Pathways of 4-Hydroxy-2-Nonenal Detoxification in a Human Astrocytoma Cell Line.

One of the consequences of the increased level of oxidative stress that often characterizes the cancer cell environment is the abnormal generation of lipid peroxidation products, above all 4-hydroxynonenal. The contribution of this aldehyde to the pathogenesis of several diseases is well known. In this study, we characterized the ADF astrocytoma cell line both in terms of its pattern of enzymatic activities devoted to 4-hydroxynonenal removal and its resistance to oxidative stress induced by exposure to hydrogen peroxide. A comparison with lens cell lines, which, due to the ocular function, are normally exposed to oxidative conditions is reported. Our results show that, overall, ADF cells counteract oxidative stress conditions better than normal cells, thus confirming the redox adaptation demonstrated for several cancer cells. In addition, the markedly high level of NADP+-dependent dehydrogenase activity acting on the glutahionyl-hydroxynonanal adduct detected in ADF cells may promote, at the same time, the detoxification and recovery of cell-reducing power in these cells.

Climate-related environmental stress in intertidal grazers: scaling-up biochemical responses to assemblage-level processes.

BACKGROUND: Organisms are facing increasing levels of environmental stress under climate change that may severely affect the functioning of biological systems at different levels of organization. Growing evidence suggests that reduction in body size is a universal response of organisms to global warming. However, a clear understanding of whether extreme climate events will impose selection directly on phenotypic plastic responses and how these responses affect ecological interactions has remained elusive. METHODS: We experimentally investigated the effects of extreme desiccation events on antioxidant defense mechanisms of a rocky intertidal gastropod (Patella ulyssiponensis), and evaluated how these effects scaled-up at the population and assemblage levels. RESULTS: With increasing levels of desiccation stress, limpets showed significant lower levels of total glutathione, tended to grow less and had reduced per capita interaction strength on their resources. DISCUSSION: Results suggested that phenotypic plasticity (i.e., reduction in adults' body size) allowed buffering biochemical responses to stress to scale-up at the assemblage level. Unveiling the linkages among different levels of biological organization is key to develop indicators that can anticipate large-scale ecological impacts of climate change.

NADP(+)-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification.

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay.

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.