Higher Levels Of Glutathione Peroxidase Research Articles

Enhanced NK cell activation via eEF2K-mediated potentiation of the cGAS–STING pathway in hepatocellular carcinoma

BackgroundLiver cancer, particularly hepatocellular carcinoma (HCC), is characterized by a high mortality rate, attributed primarily to the establishment of an immunosuppressive microenvironment. Within this context, we aimed to elucidate the pivotal role of eukaryotic elongation factor 2 kinase (eEF2K) in orchestrating the infiltration and activation of natural killer (NK) cells within the HCC tumor microenvironment. By shedding light on the immunomodulatory mechanisms at play, our findings should clarify HCC pathogenesis and help identify potential therapeutic intervention venues. MethodsWe performed a comprehensive bioinformatics analysis to determine the functions of eEF2K in the context of HCC. We initially used paired tumor and adjacent normal tissue samples from patients with HCC to measure eEF2K expression and its correlation with prognosis. Subsequently, we enrolled a cohort of patients with HCC undergoing immunotherapy to examine the ability of eEF2K to predict treatment efficacy. To delve deeper into the mechanistic aspects, we established an eEF2K-knockout cell line using CRISPR/Cas9 gene editing. This step was crucial for verifying activation of the cGAS–STING pathway and the subsequent secretion of cytokines. To further elucidate the role of eEF2K in NK cell function, we applied siRNA-based techniques to effectively suppress eEF2K expression in vitro. For in vivo validation, we developed a tumor-bearing mouse model that enabled us to compare the infiltration and activation of NK cells within the tumor microenvironment following various treatment strategies. ResultsWe detected elevated eEF2K expression within HCC tissues, and this was correlated with an unfavorable prognosis (30.84 vs. 20.99 months, P = 0.033). In addition, co-culturing eEF2K-knockout HepG2 cells with dendritic cells led to activation of the cGAS–STING pathway and a subsequent increase in the secretion of IL-2 and CXCL9. Moreover, inhibiting eEF2K resulted in notable NK cell proliferation along with apoptosis reduction. Remarkably, after combining NH125 and PD-1 treatments, we found a significant increase in NK cell infiltration within HCC tumors in our murine model. Our flow cytometry analysis revealed reduced NKG2A expression and elevated NKG2D expression and secretion of granzyme B, TNF-α, and IFN-γ in NK cells. Immunohistochemical examination confirmed no evidence of damage to vital organs in the mice treated with the combination therapy. Additionally, we noted higher levels of glutathione peroxidase and lipid peroxidation in the peripheral blood serum of the treated mice. ConclusionTargeted eEF2K blockade may result in cGAS–STING pathway activation, leading to enhanced infiltration and activity of NK cells within HCC tumors. The synergistic effect achieved by combining an eEF2K inhibitor with PD-1 antibody therapy represents a novel and promising approach for the treatment of HCC.

Effect of the addition of platelet-rich plasma to Boer buck semen on sperm quality and antioxidant activity before and after cryopreservation and in vivo fertility

The current study aimed to investigate the effects of incorporating platelet-rich plasma (PRP) into Boer buck semen at different stages of cryopreservation (dilution, equilibration, and post-thawing) on sperm quality, functionality, antioxidant activity, and in vivo fertility. One hundred sixty ejaculates were collected from eight Boer goat bucks over ten weeks during the breeding season. The semen samples were collected using an artificial vagina, evaluated, and pooled. The pooled semen was divided into four aliquots, then diluted with a Tris-based extender supplemented with varying proportions of PRP (0%, 5%, 10%, and 15%). Subsequently, the samples were equilibrated and cryopreserved. The results showed that semen supplemented with 10% PRP has significantly higher total motility (P < 0.05) compared to the control and other groups during (dilution and equilibration) and after (post-thawing) cryopreservation. Furthermore, semen supplemented with 5% and 10% PRP significantly improved (P < 0.05) rapid sperm movement compared to the control and 15% PRP groups after equilibration and post-thawing. However, compared to the control, the supplementation of 10% PRP resulted in improved sperm progressive motility and viability during and after cryopreservation (P < 0.05), as well as increased acrosome, plasma membrane integrity, and mitochondrial membrane potential after equilibration and post-thawing of the semen (P < 0.05). Moreover, 10% PRP-supplemented semen exhibited higher levels of glutathione peroxidase (GPx) and catalase (CAT) and lower levels of malondialdehyde (MDA) after equilibration and post-thawing compared to the control and 15% PRP groups. The pregnancy and kidding rates were higher (80%; 80%) for 10% PRP-supplemented semen compared to the control (48%; 40%), respectively, and other groups. In conclusion, the addition of PRP improved the cryo-tolerance of Boer buck sperm by enhancing quality, functionality, antioxidant activity, and in vivo fertility.

Quantitative Proteomics and Functional Characterization Reveal That Glutathione Peroxidases Act as Important Antioxidant Regulators in Mulberry Response to Drought Stress.

Mulberry (Morus alba L.) has been an economically important food crop for the domesticated silkworm, Bombyx mori, in China for more than 5000 years. However, little is known about the mechanism underlying mulberry response to environmental stress. In this study, quantitative proteomics was applied to elucidate the molecular mechanism of drought response in mulberry. A total of 604 differentially expressed proteins (DEPs) were identified via LC-MS/MS. The proteomic profiles associated with antioxidant enzymes, especially five glutathione peroxidase (GPX) isoforms, as a scavenger of reactive oxygen species (ROS), were systematically increased in the drought-stressed mulberry. This was further confirmed by gene expression and enzymatic activity. Furthermore, overexpression of the GPX isoforms led to enhancements in both antioxidant system and ROS-scavenging capacity, and greater tolerance to drought stress in transgenic plants. Taken together, these results indicated that GPX-based antioxidant enzymes play an important role in modulating mulberry response to drought stress, and higher levels of GPX can improve drought tolerance through enhancing the capacity of the antioxidant system for ROS scavenging.

Growth performance, physiological and antioxidant capacity responses to dietary fish meal replacement with insect meals for aquaculture: A case study in red claw crayfish ( Cherax quadricarinatus )

A 60-day feeding trial was conducted to evaluate the effects of yellow mealworm (YM) (Tenebrio molitor) (9%, 18%, 27% and 36%) and black soldier fly larvae (BSFL) (Hermetia illucens) (7%, 14%, 21% and 28%) meal replacing fish meal (FM) on growth performance and antioxidant capacity of red claw crayfish (Cherax quadricarinatus). Nine diets, including control, were fed with quadruplicate of 20 crayfish (0.20 ± 0.05 g). The highest weight gain (WG) and lowest feed conversion ratio were observed in crayfish fed YM18 and BSFL14. The highest superoxide dismutase activity and the lowest malondialdehyde content were detected in the hepatopancreas of the crayfish fed YM18 and BSFL14. Compared with the control group, crayfish fed BSFL14 had higher levels of glutathione peroxidase, catalase acid and alkaline phosphatase and the lowest malondialdehyde (MDA) content in hemocytes. The mRNA expression of CqBAF, HSP70 and MnSOD in the hepatopancreas of crayfish showed an increasing trend. Based on the second-order polynomial regression analysis of WG, the optimal dietary replacement level of FM with YM meal and BSFL was determined to be 21.9% and 17.1% for juvenile C. quadricarinatus.

Controlled expression of the migratory phenotype affects oxidative status in birds.

High caloric intake can increase production of reactive oxygen and nitrogen species. We examined whether the emergence of the migratory phenotype, primarily signalled by increased food intake and fuelling, is accompanied by changes in oxidative status. We induced autumn migration followed by a non-migratory wintering phase in common quails (Coturnix coturnix). We compared three markers of oxidative status - oxidative damage to lipids expressed as thiobarbituric acid reactive substances (TBARS); superoxide dismutase (SOD); and glutathione peroxidase (GPx) - between birds sampled during the migratory and non-migratory phase. We found that the emergence of the migratory phenotype was associated with: (i) higher levels of TBARS in the liver; (ii) lower levels of SOD in red blood cells and, marginally, in the liver; (iii) higher levels of GPx in the pectoral muscle; and (iv) sex-specific changes in red blood cells and liver. We found no link between food intake and variation in markers of oxidative status in any of the tissues examined, despite food intake being higher in the migratory birds. However, the increase in body mass was positively correlated with muscle GPx activity as birds entered the pre-migratory fattening phase, while the amount of decrease in body mass was negatively correlated with muscle GPx as birds transitioned to the non-migratory phase. Such correlations were absent in red blood cells and liver. Our work suggests that during the emergence of the migratory phenotype, birds might strategically displace oxidative costs on the liver in order to safeguard the pectoral muscles, which have a fundamental role in successfully completing the migratory flight.

Effects of vitamin E in a glucocorticoid induced cataract model in chicken embryos

ABSTRACT We investigated the antioxidant effects of vitamin E on a glucocorticoid (GC) induced model of cataracts in chick embryos. We used 70 fertilized eggs divided into seven groups as follows: phosphate-buffered saline (PBS) group, olive oil treatment (OO) group, hydrocortisone treatment (HC) group, olive oil and hydrocortisone treatment (OO + HC) group, 50 mg/kg vitamin E and hydrocortisone treatment (HC + VE (50)) group, 25 mg/kg vitamin E and hydrocortisone treatment (HC + VE (25)) group and 15 mg/kg vitamin E and hydrocortisone treatment (HC + VE (15)) group. On day 17, chick embryos were removed from the eggs and lens and liver tissues were excised. Cataract formation was evaluated and total antioxidant status (TAS), total oxidant status (TOS), malondialdehyde (MDA) and glutathione peroxidase (GPx) were measured in lens and liver tissues; MDA was measured only in liver. The lenses in the HC + VE (50) group exhibited significantly higher levels of GPx and TAS, and lower levels of TOS than for HC and OO + HC groups. The livers of the HC + VE (50) group exhibited significantly higher levels of GPx and lower levels of MDA than for the HC and OO + HC groups. The HC + VE (50) group lenses were evaluated as grade 1, because the nuclei were completely free of cataracts, likely due to the antioxidative effect of high dose VE. VE is an effective antioxidant agent that exhibits a dose-response effect, for ameliorating the negative effects of GCs.

Oxidative stress and acid–base balance during the transition period of neonatal Holstein calves submitted to different calving times and obstetric assistance

During the neonatal period, constant adaptations to the extrauterine environment are required. These adaptations are made impossible or hindered when unexpected obstetrical events take place. This study aimed to identify clinical changes of vitality, metabolic and acid-base balance, and oxidative stress of newborn calves born to assisted dams that experienced different durations of calving. A prospective and observational study was conducted with 44 Holstein calves allocated to 3 groups according to the duration of calving: 2 h (n = 16), 2-4 h (n = 16), and >4 h (n = 12), and to 2 subgroups: spontaneous calving (n = 22) and assisted calving (n = 22). We observed that obstetric assistance was beneficial when calving lasted more than 4 h, because 100% of assisted calves had a high Apgar score (≥7) at 5 min, similar to that of spontaneously born calves after 2 h of calving. Rectal temperature of all calves remained within normal values, albeit with a reduction at 20 min of life. Calves born after a calving time >4 h had the highest rectal temperatures, especially those that required obstetrical assistance. There was a progressive increase in blood oxygenation with time, but calves in the >4 h group showed the highest partial pressure of carbon dioxide at birth, whereas calves in the 2 h and 2-4 h groups had lower values. A decrease in partial pressure of CO2 was observed at 120 min in the 2-4 h and >4 h groups. In the 2-4 h group, calves with obstetrical assistance had higher oxidative stress (measured as thiobarbituric acid reactive substances, TBARS) at 120 min of life compared with that at birth. In spontaneously born calves, TBARS increased only after 240 min. In the >4 h group, TBARS increased progressively with time, regardless of obstetrical subgroup. Calves born spontaneously within the 2 h group had higher TBARS values at birth than calves in the >4 h group with obstetrical assistance. Calves in the >4 h group showed higher levels of glutathione peroxidase (an antioxidant enzyme) at birth than those in the remaining groups. Glutathione peroxidase decreased at 120 min in the >4 h group and at 240 min for the 2 h and 2-4 h groups. In conclusion, obstetric assistance did not improve neonatal vitality and blood oxygenation when calving lasted less than 2 h. Calves born after 2 h of calving showed decreased vitality, hypercapnia, hypoxemia, and increased antioxidant status (glutathione peroxidase).

Microenvironment of saphenous vein graft preservation prior to coronary artery bypass grafting.

The best preservation solution for a free vascular graft prior to coronary artery bypass grafting (CABG) remains controversial. The aim of this investigation was to evaluate the microenvironment of the human saphenous vein graft when preserved in normal saline (NS) solution or autologous heparinized whole blood (AWB). Between January 2014 and December 2014, 21 patients who underwent CABG were enrolled and a total of 162 saphenous vein graft rings were collected. NS and AWB were used to investigate the influence of the microenvironment. The hypoxia, oxidative stress and vascular apoptosis were assayed by western blot, and endothelial integrity was assessed by immunohistochemical analysis. The level of PaO2 in AWB was lower than that in NS (median: 100.5 mmHg vs 185.8 mmHg, P = 0.004). This hypoxic condition led to the production of more hypoxia-inducible factor-1 (median: 60.1% vs 15.1%, P = 0.008) and endothelial nitric oxide synthase (median: 52.6% vs 25%, P = 0.046) within 30 min of preservation time. The fact that higher levels of glutathione peroxidase resulted in the preservation of AWB suggests that it is beneficial to boost the vascular antioxidant defense with lower levels of NOX2. AWB led to increased Bcl-2, reduced cytochrome c and cleaved 85 kDa poly ADP-ribose polymerase apoptotic fragments. We concluded that AWB possesses a microenvironment that is superior to that of NS for saphenous vein graft preservation prior to CABG.

Effects of dietary Nigella sativa seed supplementation on broiler productive performance, oxidative status and qualitative characteristics of thighs meat

The objective of our study was to evaluate the effects of Nigella sativa seeds (NS) supplementation on meat quality and antioxidants content of chicken meat. Two hundred 1-d-old male broiler chicks were divided into four diet treatment groups: normal control (baseline feed only), NS supplemented (1 and 2%) groups and a standard (200 IU/kg vitamin-E) VE group. At the end of this period, 10 birds were randomly selected from each group for examination. Feed conversion ratio was significantly lower in the treatment groups than the control one. There were no significant differences in moisture or crude ash percentage in thigh muscle among groups, but dietary NS powder supplementation resulted in a significant increase in crude protein content and decrease in crude fat content relative to the control group (p < 0.05). After 24 h, thigh muscle pH was higher while drip loss, cooking loss and shear force were lower in the NS groups than the control group. Lightness values of thigh muscle colour were decreased and redness and yellowness values were increased by NS supplementation. Muscle lipidperoxidation (malondialdehyde) level, which is correlated with significantly higher levels of glutathione peroxidase and superoxide dismutase, were significantly lower (p < 0.001) in the NS groups and VE group than the normal group. These results suggest that NS supplementation is effective at improving broiler performance and meat quality by enhancing antioxidant activities and suppressing lipidperoxidation in meat.

Mitochondrial and Oxidative Stress Aspects in Hippocampus of Rats Submitted to Dietary n-3 Polyunsaturated Fatty Acid Deficiency After Exposure to Early Stress.

Chronic dietary long-chain polyunsaturated fatty acids (PUFAs) deficiency may lead to changes in cortex and hippocampus neuronal membrane phospholipids, and may be linked to impaired central nervous system function. Particularly docosahexaenoic acid deficiency appears to be involved in neuropsychiatric disorders. On the other hand, adverse events early in life may also profoundly affect brain development, leading to long-lasting effects on neurophysiology, neurobiology and behavior. This research assessed if neonatal stress and a dietary n-3 PUFAs deficiency could interact to produce hippocampal alterations related to mitochondrial functions in adult rats. There were no effects of diet, neonatal intervention or interactions on superoxide dismutase or catalase enzymatic activities, mitochondrial membrane potential and respiratory chain complexes. Rats fed n-3 PUFAs deficient diet displayed higher levels of glutathione peroxidase and catalase activity, higher free radicals production and higher thiol content compared to rats fed n-3 PUFAs adequate diet. There were interactions among diets and neonatal stress, since glutathione peroxidase, free radicals production and thiol content were increased in groups that were subjected to neonatal interventions fed n-3 PUFAs deficient diet. Additionally, reduced mitochondrial potential was observed in handled animals. Total thiol revealed a neonatal stress effect, since animals subjected to neonatal interventions displayed lower thiol content. In conclusion, we observed that a chronic treatment with deficient n-3 PUFAs diet, from the puberty period on, increased free radicals production and imbalanced antioxidant enzymes activities, and these increases were higher in animals subjected to neonatal interventions.

Vitamin D ameliorates hepatic ischemic/reperfusion injury in rats.

Vitamin D, most commonly associated with the growth and remodeling of bone, has been shown to ameliorate ischemia/reperfusion injury (IRI) in some tissues, yet its underlying mechanism remains elusive. This study was designed to examine the protective effect of vitamin D, if any, against hepatic IRI in rats and the underlying mechanism involved. Adult female Wistar rats were randomly divided into control, sham-operated (sham), ischemia/reperfusion (I/R), and ischemic-reperfused vitamin D-treated (vit D) groups. Rats in the I/R and vit D groups were subjected to partial (70%) hepatic ischemia for 45 min, followed by 1 h of reperfusion. Vitamin D was given to rats orally in a dose of 500 IU/kg daily for 2 weeks before being subjected to I/R. Markers of liver damage, oxidative stress, inflammation and apoptosis were evaluated. Hepatic morphology was also examined. Vit D-treated rats had significantly lower serum levels of alanine aminotransferase, aspartate aminotransferase, and γ glutamyl transferase compared to rats in the I/R group. Also, vit D-treated rats showed a significant decrease in malondialdehyde, interleukin-1 beta, interleukin-6, tumor necrosis factor-α, nuclear factor κB, B cell leukemia/lymphoma 2-associated X protein, cytochrome c, and caspase-3 levels, with higher levels of glutathione peroxidase and B cell lymphoma 2 protein levels in liver tissues compared to I/R rats. Histological examination showed less damaged liver tissues with amelioration of apoptotic signs in the vit D group compared to the I/R group. In conclusion, vitamin D supplementation ameliorates hepatic IRI mostly by alleviating the inflammatory-apoptotic response mediated by the oxidative reperfusion injury insult.

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts

Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.

Marcadores de desequilíbrio redox em sangue de pacientes hipertensos de uma comunidade no Nordeste do Brasil

Recent studies describe the participation of reactive oxygen and nitrogen species in hypertension. To identify the redox imbalance in the blood of hypertensive. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH), vitamin C, transferrin, ceruloplasmin, malondialdehyde (MDA) and carbonyl group were quantified in the blood of 20 hypertensives and 21 controls. The individuals had a Body Mass Index of ≥ 18.5 and ≤ 30 kg/m(2), glycemia ≤ 100 mg/dL, serum cholesterol ≤ 200 mg/dL, and were nonsmokers, non-pregnant and non-lactating women, non-users of alopurinol and probucol, with hypertensives on antihypertensive medication. All individuals underwent a preparatory period of 4 weeks without alcohol, vitamin supplements, dexamethasone and paracetamol. Reduced levels of CAT (p 0.013), GSH (p 0.003) and MDA (p 0.014), and high levels of GPx (p 0.001) and ceruloplasmin (p 0.015) were obtained in the hypertensive group compared with controls. A positive correlation between systolic pressure and MDA in hypertensive and diastolic pressure and CAT in controls was obtained. The data obtained suggest that the hypertensives were in redox imbalance, despite the possibly attenuating effect of their antihypertensive medication.

Study of Oxidative Stress in Vitiligo

Vitiligo is an idiopathic, acquired, circumscribed, hypomelanotic skin disorder, characterized by milky white patches of different sizes and shapes. It is due to the destruction of melanocytes resulting in the absence of pigment production of the skin and mucosal surfaces. Oxidative stress has been implicated in pathophysiology of vitiligo. To study the activity of blood Superoxide dismutase (SOD) and Glutathione peroxidase (GPx) in vitiligo patients. A case-control study was conducted in which 100 patients were enrolled after written consent. 50 cases were of active vitiligo and 50 served as control (25 healthy control and 25 with stable vitiligo). SOD-In our study, among the active vitiligo cases 90% had high level of SOD and 10% had normal level of SOD. Among the stable vitiligo controls, 92% had normal level of SOD and 8% had low levels of SOD.The difference between active vitiligo cases and stable vitiligo control as well as with healthy control was statistically significant (P value<0.05). GPx-Among the active vitiligo cases 74% had normal GPx levels, 22% had low and only 4% had high levels of GPx. Among the stable vitiligo controls, 64% had normal GPx levels, 16% had low, and 20% had high levels of GPx. The difference between active vitiligo cases and stable vitiligo control as well as with healthy control was statistically not significant (P value>0.05). Our study shows that oxidative stress is involved in the pathophysiology of vitiligo, as indicated by the high levels of serum superoxide dismutase activity.

Myocardial antioxidant and oxidative stress changes due to sex hormones.

The purpose of the present study was to examine myocardial antioxidant and oxidative stress changes in male and female rats in the presence of physiological sex hormone concentrations and after castration. Twenty-four 9-week-old Wistar rats were divided into four groups of 6 animals each: 1) sham-operated females, 2) castrated females, 3) sham-operated males, and 4) castrated males. When testosterone and estrogen levels were measured by radioimmunoassay, significant differences were observed between the castrated and control groups (both males and females), demonstrating the success of castration. Progesterone and catalase levels did not change in any group. Control male rats had higher levels of glutathione peroxidase (50%) and lower levels of superoxide dismutase (SOD, 14%) than females. Control females presented increased levels of SOD as compared to the other groups. After castration, SOD activity decreased by 29% in the female group and by 14% in the male group as compared to their respective controls. Lipid peroxidation (LPO) was assessed to evaluate oxidative damage to cardiac membranes by two different methods, i.e., TBARS and chemiluminescence. LPO was higher in male controls compared to female controls when evaluated by both methods, TBARS (360%) and chemiluminescence (46%). Castration induced a 200% increase in myocardial damage in females as determined by TBARS and a 20% increase as determined by chemiluminescence. In males, castration did not change LPO levels. These data suggest that estrogen may have an antioxidant role in heart muscle, while testosterone does not.

Role of oxidative stress and thiol antioxidant enzymes in nickel toxicity and resistance in strains of the green alga Scenedesmus acutus f. alternans.

Treatment with Ni(NO3)2 leads to the formation of reactive oxygen species (ROS) in the green alga Scenedesmus acutus f. alternans, causing lipid peroxidation. This effect was stronger in a Ni-sensitive strain, UTEX72, than in a Ni-resistant strain, B4. In the resistant strain, Ni induced an increased ratio of reduced to oxidized glutathione (GSH:GSSG), whereas it caused a lowered ratio in the sensitive strain. Enzymes involved in the control of ROS were studied in these strains as well as two others that have shown different degrees of nickel resistance. The resistant strain, B4, which grows while containing large amounts of internal Ni, had much higher levels of glutathione reductase and catalase than the other strains. The sensitive strain, UTEX72, had higher levels of glutathione peroxidase, superoxide dismutase, and glucose-6-phosphate dehydrogenase than did strain B4. The resistant strains, Ni-Tol and Cu-Tol, derived from strain UTEX72, which are partly able to exclude Ni, had enzyme profiles that resembled that of UTEX72 more closely than that of B4. Treatment with 10 and 100 microM Ni for 4 or 22 h had complex effects on enzyme levels in all four strains. Ni decreased glutathione reductase in B4, slightly increased it in Ni-Tol and Cu-Tol, and did not affect the low levels of this enzyme in UTEX72. Ni lowered glutathione peroxidase in B4 and either did not affect it or slightly raised it in the other strains. Ni lowered catalase in B4 and did not affect the other strains. Superoxide dismutase was raised in B4 and Ni-Tol and lowered in Cu-Tol and UTEX72, and glucose-6-phosphate dehydrogenase was lowered in all four strains. These results suggest that one major mechanism of Ni resistance, especially in strain B4, may be the ability to combat the formation of ROS when exposed to this metal, likely by maintaining a high GSH:GSSG ratio.

Immunolocalization of antioxidant enzymes in adult hamster kidney

Immunoperoxidase and immunogold techniques were used to localize the following antioxidant enzyme systems in the adult hamster kidney at the light and ultrastructural levels: superoxide dismutases, catalases, peroxidases and glutathione S-transferases. Each cell type in the kidney showed specific patterns of labelling of these enzymes. For example, proximal and distal tubular and transitional epithelial cells showed significant staining for all of these enzymes, while glomerular cells and cells of the thin loop of Henle did not show significant staining at the light microscope level. In addition, high levels of glutathione peroxidase were found in smooth muscle cells of renal arteries. At the ultrastructural level, each enzyme was found in a specific subcellular location. Manganese superoxide dismutase was found in mitochondria, catalase was localized in peroxisomes, while copper, zinc superoxide dismutase and glutathione S-transferase (liver and placental forms) were found in both the nucleus and cytoplasm. Glutathione peroxidase was found to have a broad intracellular distribution, with localization in mitochondria, peroxisomes, nucleus, and cytoplasm. Microvilli of tubular cells were labelled by antibodies to catalase, copper, zinc superoxide dismutase, glutathione peroxidase, and glutathione S-transferases. Cell types that were negative by light microscopy immunoperoxidase studies showed definite labelling with immunogold post-embedding ultrastructural techniques (glomerular cells and cells of the loop of Henle), demonstrating the greater sensitivity of the latter technique. These observations demonstrate that there are large variations in the levels of antioxidant enzymes in different cell types, and that even within a distinct cell type, the levels of these enzymes vary in different subcellular locations. Our results demonstrate for the first time the overall antioxidant enzyme status of individual kidney cell types, thereby explaining why different cell types have differing susceptibilities to oxidant stress. Possible physiological and pathological consequences of these findings are discussed.