Possible Role Of Reactive Oxygen Species Research Articles

Ambivalent Roles of Oxidative Stress in Triangular Relationships among Arthropod Vectors, Pathogens and Hosts.

Blood-feeding arthropods, particularly ticks and mosquitoes are considered the most important vectors of arthropod-borne diseases affecting humans and animals. While feeding on blood meals, arthropods are exposed to high levels of reactive oxygen species (ROS) since heme and other blood components can induce oxidative stress. Different ROS have important roles in interactions among the pathogens, vectors, and hosts. ROS influence various metabolic processes of the arthropods and some have detrimental effects. In this review, we investigate the various roles of ROS in these arthropods, including their innate immunity and the homeostasis of their microbiomes, that is, how ROS are utilized to maintain the balance between the natural microbiota and potential pathogens. We elucidate the mechanism of how ROS are utilized to fight off invading pathogens and how the arthropod-borne pathogens use the arthropods’ antioxidant mechanism to defend against these ROS attacks and their possible impact on their vector potentials or their ability to acquire and transmit pathogens. In addition, we describe the possible roles of ROS in chemical insecticide/acaricide activity and/or in the development of resistance. Overall, this underscores the importance of the antioxidant system as a potential target for the control of arthropod and arthropod-borne pathogens.

The role of mitochondrial ROS in the aging brain.

The brain is the most complex human organ, consuming more energy than any other tissue in proportion to its size. It relies heavily on mitochondria to produce energy and is made up of mitotic and postmitotic cells that need to closely coordinate their metabolism to maintain essential bodily functions. During aging, damaged mitochondria that produce less ATP and more reactive oxygen species (ROS) accumulate. The current consensus is that ROS cause oxidative stress, damaging mitochondria and resulting in an energetic crisis that triggers neurodegenerative diseases and accelerates aging. However, in model organisms, increasing mitochondrial ROS (mtROS) in the brain extends lifespan, suggesting that ROS may participate in signaling that protects the brain. Here, we summarize the mechanisms by which mtROS are produced at the molecular level, how different brain cells and regions produce different amounts of mtROS, and how mtROS levels change during aging. Finally, we critically discuss the possible roles of ROS in aging as signaling molecules and damaging agents, addressing whether age-associated increases in mtROS are a cause or a consequence of aging.

Reactive Oxygen Species-Mediated Promotion of Root Growth Under Mild Water Stress During Early Seedling Stage of Vigna radiata (L.) Wilczek

Plants often show promotion of root growth under water stress while inhibiting shoot growth. Regulation of such differential growth response is not very clearly understood. Our earlier observation on the role of reactive oxygen species (ROS) in embryonic axis growth during seed germination prompted us to explore a possible role for ROS in water stress-induced root growth promotion during early seedling development of Vigna radiata. Normal and water stress-induced root growth were dependent on generation of ROS, for example, superoxide (O 2 ·− ), hydrogen peroxide (H2O2) and hydroxyl radical (OH·), as evident from pharmacological experiments using ROS scavengers and inhibitors. However, in the case of the hypocotyl, neither normal growth nor growth inhibition under water stress was associated with ROS. Apoplastic O 2 ·− and H2O2 production monitored by spectrophotometric analysis of the bathing medium using XTT and xylenol orange, respectively and localization of O 2 ·− and H2O2 by NBT and TMB, respectively confirmed the involvement of ROS in root growth. Increased NADPH oxidase (NOX) activity (putative plasma membrane located apoplastic O 2 ·− producer) in the membrane fraction, as determined by in-gel assay and concomitant increase in apoplastic O 2 ·− and H2O2 generation, as evident by NBT and TMB stain in water stressed roots corroborated the view that promotion of root growth in the early seedling stage is effected by ROS being initiated with O 2 ·− generation by NOX in the apoplastic space.

Reactive oxygen species do not contribute to ObgE*-mediated programmed cell death.

Programmed cell death (PCD) in bacteria is considered an important target for developing novel antimicrobials. Development of PCD-specific therapies requires a deeper understanding of what drives this process. We recently discovered a new mode of PCD in Escherichia coli that is triggered by expression of a mutant isoform of the essential ObgE protein, ObgE*. Our previous findings demonstrate that ObgE*-mediated cell death shares key characteristics with apoptosis in eukaryotic cells. It is well-known that reactive oxygen species (ROS) are formed during PCD in eukaryotes and play a pivotal role as signaling molecules in the progression of apoptosis. Therefore, we explored a possible role for ROS in bacterial killing by ObgE*. Using fluorescent probes and genetic reporters, we found that expression of ObgE* induces formation of ROS. Neutralizing ROS by chemical scavenging or by overproduction of ROS-neutralizing enzymes did not influence toxicity of ObgE*. Moreover, expression of ObgE* under anaerobic conditions proved to be as detrimental to bacterial viability as expression under aerobic conditions. In conclusion, ROS are byproducts of ObgE* expression that do not play a role in the execution or progression of ObgE*-mediated PCD. Targeted therapies should therefore look to exploit other aspects of ObgE*-mediated PCD.

P61 - Characterization of the paraoxonase system in follicular fluid of women subjected to an ovarian stimulation cycle

Reactive oxygen species (ROS) and antioxidants are involved in the regulation of reproductive processes. Previous studies in infertile women undergoing an ovarian stimulation cycle have suggested a possible role for ROS in the occurrence of conception in In Vitro Fertilization. In this context the control of the redox balance of follicular fluid becomes essential for reproduction, so that the presence of enzymes with antioxidant activities, such as the paraoxonase (PON) system, would play a role in maintaining this balance. The objective of this work was a) to characterize the paraoxonase system in follicular fluid of women undergoing a controlled ovarian stimulation cycle, analysing the associated PON1, PON2, and PON3 activities, and b) to study the possible involvement of the PON system in follicular maturation. The enzyme activities were quantified in follicular fluid from large and small follicles from women undergoing an ovarian stimulation cycle in the IVI-Bilbao clinic. PON activities were quantified using spectrophotometric and HPLC techniques. Statistical comparisons were performed using the Student’s t-test for paired data. Results indicate that follicular fluid presents paraoxonase activities which are detectable by the methods developed in this study. PON activities were associated with follicular maturation, suggesting that the PON system plays a role in oocyte maturation.This work was supported by research grants from the Ministry of Health and Consumption (FIS/FEDER PI11/02559), the Basque Country Government (Dep. Education, Universities and Research ref. IT687-13, and DCIT ref. S-PE13UN063), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation.

Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Reactive oxygen species regulate hematopoietic stem cell self-renewal, migration and development, as well as their bone marrow microenvironment.

Blood forming, hematopoietic stem cells (HSCs) mostly reside in the bone marrow in a quiescent, nonmotile state via adhesion interactions with stromal cells and macrophages. Quiescent, proliferating, and differentiating stem cells have different metabolism, and accordingly different amounts of intracellular reactive oxygen species (ROS). Importantly, ROS is not just a byproduct of metabolism, but also plays a role in stem cell state and function. ROS levels are dynamic and reversibly dictate enhanced cycling and myeloid bias in ROS(high) short-term repopulating stem cells, and ROS(low) quiescent long-term repopulating stem cells. Low levels of ROS, regulated by intrinsic factors such as cell respiration or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity, or extrinsic factors such as stem cell factor or prostaglandin E2 are required for maintaining stem cell self-renewal. High ROS levels, due to stress and inflammation, induce stem cell differentiation and enhanced motility. Stem cells need to be protected from high ROS levels to avoid stem cell exhaustion, insufficient host immunity, and leukemic transformation that may occur during chronic inflammation. However, continuous low ROS production will lead to lack of stem cell function and opportunistic infections. Ultimately, balanced ROS levels are crucial for maintaining the small stem cell pool and host immunity, both in homeostasis and during stress situations. Deciphering the signaling pathway of ROS in HSC will provide a better understanding of ROS roles in switching HSC from quiescence to activation and vice versa, and will also shed light on the possible roles of ROS in leukemia initiation and development.

Effects of hydrogen peroxide in a keratinocyte-fibroblast co-culture model of wound healing

Recently, there has been renewed interest in the role of reactive oxygen species (ROS), especially H2O2, in wound healing. We previously showed that H2O2 stimulates healing in a keratinocyte scratch wound model. In this paper, we used a more complex and physiologically relevant model that involves co-culturing primary keratinocytes and fibroblasts. We found that the two main cell types within the skin have different sensitivities to H2O2 and to the widely used “antioxidant”N-acetyl-l-cysteine (NAC).Keratinocytes were very resistant to the toxicity of H2O2 (250 and 500μM) or NAC (5mM). However, the viability of fibroblasts was decreased by both compounds. Using the co-culture model, we also found that H2O2 increases re-epithelialization while NAC retards it. Our data further illustrate the possible role of ROS in wound healing and the co-culture model should be useful for screening agents that may influence the wound healing process.

Genome-wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene

AbstractWith the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34+ progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc–induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Reactive Oxygen Species Modulation of Na/K-ATPase Regulates Fibrosis and Renal Proximal Tubular Sodium Handling

The Na/K-ATPase is the primary force regulating renal sodium handling and plays a key role in both ion homeostasis and blood pressure regulation. Recently, cardiotonic steroids (CTS)-mediated Na/K-ATPase signaling has been shown to regulate fibrosis, renal proximal tubule (RPT) sodium reabsorption, and experimental Dahl salt-sensitive hypertension in response to a high-salt diet. Reactive oxygen species (ROS) are an important modulator of nephron ion transport. As there is limited knowledge regarding the role of ROS-mediated fibrosis and RPT sodium reabsorption through the Na/K-ATPase, the focus of this review is to examine the possible role of ROS in the regulation of Na/K-ATPase activity, its signaling, fibrosis, and RPT sodium reabsorption.

Reactive oxygen species in the signaling and adaptation of multicellular microbial communities.

One of the universal traits of microorganisms is their ability to form multicellular structures, the cells of which differentiate and communicate via various signaling molecules. Reactive oxygen species (ROS), and hydrogen peroxide in particular, have recently become well-established signaling molecules in higher eukaryotes, but still little is known about the regulatory functions of ROS in microbial structures. Here we summarize current knowledge on the possible roles of ROS during the development of colonies and biofilms, representatives of microbial multicellularity. In Saccharomyces cerevisiae colonies, ROS are predicted to participate in regulatory events involved in the induction of ammonia signaling and later on in programmed cell death in the colony center. While the latter process seems to be induced by the total ROS, the former event is likely to be regulated by ROS-homeostasis, possibly H2O2-homeostasis between the cytosol and mitochondria. In Candida albicans biofilms, the predicted signaling role of ROS is linked with quorum sensing molecule farnesol that significantly affects biofilm formation. In bacterial biofilms, ROS induce genetic variability, promote cell death in specific biofilm regions, and possibly regulate biofilm development. Thus, the number of examples suggesting ROS as signaling molecules and effectors in the development of microbial multicellularity is rapidly increasing.

Devil’s Triangle in Kidney Diseases: Oxidative Stress, Mediators, and Inflammation

This issue of the International Journal of Nephrology focused on kidney diseases within a devil's triangle, oxidative stress (OS), mediators, inflammation, specifically relating to the clinical significance of identification, and prevention. Every creature in need of oxygen faces OS. It has a critical role in the molecular mechanisms of renal injury in several kidney diseases, and many complications of these diseases are mediated by OS, mediators, and inflammation. There is a complex relationship between these three; mostly they induce each other. While some of the diseases themselves can contribute to OS, reactive oxygen species (ROS) produced by activated leukocytes and endothelial cells in sites of inflammation cause tissue damage. Although inflammation looks dangerous for the organism, it is a normal reaction of organs and tissues to protect themselves against several invasion(s). It enables the immune system to remove the injurious stimuli and initiate the healing process of tissues. However, the interactions between OS, mediators, and inflammation may result in glomerular damage, proteinuria, electrolyte, and volume instabilities which cause nephron loss, on the long view. Detailed studies on this topic are included in this issue. The kidney can easily be damaged by ROS, due to the rich structure of long-chain polyunsaturated fatty acids. The article by E. Ozbek summarizes the induction of OS within kidney in several conditions, including diabetes, hypertension, hypercholesterolemia, obesity, aging, urinary obstruction, environmental toxins, and molecular mechanisms of these inductions in the light of existing literature data. Diabetic nephropathy is one of the most common microvascular complications of type 1 and type 2 diabetes mellitus and the leading cause of end-stage renal disease worldwide [1]. Rojas-Rivera et al. reviewed the biological bases of oxidative stress and its role especially on diabetic nephropathy, as well as the role of the Keap1-Nrf2 pathway, and recent clinical trials targeting this pathway with bardoxolone methyl, a novel synthetic triterpenoid with antioxidant and anti-inflammatory properties. Obesity continues to be a public health problem throughout the world. Epidemiologic studies have shown that 66% of adults and 16% of children and adolescents are overweight or obese [2]. Obesity-related glomerulopathy is an increasing cause of end-stage renal diseases. J. Tang et al. stressed the chronic low-grade systemic inflammation in obesity and discussed the roles of inflammation and oxidative stress in the progression of obesity-related glomerulopathy and possible treatment modalities to prevent kidney injury in obesity, such as the usage of anti-IL-6 receptor antibody, TNF-α antagonist, adiponectin, nutritional and surgical interventions to reduce OS. Hypertension is an another important global health issue both in adults and children. It is one of the major risk factors for the progression of kidney diseases. The relationship between blood pressure and dietary sodium and salt sensitivity has been well known, and renal sodium handling is a key determinant of long-term blood pressure regulation [3]. There is a limited knowledge in the literature regarding the role of ROS-mediated fibrosis and renal proximal tubule sodium reabsorption through the Na/K-ATPase. S. Liu et al. reviewed the possible role of ROS in the regulation of Na/K-ATPase activity. The authors emphasized the importance of further researches whether ROS signaling is a link between the Na/K-ATPase/c-Src cascade and NHE3 regulation and how OS, stimulated by high salt and cardiotonic steroids, regulates Na/K-ATPase/c-Src signaling in renal sodium handling and fibrosis. Urotensin-II is the most potent mammalian vasoconstrictor identified to date, almost tenfold more potent than endothelin-I [4]. A. Balat and M. Buyukcelik discussed the role of urotensin-II on renal hemodynamics and its possible role on several kidney diseases, such as the minimal change nephrotic syndrome. The article includes a detailed discussion of urotensin-II immunoreactivity in renal biopsy specimens of children with membranoproliferative glomerulonephritis, membranous nephropathy, IgA nephropathy, Henoch-Schonlein nephritis, and focal segmental glomerulosclerosis. Because of its complex relation with OS and other mediators, authors describe it as “more than a mediator” in glomerular diseases. They briefly mention from the effectiveness of U-II antagonism, as a new promising pharmacological treatment target in some kidney diseases. Given the potential impact of OS, mediators, and inflammation trio, the importance of prevention has come into question. Strong evidence indicates the importance of new molecules that are able to diminish them which in turn may help to decrease the prevalence and/or progression of several kidney diseases. Therefore, further researches are needed to the better understanding of the molecular and clinical mechanisms of this triad. They may help to provide new therapeutical strategies to control several complications in patients with kidney diseases. Ayse Balat Halima Resic Guido Bellinghieri Ali Anarat

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.).

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen–pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O2˙− and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O2˙− and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen–pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.

Tumorigenic polyploid cells contain elevated ROS and ARE selectively targeted by antioxidant treatment

Polyploidy has been linked to tumorigenicity mainly due to the chromosomal aberrations. Elevated reactive oxygen species (ROS) generation, on the other hand, has also been associated with oncogenic transformation in most cancer cells. However, a possible link between ploidy and ROS is largely unexplored. Here we have examined the role of ROS in the tumorigenicity of polyploid cells. We show that polyploid prostate and mammary epithelial cells contain higher levels of ROS due to their higher mitochondrial contents. ROS levels and mitochondrial mass are also higher in dihydrocytochalasin B (DCB)-induced polyploid cells, suggesting that higher levels of ROS observed in polyploid cell can occur due to cytokinesis failure. Interestingly, polyploid cells were more sensitive to the inhibitory effect of the antioxidant, N-Acetyl-L-cysteine (NAC), than control diploid cells. Treatment of polyploid/diploid cells with NAC led to the selective elimination of polyploid cells over time and abrogated the tumorigenicity of polyploid cells. This effect was partially mediated via the Akt signaling pathway. We next explored a possible role for ROS in promoting chromosomal instability by analyzing the effects of ROS on the mitotic stage of the cell cycle. Enhancing ROS levels by treating cells with hydrogen peroxide delayed not only entry into and but also exit from mitosis. Furthermore, increasing ROS levels significantly increased taxol resistance. Our results indicated that increased ROS in polyploid cells can contribute to tumorigenicity and highlight the therapeutic potential of antioxidants by selectively targeting the tumorigenic polyploid cells and by reversing taxol resistance.

Abstract 4206: Tumorigenic polyploid cells contain elevated levels of reactive oxygen species and are selectively targeted by antioxidant treatment

Abstract Polyploidy has been linked to the tumorigenicity mainly due to the chromosomal aberrations. Elevated reactive oxygen species (ROS) generation, on the other hand, has also been associated with oncogenic transformation in most cancer cells. However, a possible link between ploidy and ROS is largely not known. Here we have explored the role of ROS in the tumorigenicity of polyploid cells. We show that polyploid prostate and mammary epithelial cells contain higher levels of ROS due to their higher mitochondrial contents. ROS levels and mitochondrial mass are also higher in dihydrocytochalasin B (DCB)-induced polyploid cells, suggesting that higher levels of ROS observed in polyploid cell is due to cytokinesis failure. Interestingly, polyploid cells were more sensitive to the inhibitory effect of the antioxidant, N-Acetyl-L-cysteine (NAC), than control diploid cells. Treatment of polyploid/diploid cells with NAC led to the selective elimination of polyploid cells over time and abrogated the tumorigenicity of polyploid cells. This was partially mediated via the Akt signaling pathway. We next explored a possible role for ROS in promoting chromosomal instability by analyzing the effects of ROS on the mitotic stage of the cell cycle. Enhancing ROS levels by treatment of cells with hydrogen peroxide delayed not only entry into and but also exit from mitosis. Furthermore, increasing ROS levels significantly increased taxol resistance. Our results indicate that increased ROS in polyploid cells can contribute to tumorigenicity and illuminates therapeutic potential of antioxidant by selectively targeting the tumorigenic polyploidy cells and reversing taxol resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4206. doi:10.1158/1538-7445.AM2011-4206

Intracellular Prooxidant Activity of Melatonin Induces a Survival Pathway Involving NF‐κB Activation

We have shown that melatonin exerts a prooxidant activity in U937 cells, a tumor human promonocytic cell line. (1) Here we show that melatonin induces a strong canonical activation of NF-kappaB, inducing IkappaBalpha degradation and the consequential nuclear translocation of p50/p65 subunits. The timing of NF-kappaB activation overlaps with the timing of reactive oxygen species (ROS) production due to melatonin. Overexpression of dominant-negative IkappaB, which prevents a possible NF-kappaB activation, transformed melatonin in a proapoptotic molecule. These data indicate for the first time that melatonin can trigger NF-kappaB activation and might suggest a possible role for ROS induced by melatonin. Results indicate a possible involvement in the survival pathway of melatonin-generated ROS as secondary messengers.

Selenocystine induces reactive oxygen species–mediated apoptosis in human cancer cells

Epidemiological and clinical studies have demonstrated that dietary supplementation of selenium (Se) could reduce the incidence of human cancers. In this study, selenocystine, a nutritionally available selenoamino acid, was identified as a novel agent with broad-spectrum antitumor activity. A panel of eight human caner cell lines was shown to be susceptible to selenocystine, with IC 50 values ranging from 3.6 to 37.0 μM. Selenocystine induced dose-dependent apoptosis in A375, HepG2 and MCF7 cells was evaluated by flow cytometric analysis and annexin-V staining assay. Mechanistic studies showed time- and dose-dependent increases in intracellular reactive oxygen species (ROS) in susceptible cancer cells (MCF7 and HepG2 cells) treated with selenocystine. However, selenocystine-induced ROS overproduction was not observed in non-susceptible normal human fibroblast Hs68 cells. Significant DNA strand breaks were observed in selenocystine-treated MCF7 and HepG2 cells as examined by single-cell gel electrophoresis (Comet assay). The thiol-reducing antioxidants, glutathione and N-acetylcysteine, inhibited intracellular ROS generation, DNA strand breaks and accumulation of sub-G1 population in MCF7 cells exposed to selenocystine. Our results suggest a possible role of ROS as a mediator of the signaling pathway of selenocystine-induced, DNA damage–mediated apoptosis in susceptible cancer cells.

Beneficial Effects of Exogenous Tetrahydrobiopterin on Left Ventricular Remodeling After Myocardial Infarction in Rats The Possible Role of Oxidative Stress Caused by Uncoupled Endothelial Nitric Oxide Synthase

Reactive oxygen species (ROS) is deeply involved in the process of ventricular remodeling after myocardial infarction (MI). Under oxidative stress, endothelial nitric oxide synthase (eNOS) can be converted to a ROS generator, because a relative lack of tetrahydrobiopterin (BH4), an essential cofactor for NO synthesis, leads to eNOS uncoupling. The uncoupled eNOS generates superoxide rather than NO. The possible role of ROS generated by eNOS in ventricular remodeling after MI was investigated. Rats were treated with oral BH4 supplementation starting at 3 days before coronary artery ligation. At 4 weeks after MI, there was augmented superoxide production in association with reduced BH4/dihydrobiopterin (BH2) ratio and eNOS dimer/monomer protein ratio in the heart. Treatment with BH4 increased BH4/BH2 ratio and eNOS dimer/monomer ratio, and decreased superoxide production. In BH4-treated MI rats, left ventricular size was smaller, thickness of the non-infarcted posterior wall was thinner, and cardiac function was preserved compared with the control MI rats. The present study suggested that ventricular remodeling process after MI leads to BH4 oxidation and resulted in uncoupled eNOS-derived superoxide generation, which further augmented the remodeling process and deteriorated cardiac function.

The role of reactive oxygen species in capsaicin-induced mechanical hyperalgesia and in the activities of dorsal horn neurons

Previous findings that reactive oxygen species (ROS) are involved in neuropathic pain, mainly through spinal mechanisms, suggest that ROS may be involved in central sensitization. To investigate the possible role of ROS in central sensitization, we examined in rats the effects of ROS scavengers on capsaicin-induced secondary hyperalgesia, which is known to be mediated by central sensitization. We used two different ROS scavengers: phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL). Intradermal capsaicin injection (20μg in 20μl olive oil) into the hind paw produced primary and secondary hyperalgesia. A systemic administration of PBN (100mg/kg, i.p.) or TEMPOL (200mg/kg, i.p.) alleviated capsaicin-induced secondary, but not primary, hyperalgesia. Intrathecal injection of PBN (1mg inof vertinary Surgery/anesthesiology, College of vetrinary Medic 50μl saline) greatly reduced hyperalgesia, whereas intracerebroventricular or intradermal injection of PBN produced only a minor analgesic effect, suggesting that PBN takes effect mainly through the spinal cord. Electrophysiological recordings from wide dynamic range (WDR) neurons in the dorsal horn showed that intradermal capsaicin enhanced the evoked responses to peripheral stimuli; systemic PBN or TEMPOL restored the responses to normal levels. Removal of ROS thus restored the responsiveness of spinal WDR neurons to normal levels, suggesting that ROS is involved in central sensitization, at least in part by sensitizing WDR neurons.

Alcohol Upregulation of CYP2A5: Role of Reactive Oxygen Species

Hepatic cytochrome P450 (CYP) 2E1 and CYP2A5 activate many important drugs and hepatotoxins. CYP2E1 is induced by alcohol, but whether CYP2A5 is upregulated by alcohol is not known. This article reviews recent studies on the induction of CYP2A5 by alcohol and the mechanism and role of reactive oxygen species (ROS) in this upregulation. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity and protein and mRNA levels. This induction was blunted in CYP2E1 knockout mice and by a CYP2E1 inhibitor, but was restored in CYP2E1 knockin mice, suggesting a role for CYP2E1 in the induction of CYP2A5 by alcohol. Since CYP2E1 actively generates ROS, the possible role of ROS in the induction of CYP2A5 by alcohol was determined. ROS production was elevated by ethanol treatment. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol-induced elevation of ROS and blunted the alcohol-mediated induction of CYP2A5. These results suggest that ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. Alcohol treatment activated nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), a transcription factor which up-regulates expression of CYP2A5. The antioxidants blocked the activation of Nrf2. The alcohol-induced elevation of CYP2A5, but not CYP2E1, was lower in Nrf2 knockout mice. We propose that increased generation of ROS from the alcohol-induced CYP2E1 activates Nrf2, which subsequently up-regulates the expression of CYP2A5. Thus, a novel consequence of the alcohol-mediated induction of CYP2E1 and increase in ROS is the activation of redox-sensitive transcription factors, such as Nrf2, and expression of CYP2A5. Further perspectives on this alcohol-CYP2E1-ROS-Nrf2-CYP2A5 pathway are presented.