Toxic Effects Of Nitric Oxide Research Articles

Subacute combined degeneration of the spinal cord of toxic etiology (clinical case)

For more than two hundred years after its discovery, nitric oxide (I) has been widely used in medicine as an anesthetic, in the car industry as an engine performance enhancer, in the food industry as a preservative, and, unfortunately, among young people as a mild narcotic substance. Despite the widespread opinion that there is no harm from the use of “laughing gas” for recreational purposes, in the literature and in practice, there are a lot of cases when patients suffer from various complications, including neurological ones, caused precisely by the use of nitric oxide (I). One of the main mechanisms of the pathological effects of nitrous oxide on the nervous tissue is the inactivation of vitamin B12. This has been proven by studying the physicochemical properties of the gas, the effect on the organisms of laboratory animals, the change in the concentration of certain substances in the human body, and also by the use of cyanocobalamin for the treatment of these patients. This article describes a clinical case of the development of myelopolyneuropathy caused by the toxic effects of nitric oxide (I).

Effects of Endothelin-1 and nitric oxide levels on myocardial ischemia-reperfusion injury.

To study the role of nitric oxide (NO) in myocardial ischemia-reperfusion injury (IRI) and Endothelin-1 (ET-1) in the process of reperfusion in an animal model. ET is a strong vasoconstrictor peptide, which is closely related to the physiological and pathological state of the cardiovascular system. ET not only directly stimulates and activates a variety of hormones and cytokines, but also is one of the mediators promoting myocardial remodeling, and participates in and promotes myocardial ischemia injury. Before myocardial ischemia, Krebs-Henseleit (KH) perfusion solution containing different concentrations of L-arginine (LA; substrate of NO) were given to 6 groups of rats, and ET was given at the early stage of reperfusion in 3 groups. During reperfusion, cardiac function indexes, myocardial enzyme release and NO content in coronary effluent, and the cardiac malondialdehyde (MDA) content was measured. The myocardial ultrastructure was observed by microscopy. Data of each group are expressed as mean ± standard deviation, and the baseline value of each group before ischemia was the recovery value during reperfusion. SPSS26.0 (IBM, Chicago, USA) was used for statistical processing. Before myocardial ischemia, infusion of KH solution containing a low concentration of LA (10 mmol/L) reduced myocardial IRI, whereas infusion of a KH solution containing high concentration of LA (100 mmol/L) before ischemia significantly aggravated myocardial IRI. The administration of KH solution containing LA and ET-1 (1,000 mmol/L) significantly reduced myocardial IRI. NO plays a dual role in myocardial ischemia-reperfusion, both beneficial and harmful. The combination of NO and ET-1 can reduce the toxic effect of NO.

67 - Effects of nitric oxide and sulfide donors on stored platelet bioenergetics

Platelet adhesion and aggregation are involved in hemostatic maintenance of vascular tone, regulating blood flow turbidity, and inducing inflammatory immune responses to injury. Platelets have been found to secrete redox gasotransmitters, nitric oxide (NO) and hydrogen sulfide (H2S), to regulate their thrombolytic functions. High concentrations of NO can competitively inhibit oxygen binding to cytochrome c oxidase, suppressing oxidative phosphorylation. Nitric oxide can react with superoxide radicals (O2•–) to form peroxynitrite (ONOO•–), a potent oxidant that can damage mitochondrial electron transport proteins. Conversely, H2S can antagonize the deleterious effects of NO through inhibiting O2•– formation and scavenging ONOO•–. Previous work from our lab has found that platelet activation is primarily dependent on glycolysis, but oxidative phosphorylation plays a secondary role for normal functions. Therefore, we propose the presence of a gasotransmitter-mediated mechanism protecting against spontaneous platelet activation during the induction of ROS/RNS. In this study, DetaNONOate (an NO donor) in conjunction with diallyl disulfide (DADS), a H2S donor, was used to evaluate the potential roles of redox transmitters in platelet metabolism using the Seahorse Extracellular Flux Analyser. Our findings corroborated previous understandings of the mitochondrially toxic effects of NO, as elevated DetaNONOate inhibited oxygen consumption rate (OCR) in a dose-dependent manner, suggesting that electron transport machinery was being suppressed or damaged; this dose-dependence was also found with DADS pre-treatment. Damage was reversed when platelets were pre-treated with DADS prior to DetaNONoate exposure; DADS resulted in increased glycolytic flux, leading to recovery of mitochondrial function. These data demonstrate the platelets’ abilities to compensate for acute damage to oxidative phosphorylation machinery using H2S.

Nitric oxide as a regulator of B. anthracis pathogenicity.

Nitric oxide (NO) is a key physiological regulator in eukaryotic and prokaryotic organisms. It can cause a variety of biological effects by reacting with its targets or/and indirectly inducing oxidative stress. NO can also be produced by bacteria including the pathogenic Bacillus anthracis; however, its role in the infectious process only begins to emerge. NO incapacitates macrophages by S-nitrosylating the intracellular proteins and protects B. anthracis from oxidative stress. It is also implicated in the formation of toxic peroxynitrite. In this study we further assessed the effects of B. anthracis NO produced by the NO synthase (bNOS) on bacterial metabolism and host cells in experiments with the bNOS knockout Sterne strain. The mutation abrogated accumulation of nitrite and nitrate as tracer products of NO in the culture medium and markedly attenuated growth in both aerobic and microaerobic conditions. The regulatory role of NO was also suggested by the abnormally high rate of nitrate denitrification by the mutant in the presence of oxygen. Anaerobic regulation mediated by NO was reflected in reduced fermentation of glucose by the mutant correlating with the reduced toxicity of bacteria toward host cells in culture. The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH. The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne. Our global proteomic analysis of lymph from the lymph nodes of infected mice harboring bacteria revealed numerous changes in the pattern and levels of proteins associated with the activity of bNOS influencing key cell physiological processes relevant to energy metabolism, growth, signal transduction, stress response, septic shock, and homeostasis. This is the first in vivo observation of the bacterial NO effect on the lymphatic system.

Flavodiiron nitric oxide reductases: Recent developments in the mechanistic study and model chemistry for the catalytic reduction of NO

Inducible NO synthase in mammals helps to produce up to micromolar concentration of nitric oxide (NO) which acts as a key immune defense agent to kill invading pathogens. In order to counter the toxic effects of NO, the pathogens have expressed flavodiiron nitric oxide reductases (FNORs). The FNORs reduce the toxic NO into much less toxic N2O and thus help the pathogens to survive under nitrosative stress. As a consequence, these pathogens proliferate in the human body and cause harmful infections. An appreciable amount of research work has been performed to discover the true mechanism of the FNORs. Different mechanisms involving both mononitrosyl and dinitrosyl diiron complexes as key intermediates are proposed. Evidences for the involvement of new intermediates and more and more experimental evidences for existing ones in the proposed catalytic cycle of FNORs are coming up. These interesting biochemical events have recently boosted the biomimetic chemistry of the FNOR activity as well. This article discusses the importance and the currently understood mechanistic aspects of FNORs. Structural and functional models for the active site of FNORs are discussed along with their success and limitations. Possible future prospects of the modeling chemistry are also suggested.

The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E. coli is improved by combination with miconazole and polymyxin B nonapeptide

BackgroundNitric oxide (NO) is produced as part of the host immune response to bacterial infections, including urinary tract infections. The enzyme flavohemoglobin, coded by the hmp gene, is involved in protecting bacterial cells from the toxic effects of NO and represents a potentially interesting target for development of novel treatment concepts against resistant uropathogenic bacteria. The aim of the present study was to investigate if the in vitro antibacterial effects of NO can be enhanced by pharmacological modulation of the enzyme flavohemoglobin.ResultsFour clinical isolates of multidrug-resistant extended-spectrum β-lactamase (ESBL)-producing uropathogenic E. coli were included in the study. It was shown that the NO-donor substance DETA/NO, but not inactivated DETA/NO, caused an initial growth inhibition with regrowth noted after 8 h of exposure. An hmp-deficient strain showed a prolonged growth inhibition in response to DETA/NO compared to the wild type. The imidazole antibiotic miconazole, that has been shown to inhibit bacterial flavohemoglobin activity, prolonged the DETA/NO-evoked growth inhibition. When miconazole was combined with polymyxin B nonapeptide (PMBN), in order to increase the bacterial wall permeability, DETA/NO caused a prolonged bacteriostatic response that lasted for up to 24 h.ConclusionAn NO-donor in combination with miconazole and PMBN showed enhanced antimicrobial effects and proved effective against multidrug-resistant ESBL-producing uropathogenic E. coli.

Oxidative and Nitrosative Stress and Immune-inflammatory Pathways in Patients with Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS)

Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome (CFS) has been classified as a disease of the central nervous system by the WHO since 1969. Many patients carrying this diagnosis do demonstrate an almost bewildering array of biological abnormalities particularly the presence of oxidative and nitrosative stress (O&NS) and a chronically activated innate immune system. The proposal made herein is that once generated chronically activated O&NS and immune-inflammatory pathways conspire to generate a multitude of self-sustaining and self-amplifying pathological processes which are associated with the onset of ME/CFS. Sources of continuous activation of O&NS and immune-inflammatory pathways in ME/CFS are chronic, intermittent and opportunistic infections, bacterial translocation, autoimmune responses, mitochondrial dysfunctions, activation of the Toll-Like Receptor Radical Cycle, and decreased antioxidant levels. Consequences of chronically activated O&NS and immune-inflammatory pathways in ME/CFS are brain disorders, including neuroinflammation and brain hypometabolism / hypoperfusion, toxic effects of nitric oxide and peroxynitrite, lipid peroxidation and oxidative damage to DNA, secondary autoimmune responses directed against disrupted lipid membrane components and proteins, mitochondrial dysfunctions with a disruption of energy metabolism (e.g. compromised ATP production) and dysfunctional intracellular signaling pathways. The interplay between all of these factors leads to self-amplifying feed forward loops causing a chronic state of activated O&NS, immune-inflammatory and autoimmune pathways which may sustain the disease.

A Novel Protein Protects Bacterial Iron-Dependent Metabolism from Nitric Oxide

Reactive nitrogen species (RNS), in particular nitric oxide (NO), are toxic to bacteria, and bacteria have mechanisms to allow growth despite this stress. Understanding how bacteria interact with NO is essential to understanding bacterial physiology in many habitats, including pathogenesis; however, many targets of NO and enzymes involved in NO resistance remain uncharacterized. We performed for the first time a metabolomic screen on NO-treated and -untreated bacteria to define broadly the effects of NO on bacterial physiology, as well as to identify the function of NnrS, a previously uncharacterized enzyme involved in defense against NO. We found many known and novel targets of NO. We also found that iron-sulfur cluster enzymes were preferentially inhibited in a strain lacking NnrS due to the formation of iron-NO complexes. We then demonstrated that NnrS is particularly important for resistance to nitrosative stress under anaerobic conditions. Our data thus reveal the breadth of the toxic effects of NO on metabolism and identify the function of an important enzyme in alleviating this stress.

Redox Regulation of Protein Function via Cysteine S-Nitrosylation and Its Relevance to Neurodegenerative Diseases

Debilitating neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), can be attributed to neuronal cell damage in specific brain regions. An important hallmark of these diseases is increased oxidative and nitrosative stress that occurs via overproduction of highly reactive free radicals known as reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules are normally removed by cellular antioxidant systems. Under physiological conditions, ROS/RNS are present at low levels, mediating several neurotrophic and neuroprotective signaling pathways. In contrast, under pathological conditions, there is a pronounced increase in ROS/RNS generation, impairing normal neurological function. Nitric oxide (NO) is one such molecule that functions as a signaling agent under physiological conditions but causes nitrosative stress under pathological conditions due to its enhanced production. As first reported by our group and colleagues, the toxic effects of NO can be in part attributed to thiol S-nitrosylation, a posttranslational modification of cysteine residues on specific proteins. Here, we review several reports appearing over the past decade showing that S-nitrosylation of an increasing number of proteins compromises important cellular functions, including mitochondrial dynamics, endoplasmic reticulum (ER) protein folding, and signal transduction, thereby promoting synaptic damage, cell death, and neurodegeneration.

Analysis of Neuronal Nitric Oxide Synthase Expression and Increasing Astrogliosis in the Brain of Senescence-Accelerated-Prone 8 Mice

ABSTRACTThe senescence-accelerated mouse (SAM) is an autogenic senile murine model characterized by early cognitive impairment and age-related deterioration of learning and memory. The present study investigated the alternations of neuronal nitric oxide synthase (nNOS) expression in frontal cortex and hippocampus in the aging process of SAM-prone 8 (SAMP8) and SAM-resistant 1 (SAMR1) mice. The results demonstrated that the expression of nNOS was upregulated in the frontal cortex, but downregulated in the hippocampus in SAMP8. Further, age-related increases of astrogliosis were seen in the cortex and hippocampi of aged SAMP8 and SAMR1, as revealed by the expression of the astrocyte specific marker, glial fibrillary acidic protein (GFAP). Indeed, astrogliosis in aged SAMP8 was significantly greater than that of aged SAMR1. Our results suggest the possibility of a correlation between the downregulation of nitric oxide (NO) in the hippocampus and reported learning and memory deficits in SAMP8. However, the toxic effects of NO and age-related increases of astrogliosis, may have contributed to abnormal alterations in metabolism and neurochemical mechanisms in aged SAMP8.

Drug targets for traumatic brain injury from poly(ADP‐ribose)polymerase pathway modulation

The deleterious pathophysiological cascade induced after traumatic brain injury (TBI) is initiated by an excitotoxic process triggered by excessive glutamate release. Activation of the glutamatergic N-methyl-D-aspartate receptor, by increasing calcium influx, activates nitric oxide (NO) synthases leading to a toxic production of NO. Moreover, after TBI, free radicals are highly produced and participate to a deleterious oxidative stress. Evidence has showed that the major toxic effect of NO comes from its combination with superoxide anion leading to peroxynitrite formation, a highly reactive and oxidant compound. Indeed, peroxynitrite mediates nitrosative stress and is a potent inducer of cell death through its reaction with lipids, proteins and DNA. Particularly DNA damage, caused by both oxidative and nitrosative stresses, results in activation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme implicated in DNA repair. In response to excessive DNA damage, massive PARP activation leads to energetic depletion and finally to cell death. Since 10 years, accumulating data have showed that inactivation of PARP, either pharmacologically or using PARP null mice, induces neuroprotection in experimental models of TBI. Thus TBI generating NO, oxidative and nitrosative stresses promotes PARP activation contributing in post-traumatic motor, cognitive and histological sequelae. The mechanisms by which PARP inhibitors provide protection might not entirely be related to the preservation of cellular energy stores, but might also include other PARP-mediated mechanisms that needed to be explored in a TBI context. Ten years of experimental research provided rational basis for the development of PARP inhibitors as treatment for TBI.

Necrotizing enterocolitis — bench to bedside: novel and emerging strategies

Necrotizing enterocolitis (NEC) is a devastating illness that predominantly affects premature neonates. The mortality associated with this disease has changed very little during the last two decades. Neonates with NEC fall into two categories: those who respond to medical management alone and those who require surgical treatment. The disease distribution may be focal, multifocal, or panintestinal. Surgical treatment should therefore be based on disease presentation. Recent studies have added significant insight into our understanding of the pathogenesis of NEC. Several groups have shown that upregulation of nitric oxide plays an integral role in the development of epithelial injury in NEC. As a result, some treatment strategies have been aimed at abrogating the toxic effects of nitric oxide. In addition, several investigators have reported the cytoprotective effect of epidermal growth factor, which is found in high levels in breast milk, on the intestinal epithelium. Thus, fortification of infant formula with specific growth factors could soon become a preferred strategy to accelerate intestinal maturation in the premature neonate to prevent the development of NEC. One of the most devastating complications of NEC is the development of short bowel syndrome (SBS). The current treatment of SBS involves intestinal lengthening procedures or bowel transplantation. A novel emerging method for treating SBS involves the use of tissue-engineered intestine. In laboratory animals, tissue-engineered small intestine has been shown to be successful in treating intestinal failure. This article examines recent data regarding surgical treatment options for NEC as well as emerging treatment modalities.

A new gaseous signaling molecule emerges: Cardioprotective role of hydrogen sulfide

Cardiovascular (CV) research has largely been focused on two endogenously produced gaseous signaling molecules, namely, nitric oxide (NO) and carbon monoxide (CO). These gaseous messengers are both highly conserved evolutionarily and synthesized by endogenous enzyme systems. Extensive research efforts have uniformly demonstrated that CO- and NO-based therapeutics protect the brain, heart, and circulation against a number of CV disease states (1–3). However, controversy exists regarding the therapeutic value of both CO and NO, with some studies citing NO cytotoxicity at very high levels and under certain conditions (2). The toxic effects of NO are related to the highly reactive nature of NO and its interaction with superoxide to form the potent oxidant peroxynitrite (ONOO−) (2). Very recently, a third endogenously produced gaseous signaling molecule, hydrogen sulfide (H2S), has emerged as a potentially important mediator of CV homeo stasis and cytoprotection (3). In this issue of PNAS, Benavides et al. (4) reveal that the vasculoprotective and antihypertensive effects of dietary garlic (Allium sativum) are mediated in large part via the generation of H2S.

Inhaled nitric oxide does not prevent postpneumonectomy pulmonary edema in pigs

Increase in lung permeability is an inevitable consequence of pneumonectomy in relation to inflammatory injury and increased perfusion flow. We tested whether inhaled nitric oxide, a potent vasodilatator and anti-inflammatory agent, prevents postpneumonectomy edema in the first 24 hours after pneumonectomy in pigs. We assessed hemodynamics, gas exchange, extravascular lung water estimated with the double-indicator dilution method, and lung neutrophil sequestration measured on the basis of lung myeloperoxidase activity at 1 and 24 hours after left pneumonectomy in 14 pigs randomly assigned to inhaled nitric oxide (10 ppm) or control groups. Extravascular lung water content markedly increased at 1 and 24 hours after pneumonectomy, with no difference between the 2 groups. Hemodynamics did not differ between the 2 groups. Myeloperoxidase activity was higher and PaO2 values were lower in the nitric oxide group compared with in the control group. Over the 24 hours after pneumonectomy, intraoperative inhaled nitric oxide levels neither improved gas exchange nor attenuated accumulation of lung water. On the contrary, they were associated with an increase in lung neutrophil sequestration and deterioration of arterial oxygenation, suggesting the occurrence of an early and toxic effect of nitric oxide.

Evidence for nitric oxide involvement in experimental autoimmune encephalomyelitis and adjuvant-induced arthritis in Lewis rat

Nitric oxide (NO) has been implicated as an important pathogenic mediator in several inflammatory and autoimmune diseases. We have developed experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis in the Lewis rat as experimental models for multiple sclerosis, and rheumatoid arthritis, respectively, in order to investigate the role of NO in the inflammatory process. We have used the monoclonal antibody anti-NO-cysteine (Cys)-G-bovine serum albumin (BSA) developed in our laboratory as an inhibitor of the toxic effect of NO to determine the implication of NO in the creation of neoepitopes and therefore in the symptomatology of these two diseases. In addition, we have detected a significant level of circulating endogenous antibodies directed against nitrosylated and nitrated epitopes in the sera of preimmunized rats. Our data demonstrate the ability of the monoclonal anti-NO-Cys-G-BSA antibody to modulate these diseases through NO neutralization, and further provide evidence for the in vivo synthesis of nitrosylated and nitrated neoepitopes that are stable enough to be highly immunogenic, thereby inducing inflammatory injuries and a humoral immune response. Our findings provide further evidence for the implication of the NO pathway in the pathogenesis of EAE and adjuvant-induced arthritis.

Attenuation of Reperfusion-Induced Systemic Inflammation by Preconditioning With Nitric Oxide in an In Situ Porcine Model of Normothermic Lung Ischemia

Inhalation of nitric oxide (NO) can ameliorate pulmonary ischemia/reperfusion (I/R) injury of the lung in several experimental models, but toxic effects of NO were also reported. Here we investigate whether NO inhalation for a short period prior to surgery is sufficient to prevent symptoms of lung I/R injury, especially the inflammatory response. Using an in situ porcine lung model, normothermic left lung ischemia was maintained for 90 min, followed by a 5-h reperfusion period (group 1, n = 7). In group 2 (n = 6), I/R was preceded by inhalation of NO (10 min, 15 ppm). Animals in group 3 (n = 7) underwent sham surgery without NO inhalation or ischemia. Oxygenation and hemodynamic parameters were measured as indicators of lung functional impairment. Plasma levels of interleukin (IL)-1beta, IL-6, and transforming growth factor (TGF)-beta1 were determined throughout the I/R maneuver. In addition, tissue macrophages were analyzed by lectin binding. Symptoms of I/R injury (pulmonary hypertension and decreased oxygenation) in group 1 animals were attenuated by NO inhalation. The reperfusion-induced increases of the levels of IL-1beta and IL-6 in plasma were reduced by NO pretreatment. A peak of TGF-beta1 immediately after NO administration was observed in group 2, but not in groups 1 and 3. There was no significant effect of NO on tissue macrophages. NO inhalation for a short period prior to lung I/R is sufficient to protect against pulmonary hypertension, impaired oxygenation, and the inflammatory response of pulmonary I/R injury.

Role of the thioredoxin system and the thiol-peroxidases Tpx and Bcp in mediating resistance to oxidative and nitrosative stress in Helicobacter pylori.

Helicobacter pylori possesses two distinct thioredoxin proteins (Trx1 and Trx2) which may play important roles in the ability of this bacterium to survive oxidative stress. Trx1 has previously been shown to be an electron donor in vitro for alkyl-hydroperoxide reductase (AhpC), one of three members of the peroxiredoxin family of antioxidant peroxidases present in H. pylori. In this study, mutants in the trxA1 and trxA2 genes encoding Trx1 and Trx2, respectively, and in the tpx and bcp genes, which encode the remaining two members of the H. pylori peroxiredoxin family, were constructed in order to determine their roles in resistance to damage by reactive oxygen and nitrogen species. Mutation of trxA1 led to a pronounced increase in sensitivity to oxygen, hydrogen peroxide and the superoxide generator paraquat, as well as to the nitric oxide (NO) releasers sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), consistent with an in vivo role for Trx1 as a reductant for AhpC. A trxA2 single mutant grew normally in an atmosphere of 2 % (v/v) O(2) but grew very poorly in 10 % (v/v) O(2). It showed slight increases in killing by hydrogen peroxide, paraquat, SNP and GSNO compared to the wild-type, but was significantly more sensitive to cumene hydroperoxide in disc-diffusion assays. A trxA1 trxA2 double mutant was very sensitive to all of the oxidative and nitrosative stresses applied. Comparison of the phenotypes of the tpx and bcp mutants showed that Tpx plays a significant role in peroxide and superoxide resistance in H. pylori, while the role of Bcp is minimal. No evidence was obtained for a role for either Tpx or Bcp in resistance to the toxic effects of NO. The results show that a functional thioredoxin system is necessary for both oxidative and nitrosative stress resistance in H. pylori but, surprisingly, is not essential for viability despite the absence of glutathione and a glutaredoxin system in this bacterium.

The role of nitric oxide in the pathogenesis of Chagas disease.

In this chapter we summarize the protective and toxic effects of nitric oxide (NO) that are frequently seen in parallel during the infection with Trypanosoma cruzi. The killing of trypomastigotes is dependent on the production of NO which is catalyzed by the inducible NO synthase (iNOS). The cytokines IFN-gamma and TNF-alpha and several chemoatractants molecules, which act on G protein-coupled serpentine receptors, are produced during the acute infection. They play major roles in the induction of iNOS, and in the NO production-dependent killing of T. cruzi by murine macrophages. On the other hand, TGF-beta and IL-10, which are also produced during the infection, are negative regulators of NO production. In addition to mediating resistance against the infection, NO can also suppress the immune response to T. cruzi via the induction of apoptosis of T cells. Furthermore, the expression of cardiac iNOS has been associated with myocardial dysfunction. In fact, we discuss here the evidences indicating that iNOS/NO pathway is involved in the pathogenesis of neuronal and myocardial dysfunction seen in patients and in experimental models.

Specificity of resistance to oxidative stress.

Two clonal nerve-like cell lines derived from HT22 and PC12 have been selected for resistance to glutamate toxicity and amyloid toxicity, respectively. In the following experiments it was asked if these cell lines show cross-resistance toward amyloid beta peptide (Abeta) and glutamate as well as toward a variety of additional neurotoxins. Conversely, it was determined if inhibitors of oxytosis, a well-defined oxidative stress pathway, also protect cells from the neurotoxins. It is shown that both glutamate and amyloid resistant cells are cross resistant to most of the other toxins or toxic conditions, while inhibitors of oxytosis protect from glutathione and cystine depletion and H2O2 toxicity, but not from the toxic effects of nitric oxide, rotenone, arsenite or cisplatin. It is concluded that while there is a great deal of cross-resistance to neurotoxins, the components of the cell death pathway which has been defined for oxytosis are not used by many of the neurotoxins.

Nitric oxide generated by corneas in corneal storage media.

The purpose of the study was to quantify nitric oxide release by human corneal buttons in storage media over time. Group 1 consisted of six chambers of Optisol GS corneal storage media, each containing a viable human corneal button with an attached scleral rim (unsuitable for transplantation), sampled at 1-day intervals for at least 17 days (range, 17-28 days). Group 2 consisted of 34 chambers of Optisol GS media, each used to store a corneal button for penetrating keratoplasty, sampled immediately after each surgery. An unused vial of Optisol GS storage medium was sampled daily for 17 days to serve as a background medium control. The total amount of nitrite and nitrate in each sample was determined by a spectrophotometric method based on the Griess reaction. Data from the daily sampling in group 1 showed that nitrite and nitrate concentrations in storage media containing human corneas increase from a baseline level (beginning at the time the corneas are placed in the media) to an equilibrium concentration of 2.77 microM in a mean time of 6.15 days. Seventy-six percent of the data points from group 2 fell within the 80% predictive interval derived from group 1. No nitrite or nitrate was detected in background medium control samples. The progressive increase in nitrite and nitrate in corneal storage media over time suggests that nitric oxide is continuously released by corneas during storage before transplantation. Given the toxic free radical properties of nitric oxide, corneas in storage media may be subjected to the cumulative toxic effects of nitric oxide.