Induction of oxidative stress by selenomethionine in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss).
ABSTRACT Fish are exposed to environmental selenium predominantly in the form of dietary selenomethionine (SeMet). The present study was designed to investigate the role of oxidative stress in the toxicity of SeMet using isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) as the model experimental system. Cells were exposed to an increasing range of SeMet (0-1000 μM) over 24h, and the time-dependent effects on cell viability, response of enzymatic antioxidants, thiol redox, intracellular calcium balance and caspase-mediated apoptosis were evaluated. SeMet was found to be toxic only at the highest exposure dose (1000 μM), with ∼15% decrease in cell viability. Although modest increases in the activities of antioxidant enzymes were recorded following SeMet exposure, the ratio of reduced to oxidized glutathione decreased in a dose-dependent manner, suggesting a gradual progression towards an oxidative intracellular environment. The peroxidation of membrane lipids also increased with increasing SeMet exposure dose. In addition, a rapid increase in intracellular calcium level and the activation of caspase 3/7 enzymes were recorded at the highest exposure dose, indicating that SeMet at a high exposure dose causes cell death probably via apoptosis. Overall, our study demonstrated that oxidative stress plays a key role in the cytotoxicity of SeMet in fish.
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ABSTRACT: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.Analytical Biochemistry 06/1976; 72:248-54. · 2.58 Impact Factor
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ABSTRACT: A method for measurement of both oxidized (GSSG) and reduced (GSH) glutathione has been developed, with use of o-phthalaldehyde (OPT) as a fluorescent reagent. The method takes advantage of the reaction of GSH with OPT at pH 8 and of GSSG with OPT at pH 12; GSH can be complexed to N-ethylmaleimide to prevent interference of GSH with measurement of GSSG. The method gave “recoveries” of 91 to 110% for both GSH and GSSG and was quite specific for glutathione; and none of the manipulations appeared to influence the amount of glutathione present in the tissue. Results for GSH levels agreed well with earlier reports but levels of GSSG estimated here were higher than earlier reported values. The reasons for the apparently higher levels of GSSG are discussed.Analytical Biochemistry 08/1976; 74(1):214-26. · 2.58 Impact Factor
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ABSTRACT: Sodium selenite, sodium selenate, selenocystine and selenomethionine were tested for their abilities to generate superoxide by the oxidation of glutathione and other thiols in the absence and presence of cells of the human mammary tumor cell line HTB123/DU4475. Free radical generation was measured by lucigenin- or luminol-amplified chemiluminescence. In the absence of tumor cells, lucigenin-dependent chemiluminescence was observed from the reaction of selenite with the thiols glutathione, 2-mercaptoethanol and L-cysteine, but not with oxidized glutathione. Superoxide dismutase, catalase, and glutathione peroxidase all suppressed the observed chemiluminescence; but when these enzymes were heat inactivated they had little suppressive inhibition on chemiluminescence. Luminol-dependent chemiluminescence from the reaction of selenite with glutathione was much less than that observed by lucigenin-amplified chemiluminescence. In the presence of the HTB123/DU4475 mammary tumor cells, lucigenin-dependent chemiluminescence was observed from the reactions of selenite and selenocystine with glutathione which were 5 and 23 times greater than their respective reactions with glutathione in the absence of tumor cells. The enhanced chemiluminescence generated by selenite and selenocystine in the presence of the tumor cells was also suppressed by superoxide dismutase, catalase and glutathione peroxidase. These data suggest that a free radical, the superoxide anion (O2-), and H2O2 are produced from the reaction of selenite and selenocystine with glutathione. These free radical reactions may account for the toxicity of selenite and selenocystine in vitro in comparison to a near absence of acute tumor cell toxicity and superoxide generation by selenate and selenomethionine with thiols. Enhanced chemiluminescence in the presence of tumor cells may be an expression of cellular selenium metabolism and the capability of cells to form selenium metabolites that more easily oxidize glutathione and other thiols producing reactive free radicals and peroxides.Biochemical Pharmacology 02/1993; 45(2):429-37. · 4.58 Impact Factor