Oxidation-induced Changes Research Articles

Oxidation-induced changes in human lens epithelial cells: 1. Phospholipids

Lipid compositional changes in lens epithelial cells (HLE B-3) grown in a hyperoxic atmosphere were studied to determine if oxidation could cause changes in the amount and type of phospholipid similar to those found in vivo with age and cataract. The phosphatidylcholines in HLE B-3 cells were 8 times more unsaturated than the sphingomyelins. Cell viability was the same for cells grown for up to 48 h in a normoxic or hyperoxic atmosphere. Lipid oxidation was about three times higher after growth in a hyperoxic atmosphere compared with cells grown in a normoxic atmosphere. The lack of change in the relative amount of sphingomyelin and the decrease in phosphatidylcholine coupled with the increase in lysophosphatidylcholine support the idea that similar mechanisms may be responsible for the lipid compositional changes in both lens epithelial and fiber cells. It is postulated that lipases eliminate oxidized unsaturated glycerolipids, leaving a membrane increasingly composed of more ordered and more saturated sphingolipids. Oxidative stress leads to changes in membrane composition that are consistent with those seen with age in human epithelial cells. Oxidation-induced epithelial phospholipid change is an area of research that has gone virtually unexplored in the human lens and could be relevant to all cell types and may be important to lens clarity.

Prevention of oxidative degradation of polyurethane by covalent attachment of di‐tert‐butylphenol residues

Polyurethane (PU) components of cardiovascular devices are subjected to oxidation-initiated surface degradation, which leads to cracking and ultimately device failure. In the present study, we investigated a novel bromoalkylation chemical strategy to covalently attach the antioxidant, di-tert-butylphenol (DBP), and/or cholesterol (Chol) to the PU urethane nitrogen groups to hypothetically prevent oxidative degradation. These experiments compared PU, PU-DBP, PU-Chol, and PU-Chol-DBP. A series of comparative oxidative degradation studies involved exposing PU samples (modified and unmodified) to H2O2-CoCl2 for 15 days at 37 degrees C, to cause accelerated oxidative degradation. The extent and effects of degradation were assessed by attenuated total reflectance Fourier transformation infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface contact angle measurements, and mechanical testing. Both the Chol and DBP modification conferred significant resistance to oxidation related changes compared to unmodified PU per FTIR and SEM results. SEM demonstrated cavitation only in unmodified PU. However, contact angle analysis showed significant oxidation-induced changes only in the Chol-modified PU formulations. Most importantly, uniaxial stress-strain testing revealed that only PU-DBP demonstrated bulk elastomeric properties that were minimally affected by oxidation; PU, PU-Chol, PU-Chol-DBP showed marked deterioration of their stress-strain properties following oxidation. In conclusion, these results demonstrate that derivatizing PU with DBP confers significant resistance to oxidative degradation compared with unmodified PU.

Oxidation-induced changes in human lens epithelial cells 2. Mitochondria and the generation of reactive oxygen species

The relationships among reactive oxygen species (ROS) generation, lipid compositional changes, antioxidant power, and mitochondrial membrane potential were determined in a human lens epithelial cell line, HLE-B3. Cells grown in a hyperoxic atmosphere grew linearly for about 3 days, and then progressively died. Total antioxidant power and ROS generation increased by 50 and 43%, respectively, in cells grown in a hyperoxic atmosphere compared to those cultured in a normoxic atmosphere. By specifically uncoupling the mitochondrial proton gradient, we determined that the mitochondria are most likely the major source of ROS generation. ROS generation correlated inversely with mitochondrial membrane potential and the amount of cardiolipin, factors likely to contribute to loss of cell viability. Our results support the idea that hyperoxic damage to HLE-B3 cells derives from enhanced generation of ROS from the mitochondrial electron transport chain resulting in the oxidation of cardiolipin. With extended hyperoxic insult, the oxidants overwhelm the antioxidant defense system and eventually cell death ensues.

Superhard nanocomposite coatings of TiN/Si 3N 4 prepared by reactive direct current unbalanced magnetron sputtering

Approximately 1.5-μm-thick superhard nanocomposite coatings of TiN/Si 3N 4 with varying silicon contents were deposited on silicon and stainless steel (SS 304) substrates by reactive direct current (DC) unbalanced magnetron sputtering. An asymmetric bipolar-pulsed DC power supply and a DC power supply were used to sputter Si and Ti targets, respectively in Ar + N 2 plasma. Structural characterization of the coatings was done using X-ray diffraction (XRD). The bonding structure of the nanocomposite coatings was characterized by X-ray photoelectron spectroscopy (XPS). The elemental composition of the TiN/Si 3N 4 nanocomposite coatings was determined using energy dispersive X-ray analysis. The mechanical properties of the coatings were measured using a nanoindentation hardness tester. The surface morphology of the coatings was studied using atomic force microscopy. The nanocomposite coatings exhibited a broad (200) reflection of cubic TiN phase in the XRD data. There was a significant broadening of the (200) reflection with an increase in the silicon content in the nanocomposite coatings, which suggested a decrease in the average crystallite size. Nanoindentation data showed that about 1.5-μm-thick TiN/Si 3N 4 nanocomposite coatings exhibited a maximum hardness of 5200 kg/mm 2 and an elastic modulus of 340 GPa at a silicon concentration of approximately 12 at.%. The hardness and the elastic modulus of the nanocomposite coatings decreased with further increase in the silicon content. Structural changes as a result of heating (400–850 °C) of the nanocomposite coatings in air were characterized using micro-Raman spectroscopy. The Raman data showed that the nanocomposite coatings started to oxidize at 800 °C as compared to TiN, which have been oxidized even at 500 °C. To isolate the oxidation-induced spectral changes as a result of heating of the coatings in air, the samples were also annealed in vacuum at 850 °C under similar conditions. The Raman data of the vacuum-annealed coatings showed no phase transformation even after annealing up to 850 °C. The corrosion behaviors of single-phase TiN and TiN/Si 3N 4 nanocomposite coatings deposited on stainless steel substrates were investigated using potentiodynamic polarization in 3.5% NaCl solution. The results indicated that the nanocomposite coatings exhibited superior corrosion resistance as compared to the uncoated substrate. The wear data showed that the nanocomposite coatings also exhibited better wear resistance as compared to the uncoated substrate.

Protein oxidation injury occurs during pediatric cardiopulmonary bypass

Proteins are the major effectors of biological structure and function. Oxidation-induced changes to protein structure can critically impair protein function, with important pathologic consequences. This study was undertaken to examine whether oxidation-induced changes to protein structure occur during pediatric cardiopulmonary bypass and to examine the association with postoperative outcome. Elevation of the 3,4-dihydroxyphenylalanine content of a protein relative to its native tyrosine content indicates structural damage due to oxidation. Protein 3,4-dihydroxyphenylalanine/native tyrosine ratios were measured before surgery and up to 6 hours after institution of cardiopulmonary bypass in 24 children undergoing repair of congenital heart disease, who were prospectively selected to form a cyanotic and comparable acyanotic control group. Results were correlated with perioperative variables and postoperative outcomes. Elevation of protein 3,4-dihydroxyphenylalanine/tyrosine ratios above baseline (0.48 mmol/mol [SD, 0.11 mmol/mol] vs 0.36 mmol/mol [SD, 0.13 mmol/mol]; P = .001) occurred within 30 minutes of initiating cardiopulmonary bypass in cyanotic but not in acyanotic children and correlated inversely with preoperative arterial oxygen saturation (R = -0.52; P = .03). Protein 3,4-dihydroxyphenylalanine/tyrosine ratios were also increased above baseline at 120 minutes (0.44 mmol/mol [SD, 0.12 mmol/mol]; P = .007) and 180 minutes (0.40 mmol/mol [SD, 0.14 mmol/mol]; P = .01) after the institution of cardiopulmonary bypass in children who underwent prolonged procedures. Elevation of 3,4-dihydroxyphenylalanine/tyrosine during prolonged procedures was associated with postoperative arrhythmias and the need for increased inotropic support (P = .001). Oxidative injury to proteins occurs during pediatric cardiopulmonary bypass. Cyanotic children are most at risk, particularly those undergoing prolonged procedures, in whom elevation of the protein 3,4-dihydroxyphenylalanine/tyrosine ratio is associated with increased postoperative morbidity.

Elimination of D-band in Raman spectra of double-wall carbon nanotubes by oxidation

In this Letter, we present an in situ Raman spectroscopy study of oxidation-induced changes in the structure and composition of double-wall carbon nanotubes (DWCNTs). Above 480 °C, the intensity of the D band decreases to less than 0.01% of the G band intensity, when measured using the 780 nm laser excitation. The D band was absent from the Raman spectra recorded with the 514.5 nm excitation. Thermogravimetric analysis and high-resolution transmission electron microscopy are used to explain the observed results. We conclude that oxidation provides a purification method for the DWCNT which leads to a sample containing tubes having nearly clean surfaces without disordered carbon.

Oxidation of reduced Y-doped semiconducting barium titanate ceramics

Oxidation-induced microstructural changes in reduced yttrium-doped barium titanate (Ba1-xYxTi 1-x 4+ Ti x 3+ O3) are studied using samples sintered in a reducing atmosphere (\(p_{O_2}\) = 10−4Pa) and then oxidized in air at 1150 and 1350°C. The results indicate that oxidation leads to precipitation of Ba6Ti17O40 and, at relatively high doping levels, Y2Ti2O7. These phases increase the electrical resistance of the outer layer of the grains in the ceramics.

Raman spectroscopy studies on the thermal stability of TiN, CrN, TiAlN coatings and nanolayered TiN/CrN, TiAlN/CrN multilayer coatings

About 1.5-μm-thick single-layer TiN, CrN, TiAlN coatings and nanolayered TiN/CrN, TiAlN/CrN multilayer coatings were deposited on silicon (111) substrates using a reactive direct current magnetron sputtering process. Structural characterization of the coatings was done using x-ray diffraction (XRD) and micro-Raman spectroscopy. All the coatings exhibited NaCl B1 structure in the XRD data. Raman spectroscopy data of as-deposited coatings exhibited two broad bands centered at 230–250 and 540–630 cm-1. These bands have been assigned to acoustical and optical phonon modes, respectively. Thermal stability of the coatings was studied by heating the coatings in air in a resistive furnace for 30 min in the temperature range 400–900 °C. Structural changes as a result of heating were characterized using Raman spectroscopy and XRD. Raman data showed that TiN, CrN, TiN/CrN, TiAlN, and TiAlN/CrN coatings started to oxidize at 500, 600, 750, 800, and 900 °C, respectively. To isolate the oxidation-induced spectral changes as a result of heating of the coatings in air, samples were also annealed in vacuum at 800 °C under similar conditions. The Raman data of vacuum-annealed coatings showed no phase transformation, and intensity of the optical phonon mode increased and shifted to lower frequencies. The origin of these spectral changes is discussed in terms of defect structure of the coatings. Our results indicate that the thermal stability of nanolayered multilayer coatings is superior to the single-layer coatings.

EPR investigation of cell membrane fluidity upon external oxidative stimulus

The perturbation of the physical state of cell membranes triggered by an external oxidative stimulus has been studied with sperm cells which were chosen as a model system. Electron paramagnetic resonance (EPR) spectroscopy was applied and three different nitroxides bearing a paramagnetic group on the 5th, 7th and 13th carbon of the stearic acid acyl chain were used to probe different regions of the membrane. The theoretical simulations of the experimental spectra indicate distinct domains in the intact cell plasma membrane where local steric constraints impose different degrees of motional averaging experienced by the reporter group. Upon the external oxidative stimulus the spectral changes were proportional to the accumulated oxidation products and were detected only for the reporter group residing close to the lipid-water interface. The EPR spectra reveal that the motion of the reporter group has slowed down in the oxidized cells and that the oxidatively modified membrane shows up as a more rigid structure as compared with the native state. The results also indicate that the oxidation-induced spectral changes are supressed in the presence of gangliosides.

The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice.

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.

Gun bore erosion mechanisms revisited with laser pulse heating

Prior work has shown that laser pulse heating with appropriate energy and pulse duration reproduces many of the important degradation effects at chromium coated gun bore surfaces. Among these effects are recrystallization and grain growth in the chromium, major cracks in the chromium, a heat-affected zone in the steel substrate, oxidation of steel at the chromium crack tips, and initiation and propagation of blunt cracks into the heat-affected zone of the steel substrate. This report describes further studies of gun steel using laser pulse heating in air, argon, oxygen, nitrogen and hydrogen. The observed features include surface roughening, melting and oxidation-induced composition changes on the steel surface. Computer modeling of the laser pulse process was performed for the specimen configuration used in these experiments. Comparisons of laser pulse heating experiments with data from damaged surfaces of fired gun barrels offer new perspectives on gun bore degradation mechanisms.

A reaction-induced FT-IR study of cyanobacterial photosystem I.

In oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB. A difference Fourier transform infrared spectrum, associated with P(700)(+)-minus-P(700), can be acquired using PSI from the cyanobacterium Synechocystis sp. PCC 6803. This spectrum reflects contributions from oxidation-sensitive modes of chlorophyll, as well as from oxidation-induced structural changes in amino acid residues and the peptide backbone. Oxidation-induced structural changes may play a role in the facilitation and control of electron-transfer reactions involving the primary donor. In this paper, we report that photooxidation of P(700) in cyanobacterial PSI perturbs a cysteine residue. At 264 and 80 K, a downshift of a SH stretching vibration from 2560 to 2551 cm(-1) is observed. Such a downshift is consistent with an increase in hydrogen bonding, with a change in C-S-H conformation, or with an electric field effect. Deuterium exchange experiments were also performed. While the perturbed cysteine is in a protein region that is resistant to exchange, other (2)H-sensitive vibrational chl and amino acid bands were observed. From the (2)H exchange experiments, we conclude that photooxidation of P(700) perturbs internal or bound water molecules in PSI and that the P(700)(+)-minus-P(700) spectrum is (2)H exchange-sensitive. The results are consistent with structural complexity in the PSI primary donor, as previously suggested [Kim, S., and Barry, B. A. (2000) J. Am. Chem. Soc. 122, 4980-4981]. Possible explanations, including a partial enolization of P(700)(+), are discussed.

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity.

During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.

Comparison of the Surface Activity and Bulk Aggregation of Ferrocenyl Surfactants with Cationic and Anionic Headgroups

We report the synthesis and aqueous solution properties of a novel redox-active surfactant, Fc(CH2)11SO3-Na+, where Fc is ferrocene. Electrochemical oxidation of Fc to the ferrocenium cation (Fc+) reversibly transforms the anionic surfactant (Fc(CH2)11SO3-) into a zwitterionic species (Fc+(CH2)11SO3-). When dissolved at concentrations between 0.15 and 0.4 mM in aqueous solutions containing 10 mM Li2SO4, oxidation is accompanied by the reversible transformation of a solution of monomeric species of Fc(CH2)11SO3- into vesicle-like (Dh = 70 ± 8 nm, Rg/Rh = 1.0 ± 0.3) surfactant assemblies of Fc+(CH2)11SO3-. In contrast, at concentrations above 0.4 mM, oxidation reversibly transforms a solution of globular (Dh = 6 ± 2 nm) micelles into vesicle-like assemblies of Fc+(CH2)11SO3-. These various oxidation-induced changes in aggregation are reflected in the interfacial properties of this surfactant system, which are substantially different from those of ferrocenyl surfactants with cationic headgroups (Fc(CH2)11N+(...

Effect of oxidation on Ca 2+-ATPase activity and membrane lipids in lens epithelial microsomes

Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca 2+-ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca 2+-ATPase activity decreased as a function of ascorbate concentration between 0 and 200 μM. The level of Ca 2+-ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25°C when lipids are more ordered, the Ca 2+-ATPase activity was similar to that observed in the oxidized system measured at 37°C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca 2+-ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca 2+-ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca 2+-ATPase pump by 60–80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.

Elastic scattering of Si 2p photoelectrons in silicon oxide

The cross-sections for elastic and inelastic scattering of Si 2p photoelectrons in silicon oxide were uniquely determined from the analysis of oxidation-induced changes in Si 2p photoelectron diffraction patterns arising from single crystalline Si substrate under the assumption that the structure of hydrogen-terminated Si substrate does not change by the oxidation. It was found from the Monte Carlo calculation of path of elastically and inelastically scattered Si 2p photoelectrons for the simulation of oxidation-induced changes in photoelectron diffraction patterns that the total elastic scattering cross-section and the inelastic scattering cross-section are 1.54×10 −20 and 1.26×10 −20 m 2, respectively.

Mechanical properties of oxidized silicon nitride ceramics

The effect of oxidation on the room-temperature mechanical properties of silicon nitride ceramics with four different compositions is studied. Room temperature values of bending strength and elastic modulus of the whole oxidized material and of hardness, the fracture toughness and elastic modulus of the internal region (without the strongly oxidized surface layer) are determined for the materials oxidized at five different temperatures up to 1400°C. The strength decreases with increasing oxidation temperature for all the materials investigated. The fracture toughness of the internal region decreases upon oxidation at 1000°C. A reduction of the elastic modulus of the materials oxidized at 1400°C is also observed. The oxidation-induced changes in the mechanical properties are partially explained by the changes in the phase composition.

Mild Oxidation of Lipoproteins Increases Their Affinity for Surfaces Covered by Heparan Sulfate and Lipoprotein Lipase

Lipoprotein lipase (LPL) is present in cells involved in development of atherosclerosis (endothelial cells, smooth muscle cells, and macrophages). A direct involvement of LPL in atherogenesis has been suggested. Previously we used the surface plasmon resonance technique to study the interaction of lipoproteins with surfaces covered by heparan sulfate proteoglycans (HSPG) and LPL [A. Lookeneet al.(1997)Biochemistry36, 5267–5275]. The binding was much increased by the presence of LPL. Here we demonstrate that mild oxidation of low-density-lipoprotein (LDL) and very-low-density lipoprotein (VLDL)in vitroincreases their binding to surfaces covered by HSPG and LPL, while extensive oxidation decreases it. Similar results were obtained with a lipid emulsion (Intralipid), indicating that oxidation-induced changes of the lipid part could explain the effects. LPL increased binding and uptake of the mildly oxidized (compared to nonoxidized) LDL by THP-I monocyte-derived macrophages. Our studies indicate that LPL has the highest affinity for mildly oxidized LDL and support its involvement in development of atherosclerosis.

Infrared Analysis of Ligand- and Oxidation-Induced Conformational Changes in Hemoglobins and Myoglobins

Effects of the binding of O2 and CO to heme iron (II) of deoxy forms and of the oxidation of deoxy forms to aquoiron (III) complexes on the infrared spectra of hemoglobins and myoglobins have been examined, Spectra were measured for aqueous solutions 3-4 mM in heme of human, bovine, and equine hemoglobins and sperm whale, bovine, and equine myoglobins in 10 mM sodium phosphate buffer, pH 7.4, at 20°C. All ligand binding and oxidation reactions resulted in similar spectral shifts in the region 1665 to 1670 cm−1, a portion of the amide I region assignable to β-turn structure. There were no other significant changes in the amide I region, a finding consistent with no other alterations in secondary structure, The major bands near 1655 cm−1 associated with α-helices were consistently at 2 cm−1 lower wavenumber for myoglobins than for hemoglobins. The changes in solution infrared spectra observed in this study may result at least in part from conformational changes at the FG corner associated with movements of F and E helices that have been noted previously in crystal structures.

Protein thiol modification and apoptotic cell death as cGMP-independent nitric oxide (NO) signaling pathways.

Nitric oxide signaling is achieved through both cGMP-dependent and cGMP-independent mechanisms. The latter are exemplified by protein thiol modification followed by subsequent NAD(+)-dependent automodification of the glycolytic enzyme GAPDH, or by mechanisms inducing accumulation of the tumor suppressor gene p53 and causing apoptotic cell death. Both cGMP-independent actions are initiated using NO-releasing compounds and an active LPS/cytokine-inducible NO synthase. NO-synthase inhibitors block the release of NO and hinder downstream signaling mechanisms; they are therefore valuable pharmacological tools linking a defined cellular response to various NO actions. Signal transducing mechanisms elicited by NO can be studied using GAPDH as a representative example of NO-induced protein modification and are grouped as follows: --S-Nitrosylation reactions initiated by NO+ --NAD(+)-dependent, post-translational covalent automodification of GAPDH --Oxidative modification (thiol oxidation) and inhibition of GAPDH by NO-related agents, probably ONOO- GAPDH and several other protein targets may serve as molecular sensors of elevated NO concentrations and may transmit this message through posttranslational modification and oxidation-induced conformational changes as cGMP-independent NO signaling pathways. Toxicity of NO seems to be linked to both apoptosis and necrosis, depending on the chemistry of NO it undergoes in a given biological milieu. Toxicity manifests as a relative excess of NOx, metal-NO interactions, and ONOO- formation in relation to cellular defense systems. Although accumulation of the tumor-suppressor gene product p53 in response to NO opens a regulatory mechanism known to be involved in apoptotic cell death, cGMP-independent signaling pathways remain to be elucidated. As NO-dependent modification of GAPDH would imply down-regulation of glycolysis and concomitant energy production followed by cell death, our data so far do not support this assumption. In recent years, NO has proved to be a beneficial messenger with a potentially toxic activity. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to its pathophysiological role.