Abstract:The erythrocytes of 12-month old Sod1 -/-mice showed an increased level of reactive oxygen species (ROS), as estimated by the degree of dihydroethidine and dihydrorhodamine oxidation, and the increased level of Heinz bodies. No indices of severe oxidative stress were found in the red blood cells and blood plasma of Sod1 -/-mice as judged from the lack of significant changes in the levels of erythrocyte and plasma glutathione, plasma protein thiol and carbonyl groups and thiobarbituric-acid reactive substances in the blood plasma. However, a decreased erythrocyte lifespan, increased reticulocyte count and splenomegaly were noted, indicating the importance of superoxide dismutase for maintaining erythrocyte viability. The levels of erythrocyte ROS and Sod1 +/-mice, suggesting that a superoxide dismutase activity decrease to half of its normal value may be sufficient to secure the protective effects of the enzyme.

In mammals, NO•, a signaling molecule is implicated in the regulation of vasodilation, neurotransmission and immune response. It is believed that NO• is a signaling molecule also in unicellular organism like yeast and may be involved in the regulation of apoptosis and sporulation. It has been reported that NO• is produced during chronological aging (CA) leading to an increase of the superoxide level, which in turn mediates apoptosis. Since this conclusion was based on indirect measurements of NO• by the Griess reaction, the role of NO• signaling during CA in the yeast remains uncertain. We investigated this issue more precisely using different genetic and biochemical methodologies. We used cells lacking the factors influencing nitrosative stress response like flavohemoglobin metabolizing NO•, S-nitrosoglutathione reductase metabolizing S-nitrosoglutathione and the transcription factor Fzf1p mediating NO• response. We measured the standard parameters describing CA and found an elevation in the superoxide level, percentage of death cells, the level of TUNEL positive cells and a decrease in proliferating potential. These observations showed no significant differences between wild type cells and the disruptants except for a small elevation of the superoxide level in the Δsfa1 mutant. The intracellular NO• level and flavohemoglobin expression decreased rather than increased during CA. Products of general nitrogen metabolism and protein tyrosine nitration were slightly decreased during CA, the magnitude of changes showing no differences between the wild type and the mutant yeast. Altogether, our data indicate that apoptosis during yeast CA is mediated by superoxide signaling rather than NO• signaling.

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Abbreviations used: DTNB -5,5'-dithiobis-(2-nitrobenzoic acid); H 2 R 123 -dihydrorhodamine 123; MES -2-morpholinoethanesulfonic acid; ROS -reactive oxygen species; SOD -superoxide dismutase; YPD medium -1% yeast extract, 1% Bacto-peptone, 2% glucose, YPG medium, 1% yeast extract, 1% Bacto-peptone,2% glycerol; YPE medium -1% yeast extract, 1% Bacto-peptone, 3% ethanol Abstract: We compared the oxidation of dihydrorhodamine 123, glutathione contents and activities of superoxide dismutase (SOD) and catalase for three wild-type strains of Saccharomyces cerevisiae grown on media with different carbon sources. The rate of oxidation of dihydrorhodamine 123 was much higher in respiring cells grown on ethanol or glycerol media than in fermenting cells grown on glucose medium. The total SOD activity was highest on glycerol medium and lowest on ethanol medium, while the catalase activity was highest on glycerol medium. The sequence of glutathione content values was: glucose > ethanol > glycerol.

It has been claimed that melatonin reacts directly with hydrogen peroxide with a very high rate constant (2.5 x 10(5)-2.3 x 10(6) M(-1) s(-1)) Both these values were derived from inhibition by melatonin of peroxidase-catalyzed oxidation of Phenol Red by hydrogen peroxide, assuming that this inhibition is due to direct scavenging of hydrogen peroxide by melatonin. In this study, we show that this reasoning is illegitimate and melatonin decreases the yield of oxidation of Phenol Red as a competitive substrate. Monitoring changes of concentration of hydrogen peroxide incubated with melatonin using Xylenol Orange points to poor reactivity of melatonin with H2O2.