxidative damage. This study was conducted to examine if the relationship between dietary supplementation of selenium to turkey (Meleagris gallopavo) hens and the embryonic expression of hsp70 and GSHpx activity in heat stressed embryos. Livers of embryos developing in eggs from turkey hens fed diets with or without supplemental Se were analyzed for hsp70 concentration and GSHpx activity before and after recovery from a heating episode. Before heat stress, hsp70 concentrations were equivalent in each treatment, but GSHpx activity was maximized in the SE treatment group. After recovery from the heating episode, hsp70 concentrations were significantly higher (P<0.05) in the non-Se-supplemented groups, but in the Se-supplemented groups the hsp70 concentrations were not different from pre-stress concentrations. In the pre-stress Se-supplemented group, liver GSHpx activity was significantly higher than GSHpx activity in the non-Se-supplemented embryo livers, and in the livers from embryos recovering from heat stress, GSHpx activity in the non-Se-supplemented group was lower than the pre-stress activity and significantly lower than the GSHpx activity in liver from Se-supplemented embryos recovering from heat distress. Se supplementation to the dams resulted in a significant increase in their embryos and that condition would facilitate a decreased incidence of oxidative damage to cells. A more reduced redox status in embryos from Se-supplemented dams decreased the need for cellular protection attributed to stress induced hsp70 and presumably allows heat distressed embryos to resume normal growth and development than embryos from dams with inadequate selenium nutrition.     

Age-related changes in the glutathione redox system

The effect of aging on the glutathione redox system was evaluated in this study. For this purpose, we determined reduced glutathione (GSH) and oxidized glutathione (GSSG) in whole blood, glutathione peroxidase (GPx) and glutathione reductase (GSSGR) in erythrocytes and selenium (Se) in plasma in 176 healthy individuals. We also calculated GSH/GSSG molar ratios. These subjects were divided into five groups: group 1 (n=25; 0.2-1 years old); group 2 (n=28; 2-11 years old); group 3 (n=23; 12-24 years old); group 4 (n=40; 25-40 years old); group 5 (n=60; 41-69 years old). GSH levels in groups 1 and 5 were significantly lower than the other groups (p<0.001). Conversely, GSSG levels were significantly high in these periods (p<0.001). The GSH/GSSG molar ratio was found to be low both in the first year of life and in the oldest group (p<0.001, respectively). GPx activity in group 5 was increased as compared to the other groups (p<0.001). GSSGR activity was significantly lower in the oldest groups than in the other groups (p<0.001). Se levels were found to be low in the oldest group (p<0.001). Selenium levels of women in group 5 were significantly high as compared to the men (p<0.01). We found negative correlations between age and GSH levels (r=0.402; p<0.001), selenium levels (r=0.454; p<0.001), GSH/GSSG molar ratio (r=0.557; p<0.001) and GSSGR activity (r=0.556; p<0.001). There were positive correlations between age and GPx (r=0.538; p<0.001) and GSSG level (r=0.551; p<0.001). In conclusion, our findings show that the glutathione redox system is affected by age. Oxidative stress increases during the aging process. There is no effect of aging on the glutathione redox system according to sex except for the Se level.     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

Response to natural and laboratory selection at the Drosophila hsp70 genes

Abstract.— To determine whether and how laboratory and natural selection act on the hsp70 (70-Kd heat-shock protein) genes of Drosophila melanogaster , we examined hsp70 allele frequencies in two sets of populations. First, five populations reared at different temperatures for more than 20 years differentially fixed both a large insertion/deletion (indel) polymorphism at the 87A7 hsp70 cluster ("56H8"/"122") and a single nucleotide polymorphism at the 87C1 hsp70 cluster. In both cases, the 18°C and 25°C populations fixed one allele and the 28°C populations the other, consistent with previously described evolved differences among these populations in Hsp70 expression and thermo-tolerance. Second, we examined 56H8 and 122 frequencies in a set of 11 populations founded from flies collected along a latitudinal transect of eastern Australia. The 56H8 allele frequencies are positively associated with latitude, consistent with maintenance of the 56H8/122 polymorphism by natural selection. Thermal extremes and average values are negatively correlated with latitude. These results suggest that natural selection imposed by temperature and thermal variability may affect hsp70 allele frequencies.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

Effect of different selenium source (sodium selenite and selenium yeast) on broiler chickens

A feeding experiment was carried out to compare the effects of supplementing a poultry meal-based diet with selenium as sodium selenite or selenium yeast on broiler chickens. Three groups with three replicates of broiler chickens (mean weight 710 ± 5.3 g) were given a basal diet either unsupplemented (control) or supplemented with 0.2 mg Se kg⁻¹ as sodium selenite (trial 1) or selenium yeast (trial 2) respectively, for 21 days. There was significant difference (P<0.05) in Feed Conversion Ratio (FCR) of trials 1 and 2 compared with the control. However, there were no significant differences (P>0.05) in FCR between trials 1 and 2. Final weight, survival rate and Daily Gain (DG) were not affected by the dietary Se source. Chickens fed the basal diet showed lower (P<0.05) selenium content in muscle, kidney, liver and pancreas compared to that fed selenium supplements (trials 1 and 2). Furthermore, trial 2 showed the highest value (P<0.05) among these treatments. However, there was no significant difference (P>0.05) in muscle selenium content of chickens between trials 1 and 2. Glutathione peroxidase (GSH-Px) activities in broiler chickens plasma and liver of all selenium treatment groups (trials 1 and 2) were significantly different (P<0.05) from that of the control. The GSH-Px activity in plasma was higher (P<0.05) in trial 2 compared with trial 1 and the control. However, there was no difference (P>0.05) in hepatic glutathione peroxidase between trials 1 and 2 although the average value of GSH-Px activity in trial 2 presented the trend of increase.     

Effect of dietary selenium concentration and duration of selenium feeding on hepatic glutathione concentrations in rats

Studies were conducted in rats to determine the effect of dietary selenium (Se) concentration on hepatic glutathione concentrations and enzyme activities associated with the maintenance of the cellular glutathione status. Male rats were fed 0.1, 3.0, or 6.0 ppm Se as Na2SeO3 for 2, 4, or 6 weeks at which time they were killed and analyses were performed. Both 3.0 and 6.0 ppm Se caused a significant dose-dependent increase in hepatic-reduced glutathione (GSH) by 4 weeks of feeding compared to 0.1 ppm Se. The increase in GSH was preceded by significant, dose-dependent increases in oxidized glutathione (GSSG) as well as the GSSG to GSH ratio. Increases in GSSG and the GSSG to GSH ratio as well as in glutathione reductase and glucose-6-phosphate dehydrogenase activities were observed by 2 weeks of high Se feeding. The current findings substantiate previous results demonstrating effects of high Se on hepatic glutathione concentrations (R. A. LeBoeuf and W. G. Hoekstra, J. Nutr. 113:845-854, 1983) and further suggest that increased cellular GSSG concentrations or the GSSG to GSH ratio caused by 3.0 and 6.0 ppm dietary Se signals for "adaptive" changes in hepatic glutathione metabolism.     

Temporal changes in tissue glutathione in response to chemical form,dose, and duration of selenium treatment

Selenium has been reported to affect glutathione (GSH) concentrations in short-term animal-feeding experiments. Given the central role that this tripeptide plays in maintaining cellular homeostasis, it was hypothesized that perturbations in glutathione metabolism induced by selenium might account for its cancer chemopreventive activity. In the present study, four experiments were conducted in which the effect of acute, short-, or long-term exposure to selenium was assessed. Selenium was provided as either sodium selenite or D,L-selenomethionine. Selenite was observed to induce a biphasic response in total liver GSH. Injected selenium caused an acute reduction in GSH, whereas short-term feeding (up to 8 wk) increased both total GSH and oxidized glutathione (GSSH), an effect that gradually diminished in magnitude with prolonged feeding. Our data suggest that such changes are unlikely to account for the chemopreventive activity of selenium for the following reasons: Perturbations in glutathione metabolism occurred only at doses of selenite that approached toxicity. These doses are higher than what would be required for producing cancer chemoprevention. The transient nature of these changes also contrasts with the need for a continuous supplementation of selenite in suppression of tumorigenesis. Furthermore, selenomethionine was found to have little activity in altering glutathione metabolism, even though it compares favorably with selenite as a cancer chemopreventive agent. Nonetheless, these findings do not discount the possibility that sulfhydryl compounds, such as glutathione, might be used to modify the toxicity and/or enhance the cancer prophylactic activity of selenium compounds.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Dietary selenium levels modulates antioxidant,cytokine and immune response and selenoproteins mRNA expression in rats under heat stress condition

BackgroundHigher environmental temperature is a major abiotic stress factor for animals and human beings. The selenium (Se) is an important trace mineral having diverse health promoting effects under stress conditions. However, studies on dietary requirement of selenium under prolonged heat stress condition are lacking. Present study discern the effect of higher dietary Se levels on antioxidant, cytokine, haemato-biochemical profile, and immune response, and the selenoproteins mRNA expression in rats under prolonged heat stress (HS) condition.MethodsWeaned Wistar rats (4 wk age; 67.6 ± 1.53 g BW; n = 72) housed under thermoneutral (TN) or HS conditions and fed with purified diets containing three graded Se levels were divided in six experimental groups. The groups were 1) TN control with 138 ppb Se (TN_CON), 2) HS control with 138 ppb Se (HS_CON), 3) TN with higher Se @ 291 ppb (TN_Se1), 4) HS with higher Se @ 291 ppb (HS_Se1) 5) TN with higher Se @ 460 ppb (TN_Se2), 6) HS with higher Se @ 460 ppb (HS_Se2). Rats in all the six groups were maintained in TN environmental conditions (57.3 ± 0.22 temperature humidity index; THI) for initial 28 days period. Subsequently, rats of HS groups were exposed to 77.0 ± 0.11 THI for 6 h/d in a psychrometric chamber for last fourteen days.ResultsHigher dietary Se (291 and 460 ppb) significantly improved the blood hemoglobin concentration and reduced serum alanine aminotransferase activity of rats under HS conditions. The serum triiodothyronine and insulin levels were significantly higher in high dietary Se groups irrespective of the environmental conditions. Similarly, the serum reduced glutathione levels, and catalase and superoxide dismutase enzyme activity were increased and malondialdehyde levels were reduced in high dietary Se groups irrespective of stress conditions. The glutathione peroxidase (GPx) activity was significantly higher in 460 ppb dietary Se groups as compared to other groups. The serum pro-inflammatory cytokine interleukin (IL)− 1 was declined, whereas the anti-inflammatory cytokine IL-10 level was increased in high dietary Se fed rats under both HS and TN conditions with 460 ppb dietary Se groups showing pronounced effects. Further, there was heat stress- and dietary Se level dependent- up regulation in hepatic GPx and iodothyronine deiodinase-II mRNA expression and similar pattern was noticed in hepatic thioredoxin reductase mRNA expression. The selenoprotein-P mRNA expression was up regulated in 460 ppb Se fed HS group as compared to CON and Se1_C groups. High dietary Se improved the humoral immune response 7d after antigen inoculation under HS conditions whereas cell-mediated immune response was augmented in rats fed higher Se under TN condition.ConclusionIt is concluded that under prolonged heat stress conditions the dietary requirement of Se may be increased to 460 ppb for improving the antioxidant status and humoral immune response, cytokine levels, modulating the thyroid and insulin hormone, and the selenoproteins mRNA expression of rats.     

Effect of selenium deficiency on the antioxidative status and muscle damage in growing turkeys

An experiment investigated the effect of different selenium supplementations on the antioxidant defence system and on the occurrence of muscle dystrophy in growing turkeys. Newly hatched male turkeys (B.U.T. Big 6) were divided into eight groups of 18 turkeys each and fed either a basal diet (selenium <0.010 mg/kg diet), or the basal diet supplemented with 0.10; 0.15; 0.20; 0.25; 0.30; 0.35 or 0.40 mg selenium/kg diet in the form of sodium selenate. Vitamin E was adequately supplemented in all diets. After 35 days, muscle damage parameters including aspartate aminotransferase, creatine kinase, creatine kinase M and B were significantly increased in the selenium deficient Group I. A significant reduction of weight gain, feed consumption and selenium dependent glutathione peroxidase activity was also observed in the liver of selenium deficient birds. The ratio of oxidised glutathione (GSSG) to total glutathione (tGSH) was substantially altered in the selenium deficient Group I as well as in Group II (0.10 mg selenium/kg feed). The activity of glutathione reductase (GR) and glutathione-S-transferase (GST) was not affected by selenium deficiency.     

Effect of ascorbic acid and acute heat exposure on heat shock protein 70 expression by young white Leghorn chickens

Dietary ascorbic acid (AA) and heat stress (HS) affect heat shock protein 70 (hsp70) and body temperature (BT) of strain cross white Leghorn chickens. At five weeks of age, chicks fed either no supplemental AA (N-AA) or 200 mg/kg AA (AA) were subjected to either HS (42 degrees C) or maintained in control (CN, 23 degrees C) ambient temperature (T(a)) for 1 h. Body temperature (BT) was recorded for each bird before collection of heart and liver for hsp70 assay. In the CN AA-fed groups, neither the lower constitutive hsc70 nor the decreased hsp70 response to HS in the heart and liver were sex-dependent. The BT was increased by HS, but neither AA nor sex of the bird affected BT response. A diet X T(a) interaction revealed that BT of CN AA-fed chickens was lower than in N-AA-fed chickens, but BT of HS AA-fed chicks was greater than BT in HS N-AA-fed chickens. The BT and hsp70 responses were positively correlated. A lower expression of hsp70 indicated less of a stress response in the AA-fed chickens.     

Influence of R genome on the nutritional value of triticale for broiler chicks

The following grain characteristics: protein, arabinoxylan and dietary fibre content, viscosity and water holding capacity of wheat, rye and triticale of different ploidy levels were studied as to their effect on body weight gain (BWG), feed to gain ratio (FCR), apparent metabolizable energy (AME_n), dry matter digestibility (DMD) and apparent protein retention (APR) in young broiler chicks fed isograin and isoprotein diets based on these cereals. Highly significant correlations (p≤0.01) were found between physicochemical and biological quality indicators when all cereals were taken into account. A negative response of chicks to triticale was obtained only when chicks were fed diets containing the tetraploid forms, while the nutrition parameters of chicks fed diets containing the octo- and hexaploid triticale, with rye genome shares of 1 : 3 and 1 : 2, did not differ (p≥0.05) from those fed a wheat diet. Rye diets yielded the lowest BWG, AME_n and DMD and the poorest FCR. The results indicate that as long as the share of the R genome is a minor component of the total triticale genome pool, its antinutritional effect is masked by the wheat genome. The results also indicate that hexaploid triticale can constitute the sole cereal component in the diets of young broiler chicks.     

Effect of cholesterol and 7beta-hydroxycholesterol on glutathione status and expression of Hsp70 in cultured murine peritoneal macrophages

Using cultured murine peritoneal macrophages, the change in redox ratio (oxidized/reduced glutathione) was studied at different incubation intervals (6, 12, 18 and 24 hr) with different concentrations (2.5, 5 and 7.5 microg/ml) of cholesterol and 7beta-hydroxycholesterol (7beta-OH), using fluorimeter. The changes in the levels of heat shock protein, hsp70 was determined using ELISA. Both cholesterol/7beta-OH caused a decrease in hsp70 protein levels at all the incubation intervals in dose dependent manner but the decrease was significantly higher with 7beta-OH. Treatment with 7beta-OH also resulted in significantly increased levels of oxidized glutathione (GSSG) and decreased reduced glutathione (GSH) while cholesterol showed no effect on GSSG levels. Moreover, GSH levels were lowered only at the highest concentration (7.5 microg/ml) at longer incubation intervals (18 and 24 hr) with cholesterol exposure. This altered the redox status in both cholesterol/7beta-OH treated macrophages. These results suggest that cholesterol and more likely 7beta-OH may exert their pro-atherogenic effects by lowering hsp70 protein production and inhibiting glutathione synthesis by macrophages present in the arterial wall.