Cellular Reactive Oxygen Species Content Research Articles

Anti‑epileptic mechanism of isopimaric acid from Platycladi cacumen based on network pharmacology, molecular docking and biological validation.

Platycladi cacumen (PC) is derived from the dry twigs and leaves of Platycladi orientalis (L.) Franco and exerts anti-epileptic effects. However, its mechanism of action remains unknown. The present study explored the potential anti-epileptic components and mechanisms of PC. The primary active components and targets of PC were analyzed using network pharmacology and a lipopolysaccharide (LPS)-induced murine microglial cell line (BV2) was used to confirm anti-epileptic effects by detecting reactive oxygen species (ROS), apoptosis, inflammatory markers, cell migration and signaling pathways. A total of 13 core active components showed druggable properties, of which deoxypicrop odophyllotoxin, hinokinin and isopimaric acid (IPA) were predicted to cross the blood-brain barrier. In total, 255 potential targets of these three compounds were predicted using SwissTargetPrediction and Similarity Ensemble Approach websites and 150 were associated with epilepsy. In vitro experiments confirmed that IPA significantly inhibited LPS-induced microglial oxidative stress and inflammation by decreasing the migration area, cellular ROS content, lactate dehydrogenase release and early phase of apoptosis. IPA also increased the mRNA expression of anti-oxidative enzymes (superoxide dismutase-1 and -2) and suppressed inflammatory cytokines (interleukin-1β and tumor necrosis factor-α). Furthermore, IPA phosphorylated AKT and mTOR proteins. Taken together, the present findings suggested that IPA is a potential anti-epileptic compound derived from PC.

Retrograde response to mitochondrial dysfunctions associated to LOF variations in FLAD1 exon 2: unraveling the importance of RFVT2

Flavin adenine dinucleotide (FAD) synthase (EC 2.7.7.2), encoded by human flavin adenine dinucleotide synthetase 1 (FLAD1), catalyzes the last step of the pathway converting riboflavin (Rf) into FAD. FLAD1 variations were identified as a cause of LSMFLAD (lipid storage myopathy due to FAD synthase deficiency, OMIM #255100), resembling Multiple Acyl-CoA Dehydrogenase Deficiency, sometimes treatable with high doses of Rf; no alternative therapeutic strategies are available. We describe here cell morphological and mitochondrial alterations in dermal fibroblasts derived from a LSMFLAD patient carrying a homozygous truncating FLAD1 variant (c.745C > T) in exon 2. Despite a severe decrease in FAD synthesis rate, the patient had decreased cellular levels of Rf and flavin mononucleotide and responded to Rf treatment. We hypothesized that disturbed flavin homeostasis and Rf-responsiveness could be due to a secondary impairment in the expression of the Rf transporter 2 (RFVT2), encoded by SLC52A2, in the frame of an adaptive retrograde signaling to mitochondrial dysfunction. Interestingly, an antioxidant response element (ARE) is found in the region upstream of the transcriptional start site of SLC52A2. Accordingly, we found that abnormal mitochondrial morphology and impairments in bioenergetics were accompanied by increased cellular reactive oxygen species content and mtDNA oxidative damage. Concomitantly, an active response to mitochondrial stress is suggested by increased levels of PPARγ-co-activator-1α and Peroxiredoxin III. In this scenario, the treatment with high doses of Rf might compensate for the secondary RFVT2 molecular defect, providing a molecular rationale for the Rf responsiveness in patients with loss of function variants in FLAD1 exon 2. HIGHLIGHTS FAD synthase deficiency alters mitochondrial morphology and bioenergetics; FAD synthase deficiency triggers a mitochondrial retrograde response; FAD synthase deficiency evokes nuclear signals that adapt the expression of RFVT2.

All-trans-retinoic acid induces RARB-dependent apoptosis via ROS induction and enhances cisplatin sensitivity by NRF2 downregulation in cholangiocarcinoma cells.

All-trans-retinoic acid (ATRA) has been clinically used to treat acute promyelocytic leukemia and is being studied to treat other types of cancer; however, the therapeutic role and mechanism of ATRA against cholangiocarcinoma (CCA) remain unclear. The present study investigated the cytotoxic effect and underlying mechanisms of ATRA on CCA cell lines. Cell viability was evaluated by sulforhodamine B assay. Intracellular reactive oxygen species (ROS) levels were assessed by dihydroethidium assay. Apoptosis analysis was performed by flow cytometry. The pathways of apoptotic cell death induction were examined using enzymatic caspase activity assay. Proteins associated with apoptosis were evaluated by western blotting. The effects on gene expression were analyzed by reverse transcription-quantitative PCR analysis. ATRA induced a concentration- and time-dependent toxicity in CCA cells. Furthermore, when the cytotoxicity of ATRA against retinoic acid receptor (RAR)-deficient cells was assessed, it was revealed that ATRA cytotoxicity was RARB-dependent. Following ATRA treatment, there was a significant accumulation of cellular ROS and ATRA-induced ROS generation led to an increase in the expression levels of apoptosis-inducing proteins and intrinsic apoptosis. Pre-treatment with ROS scavengers could diminish the apoptotic effect of ATRA, suggesting that ROS and mitochondria may have an essential role in the induction of apoptosis. Furthermore, following ATRA treatment, an increase in cellular ROS content was associated with suppressing nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2) and NRF2-downstream active genes. ATRA also suppressed cisplatin-induced NRF2 expression, suggesting that the enhancement of cisplatin cytotoxicity by ATRA may be associated with the downregulation of NRF2 signaling. In conclusion, the results of the present study demonstrated that ATRA could be repurposed as an alternative drug for CCA therapy.

Effects of shikonin on the development of ovarian follicles and female germline stem cells

ObjectiveTo investigate the effects and potential mechanism of action of shikonin (SHK) on the development of ovarian follicles and female germline stem cells (FGSCs).MethodsFemale Kunming adult mice were administered SHK (0, 20 and 50 mg/kg) by oral gavage. Cultures of FGSCs were treated with SHK 32 μmol/l for 24 h. The ovarian index in mouse ovaries was calculated. Numbers of primordial, primary and atretic follicles were counted. Germline stem cell markers and apoptosis were examined. Levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) were measured.ResultsBoth doses of SHK significantly decreased the ovarian index, the numbers of primordial follicles, primary follicles and antral follicles in mice. SHK significantly increased the numbers of atretic follicles and atretic corpora lutea. SHK promoted apoptosis in vivo and in vitro. SHK significantly decreased the levels of the germline stem cell markers. SHK significantly lowered GSH levels and the activity of SOD in the peripheral blood from mice, whereas SHK significantly elevated cellular ROS content in FGSCs.ConclusionsThese current results suggested that follicular development and FGSCs were suppressed by SHK through the induction of apoptosis and oxidative stress might be involved in this pathological process.

Astaxanthin Decreases Spatial Memory and Glutamate Transport Impairment Induced by Fluoride.

Excessive exposure to the sources of fluoride in drinking water, oral care products, and food is a widespread problem. Fluoride is associated with impairment in child intelligence development. It causes DNA damage, oxidative stress, and mitochondrial dysfunction, mainly due to the production of reactive oxygen species (ROS). It has been postulated that the use of antioxidants such as astaxanthin, may alleviate fluoride’s adverse effects. This study assessed the effects of fluoride on cellular ROS content and rat’s learning and memory ability and investigated the protective potency of astaxanthin with emphasis on the role of glutamate using the Morris Water Maze test, glutamate concentration determination, and western blot techniques. The fluoride treatment of cells results in an increment of cellular ROS, whereas astaxanthin inhibits lipid peroxidation. Fluoride significantly decreases the cellular glutamate uptake and glutamate transporter, protein level, possibly due to the disruption of mitochondrial energy metabolism and defect of the transporter recycle, respectively. The in-vivo study indicated that the treatment of rats with fluoride led to a loss of learning, while astaxanthin improved memory dysfunction. Measurement of ROS and glutamate levels of rat brain hippocampus showed that fluoride increased the ROS but decreased the glutamate. On the other hand, the utilization of astaxanthin decreased the brain ROS content and increased the glutamate level. It seems that fluoride disrupts the normal function of neurons via increment of ROS production and decrement of glutamate level, whereas astaxanthin has neuroprotective potency due to the ROS scavenging ability.

ROS Dynamics Delineate Functional States of Hippocampal Neural Stem Cells and Link to Their Activity-Dependent Exit from Quiescence.

SummaryCellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of intracellular reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we here show that in the hippocampus of adult mice, quiescent neural precursor cells (NPCs) maintain the highest ROS levels (hiROS). Classifying NPCs on the basis of cellular ROS content identified distinct functional states. Shifts in ROS content primed cells for a subsequent state transition, with lower ROS content marking proliferative activity and differentiation. Physical activity, a physiological activator of adult hippocampal neurogenesis, recruited hiROS NPCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results provide a metabolic classification of NPC functional states and describe a mechanism linking the modulation of cellular ROS by behavioral cues to the activation of adult NPCs.

Targeted Inhibition of P4HB Promotes Cell Sensitivity to Gemcitabine in Urothelial Carcinoma of the Bladder.

BackgroundBladder cancer (BC) is a common malignancy worldwide that accounts for 3% of global cancer diagnoses. Chemotherapy resistance limits the therapeutic effect of chemotherapeutic agents in patients with BC. Prolyl 4-hydroxylase, beta polypeptide (P4HB) is an endoplasmic reticulum (ER) chaperone that is upregulated in bladder cancer tissues (The Cancer Genome Atlas, TCGA datasets). Knockdown or suppression of P4HB exerts anticancer activity and sensitizes cells to chemotherapy in various types of cancer.PurposeWe aimed to investigate whether the inhibition of P4HB enhances the anticancer efficacy of gemcitabine (GEM) in BC cells and to study the underlying molecular mechanisms.Patients and MethodsThe P4HB mRNA expression levels of 411 BC patients from the TCGA database and P4HB expression level of eighty BC paraffin-embedded samples detected by immunohistochemistry (IHC) staining were used for clinical feature and prognostic analyses. Bioinformatics analysis was utilized for the mechanistic investigation. Highly P4HB-expressed BC cell lines (T24 and 5637) treated with P4HB inhibitor (Bacitracin, BAC) were used to study the effects of BAC on the sensitivity of BC cells to GEM and the potential mechanism. P4HB inhibition experiments were performed in highly P4HB-expressed BC cells, and cell viability, colony formation, cell cycle, reactive oxygen species (ROS), apoptosis and pathway proteins were assessed in T24 and 5637 cells.ResultsWestern blot analysis showed that P4HB expression was significantly higher in BC tissues than in paired normal tissues. IHC showed that patients with high P4HB expression had a poorer overall survival (OS) rate than those with low P4HB expression. Furthermore, increased P4HB expression was demonstrated to be an independent prognostic marker for BC. Functionally, P4HB inhibition by BAC decreased the cell proliferation ability in vitro. Moreover, BAC treatment sensitized BC cells to GEM. Molecular mechanism analysis indicated that inhibition of P4HB by BAC treatment enhanced the anticancer effects of GEM through increasing cellular ROS content and promoting cell apoptosis and PERK/eIF2α/ATF4/CHOP signaling.ConclusionHigh P4HB expression was significantly correlated with poor prognosis in BC patients. Inhibition of P4HB by BAC decreased the cell proliferation ability and sensitized BC cells to GEM by activating apoptosis and the PERK/eIF2α/ATF4/CHOP pathways.

Synthesis of Hydrophobic Propionyl Neohesperidin Ester Using an Immobilied Enzyme and Description of Its Anti-proliferative and Pro-apoptotic Effects on MCF-7 Human Breast Cancer Cells.

Neohesperidin (NH) is a natural flavonoid glycoside compound with considerable physiological and pharmacological activities. However, its bioavailability is limited due to poor solubility, and few studies have so far attempted improve the solubility and bioavailability of NH. In this study, we structurally modified NH using an immobilized lipase to improve lipophilicity and therefore expand its applicability in lipophilic media as well as enhance its bioavailability in vivo. In addition, we aimed investigated the pro-apoptoptotic activity of this new compound (propionyl neohesperidin ester, PNHE) in MCF-7 breast cancer cells using a variety of cellular assays, including the MTT (3-(4, 5-dimethyl- 2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay, assessment of intracellular reactive oxygen species (ROS) levels, and flow cytometry. We successfully synthesized PNHE using immobilized lipases, and the esterification of NH was confirmed by Fourier transform-infrared spectroscopy (FT-IR). Compared to NH, HNPE showed higher anti-proliferative and pro-apoptotic in MCF-7 breast cancer cells, which may be explained by its increased lipophilicity compared to neohesperidin, benefiting to the action of NH on the cancer cell wall. The IC50 of PNHE for inducing apoptosis of MCF-7 cells was 185.52 μg/mL. PNHE increased both the proportion of cells in Sub-G1 phase and the cellular ROS content, indicating a certain therapeutic effect of HNPE on breast cancer.

Isoliquiritigenin Induces Mitochondrial Dysfunction and Apoptosis by Inhibiting mitoNEET in a Reactive Oxygen Species-Dependent Manner in A375 Human Melanoma Cells.

The mitochondrial protein mitoNEET is a type of iron-sulfur protein localized to the outer membrane of mitochondria and is involved in a variety of human pathologies including cystic fibrosis, diabetes, muscle atrophy, and neurodegeneration. In the current study, we found that isoliquiritigenin (ISL), one of the components of the root of Glycyrrhiza glabra L., could decrease the expression of mitoNEET in A375 melanoma cells. We also demonstrated that mitoNEET could regulate the content of reactive oxygen species (ROS), by showing that the ISL-mediated increase in the cellular ROS content could be mitigated by the mitoNEET overexpression. We also confirmed the important role of ROS in ISL-treated A375 cells. The increased apoptosis rate and the decreased mitochondrial membrane potential were mitigated by the overexpression of mitoNEET in A375 cells. These findings indicated that ISL could decrease the expression of mitoNEET, which regulated ROS content and subsequently induced mitochondrial dysfunction and apoptosis in A375 cells. Our findings also highlight mitoNEET as a promising mitochondrial target for cancer therapy.

Manipulation of Cellular Redox State was Essential for Triggering Erythroid Differentiation of K562 Cells by Mycophenolic Acid

Reactive Oxygen Species (ROS) play important roles in a variety of normal cellular functions and various pathological events. Uncontrolled ROS production in various types of cancer cells has been globally reported and considered as a hallmark for new drug developments. Accordingly, in this study, we planned to investigate the probable roles of oxidants on erythroid differentiation of K562 cells under the influence of Mycophenolic acid (MPA). Based on our results, MPA attenuated the intracellular SOD activity to provide a suitable redox environment for hemoglobinization of the exposed cells, while the cellular content of glutathione and glutathione peroxidase were augmented at the initial times of MPA-induced erythroid differentiation. These variations were accompanied by the significant reduction in the cellular ROS content in the first 8 h of MPA exposure followed by an enormous increase in ROS content by the 24 h of exposure. Furthermore, while the cells were undergoing erythroid differentiation by MPA, it became evident that p27kip1, apart from its role in cell cycle arrest, was functioning as a sensor of the intracellular oxidants. Overall, it might be concluded that MPA-guided erythroid differentiation was strongly associated with manipulation of ROS content of the chronic myelogenous leukemia cells.

Effects of NOX1 on fibroblastic changes of endothelial cells in radiation‑induced pulmonary fibrosis.

Lung fibrosis is a major complication in radiation-induced lung damage following thoracic radiotherapy, while the underlying mechanism has remained to be elucidated. The present study performed immunofluorescence and immunoblot assays on irradiated human pulmonary artery endothelial cells (HPAECs) with or without pre-treatment with VAS2870, a novel NADPH oxidase (NOX) inhibitor, or small hairpin (sh)RNA against NOX1, -2 or -4. VAS2870 reduced the cellular reactive oxygen species content induced by 5 Gy radiation in HPAECs and inhibited phenotypic changes in fibrotic cells, including increased alpha smooth muscle actin and vimentin, and decreased CD31 and vascular endothelial cadherin expression. These fibrotic changes were significantly inhibited by treatment with NOX1 shRNA, but not by NOX2 or NOX4 shRNA. Next, the role of NOX1 in pulmonary fibrosis development was assessed in the lung tissues of C57BL/6J mice following thoracic irradiation using trichrome staining. Administration of an NOX1-specific inhibitor suppressed radiation-induced collagen deposition and fibroblastic changes in the endothelial cells (ECs) of these mice. The results suggested that radiation-induced pulmonary fibrosis may be efficiently reduced by specific inhibition of NOX1, an effect mediated by reduction of fibrotic changes of ECs.

Can multi-generational exposure to ocean warming and acidification lead to the adaptation of life history and physiology in a marine metazoan?

Ocean warming and acidification are concomitant global drivers that are currently threatening the survival of marine organisms. How species will respond to these changes depends on their capacity for plastic and adaptive responses. Little is known about the mechanisms that govern plasticity and adaptability or how global changes will influence these relationships across multiple generations. Here, we exposed the emerging model marine polychaete Ophryotrocha labronica to conditions simulating ocean warming and acidification, in isolation and in combination over five generations to identify: (i) how multiple versus single global change drivers alter both juvenile and adult life-history traits; (ii) the mechanistic link between adult physiological and fitness-related life-history traits; and (iii) whether the phenotypic changes observed over multiple generations are of plastic and/or adaptive origin. Two juvenile (developmental rate; survival to sexual maturity) and two adult (average reproductive body size; fecundity) life-history traits were measured in each generation, in addition to three physiological (cellular reactive oxygen species content, mitochondrial density, mitochondrial capacity) traits. We found that multi-generational exposure to warming alone caused an increase in juvenile developmental rate, reactive oxygen species production and mitochondrial density, decreases in average reproductive body size and fecundity, and fluctuations in mitochondrial capacity, relative to control conditions. Exposure to ocean acidification alone had only minor effects on juvenile developmental rate. Remarkably, when both drivers of global change were present, only mitochondrial capacity was significantly affected, suggesting that ocean warming and acidification act as opposing vectors of stress across multiple generations.

Peromyscus leucopus mice: a potential animal model for haematological studies.

Peromyscus leucopus mice share physical similarities with laboratory mice Mus musculus (MM) but have higher agility and longer lifespan. We compared domesticated P. leucopus linville (PLL) and M. musculus C57BL/6 (MMB6) mice for cellular composition of peripheral blood (PB), bone marrow (BM) and spleen. PLL mice had significantly fewer platelets and significantly more monocytes in the blood, and notably fewer megakaryocytes in the BM. Spleens of PLL mice were significantly smaller, with 50% fewer cells and reduced 'red pulp'. There was no obvious haematological change in PLL mice between 2-8 and 16-26 months of age, except for a significant increase in blood monocytes. Cellular reactive oxygen species (ROS) content showed no change with age but differed significantly between different cell types. Treating two to eight month-old PLL mice with antioxidant N-acetylcysteine in drinking water for three months did not affect cellular ROS content, but increased blood leucocytes especially the concentration of monocytes. The low platelets, low megakaryocytes, high monocytes and low splenic erythropoiesis in PLL mice resemble human measurements better than the values seen in MMB6.

Chronic nitric oxide deprivation induces an adaptive antioxidant status in human endothelial cells

In a previous work, we showed an increased cell motility due to the accumulation and transcriptional activation of the Hypoxia Inducible Factor-1α (HIF-1α) and a reduced mitochondrial energy production in an in vitro model of endothelial dysfunction (ED) represented by human endothelial cells (ECs) chronically deprived of nitric oxide (NO) by L-NAME treatment. In the present study, in the attempt to unravel the pathway(s) linking NO deficiency to HIF-1α accumulation and activation, we focused our attention on Reactive Oxygen Species (ROS). We found that ROS were partially involved in HIF-1α stabilization, but not in the pro-migratory phenotype. Regarding mitochondrial dysfunction, it did not require neither ROS generation nor HIF-1α activity, and was not due to autophagy. Very interestingly, while acute treatment with L-NAME induced a transient increase in ROS formation, chronic NO deprivation by long term L-NAME exposure drastically reduced cellular ROS content giving rise to an antioxidant environment characterized by an increase in superoxide dismutase-2 (SOD-2) expression and activity, and by nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2). These results might have important implications for our understanding of the consequences of NO deprivation in endothelium behavior and in the onset of cardiovascular diseases.

THE ROLE OF REACTIVE OXYGEN SPECIES IN COPPER TOXICITY TO TWO FRESHWATER GREEN ALGAE(1).

The role of reactive oxygen species (ROS) in copper (Cu) toxicity to two freshwater green algal species, Pseudokirchneriella subcapitata (Korshikov) Hindák and Chlorella vulgaris Beij., was assessed to gain a better mechanistic understanding of this toxicity. Cu-induced formation of ROS was investigated in the two algal species and linked to short-term effects on photosynthetic activity and to long-term effects on cell growth. A light- and time-dependent increase in ROS concentrations was observed upon exposure to environmentally relevant Cu concentrations of 50 and 250 nM and was comparable in both algal species. However, effects of 250 nM Cu on photosynthesis were different, leading to a 12% reduction in photosynthetic activity in P. subcapitata, but not in C. vulgaris. These results indicate that differences in species-specific sensitivities measured as photosynthetic activity were not caused by differences in the cellular ROS content of the algae, but probably by different species-specific ROS defense systems. To investigate the role of ROS in Cu-mediated inhibition of photosynthesis, the ROS scavenger N-tert-butyl-α-phenylnitrone (BPN) was used, resulting in a reduction of Cu-induced ROS production up to control level and a complete restoration of photosynthetic activity of Cu-exposed P. subcapitata. This finding implied that ROS play a primary role in Cu toxicity to algae. Furthermore, we observed a time-dependent ROS release process across the plasma membrane. More than 90% of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient method of cellular protection against oxidative stress.

Age-dependent accumulation of mtDNA mutations in murine hematopoietic stem cells is modulated by the nuclear genetic background

Alterations in mitochondrial DNA (mtDNA) and consequent loss of mitochondrial function underlie the mitochondrial theory of aging. In this study, we systematically analyzed the mtDNA control region somatic mutation pattern in 2864 single hematopoietic stem cells (HSCs) and progenitors, isolated by flow cytometry sorting on Lin(-)Kit(+)CD34(-) parameters from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, to test the hypothesis that the accumulated mtDNA mutations in HSCs were strain-correlated and associated with HSC functional senescence during aging. An increased level of mtDNA mutations in single HSCs was observed in old B6 when compared with young B6 mice (P=0.003); in contrast, no significant age-dependent accumulation of mutations was observed in BALB mice (old versus young, P=0.202) and the level of mutations in both young and old BALB mice was close to that of old B6 mice (P>0.280). Cellular reactive oxygen species (ROS) in mouse HSCs could not be correlated with the level of mtDNA mutations in these cells, although B6 mice had a higher proportion of ROS(-) cells when compared with the BALB mice. Propagation assays of single HSCs showed B6 cells form larger colonies compared with cells from BALB mice, irrespective of age and mtDNA mutation load. We infer from our data that age-related mtDNA somatic mutation accumulation in mouse HSCs is influenced by the nuclear genetic background and that these mutations may not obviously correlate to either cellular ROS content or HSC senescence.

Rac1 GTPase Regulates Cell Genomic Stability and Senescence

The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-kappaB.

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor-kappaB (NF-kappaB) and how this activation contributes to preconditioning-induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine-induced (200 nM, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF-kappaB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IkappaBalpha as well as by electrophoretic mobility shift assay. Preconditioning-mediated neuroprotection was abolished by antioxidants, inhibitors of NF-kappaB activation and cycloheximide suggesting the involvement of ROS, an activation of NF-kappaB and de novo protein synthesis in preconditioning-mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn-superoxide dismutase which could be blocked by antioxidants, cycloheximide and kappaB decoy DNA. Our data suggest that inhibition of staurosporine-induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF-kappaB and an increase in the protein level of Mn-superoxide dismutase.