Taking EPR "snapshots" of the oxidative stress status in human blood

Assessment of oxidative stress status (OSS) in human tissues is still troublesome. Using an innovative EPR-radical-probe we successfully measured the instantaneous concentration of ROS directly in peripheral blood of athletes and normally active workers during 60 min controlled exercise. The probe employed was bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate, which quantitatively and instantaneously reacts with oxygen-centered radicals (including superoxide) to yield the parent nitroxide, which is sufficiently persistent to be measured by EPR. Our measurements suggest that while at rest normally active individuals may benefit more from antioxidant supplementation than athletes; conversely, during exercise athletes may benefit more from supplementation. Our method allows reliable, quick, and non-invasive quantitative determination of OSS in human peripheral blood.     

"Action-at-a distance" of a new DNA oxidative damage product 6-furfuryl-adenine (kinetin) on template properties of modified DNA

N(6)-furfuryladenine (kinetin, K) was shown to have cytokinin activity and antiageing effects. It also appears to protect DNA against oxidative damage mediated by the Fenton reaction. Kinetin was identified as a natural component of DNA in plant extract, calf thymus DNA, fresh DNA preparations from human cell culture, as well as in human urine. A proposed mechanism of kinetin synthesis includes furfural, the oxidative damage product of a 2-deoxyribose moiety of DNA, which reacts with an adenine residue to form N(6)-furfuryladenine at DNA level. The identification of kinetin in plant cell extracts, as well as human urine, suggests its excision from DNA by repair mechanisms. Since such a bulky modification as kinetin induces conformational changes of DNA, this could lead to mutations. Therefore, it was interesting to analyze an effect of kinetin on coding properties of DNA. Chemically synthesized oligodeoxynucleotide (20-mer) containing kinetin AAAACTGCCGTCCTGAKGAT was used as a primer. It was elongated in a polymerase chain reaction (PCR) on a template plasmid pEW1 harboring a 210-bp fragment of DNA derived from the 5' end of HIV mRNA. The PCR product of that length containing kinetin in position 17 from the 5' end was isolated and sequenced. Interestingly, DNA polymerase correctly incorporates thymine opposite of kinetin (an adenine derivative) on the complementary strand, but the misincorporations occur in a vicinity of the modified base.     

Damage to cultivated Japanese pearl oysters by oxidative stress that was related to "mass mortality"

Increased blood-DNA breakage was observed in diseased pearl oysters. They showed significant formation of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA), whereas the oysters that had a low mortality rate from the disease had high activity of superoxide dismutase (SOD) and low amounts of 8-OHdG and MDA. These results suggest that radical damage had occurred only in the diseased pearl oysters with the cytolysis of their haemocytes, which was related to the mass mortality of the Japanese pearl oysters.     

Tetrahydrophthalazine derivative "sodium nucleinate" exert its anti-inflammatory effects through inhibition of oxidative burst in human monocytes

We described the use of a new chemical substance Sodium nucleinate (SN) as an immunomodulatory substance exhibiting antiinflammatory properties. Sodium nucleinate (SN) registrated in Russian Federation as Tamerit, is 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, derivative of well known chemical substance luminol. To comprehend the mechanisms of SN immunomodulatory activity, we examined the SN modulation of the oxidative burst responses of whole blood human monocytes and polimorphonuclear cells (PMC) stimulated with phorbol 12-myristate 13-acetate (PMA) or E. coli suspension in vitro. SN did not inhibit the proportion of neutrophils and monocytes phagocytosing E. coli. Oxidative burst responses of monocytes stimulated with PMA were strongly inhibited at SN concentration ranging from 10-500 mg/ml, less efficient inhibitor was SN in E. coli stimulated monocytes (inhibition range was from 50-500 mg/ml SN). SN inhibited PMC oxidative burst only in range 100-500 mg/ml SN. In conclusion, we found SN as an efficient inhibitor of oxidative burst in monocytes. Since ROS generation in monocytes/macrophages has been found to be important for LPS-driven production of several proinflammatory cytokines, SN may exsert its antiinflammatory effects through monocyte/macrophage oxidative burst inhibition.     

Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry"

Induction of DNA damage by oxidants such as H(2) O(2) activates the complex network of DNA damage response (DDR) pathways present in cells to initiate DNA repair, halt cell cycle progression, and prepare an apoptotic reaction. We have previously reported that activation of Ataxia Telangiectasia Mutated protein kinase (ATM) and induction of γH2AX are among the early events of the DDR induced by exposure of cells to H(2) O(2) , and in human pulmonary carcinoma A549 cells, both events were expressed predominantly during S-phase. This study was designed to further explore a correlation between these events and DNA replication. Toward this end, we utilized 5-ethynyl-2'deoxyuridine (EdU) and the "click chemistry" approach to label DNA during replication, followed by exposure of A549 cells to H(2) O(2) . Multiparameter laser scanning cytometric analysis of these cells made it possible to identify DNA replicating cells and directly correlate H(2) O(2) -induced ATM activation and induction of γH2AX with DNA replication on a cell by cell basis. After pulse-labeling with EdU and exposure to H(2) O(2) , confocal microscopy was also used to examine the localization of DNA replication sites ("replication factories") versus the H2AX phosphorylation sites (γH2AX foci) in nuclear chromatin in an attempt to observe the absence or presence of colocalization. The data indicate a close association between DNA replication and H2AX phosphorylation in A549 cells, suggesting that these DNA damage response events may be triggered by stalled replication forks and perhaps also by induction of DNA double-strand breaks at the primary DNA lesions induced by H(2) O(2) .     

"Effect of resveratrol on oxidative and inflammatory stress in liver and spleen of streptozotocin-induced type 1 diabetic rats"

"It has been well known that both oxidative stress and inflammatory activity play crucial roles in the pathogenesis of type 1 diabetes mellitus (T1DM). Resveratrol (RSV), a naturally occurring polyphenol found in grapes and red wine, has recently been shown to exert potent anti-diabetic, anti-oxidative and anti-inflammatory actions. In the present study, we investigated the effect of RSV on oxidative stress and inflammatory response in the liver and spleen of streptozotocin (STZ)-induced type 1 diabetic animal models. Male Long-Evans rats were injected with 65 mg/kg STZ to induce diabetes for 2 weeks, and subsequently administrated with the dosage of 0.1 or 1 mg/kg/day RSV for 7 consecutive days. Hepatic and splenic tissues were dissected for evaluation of oxidative and inflammatory stress. Oxidative stress was assessed by quantification of oxidative indicators including superoxide anion content, lipid and protein oxidative products, as well as manganese superoxide dismutase (Mn-SOD) and nitro-tyrosine protein expression levels. Inflammatory stress was evaluated by the levels of nuclear factor κB (NF-κB) and the proinflammatory cytokine tumor necrosis factorα (TNF-α), interleukin 1 β (IL-1 β ) and IL-6. The experimental results indicated that RSV significantly decreased oxidative stress (superoxide anion content, protein carbonyl level and Mn-SOD expression) in both tissues and hepatic inflammation (NF- κB and IL-1 β ), but implicated proinflammatory potential of RSV in diabetic spleen (TNF-α and IL-6). The results of this study suggest that RSV may serve as a potent antioxidant, but RSV possesses a proinflammatory potential in certain circumstances in diabetes."     

Motile plant cell body: a "bug" within a "cage"

Analysis of the cytoskeleton in morphogenetically active plant cells allows us to propose a unified concept for the structural organization of eukaryotic cells. Their cytoarchitecture is determined by two principal structural complexes: nucleus-microtubule-based cell bodies ("bugs") and plasma-membrane-F-actin-based cell periphery complexes ("cages"). There are dynamic interactions between each of these entities in response to extracellular and intracellular signals. In the case of the cell body, these signals determine its polarization, rotation and migration. Interactions between cell body and cell periphery complexes determine cell growth polarity and morphogenesis throughout the eukaryotic kingdom.     

Calcein-AM is a detector of intracellular oxidative activity

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H₂O₂ and xanthine–xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H₂O₂, xanthine–xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.     

Compensatory growth and oxidative stress in a damselfly

Physiological costs of compensatory growth are poorly understood, yet may be the key components in explaining why growth rates are typically submaximal. Here we tested the hypothesized direct costs of compensatory growth in terms of oxidative stress. We assessed oxidative stress in a study where we generated compensatory growth in body mass by exposing larvae of the damselfly Lestes viridis to a transient starvation period followed by ad libitum food. Compensatory growth in the larval stage was associated with higher oxidative stress (as measured by induction of superoxide dismutase and catalase) in the adult stage. Our results challenge two traditional views of life-history theory. First, they indicate that age and mass at metamorphosis not necessarily completely translate larval stress into adult fitness and that the observed physiological cost may explain hidden carry-over effects. Second, they support the notion that costs of compensatory growth may be associated with free-radical-mediated trade-offs and not necessarily with resource-mediated trade-offs.     

Calcein-AM is a detector of intracellular oxidative activity

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine-xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine-xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.