Inducible nitric oxide synthase-mediated injury in a mouse model of acute salivary gland dysfunction.

AIM: Inducible nitric oxide synthase (iNOS) is a key regulator of the innate immune system. The aim of the current study was to explore whether innate immune-mediated iNOS and reactive nitrogen species acutely perturb acinar cell physiology and calcium homeostasis of exocrine salivary tissues. METHODS: Innate immunity in the submandibular gland of C57BL/6 mice was locally activated via intraductal retrograde infusion of polyinosinic:polycytidylic acid (poly (I:C). Expressions of iNOS and the activity of the reactive nitrogen species peroxynitrite, were evaluated by immunohistochemistry. Mice were pre-treated with the selective iNOS inhibitor aminoguanidine in order to substantiate the injurious effect of the nitrosative signal on the key calcium regulator sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2b) and calcium signalling. RESULTS: Challenging salivary gland innate immunity with poly (I:C) prompted upregulated expression of iNOS and the generation of peroxynitrite. Inhibition of iNOS/peroxynitrite revealed the role played by upregulated nitrosative signalling in: dysregulated expression of SERCA2b, perturbed calcium homeostasis and loss of saliva secretion. CONCLUSION: iNOS mediates disruption of exocrine calcium signalling causing secretory dysfunction following activation of innate immunity in a novel salivary gland injury model.

Tyrosine oxidation and nitration in transmembrane peptides is connected to lipid peroxidation.

Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.

Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment.

Mechanism of cysteine oxidation by peroxynitrite: An integrated experimental and theoretical study.

Since peroxynitrite was identified as a pathophysiological agent it has been implicated in a great variety of cellular processes. Particularly, peroxynitrite mediated oxidation of cellular thiol-containing compounds such as Cys residues, is a key event which has been extensively studied. Although great advances have been accomplished, the reaction is not completely understood at the atomic level. Aiming to shed light on this subject, we present an integrated kinetic and theoretical study of the oxidation of free Cys by peroxynitrite. We determined pH-independent thermodynamic activation parameters, namely those corresponding to the reaction between the reactive species: Cys thiolate and peroxynitrous acid. We found a pH-independent activation energy of 8.2 ± 0.6 kcal/mol. Simulations were performed using state of the art hybrid quantum-classical (QM-MM) molecular dynamics simulations. Our results are consistent with a SN2 mechanism, with Cys sulfenic acid and nitrite anion as products. The activation barrier is mostly due to the alignment of sulfur's thiolate atom with the oxygen atoms of the peroxide, along with the concomitant charge reorganization and important changes in the solvation profile. This work provides an atomic detailed description of the reaction mechanism and a framework to understand the environment effects on peroxynitrite reactivity with protein thiols.

Polymer nanoreactors with dual functionality: simultaneous detoxification of peroxynitrite and oxygen transport.

The design of multifunctional systems is in focus today as a key strategy for coping with complex challenges in various domains that include chemistry, medicine, environmental sciences, and technology. Herein, we introduce protein-containing polymer nanoreactors with dual functionality: peroxynitrite degradation and oxygen transport. Vesicles made of poly-(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) successfully encapsulated hemoglobin (Hb), which serves as a model protein because of its dual function in oxygen transport and peroxynitrite degradation. By inserting channel proteins, the polymer membranes of vesicles permitted passage of various compounds that served for the assessment of in situ Hb activity. The requisite conformational changes in the protein structure and the change in oxidation states that took place within the confined space of the vesicle cavity demonstrated that Hb preserved its dual functionality: peroxynitrite degradation and oxygen transport. The functionality of our nanoreactor, combined with its simple procedure of production and extensive stability over several months, supports it as a promising system for further medical applications.

Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: role of protein kinase G nitration.

Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO(-) ) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO(-) donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24 h (with exogenous activation of cGMP) revealed increased ONOO(-) levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO(-) and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.

Salivary gland dysfunction and salivary redox imbalance in patients with Alzheimer's disease.

Alzheimer's disease (AD) is associated with the deposition of ß-amyloid in the brain. AD accounts for over 50% of cases of dementia which results from disturbances in redox homeostasis. Indeed, increased intensity of protein oxidation and nitration as well as lipid peroxidation is observed in brain areas with considerable amounts of amyloid plaques and neurofibrillary tangles. However, little is known about the oxidoreductive balance of salivary glands in AD patients. Therefore, the aim of this study was to evaluate the antioxidant barrier and oxidative/nitrosative stress biomarkers in stimulated saliva and blood of AD patients. The study was participated by 25 AD patients and 25 non-demented controls without neurological diseases or cognitive impairment, matched by age and gender to the study group. The number of patients was determined based on a previous pilot study (test power = 0.9). We found a significant decrease in the activity of erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx), increased activity of catalase (CAT) and reduced concentration of plasma non-enzymatic antioxidants (uric acid, UA and reduced glutathione, GSH). In contrast, in the stimulated saliva of AD patients we observed significantly decreased activity of all antioxidant enzymes (SOD, CAT and GPx) as well as concentration of GSH compared to the control group. The content of lipid (malondialdehyde, MDA) and protein (advanced oxidation protein products, AOPP; advanced glycation end-products, AGE) oxidation products as well as biomarkers of nitrosative stress (peroxynitrite, nitrotyrosine) was significantly higher in both saliva and plasma of AD patients compared to the controls. In AD patients, we also observed a considerable decrease in stimulated saliva secretion and salivary total protein content, and an increase in salivary ß-amyloid concentration. In conclusion, AD results in redox imbalance towards oxidative reactions, both at the level of the oral cavity and the entire body. General redox balance disturbances do not coincide with salivary redox balance disturbances. Reduction in stimulated saliva secretion in AD patients reflects secretory dysfunction of the parotid glands.

Application of Molecular Hydrogen as a Novel Antioxidant in Sports Science.

Molecular hydrogen (H2) is a colorless, tasteless, odorless, and minimal molecule with high flammability. Although H2 has been thought to be an inert gas in living bodies for many years, an animal study reported that inhalation of H2 gas decreased oxidative stress and suppressed brain injury caused by ischemia and reperfusion injury due to its antioxidant action. Since then, the antioxidant action of H2 has attracted considerable attention and many studies have reported on its benefits. Most studies have reported the effects of H2 on diseases such as cancer, diabetes, cerebral infarction, and Alzheimer's disease. However, little is known regarding its effects on healthy subjects and exercise. Thus far, including our study, only 6 studies have explored the effect of H2 on exercise. H2 is the smallest molecule and therefore can easily penetrate the cellular membrane and rapidly diffuse into organelles. H2 is thought to be able to selectively reduce hydroxyl radicals and peroxynitrite and does not affect physiologically reactive species. H2 can be supplied to the body through multiple routes of administration, such as oral intake of H2 water and H2 bathing. Therefore, H2 may be a potential alternative strategy for conventional exogenous antioxidant interventions in sports science. The purpose of this review is to provide evidence regarding the effects of H2 intake on changes in physiological and biochemical parameters, centering on exercise-induced oxidative stress, for each intake method. Furthermore, this review highlights possible future directions in this area of research.

A Dual-Emissive Phosphorescent Polymeric Probe for Exploring Drug-Induced Liver Injury via Imaging of Peroxynitrite Elevation In Vivo.

Drug-induced liver injury (DILI) is a widespread clinical problem. The pathophysiological mechanisms of DILI are complicated, and the traditional diagnostic methods for DILI have their limitations. Owing to its convenient operation, high sensitivity, and high specificity, luminescent sensing and imaging as an indispensable tool in biological research and clinical trials may provide an important means for DILI study. Herein, we report the rational design and preparation of a near-infrared dual-phosphorescent polymeric probe (P-ONOO) for exploring the DILI via specific imaging of peroxynitrite (ONOO-) elevation in vivo, which was one of early markers of DILI and very difficult to be detected due to its short half-life and high reactive activity. With the utilization of P-ONOO, the raised ONOO- was visualized successfully in the drug-treated hepatocytes with a high signal-to-noise ratio via ratiometric and time-resolved photoluminescence imaging. Importantly, the ONOO- boost in the acetaminophen-induced liver injury in real time was verified, and the direct observation of the elevated ONOO- production in ketoconazole-induced liver injury was achieved for the first time. Our findings may contribute to understanding the exact mechanism of ketoconazole-induced hepatotoxicity that is still ambiguous. Notably, this luminescent approach for revealing the liver injury works fast and conveniently.

Tyrosine nitration, dimerization, and hydroxylation by peroxynitrite in membranes as studied by the hydrophobic probe N-t-BOC-l-tyrosine tert-butyl ester.

Protein tyrosine oxidation mechanisms in hydrophobic biocompartments (i.e., biomembranes, lipoproteins) leading to nitrated, dimerized, and hydroxylated products are just starting to be appreciated. This chapter reports on the use of the hydrophobic tyrosine analog N-t-BOC-l-tyrosine tert-butyl ester (BTBE) incorporated to phosphatidyl choline liposomes to study peroxynitrite-dependent tyrosine oxidation processes in model biomembranes. The probe proved to be valuable in defining the role of biologically relevant variables in the oxidation process, including the action of hydrophilic and hydrophobic peroxynitrite and peroxynitrite-derived free radical scavengers, transition metal catalysts, carbon dioxide, molecular oxygen, pH, and fatty acid unsaturation degree. Moreover, detection of the BTBE phenoxyl radical and relative product distribution yields of 3-nitro-, 3,3'-di-, and 3-hydroxy-BTBE in the membrane fully accommodate with a free radical mechanism of tyrosine oxidation, with physical chemical and biochemical determinants that in several respects differ of those participating in aqueous environments. The methods presented herein can be extended to explore the reaction mechanisms of tyrosine oxidation by other biologically relevant oxidants and in other hydrophobic biocompartments.

A liposomal formulation study of 2,7-dichlorodihydrofluorescein for detection of reactive oxygen species.

It is known that reactive oxygen species (ROS) are connected with various diseases, and many studies have examined redox conditions in the body. However, there is a problem with stability of ROS and selectivity of detection probe. In this study, we aimed to stabilize the detection probe, and have tried to encapsulate the probe in polyethyleneglycol (PEG)-modified liposomes. Dichlorodihydrofluorescein (DCDHF) is oxidized to dichlorofluorescein (DCF), a highly fluorescent product, by ROS. We tried liposomalization of DCDHF probes by the Bangham method. However, it was found that DCDHF was oxidized during preparation and converted to DCF. In contrast, when we performed to encapsulate DCDHF in the liposome after the preparation of empty liposome, the stabilized DCDHF encapsulated liposome was successfully proposed by the addition of DCDHF solution by freeze-drying and re-hydration. Furthermore, the encapsulated efficacy of PEG-modified liposomes was higher than unmodified liposomes. This DCDHF liposome was examined for reactivity with hydroxyl radical and peroxynitrite as highly ROS. It was confirmed that DCDHF liposome had a protective effect on the hydroxyl radical, though an effect of the liposomalization of DCDHF was not shown on reactivity of the peroxynitrite. Therefore, it is likely that the liposomalization of DCDHF has selectivity for certain radical species. It is hoped that these results can be applied to novel and simple diagnostics for redox detection of conditions in the body.

Comparison of total antioxidant capacity of salivary, capillary and venous samplings: interest of the salivary total antioxidant capacity on triathletes during training season.

AIM: Total antioxidant capacity (TAC) is an essential parameter to watch over defense system of athletes exposed to an oxidant stress during intensive periods of training. To control this parameter throughout the training period, repetitive biological samples are required. The TAC is usually investigated in venous blood which needs invasive withdrawings. Thus, we proposed to find alternatives to venous blood analysis by venepuncture, which is invasive, stressful and not allow a regular follow-up on athletes during annual training season. METHODS: We measured capillary and salivary TAC in 65 physically active subjects at rest and compared them to the venous TAC. We followed the evolution of venous and salivary TAC in 7 triathletes throughout an annual training period (March and June) corresponding to two different types of training. RESULTS: There was a good correlation between plasma venous and capillary TAC values (r=0.77; P<0.0001), but salivary TAC were significantly lower than the plasma ones and did not correlate. Venous and saliva TAC of triathletes were significantly higher in March compared to June. The variations of plasma and salivary TAC between the two periods of training were correlated (r=0.96; P<0.01). CONCLUSION: The capillary sampling can replace the venous one for TAC evaluation in routine assays for the follow-up of athletes. Even if saliva TAC did not reflect plasma TAC, it could be used in the follow-up of athletes since a strong correlation is found between the variation of saliva and plasma TAC during the training season.

Functions of transcription factors NF-κB and Nrf2 in the inhibition of LPS-stimulated cytokine release by the resin monomer HEMA.

OBJECTIVE: Resin monomers like 2-hydroxyethyl methacrylate (HEMA) interfere with effects induced by stressors such as lipopolysaccharide (LPS) released from cariogenic microorganisms. In this study, mechanisms underlying monomer-induced inhibition of the LPS-stimulated secretion of inflammatory cytokines from immunocompetent cells were investigated. METHODS: Secretion of pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory IL-10 from RAW264.7 mouse macrophages exposed to LPS and HEMA (0-6-8mM) was determined by ELISA. The formation of reactive oxygen (ROS) and nitrogen species (RNS) was determined by flow cytometry (FACS) after staining of cells with specific fluorescent dyes. Cell viability was analyzed by FACS, and protein expression was detected by Western blotting. RESULTS: Secretion of TNF-α, IL-6 and IL-10 from LPS-stimulated cells increased after a 24h exposure. A HEMA-induced decrease in cytokine secretion resulted from the inhibition of LPS-stimulated NF-κB activation. Nuclear translocation of NF-κB was inhibited possibly as a result of enhanced levels of hydrogen peroxide (H2O2) and nitric oxide (NO) in HEMA-exposed cells. Oxidative stress caused by HEMA-induced formation of H2O2 and LPS-stimulated peroxynitrite (ONOO) also enhanced nuclear expression of Nrf2 as the major regulator of redox homeostasis, as well as Nrf2-controlled stress protein HO-1 to inhibit NF-κB activity. HEMA inhibited the LPS-stimulated expression of NOS (nitric oxide synthase) to produce NO but counteracted the expression of Nox2, which forms superoxide anions that combine with NO to peroxynitrite. CONCLUSIONS: Resin monomers like HEMA inhibit LPS-stimulated NF-κB activation essential for cytokine release as a crucial response of immunocompetent cells of the dental pulp to invading cariogenic pathogens.

iNOS-mediated generation of reactive oxygen and nitrogen species by biomaterial-adherent neutrophils.

Infection due to implanted cardiovascular biomaterials is a serious complication initiated by bacterial adhesion to the surface of the implant. The release of reactive oxygen species by neutrophils, particularly superoxide anion, is a well-known bactericidal mechanism. Additionally, nitric oxide (NO) has also been identified as an important cytotoxic mediator in acute and chronic inflammatory responses with enhanced NO production by upregulation of inducible nitric oxide synthase (iNOS). The interaction of NO and superoxide anion will result in the formation of peroxynitrite (OONO-), a potent cytotoxic oxidant. In this study, we have shown that biomaterial-induced neutrophil activation does not cause upregulation of iNOS and activation of iNOS-mediated pathways. However, NO and O2- production does occur over time upon adhesion to a biomaterial and is modulated by biomaterial surface chemistry. With no stimulus, the polyethylene oxide-modified polyurethane induced greater neutrophil activation than did the control as indicated by the increased production of NO and O2- over time. Adherent-stimulated neutrophils generally produced lower amounts of NO over time in comparison with unstimulated cells. Furthermore, there is no evidence of peroxynitrite activity in unstimulated neutrophils adherent to the Elasthane 80A. However, upon stimulation with adherent Staphylococcus epidermidis, peroxynitrite formation did occur. Our results suggest that bactericidal mechanisms in neutrophils involving NO generation (NOS pathway) are further compromised than O2- producing pathways (NADPH oxidase) upon exposure to biomaterials, resulting in a diminished microbial killing capacity, which can increase the probability of device-centered infections.

Endothelial cell activation by endotoxin involves superoxide/NO-mediated nitration of prostacyclin synthase and thromboxane receptor stimulation.

In bovine coronary artery segments, peroxynitrite inhibits prostacyclin (PGI2) synthase by tyrosine nitration. Using this pharmacological model, we show that a 1 h exposure of bovine coronary artery segments to endotoxin (lipopolysaccharide [LPS]) inhibits the relaxation phase following angiotensin II (Ang II) stimulation and causes a vasospasm that can be suppressed by a thromboxane A2 (TxA2) receptor blocker. In parallel, PGI2 synthesis decreases in favor of prostaglandin E2 formation. Immunoprecipitation and costaining with an anti-nitrotyrosine antibody identified PGI2 synthase as the main nitrated protein in the endothelium. All effects of LPS could be prevented in the presence of the nitric oxide (NO) synthase inhibitor Nomega-mono-methyl-L-arginine and polyethylene-glycolated Cu/Zn- superoxide dismutase. Thus, the early phase of endothelial cell activation in bovine coronary arteries by inflammatory agents proceeds by a protein synthesis-independent priming process for a source of superoxide that we tentatively attribute to xanthine oxidase. Upon receptor activation, Ang II stimulates NO and superoxide production, resulting in a peroxynitrite-mediated nitration and inhibition of PGI2 synthase. The remaining 15-hydroxy-prostaglandin 9,11-endoperoxide (PGH2) first activates the TxA2/PGH2 receptor and then is converted to prostaglandin E2 (PGE2) by smooth muscle cells. PGE2 together with a lack of NO and PGI2 is known to promote the adhesion of white blood cells and their immigration to the inflammatory locus.

Using nanosensors for in situ monitoring and measurement of nitric oxide and peroxynitrite in a single cell.

The cytotoxic peroxynitrite (ONOO(-)) is an oxidation product of the cytoprotective nitric oxide (NO). Our studies support the hypothesis that the concentration ratio of NO and ONOO(-), [NO]/[ONOO(-)] can be a marker of nitroxidative imbalance, which subsequently correlates well with endothelial dysfunction and dysfunction of the cardiovascular system. Nanosensors, described here, have been used for simultaneous monitoring and measurement of NO and ONOO(-) release from a single endothelial cell. These nanosensors, with a diameter of 200-300 nm, can be positioned accurately in close proximity of 5-10 µm from the endothelial cell membrane. The response time of the sensors is better than a millisecond and the detection limit is 10(-9) M, with a linear concentration response of up to about 2 µM. The application of these sensors for the measurement of the balance and imbalance of [NO]/[ONOO(-)] in normal and dysfunctional endothelium is demonstrated.

Polyphenols and red wine as peroxynitrite scavengers: a chemiluminescent assay.

A novel chemiluminescent assay for evaluating peroxynitrite (ONOO(-))-scavenging capacity was developed. The experimental protocol ensures sensitivity and reproducibility of measurements. The addition of 0-500 microM ONOO(-) to rat liver homogenate generated a luminous signal that was analyzed by chemiluminescence in a LKB Wallac liquid scintillation counter. The obtained optimal conditions were: 1-2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 220 microM ONOO(-) at 30 degrees C. As polyphenols we used (+)-catechin, (-)-epicatechin, and myricetin. The most efficient of the compounds tested was myricetin with an IC(50) of 20 microM. The effectiveness of this method was verified by evaluating the antioxidant ability of three red wine samples to decrease peroxynitrite-initiated chemiluminescence. The ONOO(-)-scavenging activity of wines measured by this assay was related to the phenolic level of the samples. The quickness and reliability of this assay makes it particularly suitable for a large-scale screening of watery food extracts.

Oxidative stress-induced DNA damage in the synovial cells of the temporomandibular joint in the rat.

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.

Reactive oxygen species inhibited by titanium oxide coatings.

Titanium is a successful biomaterial that possesses good biocompatibility. It is covered by a surface layer of titanium dioxide, and this oxide may play a critical role in inhibiting reactive oxygen species, such as peroxynitrite, produced during the inflammatory response. In the present study, titanium dioxide was coated onto silicone substrates by radio-frequency sputtering. Silicone coating with titanium dioxide enhanced the breakdown of peroxynitrite by 79%. At physiologic pH, the peroxynitrite donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) was used to nitrate 4-hydroxyphenylacetic acid (4-HPA) to form 4-hydroxy-3-nitrophenyl acetic acid (NHPA). Titanium dioxide-coated silicone inhibited the nitration of 4-HPA by 61% compared to aluminum oxide-coated silicone and 55% compared to uncoated silicone. J774A.1 mouse macrophages were plated on oxide-coated silicone and polystyrene and stimulated to produce superoxide and interleukin-6. Superoxide production was measured by the chemiluminescent reaction with 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA). Titanium dioxide-coated silicone exhibited a 55% decrease in superoxide compared to uncoated silicone and a 165% decrease in superoxide compared to uncoated polystyrene. Titanium dioxide-coated silicone inhibited IL-6 production by 77% compared to uncoated silicone. These results show that the anti-inflammatory properties of titanium dioxide can be transferred to the surfaces of silicone substrates.

Caroverine, a multifunctional drug with antioxidant functions.

Here we show that lipid peroxidation of liposomal membranes was suppressed in the presence of Caroverine, a spasmolytic drug used in some countries. In order to understand the mechanism of this antioxidant action of Caroverine we studied the interaction of Caroverine with superoxide radicals, hydroxyl radicals and peroxynitrite. The results of the study show that the reaction of Caroverine with O2-* radicals is of marginal significance. However, it is efficient in removing peroxynitrite and a particular high reaction constant was found for reaction with hydroxyl radicals. The strong antioxidant activity of Caroverine is therefore based both on the partial prevention of the formation and the highly active scavenging of hydroxyl radicals.