Evaluation of Dynamic Disulphide/Thiol Homeostasis in Silica Exposed Workers.

BACKGROUND: Oxidative stress is implicated as one of the main molecular mechanism underlying silicosis. AIMS: In this study, our aim was to asses the redox status in occupationally silica-exposed workers, by evaluating the dynamic thiol-disulphide homeostasis. STUDY DESIGN: Case-control study. METHODS: Thirty-six male workers occupationally exposed to silica particles and 30 healthy volunteers, working as office workers were included to the study. Posteroanterior chest radiographs and pulmonary function tests of both groups were evaluated. Also serum thiol disulphide levels were measured using the spectrophotometric method described by Erel and Neselioglu. RESULTS: Among the 36 workers that underwent pulmonary function tests 6 (17%) had obstructive, 7 (19%) had restrictive, 6 (17%) had obstructive and restrictive signs whereas 17 (47%) had no signs. The mean PFTs results of silica-exposed workers were significantly lower than control subjects. The serum disulphide levels of silica-exposed workers were significantly higher than control subjects (23.84±5.89 µmol/L and 21.18±3.44 µmol/L, respectively p=0.02). CONCLUSION: The serum disulphide levels, a biomarker of oxidative stress, are found to be higher in silica-exposed workers.

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide.

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide / Níveis de glutationa e capacidade antioxidante total em células de Candida sp. expostas a estresse oxidativo causado por peróxido de hidrogênio

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

INTRODUÇÃO: A capacidade de suportar o estresse oxidativo imposto por fagócitos parece ser crítica para que espécies de Candida causem candidíase invasiva. MÉTODOS: Para melhor caracterizar a resposta ao estresse oxidativo (REO) de oito Candida sp. clinicamente relevantes, um componente vital do balanço redox intracelular, a glutationa, foi mensurada pelo método de reconversão DTNB-GSSG redutase e a capacidade antioxidante total (CAT) foi mensurada por um método modificado baseado na descoloração do ABTS*+. Ambos os métodos foram utilizados em extratos celulares das espécies de Candida tratadas ou não com peróxido de hidrogênio (0,5mM). RESULTADOS: O estresse oxidativo induzido pelo peróxido de hidrogênio claramente reduziu os níveis intracelulares de glutationa. Esta diminuição foi mais intensa em C. albicans e os níveis de glutationa em células não tratadas foram também maiores nesta espécie. A capacidade antioxidante total demonstrou variação intraespecífica na capacidade antioxidante. CONCLUSÕES: Os níveis de glutationa não se correlacionaram com a capacidade antioxidante total mensurada, apesar desta ser a defesa antioxidante intracelular não-enzimática mais importante. Os resultados indicam que a medição isolada da CAT não fornece um quadro claro da habilidade de certa espécie de Candida responder ao estresse oxidativo.

Selection of a suitable mobile phase for the speciation of four arsenic compounds in drinking water samples using ion-exchange chromatography coupled to inductively coupled plasma mass spectrometry.

The performance of two mobile phase buffers, phosphate and TRIS, were compared for the speciation of four arsenic species: arsenate (As(V)), arsenite (As(III)), mono methylarsonic acid (MMA), and dimethyl arsinic acid (DMA) in drinking water, using ion-exchange chromatography inductivelycoupled plasma mass spectrometry (IEC-ICP-MS). The mobile phase containing TRIS acetate buffer ("TRIS") demonstrated superior perfomance in baseline separation of all four arsenic species and the internal standard. It is also applicable to high-throughput sample analysis as it minimized the frequency required to clean the sampling interface due to salt build-up when compared to the phosphate mobile phase. The method was evaluated for its precision, accuracy, linearity and detection limits. The method was successfully applied for the analysis of drinking water samples.

Changes in indices of antioxidant status, lipid peroxidation and inflammation in human skeletal muscle after eccentric muscle actions.

This study investigated the effects of chronic muscle inflammation on indices of antioxidant status and muscle injury after eccentric exercise. Eight subjects each performed 70 maximal voluntary eccentric muscle actions on an isokinetic dynamometer, using the knee extensors of a single leg. Venous blood samples were collected into serum and EDTA tubes 5 and 3 days before exercise, immediately before exercise, and then again on days 3, 4, 5, 6, 7, 10 and 12 after the bout. Needle biopsies were taken from the vastus lateralis of six subjects, a week before exercise (baseline), and again on days 4 and 7 post-exercise. The concentrations of malondialdehyde in plasma and muscle were used as markers of lipid peroxidation. Creatine kinase activity, beta-glucuronidase activity and total antioxidant capacity were determined in serum. In muscle, aqueous and bound total antioxidant capacity, the aqueous sulphydryl concentration, and beta-glucuronidase and glucose-6-phosphate dehydrogenase activity were determined. No changes were detected in serum total antioxidant capacity, serum creatine kinase and beta-glucuronidase after the baseline biopsy. After exercise serum creatine kinase and beta-glucuronidase were elevated although other serum measures were unchanged. In muscle, aqueous and bound total antioxidant capacity, sulphydryls, glucose-6-phosphate dehydrogenase and beta-glucuronidase were all elevated. Despite evidence of inflammation in this study, muscle antioxidant status was not compromised, and malondialdehyde was unaltered in muscle and plasma. Therefore, this study provides no evidence that chronic muscle inflammation compromises antioxidant status or increases lipid peroxidation.

Distribution of free seleno-amino acids in plant tissue of Melilotus indica L. grown in selenium-laden soils.

Accumulation of specific groups of seleno-amino acids in plant tissue reflects not only the Se tolerance of a plant species, but also Se toxicity to animals. The distribution of seleno-amino acids in a Se-tolerant grassland legume species (Melilotus indica L.) grown in Se-laden soils was studied using high-resolution gas chromatography- and gas chromatography-mass spectrometry. Five seleno-amino acids including selenocystine, selenomethionine, selenocysteine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine were identified and measured for their plant tissue concentrations. Se-methylselenocysteine, a nonprotein seleno-amino acid, was found in the plant tissue. Its concentration ranged from 15.3 mumol kg-1 for the plants growing in soil of low Se concentration to 109.8 mumol kg-1 for the plants grown in soil of high Se concentration. Accumulation of the nonprotein seleno-amino acid in this species resembles that in Se accumulator plants. gamma-Glutamyl-Se-methylselenocysteine was detected in the plant. However, its concentration was very low. It might not become a toxic element in the food chain. Results of plant tissue Se accumulation analysis indicated that there was a five-fold increase in tissue selenocysteine concentration when the total tissue Se increased from 5.07 to 22.02 mg kg-1, but there was no further increase in tissue selenocysteine concentration when the tissue total Se concentration increased from 22.0 to 117.4 mg kg-1. Selenomethinone constituted more than 50% of the total seleno-amino acid in the plant. More research is needed to reveal whether the mechanisms limiting the accumulation of selenocysteine and preferential accumulation of selenomethionine found in this study play any role in Se tolerance in this species.

Oxidative activation of K-Cl cotransport by diamide in erythrocytes from humans with red cell disorders, and from several other mammalian species.

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0-1.0 mm) in K-free Cl or NO3 media of variable osmolalities (200-450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO3) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) approximately rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0. 00001) and in NO3 (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity.

Tolerance to nitroglycerin in vascular smooth muscle cells is not affected by the level of intracellular glutathione or L-cysteine.

A major hypothesis for the mechanism of tolerance to nitroglycerin (NTG) is that continued use causes a decrease in thiol donors within the vascular smooth muscle cell that are essential for the effect of NTG. We tested this idea directly in the target cell. NTG tolerance, measured as reduced formation of intracellular cyclic guanosine monophosphate (cGMP), was induced in pig coronary smooth muscle cells. The consequence of altering intracellular levels of the thiol donors, glutathione (GSH) and L-cysteine (L-cys), was determined. Incubating cells with 100 microM NTG for 1 h caused an 83% reduction in cGMP formation in response to acute readministration of 200 microM NTG for 2 min but was not associated with a reduction in intracellular GSH or L-cys. This result was not altered when intracellular GSH levels were increased three-fold by including 1 mM GSH in the incubation buffer. Also, recovery from tolerance was not affected by supplementation with GSH. Further, the response of cGMP to NTG was not altered by inhibiting the synthesis of GSH and lowering intracellular levels of GSH by 77%. Similar findings were made with supplemental L-cys or N-acetyl-L-cysteine. These results do not support the hypothesis that tolerance to NTG is the result of a reduction of the thiol donors GSH and L-cys within vascular smooth muscle cells.

[Study of reactivity of sulfhydryl groups of troponin]. / Izuchenie reaktsionnoi sposobnosti sul'fgidril'nykh grupp troponina.

The reactivities of SH-groups of troponin and its components were studied, using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). At low concentrations of Ca2+ (pCa greater than 8) one rapidly- and two slowly-reacting SH-groups of troponin I and one SH-group of troponin C slowly reacting with NBD-chloride are titrated in the whole troponin complex. When Ca2+ concentration is increased up to pCa less than 5, only one slowly-reacting and one rapidly-reacting with NBD-chloride SH-groups of troponin I are titrated. The increase of Ca2+ concentration from pCa greater than 8 to pCa less than 5 results in a change of the environment polarity for the highly reactive SH-group of troponin I in the whole troponin complex. This phenomenon may suggest that the changes in troponin C structure during Ca2+ binding somehow induce changes in that of troponin I. The half-maximal change of troponin SH-groups reactivity is found at pCa 6.8. No cooperativity for Ca2+ binding by the troponin complex is observed using SH-groups titration by NBD-Cl.

Effects of sulfhydryl reagents on basal and vasopressin-stimulated Na+ transport in the toad bladder.

The role of reactive SH groups (presumably in proteins) of the apical plasma membrane in transepithelial Na+ transport was studied in the isolated urinary bladder of the toad. On the basis of assays for TCA-soluble SH compounds (e.g., glutathione, methionine), PCMB, PCMPS, NTCB, and DTNB did not penetrate the intracellular compartment from the luminal media either in control or vasopressin-treated bladders. In contrast, PCMB from the serosal side and NEM from the luminal side titrated significant fractions of the TCA-soluble SH compounds. We conclude, therefore, the PCMB, PCMPS, NTCB, and DTNB are suitable reagents for studies on the physiological properties of apical plasma membrane SH groups. Titration of apical membrane SH groups with PCMPS, NTCB, and DTNB revealed heterogeneity in functional responses: PCMPS and NTCB elicited transient, 25-60% increases in SCC. In substrate-free media, pretreatment with these reagents inhibited the increase in SCC produced by vasopressin or cyclic AMP (+ theophylline). In glucose-enriched media, the responses to combinations of vasopressin and PCMPS or NTCB were additive, implying activation via parallel pathways. Simultaneous addition of vasopressin or cyclic AMP (+ theophylline) and NTCB resulted in marked synergism, presumably as a result of unmasking of SH groups by the the hormone (or the intermediate). These results suggest that basal Na+ transport is regulated in part by SH compounds in the apical membrane that are distinct, although not necessarily different in kind, from those involved in the response to vasopressin.