Agnieszka Grzelak

Hemoglobin can nitrate itself and other proteins.

Incubation of human hemoglobin with nitrite and hydrogen peroxide was found to induce autonitration and nitration of another protein (bovine serum albumin), as demonstrated by detection of nitrotyrosine residues in Western blots of separated membrane proteins. Inhibition of nitration by conversion of hemoglobin into the cyanmet form demonstrates that nitration is due to the pseudoperoxidase activity of hemoglobin. Incubation of whole erythrocytes with nitrite and hydrogen peroxide induces nitration of erythrocyte membrane proteins, much stronger when cellular catalase was inhibited with azide. These results suggest that hemoglobin and other hemoproteins may contribute to the tyrosine nitration in vivo.

Oxidative DNA damage corresponds to the long term survival of human cells treated with silver nanoparticles

We examined the relation between DNA damage and the clonogenic potential of 3 human cell lines, HepG2, HT29 and A549, treated with bare 20 nm or 200 nm silver nanoparticles (AgNPs). The endpoints examined were the DNA breakage estimated by the comet assay, the oxidative base damage recognized by formamido-pyrimidine glycosylase (FPG) and estimated with the FPG+comet assay, and the frequencies of histone γH2AX foci and micronuclei. Each cell line studied had a different pattern of DNA breakage and base damage versus the NPs concentration and time of treatment. The overall pattern of DNA breakage and base damage induction corresponded to the intracellular generation of reactive oxygen species. There was no increase in the frequencies of histone γH2AX foci and micronuclei as compared to those in the untreated cells. The reported experiments suggest that only the oxidative DNA damage corresponds to the loss of the clonogenic ability of cells treated with AgNPs.

Pro-oxidative activity of nitroxides in their reactions with glutathione

Nitroxides are unreactive towards glutathione in vitro. Interaction of nitroxides with peroxynitrite does not lead to a significant loss of their electron paramagnetic resonance (EPR) signal. However, addition of peroxynitrite to a solution containing glutathione and nitroxides induces an irreversible disappearance of EPR signal of nitroxides and augmentation of glutathione oxidation which is a pro-oxidant effect of these compounds. Nitroxide loss leading to the formation of amine derivatives is initiated by products of glutathione oxidation by peroxynitrite. The pro-oxidant action of nitroxides at micromolar concentrations may be important in view of the proposed use of these compounds as antioxidants.

Interaction between antioxidants in assays of total antioxidant capacity.

Sub-additivity of antioxidant activities in assays of total antioxidant capacity has been reported previously and ascribed to binding of low-molecular weight antioxidants such as flavonoids by proteins. We demonstrate that this phenomenon is much more common and concerns also interactions between typical low-molecular weight oxidants in the assays of ABTS- decolorization and protection of fluorescein from AAPH-induced bleaching. The subadditive interactions between antioxidants may affect quantitative considerations drawn from in vitro assays of antioxidant capacity of biological samples.

Dopamine-melanin protects against tyrosine nitration, tryptophan oxidation and Ca2+-ATPase inactivation induced by peroxynitrite

The effects of dopamine-melanin (DA-melanin), a synthetic model of neuromelanin, on peroxynitrite-mediated 3-nitrotyrosine formation, oxidation of tryptophan in bovine serum albumin and inactivation of erythrocyte membrane Ca(2+)-ATPase activity were investigated in the absence and in the presence of bicarbonate. DA-melanin inhibited nitration of free tyrosine, loss of tryptophan residues and Ca(2+)-ATPase inactivation by peroxynitrite in a dose dependent manner. In the presence of bicarbonate, this inhibitory effect was lower for nitration and insignificant for oxidative protein modifications. These results suggest that neuromelanin can protect against nitrating and oxidizing action of peroxynitrite but is a worse protector against the peroxynitrite-CO(2) adduct. As peroxynitrite may be a mediator of neurotoxic processes, the obtained results suggest that neuromelanin may be important as a physiological protector against peroxynitrite.

Nucleolus as an oxidative stress sensor in the yeast Saccharomyces cerevisiae

Abstract In mammals, the nucleolus is thought to be a stress sensor; upon cellular stress conditions, a release of nucleolar proteins and down-regulation of rDNA transcription occurs. Since yeast Rrn3p is a homolog of the mammalian RNA polymerase I (Pol I)-specific transcription factor TIF-IA, we decided to investigate the role of Rrn3p in oxidant-induced nucleolar stress in yeast. We show that, after oxidant treatment, the level of Rrn3p is unaffected but Rrn3p is translocated from the nucleolus into the cytoplasm and a point mutation in the RRN3 gene leads to hypersensitivity of the yeast to oxidants. This hypersensitivity can be abolished by re-introduction of the active RRN3 gene, antioxidant supplementation and anoxic atmosphere. Additionally, we employed the PRINS technique to monitor oxidant-mediated changes in the nucleolar structure. Taken together, our results suggest the role of the yeast nucleolus in the response to oxidative stress signals.

The effects of superoxide dismutase knockout on the oxidative stress parameters and survival of mouse erythrocyt

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 Heinz bodies and the reticulocyte count were indistinguishable in Sod1+/+ 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.

Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress

Multidrug resistance (MDR), which is the main obstacle to cancer chemotherapy, is mainly due to overexpression of ATP-binding cassette (ABC) transporters, especially ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP). A novel idea to overcome MDR is that of collateral sensitivity, i.e., finding a treatment to which cells overexpressing ABC transporters are more sensitive than cells that do not overexpress them. In this study we demonstrate for the first time that MDCKII-BCRP cells, overexpressing ABCG2, are more vulnerable to exogenous oxidative stress induced by several oxidants, viz. paraquat, menadione, hydrogen peroxide, tert-butylperoxide, and 2,2-azobis(2-methylpropionamidine) dihydrochloride. MDCKII-BCRP cells have significantly decreased glutathione level and decreased activities of glutathione S-transferase and glutathione reductase, which may underlie their augmented vulnerability to oxidative stress. These results suggest the possibility of using agents that induce oxidative stress to selectively kill cells overexpressing BCRP.

A genetic analysis of nitric oxide-mediated signaling during chronological aging in the yeast.

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.

Decreased antioxidant defense during replicative aging of the yeast Saccharomyces cerevisiae studied using the ‘baby machine’ method

Replicatively senescent cells of Saccharomyces cerevisiae were obtained using the ‘baby machine’ method by immobilizing cells on CovaLink™ NH2 plates and allowing them to divide while exchanging medium and removing daughter cells. Centrifugation in a Percoll density gradient was employed for further purification of replicatively old yeast cells. Comparison of senescent cells showing more than 20 bud scars with cells from early stationary culture demonstrated a significant reduction of total and reduced glutathione and decrease of superoxide dismutase activity during replicative aging of yeast cells.