Sylvia Pietri

Nitric oxide, superoxide and peroxynitrite: putative mediators of NMDA-induced cell death in cerebellar granule cells.

In this study, we analysed the implication of superoxide (O2-.) and nitric oxide (NO.) free radicals and their resulting product peroxynitrite (ONOO-) in the neuronal death induced by the activation of the glutamatergic receptor of the N-methyl-D-aspartate (NMDA) subtype using cultured cerebellar granule cells. The NOl donor SIN-1 (3-morpholinosydnonimine N-ethylcarbamide), at concentrations which produced a much higher guanylate cyclase activation (i.e. NO. concentration) than NMDA, was not neurotoxic and did not increase the NMDA-induced neuronal death. The absence of involvement of NO. in NMDA-induced neuronal death was confirmed by the ineffectiveness of L-NG-nitroarginine (L-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) experiments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, indicated that NMDA receptor stimulation led to the generation of O2-. from at least 15-30 min. The generation of O2-. by xanthine (XA)-xanthine oxidase (XO) induced a neuronal death similar to that of NMDA. XA-XO-induced neuronal death was suppressed by addition of either superoxide dismutase (SOD) plus catalase (CAT), or DMPO in the incubation medium. In contrast, NMDA-induced neuronal death was widely blocked by DMPO and other spin trap compounds, but not by SOD +/- CAT. XA-XO-induced neuronal death was not potentiated by SIN-1 indicating that ONOO- is not more toxic than O2-. in our neuronal model.

Tumor Protein 53-Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

p53 exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints, apoptosis, and regulation of cell redox status. p53 antioxidant function is dependent on its transcriptional activity and proceeds by sequential induction of antioxidant and proapoptotic targets. However, none of the thus far renowned p53 targets have proved able to abolish on their own the intracellular reactive oxygen species (ROS) accumulation caused by p53 deficiency, therefore pointing to the existence of other prominent and yet unknown p53 antioxidant targets. Here, we show that TP53INP1 represents such a target. Indeed, TP53INP1 transcript induction on oxidative stress is strictly dependent on p53. Mouse embryonic fibroblasts (MEF) and splenocytes derived from TP53INP1-deficient (inp1(-/-)) mice accumulate intracellular ROS, whereas overexpression of TP53INP1 in p53-deficient MEFs rescues ROS levels to those of p53-proficient cells, indicating that TP53INP1 antioxidant function is p53 independent. Furthermore, accumulation of ROS in inp1(-/-) cells on oxidant challenge is associated with decreased expression of p53 targets p21/Cdkn1a, Sesn2, TAp73, Puma, and Bax. Mutation of p53 Ser(58) (equivalent to human p53 Ser(46)) abrogates transcription of these genes, indicating that TP53INP1-mediated p53 Ser(58) phosphorylation is implicated in this process. In addition, TP53INP1 deficiency results in an antioxidant (N-acetylcysteine)-sensitive acceleration of cell proliferation. Finally, TP53INP1 deficiency increases oxidative stress-related lymphoma incidence and decreases survival of p53(+/-) mice. In conclusion, our data show that TP53INP1 is a major actor of p53-driven oxidative stress response that possesses both a p53-independent intracellular ROS regulatory function and a p53-dependent transcription regulatory function.

Ginkgo biloba extract (EGb 761) pretreatment limits free radical-induced oxidative stress in patients undergoing coronary bypass surgery.

A growing body of evidence supports the trigger role of free radicals in the delayed functional and metabolic myocardial recovery following cardiopulmonary bypass (CPB) in humans, thus opening the field to specific therapies. This clinical study was designed to evaluate, in 15 patients undergoing aortic valve replacement, whether the extent of CPB- and reperfusion-induced lipid peroxidation, ascorbate depletion, tissue necrosis, and cardiac dysfunction is reduced by orally administered EGb 761, a Ginkgo biloba extract withpotent in vitro antiradical properties. Patients received either EGb 761 (Tanakan, 320 mg/day, n = 8) or a matching placebo (n = 7) for 5 days before surgical intervention. Plasma samples were obtained from the peripheral circulation and the coronary sinus at crucial stages of the operation (i.e., before incision, during ischemia, and within the first 30 minutes post-unclamping), and up to 8 days postoperatively. Upon aortic unclamping, EGb 761 inhibited the transcardiac release of thiobarbituric acid species (p ` 0.05), as assessed by high-performance liquid chromatography, and attenuated the early (5–10 minute) decrease in dimethylsulfoxide/ascorbyl free radical levels, an electron spin resonance index of the plasma ascorbate pool (p ` 0.05). EGb 761 also significantly reduced the more delayed leakage of myoglobin (p = 0.007) and had an almost significant effect on ventricular myosin leakage (p = 0.053, 6 days postoperatively). The clinical outcome of recovery of treated patients was improved, but not significantly, compared with untreated patients. Our results demonstrate the usefulness of adjuvant EGb 761 therapy in limiting oxidative stress in cardiovascular surgery and suggest the possible role of highly bioavailable terpene constituents of the drug.

Colitis and Colitis-Associated Cancer Are Exacerbated in Mice Deficient for Tumor Protein 53-Induced Nuclear Protein 1

ABSTRACT Tumor protein 53-induced nuclear protein 1 (TP53INP1) is an antiproliferative and proapoptotic protein involved in cell stress response. To address its physiological roles in colorectal cancer and colitis, we generated and tested the susceptibility of Trp53inp1-deficient mice to the development of colorectal tumors induced by injection of the carcinogen azoxymethane followed by dextran sulfate sodium (DSS)-induced chronic colitis. Trp53inp1-deficient mice showed an increased incidence and multiplicity of tumors compared to those of wild-type (WT) mice. Furthermore, acute colitis induced by DSS treatment was more severe in Trp53inp1-deficient mice than in WT mice. Treatment with the antioxidant N-acetylcysteine prevented colitis and colitis-associated tumorigenesis more efficiently in WT mice than in Trp53inp1-deficient mice, suggesting a higher oxidative load in the latter. Consistently, we demonstrated by electron spin resonance and spin trapping that colons derived from deficient mice produced more free radicals than those of the WT during colitis and that the basal blood level of the antioxidant ascorbate was decreased in Trp53inp1-deficient mice. Collectively, these results indicate that the oxidative load is higher in Trp53inp1-deficient mice than in WT mice, generating a more-severe DSS-induced colitis, which favors development of colorectal tumors in Trp53inp1-deficient mice. Therefore, TP53INP1 is a potential target for the prevention of colorectal cancer in patients with inflammatory bowel disease.

Ascorbyl free radical: A noninvasive marker of oxidative stress in human open-heart surgery

To assess the development of oxidative stress in cardiac ischemia/reperfusion, the resulting depletion of plasma ascorbate was monitored by electron spin resonance spectroscopic detection of ascorbyl free radical (AFR) in a homogeneous group of 12 patients undergoing aortic valve replacement. Dimethyl sulfoxide (DMSO) was used as an enhancer and stabilizer for AFR in plasma separated from blood samples collected 15 min before incision, 10 min before aortic declamping, and sequentially during the initial 30 min of reperfusion. Plasma DMSO/AFR levels of patients were found to be significantly lower than in healthy subjects (-25%), further decreased upon ischemia (-35%), dropped to their lowest values within the first 10 min of reperfusion (-46%), and did not recover their initial values within 30 min following reflow. Cardiac index measurements revealed a still depressed heart function 4 h postdeclamping and a more delayed tissue injury was evidenced by cardiac myosin and myoglobin release in plasma. DMSO/AFR levels at early reperfusion were slightly (+ 12%) higher in plasma obtained from coronary sinus samples than in plasma from peripheral blood, suggesting an extra ascorbate release from the injured heart tissue. The close analogy between these results and the reported measurements of other plasma markers of oxidative stress, including ascorbate, indicates that the present method could be of great value in clinical practice.

Plastid Alternative Oxidase (PTOX) Promotes Oxidative Stress When Overexpressed in Tobacco

Photoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b6f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant system.

Sedentary aging increases resting and exercise-induced intramuscular free radical formation

Mitochondrial free radical formation has been implicated as a potential mechanism underlying degenerative senescence, although human data are lacking. Therefore, the present study was designed to examine if resting and exercise-induced intramuscular free radical-mediated lipid peroxidation is indeed increased across the spectrum of sedentary aging. Biopsies were obtained from the vastus lateralis in six young (26 + or - 6 yr) and six aged (71 + or - 6 yr) sedentary males at rest and after maximal knee extensor exercise. Aged tissue exhibited greater (P < 0.05 vs. the young group) electron paramagnetic resonance signal intensity of the mitochondrial ubisemiquinone radical both at rest (+138 + or - 62%) and during exercise (+143 + or - 40%), and this was further complemented by a greater increase in alpha-phenyl-tert-butylnitrone adducts identified as a combination of lipid-derived alkoxyl-alkyl radicals (+295 + or - 96% and +298 + or - 120%). Lipid hydroperoxides were also elevated at rest (0.190 + or - 0.169 vs. 0.148 + or - 0.071 nmol/mg total protein) and during exercise (0.567 + or - 0.259 vs. 0.320 + or - 0.263 nmol/mg total protein) despite a more marked depletion of ascorbate and uptake of alpha/beta-carotene, retinol, and lycopene (P < 0.05 vs. the young group). The impact of senescence was especially apparent when oxidative stress biomarkers were expressed relative to the age-related decline in mitochondrial volume density and absolute power output at maximal exercise. In conclusion, these findings confirm that intramuscular free radical-mediated lipid peroxidation is elevated at rest and during acute exercise in aged humans.

Calcium-dependent free radical generation in cultured retinal neurons injured by kainate

Cultured rat retinal neurons exposed to kainate produced free radicals, as demonstrated by electron spin resonance (ESR) spin trapping using the nitrone 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the generation of DMPO hydroxyl adduct (DMPO-OH). This DMPO-OH production was abolished by EGTA, nitro-arginine and oxypurinol, suggesting that it was dependent on Ca2+ influx and subsequent activation of nitric oxide synthase and xanthine oxidase. Moreover, kainate induced a receptor-mediated Ca2+ influx and neuronal injury assessed by lactate dehydrogenase release. Neuroprotection afforded by nitro-arginine and oxypurinol shows that calcium-dependent free radical production plays a major role in kainate retinal toxicity.

Anticancer Activities of Essential Oils Constituents and Synergy with Conventional Therapies: A Review

Many studies have shown that a large number of terpenoids and aromatic compounds contained in essential oils have significant anticancer activities, both on cell lines and on tumors in animals. The activity of these constituents is related to the activation of cell death (apoptosis) induced by the caspases proteins in cancer cells, with minor modifications of healthy cells. Many phenomena seem to occur, among which are as follows: overexpression and regulation of liver detoxification enzymes, changes in the membrane potential of cancer cells and mitochondria, production of free radicals in cancer cells, inhibition of angiogenesis, and modification of tumor‐inducing genes. These active essential oil constituents appear to act synergistically with conventional chemotherapy and radiotherapy, and some clinical studies in humans are beginning to be realized. In this review, we discuss about the antitumoral activity of 13 essential oil components selected among the most studied in the literature, with a focus on their possible mode of action. We also report current data on the anticancer properties of several total essential oils. Copyright

Exercise‐induced oxidative–nitrosative stress is associated with impaired dynamic cerebral autoregulation and blood–brain barrier leakage

The present study examined whether dynamic cerebral autoregulation and blood–brain barrier function would become compromised as a result of exercise‐induced oxidative–nitrosative stress. Eight healthy men were examined at rest and after an incremental bout of semi‐recumbent cycling exercise to exhaustion. Changes in a dynamic cerebral autoregulation index were determined during recovery from continuous recordings of blood flow velocity in the middle cerebral artery (MCAv) and mean arterial pressure during transiently induced hypotension. Electron paramagnetic resonance spectroscopy and ozone‐based chemiluminescence were employed for direct detection of spin‐trapped free radicals and nitric oxide metabolites in venous blood. Neuron‐specific enolase, S100β and 3‐nitrotyrosine were determined by ELISA. While exercise did not alter MCAv, it caused a mild reduction in the autoregulation index (from 6.9 ± 0.6 to 5.5 ± 0.9 a.u., P < 0.05) that correlated directly against the exercise‐induced increase in the ascorbate radical, 5‐(diethoxyphosphoryl)‐5‐methyl‐1‐pyrroline N‐oxide and N‐tert‐butyl‐α‐phenylnitrone adducts, 3‐nitrotyrosine and S100β (r=–0.66 to –0.76, P < 0.05). In contrast, no changes in neuron‐specific enolase were observed. In conclusion, our findings suggest that intense exercise has the potential to increase blood–brain barrier permeability without causing structural brain damage subsequent to a free radical‐mediated impairment in dynamic cerebral autoregulation.