BioMicro World2007 - Evidence of a bimodal effect on Saccharomyces cerevisiae UE-ME3 by vanadium (V) Stress - a dual response to different V2O5 medium concentration detected in the rate growth, G6PD, CAT T and GR enzymatic activities

Abstract

Vanadium, widely distributed in nature is a major trace metal in fossil fuels, and combustion of these materials provides a significant environmental source of this element. Environmental scientists have declared vanadium as a potentially dangerous chemical pollutant. Despite metal V is essential for living organisms, including fungi, but elevated concentrations of this metal can result in growth inhibition and toxicity. The vanadium oxide (V2O5) is described in literature as very toxic compound which molecular effects are poorly studied. In general, the toxicity of metals can be result from generation of reactive oxygen species (ROS) that may cause wide-ranging damage in biomolecules, eventually leading to cell death. Fungi display several antioxidant enzymes against ROS, including catalase T (CAT T, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2), capable of removing oxygen radicals and their products and/or repairing oxidative damage. Glutathione recycling is dependent on the maintenance of an intracellular pool of NADPH, mainly via the pentose phosphate pathway, where glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) is the regulator step. An adequate level of NADPH is also required to protect cells against oxidative stress. Cells that are exposed to oxidative stress as well as rapidly proliferating cells particularly depend on the regeneration of GSH. GR carries out the enzymatic reduction of glutathione disulfide (GSSG) to glutathione (GSH) at the expense of the reducing power of NADPH. Subsequently, the main purpose of this work was to evaluate the effects of five medium [V2O5] (0.5, 1.0, 1.5 and 2.0 mM) on cell viability, G6PD, CAT T and GR activities of wild wine yeast Saccharomyces cerevisiae UE-ME3. The cells were grown to mid-exponential phase in a water bath, with orbital stirring, at 28ºC, in 250-ml flasks containing 100 ml of YEPD medium with 2% (w/v) of glucose. The cells (106 cells ml-1) at mid-exponential phase were inoculated in the same condition in the absence or presence of V2O5, during 200 min. At final time, samples from each treatment were diluted and plated on YEPD medium, in order to obtain viable counts. The cells were harvested and ultra-sonic disrupt. The cellular extract obtained were centrifuged at 3000 and 12000 g to obtain the post-peroxissomal supernatant which were used for determination of G6PD and antioxidant activities CAT T and GR. G6PD activity was determined measuring the increase in absorbance at 340 nm, resulting from NADP reduction, according to Postma et al.(1989). GR were determined by a NADPH-coupled assay followed by the decrease in absorbance at 340 nm according Goldberg and Spooner (1987). CAT T activity was determined by measuring the decrease in absorbance at 240 nm due to H2O2 consumption, according to Beers and Sizer (1952). Protein concentration was determined according to Lowry et al. (1951) using bovine serum albumin (BSA) as standard. Enzymatic activities were compared by one-way ANOVA, followed by a Dunnett's test to identify significant differences (p<0.01). Our results show a bimodal response of S. cerevisiae to [V2O5], with slightly effect of yeast cells growth rate in the range from 0 to 1.0 mM, and a significantly decrease (58% and 14% of control) for 1.5 and 2.0 mM, respectively. Conversely, we also observed a significant increase of G6PD activity (2 fold) for 0.5 mM V2O5, and a decrease of GR and CAT T activities for 0.5 and 1.0 mM. These facts appoint us to a reductive inactivation mechanism of antioxidants enzymes, which depend [V2O5] and is eventually mediated by intracellular level of NADPH. Furthermore, for [V2O5], ranging between 1.0 and 2.0 mM, we observed a decrease of G6PD (50% lower than control) occurring, at the same time, a make up of GR and CAT activity, which in the case of GR we observed an increase of 29 % and 54 % for 1.5 and 2.0 mM, respectively. Again, the reverse effects, which occur for elevated [V2O5], appoints us that ROS generate by vanadium pentoxide bring cell death.