Central Role Of Oxidative Stress Research Articles

Gcn5 Modulates the Cellular Response to Oxidative Stress and Histone Deacetylase Inhibition.

To identify chemical genetic interactions underlying the mechanism of action of histone deacetylase inhibitors (HDACi) a yeast deletion library was screened for hypersensitive deletion mutants that confer increased sensitivity to the HDACi, CG-1521. The screen demonstrated that loss of GCN5 or deletion of components of the Gcn5 histone acetyltransferase (HAT) complex, SAGA, sensitizes yeast to CG-1521-induced cell death. Expression profiling after CG-1521 treatment reveals increased expression of genes involved in metabolism and oxidative stress response, and oxidative stress response mutants are hypersensitive to CG-1521 treatment. Accumulation of reactive oxygen species and increased cell death are enhanced in the gcn5Δ deletion mutant, and are abrogated by anti-oxidants, indicating a central role of oxidative stress in CG-1521-induced cell death. In human cell lines, siRNA mediated knockdown of GCN5 or PCAF, or chemical inhibition of GCN5 enzymatic activity, increases the sensitivity to CG-1521 and SAHA. These data suggest that the combination of HDAC and GCN5/PCAF inhibitors can be used for cancer treatment.

Beneficial Effects of Melatonin on Oxidative Damage Observed During Whole Blood Storage.

It is well known that oxidative stress rises during the storing period of whole blood and there has been a great interest to improve the quality of stored blood for longer periods. In the present study, we determine the effects of melatonin as an antioxidant on the whole blood indices stored under blood bank conditions. Human blood was collected by venipuncture into citrate-dextrose-phosphate (CDP) bags from healthy volunteers. Immediately after collection, blood was subdivided into two parts and exposed to either melatonin or control saline solutions. Several biochemical parameters were measured on the day of collection and at weekly intervals up to 3 weeks. Mean corpuscular and platelet volumes tended to increase during storing process and with melatonin, these indices remained near to physiologic levels. As expected, acidity, oxidative damage and osmotic fragility increased in stored blood. Interestingly addition of melatonin reduced acidity, oxidative damage by lowering malondialdehyde levels and by increasing superoxide dismutase activity, and osmotic fragility during storage without adversely affecting other biochemical parameters. pH was 6.68 ±0.04 in day 14 of control group while it was 7.00 ± 0.03 in melatonin group. Malondialdehyde level was 1.223±0.05 in day 21 of control group while it was 0.723±0.04 in melatonin group (p< 0.05). Superoxide dismutase activity was significantly higher in melatonin group at days 14 and 21 (151.3±12.2 and 82.8±9.2) compared to saline (111.8±6.5 and 44.8±3.0) (p< 0.05). Through these results, we could confirm a central role for oxidative stress in the mechanism of the most evident blood storage lesions and melatonin seems to exhibit beneficial effects on these lesions.

Mitochondrial Haplogroup Clusters Modify the Effects of Black Carbon Exposure on Age Related Cognitive Impairment.

Background: Recent observational studies have linked traffic-related air pollution to loss of cognitive function in the elderly, an effect potentially mediated by oxidative-stress actions on the brain. Mitochondria are the main cellular source of oxidative stress. Haplogroups in mitochondrial DNA (mtDNA) determine between-subject differences in oxidative potential and have been linked with neurodegerative disease. No study has yet investigated whether mtDNA haplogroups influence susceptibility to pro-oxidant exposures, including traffic-related pollution. Aims: To investigate whether mtDNA haplogroups determine differential susceptibility to the effects of long-term exposure to black carbon (BC), a marker of traffic-related air pollution, on cognitive function in older men. Methods: A cohort of 616 men (mean±SD, 71±7 years of age) with an average 1.8 visits (from 1 to 4) per participant between 1996-2007 was drawn from the VA Normative Aging Study. We focused on low (?25) Mini Mental State Examination (MMSE) score as proxy of impaired cognition in multiple domains. We fitted repeated-measure logistic regression using BC estimated at participant’s address in the year before the first visit through a validated spatio-temporal land-use regression model. Results: In models adjusted for clinical features and socio-economics factors, each doubling in BC was associated with 1.2 (95%CI 0.9-1.5) times higher odds of low MMSE score. BC effect on MMSE differed by haplogroup, with most of the effect found in carriers of haplogroups I (OR=3.4; 95%CI 0.8-13.7) or X (OR=2.3; 95%CI 0.9-5.5). Carriers of haplotypes J, T, H V, K, U and W did not show any effect from BC. Conclusions: Susceptibility to the adverse cognitive effects of BC was limited to carriers of mtDNA haplogroups I and X. mtDNA haplogroups may represent a novel susceptibility factor that, due to the central role of oxidative stress in mediating air pollution risks, may also modify effects on other systems.

Abstract 846: ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells .

Abstract Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as cell migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS increased expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, with concomitant decreased expression of HIF1α. Further investigation into pVHL during apoptosis revealed that the tumor suppressor becomes phosphorylated at S111 prior to activity; S111 is within a target motif (RXXS) forphosphorylation by AKT kinase. We then determined that pVHL and p-AKT associated in vitro via co-immunoprecipitation assay. Moreover, knockdown of pVHL by siRNA rescued HIF1α protein expression and rescued cells from apoptosis, which was determined by Annexin-V and cleaved-PARP. Collectively, our study suggests that in the context of oxidative stress, activatedAKT facilitated apoptosis by inducing pVHL function. Moreover, our studies suggest a novel paradigm for AKT-that it may participate in tumor suppressor rather than oncogenesis. Citation Format: Mahandranauth A. Chetram, Kia J. Jones, Christopher Coke, Ayesha Don-Salu-Hewage, Danaya A. Bethea, Cimona V. Hinton. ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 846. doi:10.1158/1538-7445.AM2013-846

Programming of respiratory health in childhood

This overview highlights recent experimental and epidemiological evidence for the programming effects of outdoor air pollution exposures during early development on lung function and chronic respiratory disorders, such as asthma and related allergic disorders. Air pollutants may impact anatomy and/or physiological functioning of the lung and interrelated systems. Programming effects may result from pollutant-induced shifts in a number of molecular, cellular, and physiological states and their interacting systems. Specific key regulatory systems susceptible to programming may influence lung development and vulnerability to respiratory diseases, including both central and peripheral components of neuroendocrine pathways and autonomic nervous system (ANS) functioning which, in turn, influence the immune system. Starting in utero, environmental factors, including air pollutants, may permanently organize these systems toward trajectories of enhanced pediatric (e.g., asthma, allergy) as well as adult disease risk (e.g., chronic obstructive pulmonary disease). Evidence supports a central role of oxidative stress in the toxic effects of air pollution. Additional research suggests xenobiotic metabolism and subcellular components, such as mitochondria are targets of ambient air pollution and play a role in asthma and allergy programming. Mechanisms operating at the level of the placenta are being elucidated. Epigenetic mechanisms may be at the roots of adaptive developmental programming. Optimal coordinated functioning of many complex processes and their networks of interaction are necessary for normal lung development and the maintenance of respiratory health. Outdoor air pollution may play an important role in early programming of respiratory health and is potentially amenable to intervention.

ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.

Molecular Neuropathogenesis of Alzheimer‘s Disease: An Interaction Model Stressing the Central Role of Oxidative Stress

Alzheimer's disease (AD) exhibits a complex etiology that simultaneously manifests as a complex cellular, neurobiological, molecular, anatomic-physiological and clinical entity. Other significant psychiatric conditions, such as depression and schizophrenia, may also present with complex and concurrent clinical and/or molecular phenotypes. These neuropsychiatric pathologies also originate from both environmental and genetic factors. We analyzed the molecular phenotypes of AD and discuss them with respect to the classical theories, which we integrated into mechanisms that share molecular and/or anatomical connections. Based on these mechanisms, we propose an interaction model and discuss the model in light of studies that refute or support it. Given the spectrum of AD phenotypes, we limit the scope of our discussion to a few, which facilitates concrete analysis. In addition, the study of specific, individual pathogenic phenotypes may be critical to defining the complex mechanisms leading to AD, thereby improving strategies for developing novel therapies.

Health and cellular impacts of air pollutants: from cytoprotection to cytotoxicity.

Air pollution as one of the ravages of our modern societies is primarily linked to urban centers, industrial activities, or road traffic. These atmospheric pollutants have been incriminated in deleterious health effects by numerous epidemiological and in vitro studies. Environmental air pollutants are a heterogeneous mixture of particles suspended into a liquid and gaseous phase which trigger the disruption of redox homeostasis—known under the term of cellular oxidative stress—in relation with the establishment of inflammation and cell death via necrosis, apoptosis, or autophagy. Activation or repression of the apoptotic process as an adaptative response to xenobiotics might lead to either acute or chronic toxicity. The purpose of this paper is to highlight the central role of oxidative stress induced by air pollutants and to focus on the subsequent cellular impacts ranging from cytoprotection to cytotoxicity by decreasing or stimulating apoptosis, respectively.

Central role of oxidative stress and its signaling pathways in causing and preventing acute lung injury*

Central role of oxidative stress and its signaling pathways in causing and preventing acute lung injury*

Antioxidant Vitamins and Cardiovascular Disease

Cardiovascular disease represents an unparalleled proportion of the global burden of disease and will remain the main cause of mortality for the near future. Fortunately, most premature cardiovascular deaths are preventable. Therefore, prevention becomes vital and diet has shown beneficial effects to protect from CVD (CVD). Fruits and vegetables are dietary sources of natural antioxidants and it is generally accepted that antioxidants in these foods are key in explaining the inverse association between fruits and vegetables intake and the risk of developing a cardiovascular event or having elevated levels of cardiovascular risk factors. Available evidence supports the central role of oxidative stress in the atherosclerosis process and the correlation between increased oxidative stress and vascular disease. Theoretically, antioxidants in fruits and vegetables are important in inhibiting oxidative mechanisms that lead to various degenerative diseases including CVD. However, results from many interventional trials using antioxidants given as supplements have not been concordant with previous positive findings from observational epidemiologic cohort studies. The present manuscript gives a brief overview of the relationship between natural antioxidants (specially vitamin C, vitamin E and beta-carotene) intake and the risk of CVD.

Antioxidants in the Treatment of Diabetes

Diabetes is a chronic metabolic disorder that continues to present as a major health problem worldwide. It is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action and is associated with chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolism. Many studies suggest a central role for oxidative stress in the pathogenesis of this multi-faceted metabolic disorder. This has prompted investigations in the use of antioxidants as a complementary therapeutic approach. In this review we briefly summarize oxidative mechanisms implicated in diabetic complications and then focus on the findings resulting from human clinical trials where antioxidants were studied as an adjuvant to standard diabetes treatment during the last ten years. A literature search using PubMed (last ten years) was performed using the following terms: vitamin E, vitamin C, coenzyme Q10, alpha lipoic acid, L-carnitine, ruboxistaurin or LY 333531 and diabetes. This search was limited to human clinical trials. We concluded there is not any established benefit for antioxidants use in the management of diabetic complications. Therefore, routine vitamin or mineral supplementation is not generally recommended.

Amelioration of nephropathy with apoA-1 mimetic peptide in apoE-deficient mice

There is mounting evidence that dyslipidaemia may contribute to development and progression of renal disease. For instance, hyperlipidaemia in apolipoprotein E-deficient (apoE(-/-)) mice is associated with glomerular inflammation, mesangial expansion and foam cell formation. ApoA-1 mimetic peptides are potent antioxidant and anti-inflammatory compounds which are highly effective in ameliorating atherosclerosis and inflammation in experimental animals. Given the central role of oxidative stress and inflammation in progression of renal disease, we hypothesized that apoA-1 mimetic peptide, D-4F, may attenuate renal lesions in apoE(-/-) mice. Twenty-five-month-old female apoE(-/-) mice were treated with D-4F (300 µg/mL in drinking water) or placebo for 6 weeks. Kidneys were harvested and examined for histological and biochemical characteristics. Compared with the control mice, apoE(-/-) mice showed significant proteinuria, tubulo-interstitial inflammation, mesangial expansion, foam cell formation and up-regulation of oxidative [NAD(P)H oxidase subunits] and inflammatory [NF-κB, MCP-1, PAI-1 and COX-2] pathways. D-4F administration lowered proteinuria, improved renal histology and reversed up-regulation of inflammatory and oxidative pathways with only minimal changes in plasma lipid levels. The apoE(-/-) mice develop proteinuria and glomerular and tubulo-interstitial injury which are associated with up-regulation of oxidative and inflammatory mediators in the kidney and are ameliorated by the administration of apoA-1 mimetic peptide. These observations point to the role of oxidative stress and inflammation in the pathogenesis of renal disease in hyperlipidaemic animals and perhaps humans.

Pathophysiology of cell phone radiation: oxidative stress and carcinogenesis with focus on male reproductive system

Hazardous health effects stemming from exposure to radiofrequency electromagnetic waves (RF-EMW) emitted from cell phones have been reported in the literature. However, the cellular target of RF-EMW is still controversial. This review identifies the plasma membrane as a target of RF-EMW. In addition, the effects of RF-EMW on plasma membrane structures (i.e. NADH oxidase, phosphatidylserine, ornithine decarboxylase) and voltage-gated calcium channels are discussed. We explore the disturbance in reactive oxygen species (ROS) metabolism caused by RF-EMW and delineate NADH oxidase mediated ROS formation as playing a central role in oxidative stress (OS) due to cell phone radiation (with a focus on the male reproductive system). This review also addresses: 1) the controversial effects of RF-EMW on mammalian cells and sperm DNA as well as its effect on apoptosis, 2) epidemiological, in vivo animal and in vitro studies on the effect of RF-EMW on male reproductive system, and 3) finally, exposure assessment and dosimetry by computational biomodeling.

Molecular mechanisms of cardiovascular disease in OSAHS: the oxidative stress link

Obstructive sleep apnoea/hypopnoea syndrome (OSAHS) is a highly prevalent breathing disorder in sleep that is an independent risk factor for cardiovascular morbidity and mortality. A large body of evidence, including clinical studies and cell culture and animal models utilising intermittent hypoxia, delineates the central role of oxidative stress in OSAHS as well as in conditions and comorbidities that aggregate with it. Intermittent hypoxia, the hallmark of OSAHS, is implicated in promoting the formation of reactive oxygen species (ROS) and inducing oxidative stress. The ramifications of increased ROS formation are pivotal. ROS can damage biomolecules, alter cellular functions and function as signalling molecules in physiological as well as in pathophysiological conditions. Consequently, they promote inflammation, endothelial dysfunction and cardiovascular morbidity. Oxidative stress is also a crucial component in obesity, sympathetic activation and metabolic disorders such as hypertension, dyslipidaemia and type 2 diabetes/insulin resistance, which aggregate with OSAHS. These conditions and comorbidities could result directly from the oxidative stress that is characteristic of OSAHS or could develop independently. Hence, oxidative stress represents the common underlying link in OSAHS and the conditions and comorbidities that aggregate with it.

UV-light-induced conversion and aggregation of prion proteins

Increasing evidence suggests a central role for oxidative stress in the pathology of prion diseases, a group of fatal neurodegenerative disorders associated with structural conversion of the prion protein (PrP). Because UV-light-induced protein damage is mediated by direct photo-oxidation and radical reactions, we investigated the structural consequences of UVB radiation on recombinant murine and human prion proteins at pH 7.4 and pH 5.0. As revealed by circular dichroism and dynamic light scattering measurements, the observed PrP aggregation follows two independent pathways: (i) complete unfolding of the protein structure associated with rapid precipitation or (ii) specific structural conversion into distinct soluble β-oligomers. The choice of pathway was directly attributed to the chromophoric properties of the PrP species and the susceptibility to oxidation. Regarding size, the oligomers characterized in this study share a high degree of identity with oligomeric species formed after structural destabilization induced by other triggers, which significantly strengthens the theory that partly unfolded intermediates represent initial precursor molecules directing the pathway of PrP aggregation. Moreover, we identified the first suitable photo-trigger capable of inducing refolding of PrP, which has an important biotechnological impact in terms of analyzing the conversion process on small time scales.

Mass spectrometric quantification of amino acid oxidation products identifies oxidative mechanisms of diabetic end-organ damage

Diabetes mellitus is increasingly prevalent worldwide. Diabetic individuals are at markedly increased risk for premature death due to cardiovascular disease. Furthermore, substantial morbidity results from microvascular complications which include retinopathy, nephropathy, and neuropathy. Clinical studies involving diabetic patients have suggested that degree of diabetic hyperglycemia correlates with risk of complications. Recent evidence implicates a central role for oxidative stress and vascular inflammation in all forms of insulin resistance, obesity, diabetes and its complications. Although, glucose promotes glycoxidation reactions in vitro and products of glycoxidation and lipoxidation are elevated in plasma and tissue in diabetics, the exact relationships among hyperglycemia, the diabetic state, and oxidative stress are not well-understood. Using a combination of in vitro and in vivo experiments, we have identified amino acid oxidation markers that serve as molecular fingerprints of specific oxidative pathways. Quantification of these products utilizing highly sensitive and specific gas chromatography/mass spectrometry in animal models of diabetic complications and in humans has provided insights in oxidative pathways that result in diabetic complications. Our studies strongly support the hypothesis that unique oxidants are generated in the microenvironment of tissues vulnerable to diabetic damage. Potential therapies interrupting these reactive pathways in target tissue are likely to be beneficial in preventing diabetic complications.

Why Have Antioxidants Failed in Clinical Trials?

Antioxidant therapies have been evaluated in placebo-controlled trials involving tens of thousands of patients. Despite pathophysiologic, epidemiologic, and mechanistic data suggesting otherwise, these clinical trial results have been, to date, mostly negative in the setting of chronic preventative therapy. On the other hand, a much smaller number of trials involving handfuls of patients have been much more encouraging in terms of the acute benefit of antioxidants reflected by the data on N-acetylcysteine. However, the seemingly overwhelmingly data not supporting a role for antioxidants in the chronic suppression of atherosclerosis must be kept in perspective. Most antioxidant therapies that have been tested were not chosen because they were proved to be the best antioxidants, but rather because of their easy availability. An excellent example is vitamin E. Although easily available, it has many limitations as an antioxidant. In fact, in some studies, vitamin E has been shown to have some prooxidant effects. Another possible explanation for the lack of benefit in clinical trials is that the trials have not lasted long enough. It may be impossible to show the benefits of antioxidant therapy over several years if the therapy is trying to reverse the results of several decades of oxidative stress. It is critical to remember that the lack of benefits seen in clinical trials to date does not disprove the central role of oxidative stress in atherosclerosis. Rather, these results challenge us to evaluate optimal antioxidant therapies, the ideal study patients to study, and the appropriate trial duration.

Amyloidogenic lambda Light Chain Renal Toxicity: Oxidative Stress Implicated.

The kidney is the most common organ involved in systemic light-chain amyloidosis (AL), seen in up to 40% of patients. Renal involvement can result in proteinuria and/or renal insufficiency. The mechanism of renal injury in amyloidosis remains unclear. We present data supporting a central role for oxidative stress in amyloid-induced renal damage. Soluble free light chains(LCs) were isolated from plasma of AL and myeloma patients obtained at the time of stem cell collection. LCs were purified by liquid-phase iso-electric fractionation of pooled plasma samples from each patient. Purified LC were incubated with cultured rat renal epithelial cells, both wild-type and heme oxygenase-1 (HO-1) over-expressing, for 16 hours. Macrophage chemoattractant protein-1 (MCP-1) induction was measured in the media by ELISA. LC from myeloma patients were used as negative controls. Significant induction of MCP-1, a sensitive indicator of oxidative stress, was seen in 4/5 patients with lambda light-chain amyloidosis and 0/4 kappa light-chain amyloidosis patients. Induction of MCP-1 was seen in only 1/8 myeloma patients tested (4 lambda and 4 kappa). Induction of MCP-1 was reduced in the presence of HO-1 overexpression, implicating oxidative stress as a critical pathway of toxicity of amyloidogenic lambda light chains in this system. These results suggest that lambda and kappa light chains may cause renal injury through distinct pathways in the kidney.

Atherosclerosis and Oxidant Stress: The End of the Road for Antioxidant Vitamin Treatment?

Extensive experimental data have revealed a central role for oxidative stress in atherogenesis and suggested a potential role for 'antioxidant' treatment in cardiovascular disease (CVD) [1-11]. Experimental data, however, have not translated into clinical benefit: most antioxidant vitamin trials have failed to reduce cardiovascular morbidity and mortality [12]. Moreover, recent clinical trials have suggested that mono-therapy with certain antioxidant vitamins like vitamin E may, in fact, be detrimental [13]. As a result of the disappointing outcome of 'antioxidant' vitamin trials, some authors have questioned both the utility of 'antioxidant' treatment in CVD and the supposedly central role of oxidative stress in atherogenesis [14-19]. Other investigators, however, sustain that the beneficial effects of lipid lowering and anti-hypertensive treatment are at least, in part, due to their 'antioxidant' properties, in addition to their specific pharmacological properties [20, 21]. Oxidant stress plays a pivotal role in atherogenesis, however, the clinical promise of antioxidant vitamins has failed to translate into clinical benefit. Increasing evidence suggests that more rigorous clinical trial designs are necessary to effectively divulge antioxidant utility and that a multifaceted antioxidant approach to atherosclerosis may yield the most clinical reward. This article reviews currently available evidence on the role of oxidant stress in atherosclerosis, analyzes the results of large anti-oxidant trials, and suggests ways to investigate the true role of antioxidant treatment in the clinical setting.

Sustained Elevation of Intracellular cGMP Causes Oxidative Stress Triggering Calpain-Mediated Apoptosis in Photoreceptor Degeneration

Sustained elevation in cGMP and a concomitant increase in intracellular Ca2+ levels in the rd1 photoreceptors are followed by a rapid loss of photoreceptors. In a murine-derived photoreceptor cell line, 661W, treated with the phosphodiesterase inhibitor IBMX or the cyclic GMP-gated channel agonist 8-bromo-cGMP, it was previously found that the induced cell death was mediated by calpain and caspase-3. Because oxidative stress is a common product of ionic imbalance or elevated Ca2+, we tested the role of oxidative stress in cGMP-induced photoreceptor cell death. In the rd1 mouse retina, oxidative stress was found to precede calpain and caspase-3 activation. In 661W cells, the increase in intracellular cGMP and Ca2+ resulted in the generation of reactive oxygen species (ROS), the activation of oxidative stress enzymes, and the activation of calpain, followed by apoptosis mediated by the effector caspase-3. All these events, including calpain activation, were ameliorated by docosahexanoic acid (DHA). The cell-permeable inhibitor of calpain, SJA6017, while inhibiting cell death, had no effect on the generation of oxidative stress. These results establish a central role for oxidative stress in cGMP-induced cell death and suggest a ROS-mediated sequential activation of signal transduction events, which provide targets for future treatment strategies.