Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells.

Exposure to high levels of ozone (O(3)) damages respiratory tract epithelial cells. This research evaluated the ability of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide free radical, to decrease O(3)-mediated injury to a respiratory tract-derived cell line (A549 cells) by monitoring in this cell system the interleukine-8 (IL-8) production. TEMPO reduced O(3)-induced IL-8 production in A549 cells, as evidenced by PCR analysis, Western blot and ELISA assays. This behaviour is explainable on the basis of the reactivity between TEMPO with O(3) and/or O(3)-derived free radicals in biological systems. The study provides evidence that TEMPO reacts with O(3) and/or its cytotoxic products and may provide protections against O(3)-induced biotoxicities.

In vitro photostability and photoprotection studies of a novel 'multi-active' UV-absorber.

This paper reports on the synthesis and properties of a new UV-absorber (OC-NO) based on the most popular UV filter worldwide, ethylhexyl methoxycinnamate (OMC) in which the methoxy group has been replaced with a pyrrolidine nitroxide bearing antioxidant activity. This sunscreen active has therefore both UV-absorbing and antioxidant properties which could ideally address both the UV-B and UV-A skin photo-damage. For broad-spectrum coverage, the combinations of OC-NO with two commonly used UV-A absorbers (BMDBM and DHHB) were also studied. The results obtained reveal that OC-NO: (a) is as photostable as OMC after UV-A exposure; (b) acts as free radical scavenger as demonstrated by EPR and chemical studies; (c) reduces UV-A and UV-A+BMDBM induced lipid peroxidation in liposomes and cells, measured as reduced TBARS levels and increased C11-BODIPY red fluorescence, respectively; (d) has comparable antioxidant activity to that of vitamin E and BHT commonly used in skin care formulations; (e) is non-cytotoxic to human skin fibroblasts as assessed with the MTT assay when exposed to increasing doses of UV-A; and (f) OC-NO+DHHB is a promising, photostable broad spectrum UV-filter combination that concomitantly reduces UV-induced free radical damage. These results suggest that nitroxide/antioxidant-based UV-absorbers may pave the way for the utilization of 'multi-active' ingredients in sunscreens thereby reducing the number of ingredients in these formulations.

Raloxifene covalently bonded to titanium implants by interfacing with (3-aminopropyl)-triethoxysilane affects osteoblast-like cell gene expression.

Since Raloxifene, a drug used in osteoporosis therapy, inhibits the osteoclast functions but not osteoblast functions, it could improve the recovery during implant surgery. This preliminary report describes a simple method to link, through a covalent bond, Raloxifene to titanium by interfacing with (3-aminopropyl)-Triethoxysilane as assessed by the IR-FT and SEM. To evaluate the biological response of osteoblast-like cells to this implant, we compared expression gene profiling of cell cultures on Raloxifene conjugated implant and normal implant by DNA microarray. By using DNA microarrays containing 19,200 genes, we identified differently expressed genes in osteoblast-like cell line (MG-63). Surface Raloxifene conjugated implants have been shown to have a relevant importance in modifying cell response. This result could be an interesting starting point for the use of an immediate functional loading of implants.

Reactivity of ubiquinones and ubiquinols with free radicals.

The reactivity of quinones 1-4 and of the corresponding quinols 5-8 towards carbon- and oxygen-centred radicals were studied. All quinones bearing at least one nuclear position free, readily react with alkyl and phenyl radicals to afford the alkylated quinones 12-24; however, quinones 1 and 3 reacted with 2-cyano-2-propyl radical to yield products (the mono- and di-ethers 9-11) derived from the attack on the carbonylic oxygen. The reactions carried out on quinones with the benzoyloxy radical led to no reaction products and in the case of Q10, the isoprenic chain also remained unchanged. Quinols 5-8 reacted only with oxygen-centred radicals (benzoyloxy and 2-cyano-2-propyl-peroxy radicals) to give the corresponding quinones. The isoprenic chain of Q10 did not undergo attack even with peroxy radicals. Carbon-centred radicals resulted unable to abstract hydrogen from the studied quinols.

The activated partial thromboplastin time in early diagnosis of myocardial infarction.

Intracoronary thrombosis is fundamental to the pathogenesis of acute myocardial infarction (MI), yet few studies have examined the diagnostic value of routine coagulability markers, such as the activated partial thromboplastin time (aPTT), in patients with chest pain. We hypothesized that the initiation of thrombosis early in MI would shorten the aPTT, and conducted a retrospective cohort study of patients admitted with a diagnosis of chest pain through the emergency department of one community hospital between 1 January and 30 August 1998. Patients were diagnosed as MI positive or negative based on World Health Organization (WHO) criteria. The aPTT obtained on arrival (prior to anticoagulation therapy) was retrieved from the electronic medical record. Of 120 eligible patients (49% female, mean age 63.7 years), 27 (23%) were diagnosed with MI. Patients with an aPTT control (RR = 2.83, 95% confidence interval 1.15 to 6.96, P = 0.013). A shortened aPTT (

High-output cardiac failure in a patient with prostate cancer.

The authors describe a case of high-output cardiac failure in a patient with rapidly progressing prostate cancer for which no previously described cause could be found. His new onset and increasingly worsening heart failure corresponded to the rapid spread of his prostate cancer. The authors hypothesize that a cytokine released from the neoplastic cells or the bone was responsible for the high-output cardiac failure observed in this patient. (c)2001 CHF, Inc.

The effects of nitroxide radicals on oxidative DNA damage.

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.

Cyto- and genotoxic effects of novel aromatic nitroxide radicals in vitro.

Because of the increasing interest in the use of nitroxide radicals as antioxidants and probes for various applications in biological systems, the question of their toxicity is of paramount importance. Cytotoxicity and mutagenicity studies have been extensively performed with the commercially available aliphatic nitroxides, and the general outcome is that these compounds are nonmutagenic and relatively noncytotoxic. In this study, the cytotoxicity and genotoxicity of a new class of aromatic nitroxides that we have synthesized (i.e., indolinonic and quinolinic nitroxides), whose antioxidant activity has been established in both chemical and biological systems, were evaluated and compared with those of two commercial nitroxides and with that of butylated hydroxytoluene (BHT). The mutagenicity assay was performed using Salmonella typhimurium tester strains TA98, TA100, and TA102, chosen on the basis of their ability to detect various types of mutations and their sensitivity to oxidative damage. None of the compounds tested were found to be mutagenic. The colony-forming assay (CFA) using Chinese hamster ovary (CHO) AS52 cells was employed for determining the cytotoxicity of the test compounds. On comparing the effective dose that inhibits the CFA by 50% (IC(50)), most of the compounds tested on an equal molar concentration basis were less toxic than BHT. Therefore, the overall results obtained correlate well with the data reported in the literature on the toxicity of aliphatic nitroxides and lend support to the possible use of these compounds as therapeutic antioxidants.

Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: protection by nitroxide radicals.

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.

Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals.

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals. Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.

Action of quinolinic and indolinonic aminoxyls as radical-scavenging antioxidants.

The kinetic studies on the actions of quinolinic and indolinonic aminoxyls in the oxidation of lipid peroxidation induced by free radicals were carried out to evaluate their antioxidant activity. These aminoxyls showed a similar reactivity toward peroxyl radical with alpha-tocopherol. The antioxidant efficacies of aminoxyls against oxidation of methyl linoleate in homogeneous solution were smaller than that of alpha-tocopherol. Hydroxylamine, a reduced form of aminoxyl, possessed a comparative antioxidant efficacy with alpha-tocopherol and was capable of suppressing the consumption of alpha-tocopherol. Aminoxyls showed more potent antioxidant activity than alpha-tocopherol against the oxidation of methyl linoleate micelles induced by peroxyl radical or by a combination of copper ion and hydrogen peroxide. These results suggest that quinolinic and indolinonic aminoxyls may act as potent antioxidants against lipid peroxidation, especially in the presence of a good reductant which reduces aminoxyl radicals to hydroxylamines.

Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient.

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.

The effect of indolinic and quinolinic nitroxide radicals on trout erythrocytes exposed to oxidative stress.

The purpose of this study was to evaluate the ability of indolinic and quinolinic nitroxide radicals to protect trout (Salmo irideus) erythrocytes against oxidative stress. By using laurdan as a fluorescence probe, it was observed that the nitroxides inhibited the shift towards a gel phase of liposomes prepared with phospholipids extracted from trout erythrocyte membranes prior to the hemolytic event. In addition, the presence of 100 microM nitroxides in these liposomes protected the latter against lipid peroxidation determined by monitoring conjugated diene formation. However, the short chain analogue of the indolinic nitroxide and the quinolinic nitroxide had a negative effect on trout hemolysis, contrary to what has already been observed in previous studies on human RBCs (red blood cells). The half-time (t1/2) of the hemolytic process was 174 +/- 4.02 min for the former and 184 +/- 4.30 min for the latter compared to the control, 283 +/- 5.05 min. Furthermore, the nitroxides remarkably increased the autoxidation rate of both trout and human hemoglobin to met-Hb. Even though protection at the membrane level is conferred by the nitroxides during the early stages of lipid peroxidation, their antioxidative ability might be overwhelmed at a later stage by other mechanisms such as the increased autoxidation of hemoglobin in the presence of the nitroxides, thus giving a possible explanation for the early induction of hemolysis induced by the nitroxides. The superoxide scavenging ability of all the nitroxides used was also evaluated through chemiluminescence.

Is amniotic fluid analysis the key to preterm labor? A model using interleukin-6 for predicting rapid delivery.

OBJECTIVE: Our purpose was to create a model for predicting amnionitis and rapid delivery in preterm labor patients by use of amniotic fluid interleukin-6 and clinical parameters. STUDY DESIGN: Amniotic fluid was cultured and analyzed, and a clinical score (incorporating gestational age, amniotic fluid Gram stain, glucose, leukocyte esterase, and maternal serum C-reactive protein) was determined in 111 patients diagnosed with preterm labor. Statistical analysis involved t tests, chi2, logarithmic regression, and multivariate regression analysis (P < or = .05). RESULTS: The incidence of positive amniotic fluid cultures was 8.7% (9 of 103 patients). Patients with positive cultures of the amniotic fluid had a shorter delivery interval (4.8 +/- 7.5 vs 28.9 +/- 25.4 days, P < .001). Patients with elevated amniotic fluid interleukin-6 (> or = 7586 pg/ml) were more likely to have a positive amniotic fluid culture (relative risk = 8.8, 95% confidence interval = 1.6 to 47.4, P < .001) and to be delivered within 2 days (relative risk = 16.8, 95% confidence interval = 4.5 to 62.7, P < .001). Stepwise multivariate regression analysis yielded a model using interleukin-6, cervical dilatation, and gestational age (r2 = 0.63, P < .001) with a specificity of 100% for predicting delivery within 2 days of amniocentesis. CONCLUSIONS: A mathematical model using maternal amniotic fluid interleukin-6 seems to be a useful clinical tool for quantifying the interval to preterm birth for patients in preterm labor.

Vitamin E consumption induced by oxidative stress in red blood cells is enhanced by melatonin and reduced by N-acetylserotonin.

The effect of melatonin and its precursor N-acetylserotonin was studied in a model of lipid peroxidation induced in human red blood cells by incubation with cumene hydroperoxide (CHP) and H2O2. The oxidative stress was expressed as vitamin E consumption in the presence of melatonin or N-acetylserotonin (concentration ranging from 0.3 to 400 microM): incubation with melatonin not only lacked any protective effect but it induced a dose-dependent extra vitamin E consumption with both CHP and H2O2. On the contrary, N-acetylserotonin showed a strong antioxidant effect at concentrations between 100 and 400 microM. The hydrogen-donating capacity of melatonin and N-acetylserotonin was also evaluated from the decay of the ESR signal of galvinoxyl radical used as hydrogen abstractor. Lack of hydrogen-donating capacity was observed with melatonin, whereas N-acetylserotonin showed a significant hydrogendonating capacity although inferior to vitamin E, thus suggesting that N-acetylserotonin acts by the classical antioxidant mechanism of hydrogen donation. The measurement of the oxidation potential and the specific molecular structure suggest that the vitamin E consumption effect observed with melatonin could be due to the interactions of its radical cation or derivatives on vitamin E.

Detection of DNA damage in stressed trout nucleated erythrocytes using the comet assay: protection by nitroxide radicals.

Because previous literature reports have demonstrated that nucleated trout erythrocytes in conditions of oxidative stress are subjected to both membrane damage and a decrease in the enzymatic defense systems (glutathione peroxidase), which in turn lead to hemolysis, the present study was undertaken to determine whether DNA may be affected too, prior to the hemolytic event. Impairment of DNA in stressed trout erythrocytes was assessed using the comet assay--a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. In addition, indolinic and quinolinic nitroxide radicals were included in the study to determine their efficacy as antioxidants against free-radical-induced DNA damage. The parameters, tail length, tail intensity, and tail moment, used as an index of DNA damage, have shown that trout erythrocytes exposed to oxidative stress experience DNA damage prior to hemolysis and that the nitroxides significantly prevent this damage. This result provides further information about the potential use of these compounds as antioxidants in biological systems.

Altered expression of alpha-actin, smooth muscle myosin heavy chain-1 and calponin in cultured smooth muscle cells by oxidized low density lipoproteins.

The expression of the contractile proteins, alpha-actin, smooth muscle myosin heavy chain-1 (SM1) and calponin present in smooth muscle cells (SMC) in the presence of oxidized low density lipoproteins (oxLDL) was investigated in two different cell cultures: the mouse smooth muscle cell line SVSC and rat smooth muscle cells (RSMC). Exposure of the cells to 187 microg protein/ml oxLDL for 24 h reduced the expression of all three contractile proteins in both cell cultures when compared to cells incubated in the presence of native LDL. This investigation of the response of SMC contractile proteins to oxLDL may provide further insights into the mechanisms by which oxidatively modified LDL is atherogenic and suggests that oxLDL may contribute to the regulation of the expression of the genes responsible for the synthesis of smooth muscle cell contractile proteins.

Effect of aromatic nitroxides on hemolysis of human erythrocytes entrapped with isolated hemoglobin chains.

An in vitro model of thalassemia was produced by entrapment of isolated hemoglobin chains in human erythrocytes, thus subjecting the loaded cells to oxidative stress. The presence of these unpaired chains induced physico-chemical modifications at the membrane level as studied by laurdan fluorescence. The polarity of the lipid bilayer was shown to decrease with a concomitant shift towards a gel phase in alpha-loaded erythrocytes. The determination of conjugated dienes before the hemolytic event was used as an oxidation index; the results obtained demonstrate that beta thalassemia is associated with oxidative stress. Furthermore, the presence of indolinic and quinolinic nitroxide radicals, a new class of antioxidants, in suspensions of alpha-loaded erythrocytes protected the erythrocytes from the hemolytic event. However, the protective effect exerted by the nitroxide radicals is not related to effects on membrane polarity and lipid peroxidation, even though a chemiluminescence study has demonstrated the superoxide scavenging activity of these nitroxide radicals.

Influence of structure on the antioxidant activity of indolinic nitroxide radicals.

An in vitro study was carried out to verify whether the chain length of a substituent on an indolinic nitroxide could influence its antioxidant activity in different biological environments subjected to oxidative stress. Three distinct indolinic nitroxides were synthesized and compared with vitamin E and Trolox (a hydrophilic analogue of vitamin E), where the only difference between the nitroxides was the length of the hydrocarbon chain in the 2-position of indole, namely 2 (C2), 10 (C10), and 18 (C18) carbons. All the nitroxides were effective in preventing oxidation of bovine serum albumin, but to different extents, with the longer chain derivatives being more efficient. However, the C2 compound was the most efficient in preventing lipid peroxidation in microsomal membranes. The C2 and C18 compounds, Trolox, and vitamin E protected microsomal protein oxidation to the same extent at the highest concentration used (13 microM). The nitroxide with a C10 chain was less effective in this system. The influence of these compounds on the enzymatic activity of two mitochondrial proteins subjected to oxidative stress was also studied by means of oxygraph measurements. Mitochondrial rotenone-sensitive NADH oxidase and succinate oxidase responded differently to BuOOH-induced radical chemistry, and the compounds under study also protected the activity of the two enzymes but to different extents. The results clearly demonstrate that indolinic nitroxides are very efficient antioxidants, protecting both lipids and proteins from peroxidation. The indole structure influences the antioxidant efficacy in biological systems.

Protective effect of 4-hydroxy-TEMPO, a low molecular weight superoxide dismutase mimic, on free radical toxicity in experimental pancreatitis.

Rats develop acute pancreatitis when infused iv for 3 h with cerulein (10 micrograms/kg/h). Autopsies of the pancreas seen by light microscope show interstitial edema, acinar cells vacuolization, and leukocyte margination in pancreatic capillaries; under electron microscope, severe damage concerning mitochondrial and zymogen granules structures are apparent. Particularly, swelling of the mitochondria and disruption of mitochondrial cristae was observed as well as formation of large vacuoles arising from zymogen granules and liposome fusion. A significant increase of lipid hydroperoxide level in the pancreatic tissue was observed. The purpose of this study was to evaluate the effect of 4-hydroxy-TEMPO--a low-mol-wt superoxide dismutase mimic--in a rat cerulein model of acute pancreatitis, with the expectation that free radical mediated hydroperoxide formation and tissue damage may be reduced significantly. Twenty-one male Wistar rats were divided into three groups: Group 1 (n = 5) served as a control and was infused iv for 3 h with physiologic saline; Group 2 (n = 8) was infused i.v. for 3 h with cerulein 10 micrograms/kg/h; and Group 3 (n = 8) infused i.v. both with cerulein and 4-hydroxy-TEMPO 22.6 mg/kg/h. Pancreatic tissue damage was quantified by measuring lipid hydroperoxide (LOOH) level, the weight of the organ, and by light and electron microscopic examination. 4-hydroxy-TEMPO penetration across cellular membrane barriers was quantified by ESR spectrometric measurements of 4-hydroxy-TEMPO concentration in pancreatic tissue samples and pancreatic juice as well. Administering 4-hydroxy-TEMPO to rats resulted in preventing both lipid hydroperoxide formation and severe morphological damage. 4-hydroxy-TEMPO crossed cellular membrane barriers and was excreted to pancreatic juice. Infusion of 4-hydroxy-TEMPO appears to prevent pancreatic injury caused by free radicals in experimental cerulein pancreatitis.