Se-dependent Glutathione Peroxidase Research Articles

Developmental pattern of reactive oxygen species generation and antioxidative defense machinery in rat cerebral microvessels

The developmental profile of certain enzymatic antioxidants as well as the generation of reactive oxygen species was studied in the rat cerebral microvessels during first three weeks of life and the levels were compared to those present in adults. The data showed a higher generation of superoxide anion (+67%) and H2O2 (+200%) at postnatal day (PND) 21. Superoxide anion production was significantly lower (−24%) at PND 14 and almost comparable to adult values at PND 7. The activity of superoxide dismutase increased with development and attained an adult level at PND 21. Catalase was higher in neonates with a maximum activity at PND 7 and 14 (+68, 69%). The measurement of microvessel glutathione and glutathione-related antioxidant enzymes showed that glutathione level was higher at PND 7, which declined to an adult level at PND 14. Se-dependent GPx showed a marked increase between PND 14 and 21, however, it declined in adults. The activity of Se-independent glutathione peroxidase was very low in cerebral microvessels. Glutathione reductase activity in 7-day-old, that was comparable to adult level, declined at PND 14 and 21. The level of glutathione S-transferase was higher (+43%) at PND 21. The activity of microvessel marker enzyme γ-glutatmyl transpeptidase increased with age, whereas, alkaline phosphatase showed a slight increase up to PND 14 and thereafter it declined. Lipid peroxidation was found to be significantly lower (−18%) at PND 21 as compared to adults. It may be concluded that developing cerebral microvessels contain high levels of several antioxidant enzymes that are more or equal to those present in adult brain microvessels.

Biomarkers of oxidative stress and genotoxicity in livers of field-collected brown bullhead, Ameiurus nebulosus.

The objective of this investigation was to determine whether differences in a suite of biomarker assays in brown bullhead liver tissues could be detected and related to the pollution histories of two Ohio locations, one a reach of the Black River that had historically been severely impacted by the effluents of a coking plant, the other at Old Woman Creek, a freshwater estuarine research preserve. There were no gross differences in pathologies detectable in fish from either site, and the major difference found in bullheads at the two sites was in the relative liver weight (RLW). Differential responses of glutathione reductase, glutathione-S-transferase, Se-dependent glutathione peroxidase, Se-independent glutathione peroxidase, and oxidized glutathione have been reported between fish from contaminated and uncontaminated sites in other studies, but no such differences were observed in the present study. Of the oxidative stress biomarkers included in this investigation, only the responses of superoxide dismutase, catalase, and total glutathione appeared to correlate with environmental exposure of brown bullhead to polycyclic aromatic hydrocarbons. Results with single-strand DNA and ethoxyresorufin-O-deethylase were the reverse of what has been reported in most other studies, and may reflect adaptation of the fish at the previously highly contaminated site. The fish at the Black River site appear to have responded to the xenobiotics present in their environment by increasing their overall liver size, thereby increasing the overall amount of enzymes rather than altering the specific activity of a select set of protective enzymes.http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p236.html

Oxidative stress and changes in liver antioxidant enzymes induced by experimental dicroceliosis in hamsters.

The purpose of our study was to assess the effects of experimental dicroceliosis on the antioxidant defense capability of the liver in hamsters. Studies were carried out at 80 and 120 days after infection with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. The parasitic pathology was ascertained by the presence of fluke eggs in feces, increased serum ALT and AST activities, and histological findings. The concentration of thiobarbituric acid-reactive substances (TBARS) and the ratio of oxidized to reduced glutathione (GSSG/GSH), measured as markers of oxidative stress, were significantly increased [TBARS: +40% and +84% at 80 and 120 days postinfection (p.i.), respectively; GSSG/GSH: +200% and +117%]. Dicroceliosis increased Se-dependent glutathione peroxidase (GPx) activity in both cytosol (+24% and +46%) and mitochondria (+73% and +41%). Superoxide dismutase activity was significantly reduced in cytosol (-19% and -38%) and mitochondria (-20% and -39%). No significant change was found in the activity of Se-independent GPx or catalase. The ratio of glutathione peroxidase/glutathione reductase at 80 and 120 days p.i. was increased by 25% and 63%, respectively. Gamma-glutamyl cysteinyl synthetase activity was increased by 27% and 20%, respectively. Our data indicate that although dicroceliosis courses with activation of antioxidant enzymes and glutathione synthesis, inefficient scavenging of reactive oxygen species takes place, resulting in oxidative liver damage.

Binding of tellurium to hepatocellular selenoproteins during incubation with inorganic tellurite: consequences for the activity of selenium-dependent glutathione peroxidase

The metallic group XVIa elements selenium and tellurium possess remarkably similar chemical properties. However, unlike selenium, tellurium is not an essential micronutrient and, indeed, induces both acute and chronic toxicity in a variety of species. Despite this, very little is known of the molecular mechanisms of toxicity of tellurium, particularly with respect to potential chemical interactions with selenium-containing components in the cell. In this work we describe a novel interaction of inorganic tellurite with hepatocellular selenoproteins, particularly with selenium-dependent glutathione peroxidase. The accumulation of ( 121Te)-tellurite into cultured primary rat liver hepatocytes was shown to be much more rapid than that of ( 75Se)-selenite on a molar basis. Neither the uptake of ( 121Te)-tellurite nor of ( 75Se)-selenite was affected by a large molar excess of the unlabelled counterpart, respectively. Interestingly, separation of the hepatocellular proteins on continuous pH denaturing gels demonstrated clear binding of radiolabelled tellurium to a number of protein bands, including one at 23 and one at 58 kDa, which corresponded to proteins readily labelled in cells treated with ( 75Se)-selenite. The binding of ( 121Te) to these proteins was insensitive to reduction with mercaptoethanol and not affected by pre-treatment of the cells with cycloheximide. When purified selenium-dependent glutathione peroxidase was treated directly with ( 121Te)-tellurite, the protein became labelled in an analogous manner to that achieved in intact cells. This was not affected by coincubation of the enzyme with ( 121Te)-tellurite and one or both of its substrates. Additionally, incubation of the peroxidase with tellurite effectively inhibited its ability to catalyse glutathione-dependent reduction of hydrogen peroxide. These data suggest that inorganic tellurite delivers tellurium to the intracellular milieu in a form capable of binding to some intracellular selenoproteins and at least in the case of glutathione peroxidase, cause inhibition of catalytic activity. The nature of the binding seems not to be due to the insertion of tellurocysteine into the protein and the insensitivity to reductive cleavage with mercaptoethanol seems to preclude the formation of stable telluro-selenides in the proteins. These data may offer alternative explanations for the established toxicity of tellurium via disruption of selenoprotein function, particularly by the induction of intracellular oxidative stress by the inhibition of Se-dependent glutathione peroxidase.

Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA.

The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an approximately 50-fold reduction in Se-GPx1 activity and an approximately 20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.

Selenium Supplementation (Se+) does not Modulate Early Anemia of Prematurity† 1569

Several investigators have documented low levels of selenium in serum and erythrocytes of low-birth weight (LBW) infants at birth or in the early postnatal period. Longitudinal studies during subsequent weeks or months have shown persistently low, and in some cases decreasing levels, of both Se and Se-dependent glutathione peroxidase (GPx) enzymes in premature infants not receiving supplementary selenium. It has been suggested that inadequate Se intake and low activity of red cell GPx might play a role in the development of early anemia of prematurity. However, there have been no published data on the effect of Se supplemented (Se+) infant formulas on anemia in prematures. We had previously documented a significant improvement in Se status of LBW infants (1.0-1.75kg B.Wt.) receiving Se+ formulas (Se content 24-29μg/L) compared with a standard (Se-) formula (Se=7.5-9μg/L) during a 20 week postnatal follow-up. Mean estimated Se intakes by 4 wks. were: Se-, 1.7μg/kg/day; Se+, 4.9μg/kg/day. We have evaluated the hematologic status of these infants as presented in the table below. Data were analyzed as “intent-to-treat” so as to include“drop-outs” until the time of their withdrawal. Reasons for withdrawal from further study included need for blood transfusion, significant illness, feeding other food (cereal.)

Antioxidant Enzyme Response to Hypericin in EMT6 Mouse Mammary Carcinoma Cells

Antioxidant enzyme activities were measured following exposure to hypericin ± irradiation in EMT6 cells. CuZnSOD and catalase activities peaked within 0.5 h following irradiation for nontoxic 0.5 μM hypericin and toxic 1.0 μM hypericin. Catalase remained elevated up to 3 h for 1.0 μM hypericin + light. MnSOD activity was elevated immediately following irradiation for both doses. These levels returned to control by 1 h for 0.5 μM hypericin, but were depressed after 1 h for 1.0 μM hypericin. This suggests that mitochondria impairment may be a critical factor in hypericin phototoxicity. Glutathione reductase was inhibited immediately following irradiation with 1.0 μM hypericin, suggesting that an altered status of the glutathione pool contributed to cytotoxicity. Glutathione peroxidase activities were elevated following irradiation but returned to control levels within 0.5 h for both doses, implicating hydroperoxide formation as an early event in hypericin phototoxicity. Inhibition by hypericin in the dark was demonstrated for purified CuZnSOD, Se-dependent glutathione peroxidase, glutathione S-transferase, and glutathione reductase activities in vitro. Irradiation did not potentiate hypericin-mediated glutathione reductase inhibition and decrease inhibition for the other enzymes. Collectively, these data demonstrate an antioxidant enzyme response to hypericin photoactivation and confirm a role for oxygen in hypericin phototoxicity.

Dietary Selenium Supplementation Is Required to Support Full Expression of Three Selenium-Dependent Glutathione Peroxidases in Various Tissues of Weanling Pigs ,

The current dietary allowance for selenium (Se) for pigs does not consider Se requirements for expression of several newly discovered Se-dependent enzymes and has raised environmental concerns. Our objective was to determine dietary Se requirements of young pigs for the full expression of cellular (GPX1), plasma (GPX3) and phospholipid hydroperoxide (GPX4) glutathione peroxidases. In Experiment 1, 18 weanling male pigs (4 wk old) were fed a corn-soybean meal basal diet (BD, 0.03 mg Se/kg) with the addition of 0, 0.1 or 0.3 mg Se/kg (Na2SeO3). In Experiment 2, 24 weanling barrows (6 wk old) were fed a similar BD with the addition of 0, 0.2, 0.3 or 0.5 mg Se/kg. Both experiments lasted for 5 wk. Pigs fed the BD had lower (P< 0.05) tissue GPX1 and GPX4 activities, plasma GPX activity, and(or) plasma Se concentrations than those fed the Se-supplemented diets. In Experiment 1, GPX1 and GPX4 activities in liver, heart and lung were lower (P< 0.05) in pigs fed 0.1 mg Se/kg than in those fed 0.3 mg Se/kg, although no such differences existed in thyroid or pituitary. Pigs fed 0.1 mg Se/kg also had lower (P< 0.05) plasma GPX3 activity at wk 5 and higher (P< 0.05) hepatic glutathione S-transferase activity than pigs fed 0.3 mg Se/kg. In Experiment 2, GPX1 and GPX4 activities in liver and heart, GPX1 and GPX4 mRNA levels in liver and GPX3 activity in plasma exhibited plateaus at 0.2 mg Se/kg. Pigs fed the BD had greater concentrations of F2-isoprostanes (a marker of in vivo lipid peroxidation) than those fed 0.2 mg Se/kg in plasma (P< 0.03) and liver (P< 0.04). We conclude that supplemental Se at 0.2 mg Se/kg of diet is required to support the full expression of three Se-dependent glutathione peroxidases in young pigs.

Activation of hepatic NF-kappaB by the herbicide Dicamba (2-methoxy-3,6-dichlorobenzoic acid) in female and male rats.

Nuclear factor-kappaB is a transcription factor that is activated in many different cell types by pathologic stimuli, such as reactive oxygen intermediates. One class of hepatocarcinogens, the peroxisome proliferators, may produce reactive oxygen intermediates, and one potent peroxisome proliferator, ciprofibrate, was recently reported to activate nuclear factor-kappaB. In this study, we investigated whether Dicamba, a broad leaf herbicide and peroxisome proliferator, could activate nuclear factor-KB in the livers of rats. Female and male Sprague Dawley rats (n = 4) were fed diets containing either 0, 1, or 3% Dicamba or 0.01% ciprofibrate for 7 days. As expected, the potent peroxisome proliferator, ciprofibrate, significantly increased fatty acyl CoA oxidase, peroxisomal beta-oxidation, and catalase activities in male rats and, except for catalase, also in female rats. Dicamba significantly increased peroxisomal fatty acyl CoA oxidase, peroxisomal beta-oxidation, and catalase activities, but decreased the activity of the cytosolic antioxidant enzyme, Se-dependent glutathione peroxidase, in both female and male rats. Dicamba increased nuclear factor-kappaB binding in the nuclear protein of livers from male rats fed both the 1 and 3% Dicamba diets. However, the highest binding was seen in nuclear protein from female rats fed 3% Dicamba. Both supershift and cold competition assays confirmed that this DNA binding activity was specific for nuclear factor-kappaB. Our results in this study suggest that the herbicide and peroxisome proliferator Dicamba has the ability to activate nuclear factor-kappaB.

Acute hexachlorocyclohexane-induced oxidative stress in rat cerebral hemisphere.

Hexachlorocyclohexane (HCH) is reported to induce oxidative stress in liver and testis of rat. With an objective to examine its effect on brain tissue acute toxicity of HCH (10 and 20 mg/kg body wt, i.p.) on the antioxidant defense system of cerebral hemisphere of rat was evaluated. Lipid peroxidation (LPX) was elevated after 24 h in the crude homogenate and sub-cellular fractions (nuclear and mitochondrial) except the microsomal fraction in which LPX was induced after 6 h and remained elevated till 24 h. The pesticide elicited decrease in the activities of cytosolic total, CN(-)-sensitive (not at 24 h) and CN-resistant superoxide dismutases; total, Se-dependent and Se-independent glutathione peroxidases; and catalase throughout the measurement period. In contrast, glutathione reductase activity was elevated till 24 h after a fall at 6 h of pesticide exposure. Cerebral contents of glutathione and ascorbic acid were decreased in response to HCH. The results suggest the possible involvement of reactive oxygen species in the mechanism of HCH-induced neurotoxicity in rat.

Dietary Selenium Increases Selenoprotein W Levels in Rat Tissues , ,

Two experiments were conducted to evaluate the influence of dietary selenium (Se) on tissue levels of selenoprotein W (Se-W) in rats. Se dependent glutathione peroxidase (GPX) activity and Se levels were also determined for comparative measurements. In the first experiment, rats were fed a basal diet deficient in Se or supplemented with either 0.1 or 4.0 mg Se (as selenite) per kg diet for 6 wk. Se-W levels were significantly higher in muscle, spleen and testes of rats fed 0.1 mg Se per kg diet compared to those fed the deficient diet (controls), and those fed 4.0 mg Se per kg diet had significantly higher levels in muscle, brain and spleen (P< 0.05) than those fed 0.1 mg Se per kg diet. No further increases, however, occurred in the tests. There was a significant increase (P< 0.05) of mRNA encoding Se-W in muscle with each increase of dietary Se. In the second experiment rats were fed the basal diet or this diet plus 0.01, 0.03, 0.06, 0.1, 1.0, 2.0 or 4.0 mg Se per kg diet. The levels of Se-W in muscle did not increase (P< 0.05) until 0.06 mg Se per kg diet were fed to rats. A very marked increase (P< 0.05) occurred when 1.0 mg Se per kg diet was fed with no further increases with higher levels. There was a linear increase of Se-W in brain (r= 0.89) and spleen (r= 0.98) with the Se concentration in the diet up to 0.1 mg Se per kg where a plateau was reached. The testes showed a different pattern in that a very marked increase (P< 0.01) occurred when only 0.01 mg Se per kg diet was fed where an inflection was reached. Except for muscle, GPX activities reached a plateau in all tissues when diets containing 0.06 to 0.1 mg supplemental Se per kg were fed. The Se concentration in these tissues increased at a linear rate with the Se concentration in the diets up to 0.1 mg Se per kg where it continued to rise at a different rate. The results indicate that in rats, the regulation of Se-W by Se is different for various tissues and differs from that for GPX.

ACTION OFCAPPARIS DECIDUAAGAINST ALLOXAN-INDUCED OXIDATIVE STRESS AND DIABETES IN RAT TISSUES

Alloxan-induced diabetic rats were treated with insulin (i.p.) or withCapparis deciduapowder as a hypoglycaemic agent mixed with diet. The effect was assessed on lipid peroxidation (LPO) and the antioxidant defense system in rat tissues. The increased levels of blood glucose in diabetes produce superoxide anions and hydroxyl radicals in the presence of transition metal ions which cause oxidative damage to cell membranes. The heart tissue showed an increased lipid peroxidation (LPO) in diabetic rats while no significant change was observed in the liver and kidney. The treatment withC. decidualowered LPO in these tissues even more effectively than insulin-treated rats. The superoxide dismutase (SOD) activity increased in the heart and kidneys in the diabetic group of rats probably to increase dismutation of superoxide anions. However, treatment withC. deciduadecreased SOD activity in the liver and kidney and was comparable to control rats. Catalase (CAT) activity was not significantly affected in any of the tissues in diabetic and insulin-treated animals, however, CAT activity markedly increased in tissues withC. deciduatreatment. Total and Se-dependent glutathione peroxidase (GSH-Px) in the heart was markedly lowered in diabetic rats which recovered with insulin as well as with C. decidua treatment. The increase in GSH-Px and CAT activity withC. deciduatreatment may lower H2O2toxicity and reduce oxidative stress in diabetes. However, glutathione (GSH) content in the heart and kidney and glutathione reductase (GSH-R) activity in all the tissues studied increased in diabetic rats while treatment with insulin lowered GSH content and GSH-R activity in these tissues. The treatment withC. deciduaalso decreased GSH-R activity in the kidney and heart which resulted in the decrease in GSH content in these tissues. The changes such as the increase in kidney and heart SOD may be an adaptive response in order to neutralize superoxide anions. The increase in GSH content and GSH-R activity in the tissue are in response to neutralize superoxide anions and to counteract oxidative stress in diabetes. GlutathioneS-transferase (GST) was not significantly affected in diabetic rat tissue, however, heart GST increased with antidiabetic treatments. The increase in glucose-6-phosphate dehydrogenase (G6PDH) in the kidney and heart of diabetic rats subsequently decreased withC. deciduatreatment. The increase in G6PDH in tissues may increase NADPH generation required for GSH-R activity and GSH production. It is suggested that these changes initially counteract the oxidative stress in diabetes, however, a gradual decrease in the antioxidative process may be one of the factors which results in chronic diabetes. The data indicate thatC. deciduamay have potential use as an antidiabetic agent and in lowering oxidative stress in diabetes.

Tissue-specific modification of selenium concentration by acute and chronic dexamethasone administration in mice.

Several clinical reports have shown changes in plasma Se concentration with corticosteroid treatments, but the results have been inconsistent. Few experimental studies have been done on this subject. In the present study the effect of dexamethasone (DEX) treatment on Se concentrations and activities of Se-dependent glutathione peroxidase (EC 1.11.1.9; SeGPx) were examined in adult male ICR mice. In the first experiment, DEX was given via drinking water containing 5 or 50 mg DEX/l. At 1 or 3 weeks of DEX treatment, mice were dissected and the Se concentrations as well as SeGPx activities in various tissues, including plasma, were determined. At 1 week the DEX-treated groups had significantly lower hepatic Se concentrations and significantly higher plasma and cerebral concentrations than the control group. The DEX-treated groups showed lower SeGPx activities in the hepatic cytosol and higher SeGPx activities in the plasma than the saline (9 g NaCl/l)-treated group, in parallel with the changes in Se concentrations. At 3 weeks, neither hepatic nor plasma Se concentrations showed a significant change. In the second experiment, mice were injected subcutaneously with DEX and, thereafter, mice were food-deprived. The DEX-injected groups had higher plasma Se concentrations. A similar finding was obtained also when the DEX- or saline-injected mice were not food-deprived. Thus, the difference between the DEX-treated and control groups was possibly caused by redistribution of tissue Se. These results suggested that the effects of DEX on Se concentrations were tissue dependent and that the higher plasma Se observed in DEX-treated groups might be explained by the release of tissue Se into plasma as plasma SeGPx.

Changes in the hepatic glutathione peroxidase redox system produced by coplanar polychlorinated biphenyls in Ah-responsive and -less-responsive strains of mice: mechanism and implications for toxicity

The alteration in hepatic glutathione peroxidase (GPx) produced by polychlorinated biphenyls (PCBs) was studied in vivo in aryl hydrocarbon (Ah)-responsive C57BL and -less-responsive DBA strains of mice. 3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126), one of the high-affinity ligands for the Ah receptor, significantly reduced Se-dependent GPx activity in C57BL mice, but not in DBA mice. A reduction in activity in C57BL mice was also observed following treatment with a high dose of 3,3′,4,4′-tetrachlorobiphenyl with lesser affinity for the Ah receptor than PCB 126, but not by 2,2′,5,5′-tetrachlorobiphenyl, a low-affinity ligand. To assess the effects on GPx in the liver, the content of reduced glutathione (GSH), an obligate co-factor for GPx, and the activity of two enzymes, γ-glutamyl transpeptidase ( γ-GTP) and glutathione reductase (GR), which play a role in supplying GSH were determined after PCB treatment. The results showed that although the hepatic activity of γ-GTP and GR was affected differently by PCB 126, the content of GSH was slightly increased rather than reduced in both strains of mice. The activity of non-Se-dependent GPx, which is due to the catalysis by some isozymes of glutathione S-transferase (GST), was significantly increased only in C57BL mice by PCB 126 treatment. Immunoblot analysis demonstrated that the induction of the class θ GST, which is a potent reducer of peroxides (Hiratsuka et al., 1995. Biochem. Biophys. Res. Commun. 212, 743) reflects the enhancement of the above activity. These results suggest that (i) the PCB-induced reduction in Se-dependent GPx activity is mediated by a mechanism involving the Ah receptor; and (ii) a concomitant increase in the class θ GST partially rescues the Ah-responsive mice from coplanar PCB-induced oxidative stress.

Antioxidant adaptive response in human blood mononuclear cells exposed to UVB

Human blood mononuclear cells exposed to UVB radiation develop increased antioxidant enzyme activities. Catalase (5.50±0.65 pmol (mg protein) −1), CuZn-superoxide dismutase (16.7±2.1 pmol (mg protein) −1), Mn-superoxide dismutase (11.3±1.7 pmol (mg protein) −1), Se-dependent glutathione peroxidase (13.2±1.5 mU (mg protein) −1) and Se-independent glutathione peroxidase (3.30±0.52 mU (mg protein) −1) activities increase by 1.3–1.5-fold from the control activities after exposure to 0.3 W m −2 of 280–315 nm light for 15 min and a 3 h dark incubation period. DT-diaphorase activity (2.86±0.21 μmol DCPIP min −1 (mg protein) −1) increases threefold from the indicated control values. In contrast, cytochrome oxidase (0.36±0.04 min −1( k′) (mg protein) −1) and succinate dehydrogenase (3.06±0.25 μmol DCPIP min −1 (mg protein) −1) activities remain unchanged during the same irradiation and incubation period. The treatment of cells with cycloheximide prevents the response triggered by UVB exposure. These findings suggest that an inducible antioxidant defence mechanism operates on photo-oxidative stress and that both superoxide dismutase and DT-diaphorase may display a concerted antioxidant role.

Relationship between changes of active oxygen metabolism and blood flow and formation, progression, and recovery of lesions is gastric mucosa of rats with a single treatment of compound 48/80, a mast cell degranulator.

The relationship between the changes of active oxygen metabolism and blood flow and the formation, progression, and recovery of lesions was examined in the gastric mucosa of rats treated once with compound 48/80, a mast cell degranulator. Gastric mucosal lesions appeared 0.5 hr after compound 48/80 treatment, became worst at 3 hr, and recovered fairly well at 12 hr. Increases in gastric mucosal lipid peroxide content and xanthine oxidase and myeloperoxidase activities and decreases in gastric mucosal vitamin E and hexosamine contents and Se-dependent glutathione peroxidase activity occurred with the formation and progression of gastric mucosal lesions. These changes were attenuated with the recovery of the lesion. Gastric mucosal nonprotein SH content decreased with the formation of gastric mucosal lesions, and this decreased SH content returned to near the original level with lesion progression. No changes in gastric mucosal superoxide dismutase and catalase activities occurred with the formation, progression, and recovery of gastric mucosal lesions. Gastric mucosal blood flow decreased with the formation of gastric mucosal lesions, and this decreased blood flow recovered with lesion progression. Serum serotonin concentration, an index of mast cell degranulation, increased with the formation of gastric mucosal lesions, and this increased serotonin level was attenuated with lesion progression and recovery. Pretreatment with ketotifen, a connective tissue mast cell stabilizer, prevented the formation of gastric mucosal lesions, the increases of gastric mucosal lipid peroxide content, xanthine oxidase and myeloperoxidase activities, and serum serotonin level; and the decreases of gastric mucosal nonprotein SH content, glutathione peroxidase activity, and blood flow found at 0.5 hr after compound 48/80 treatment. These results indicate that the changes of gastric mucosal active oxygen metabolism and blood flow are closely related to the formation, progression, and recovery of gastric mucosal lesions in rats with a single compound 48/80 treatment. The present results also suggest that this compound 48/80-induced gastric mucosal injury could be a kind of ischemia-reperfusion-induced injury occurring through degranulation of connective tissue mast cells.

Disruption of gastric mucosal antioxidant defense system and its relation to the development of gastric mucosal lesions in rats with repeated treatment of compound 48/80, a mast cell degranulator

Abstract The disruption of gastric mucosal antioxidant defense system and its relation to the development of gastric mucosal lesions was examined in Donryu rats treated once daily with compound 48/80 (0.75 mg/kg body weight), a mast cell degranulator, over a four day period. Mucosal lesions appeared after the 1st treatment and developed as the treatment was repeated. Mucosal lipid peroxide levels increased with an increase in the treatment period. Xanthine oxidase activity increased with a decrease in xanthine dehydrogenase activity in the mucosal tissue during the treatment, although the sum of both activities did not change during the treatment. Mucosal myeloperoxidase activity increased with an increase in the treatment period. Mucosal superoxide dismutase activity decreased after the 2nd treatment and a further decrease in activity occurred until the end of the treatment. Mucosal catalase and Se-dependent glutathione peroxidase activities decreased with an increase in the treatment period. A relatively constant decrease in mucosal glutathione reductase activity occurred after the 2nd, 3rd, and 4th treatment. Neither mucosal glucose-6-phosphate dehydrogenase activity nor nonprotein SH content changed during the treatment. A relatively constant decrease in mucosal vitamin E content occurred during the treatment. Mucosal hexosamine content decreased after the 1st treatment. But, mucosal hexosamine content in the compound 48/80-treated rats was equal to the control level after the 2nd treatment and was over the control level after the 3rd and 4th treatment. These results suggest that in rats with repeated compound 48/80-treatment, the disruption of gastric mucosal antioxidant defense system could be related to the development of gastric mucosal lesions.

Effects of age and dietary restriction on liver endogenous antioxidant defenses in male Lobund-Wistar rats

Dietary restriction (DR) of 30% in caloric intake extends both median and maximum life span by about 30%. DR retards the aging process, but the mechanism of action is not clearly understood. The effects of DR on major endogenous antioxidant defenses were studied in 80 male Lobund-Wistar (L-W) rats at various ages throughout the life span. Two groups of rats were fed ad libitum (AL) or restricted diet (DR) from 6 weeks of age. Adult DR rats received 30% less diet with regard to calories per day when compared to adult AL rats. Eight rats in each diet group were killed at 6, 12, 18, 24 and 30 months of age. The livers were excised and prepared for the determinations of major endogenous antioxidant defense parameters. Hepatic reduced glutathione (GSH) levels were decreased at old age in the AL group, however, DR eliminated this decrease. Activities of glutathione reductase (GR) and Se-dependent glutathione peroxidase (GPx) were not affected by age nor by DR. Superoxide dismutase (SOD) activity decreased from 6 to 12 months of age and catalase activity decreased with aging in the AL group, while DR maintained the enzyme activities at similar levels for all ages. Quinone reductase (QR) activity increased with increasing age in the AL group, and DR further increased the enzyme activity at all ages. The results suggest that 30% DR may contribute to the delaying of the aging process by improving endogenous antioxidant defense capability which decreases by 20 to 30% during aging.

Endotoxin stimulates gene expression of ROS-eliminating pathways in rat hepatic endothelial and Kupffer cells.

Reactive oxygen species (ROS) are mediators of cellular injury and play a putative role in the onset of hepatic damage during endotoxemia or sepsis. It has been suggested that induction of glucose-6-phosphate (G-6-P) dehydrogenase, the key enzyme of the hexose monophosphate shunt (HMS), may support ROS-producing or ROS-eliminating pathways in hepatic endothelial and Kupffer cells during endotoxemia. The aim of the study was to assess in vivo lipopolysaccharide (LPS)-induced alterations in rat gene expression of selected enzymes that are in functional relationship with the HMS. mRNA levels and activities of glucose transporter GLUT-1, Mn- and CuZn-dependent superoxide dismutases (Mn-SOD and CuZn-SOD), and Se-dependent glutathione peroxidase (Se-GPX) were determined. Cellular extracts were analyzed 7 or 22 h after injection of LPS (Escherichia coli, 2 mg/kg ip) or injection of saline. Exposure to LPS for 7 or 22 h caused a 10- to 25-fold increase in GLUT-1 mRNA levels in endothelial and Kupffer cells. In parenchymal cells, GLUT-1 mRNA expression was low, and LPS caused no marked changes. Cellular levels of Mn-SOD mRNA were 20-40 times greater in all hepatic cells from LPS-treated animals than in cells from control rats. LPS at 22 h increased Mn-SOD activity by 45% in endothelial cells but caused no significant changes in Kupffer or parenchymal cells. Message levels and enzyme activities of CuZn-SOD and Se-GPX were significantly elevated 22 h after LPS injection in endothelial cells only. Thus LPS results in marked upregulation of functionally related genes in hepatic cells. In endothelial cells, the simultaneous upregulation of GLUT-1, G-6-P dehydrogenase, Mn-SOD, CuZn-SOD, and Se-GPX may represent an important mechanism for accelerated elimination of ROS released from activated sinusoidal phagocytes. In Kupffer cells, upregulated GLUT-1 and G-6-P dehydrogenase, together with constitutively present SOD and lack of upregulated Se-GPX, suggest an elevated capacity to produce O2- and H2O2 that is consistent with primed bacterial killing.

Conferring drug resistance by MDR1 gene transfection increases susceptibility to irradiation and lipid peroxidation in 3T3 cell line

This study was performed to test the hypothesis that conferring multiple drug resistance reduces cell susceptibility to irradiation and iron-stimulated lipid peroxidation. Multidrug resistant (PN1A) and parental drug sensitive (PSI-2) cell lines were exposed to ADP-Fe or Ascorbate-Fe complexes at 37°C and to irradiation. Lipid peroxidation was estimated by the TBA test, whereas x-ray effect was estimated by clonogenic assay. Cell glutathione-S-transferase (GST), total and Se-dependent glutathione peroxidase (GSH-Px) activities, and glutathione and vitamin E were measured. PNIA produced more peroxides than PSI-2 after exposure to iron complexes and formed fewer colonies after irradiation. Higher activities of GST and total and Se-GSH-Px were observed in PNIA. Vitamin E and total glutathione did not differ in the two cell subclones. These data show that the induction of the mdrl phenotype by transfection of mdrl gene in 3T3 cells increases susceptibility to irradiation and iron stimulated lipid peroxidation