Rafael Cueto

Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter.

The health effects of airborne fine particles are the subject of government regulation and scientific debate. The aerodynamics of airborne particulate matter, the deposition patterns in the human lung, and the available experimental and epidemiological data on health effects lead us to focus on airborne particulate matter with an aerodynamic mean diameter less than 2.5 microm (PM(2.5)) as the fraction of the particles with the largest impact in health. In this article we present a novel hypothesis to explain the continuous production of reactive oxygen species produced by PM(2.5) when it is deposited in the lung. We find PM(2.5) contains abundant persistent free radicals, typically 10(16) to 10(17) unpaired spins/gram, and that these radicals are stable for several months. These radicals are consistent with the stability and electron paramagnetic resonance spectral characteristics of semiquinone radicals. Catalytic redox cycling by semiquinone radicals is well documented in the literature and we had studied in detail its role on the health effects of cigarette smoke particulate matter. We believe that we have for the first time shown that the same, or similar radicals, are not confined to cigarette smoke particulate matter but are also present in PM(2.5). We hypothesize that these semiquinone radicals undergo redox cycling, thereby reducing oxygen and generating reactive oxygen species while consuming tissue-reducing equivalents, such as NAD(P)H and ascorbate. These reactive oxygen species generated by particles cause oxidative stress at sites of deposition and produce deleterious effects observed in the lung.

A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide.

The reaction of ozone (approximately 5% in oxygen) with sodium azide (0.02-0.2 M in water) at pH 12 and 0-4 degrees C is shown to yield concentrated, stable peroxynitrite solutions of up to 80 mM. The product of this reaction is identified based on a broad absorption spectrum with a maximum around 302 nm and by its first-order rate of decomposition (k = 0.40 +/- 0.01 s-1 at pH 7.05 and 25 degrees C). These peroxynitrite solutions can be obtained essentially free of hydrogen peroxide (detection limit 1 microM) and only traces of azide (detection limit 0.1 mM). They are low in ionic strength and have a pH of about 12 but without buffering capacity; therefore, they can be adjusted to any pH by addition of buffer. These preparations of peroxynitrite frozen at -20 degrees C show negligible decomposition for about 3 weeks of storage and follow a first-order decomposition with a halflife of about 7 days at refrigerator temperatures (approximately 5 degrees C). These preparations give reactions that are characteristic of peroxynitrite. For example, at pH 7.0, they react with L-tyrosine to give a 7.3 mol % yield of nitrotyrosine(s), and with dimethyl sulfoxide to give a 8.2 mol % yield of formaldehyde, based on starting peroxynitrite concentration.

Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells

Human MutT homologue (hMTH1) mRNA was overexpressed in SV‐40‐transformed non‐tumorigenic human bronchial epithelial cells (BEAS‐2B cells) and in 11 out of 12 human lung cancer cell lines relative to normal human bronchial epithelial cells. Expression levels of hMTH1 mRNA were inversely proportional to cellular levels of 8‐oxo‐deoxyguanosine. Together, these results suggest that hMTH1 gene expression may represent a molecular marker of oxidative stress that could ultimately be used to elucidate the temporal relationships between oxidative stress, genomic instability and the development of lung cancer.

RAPID OXIDATION OF DL-SELENOMETHIONINE BY PEROXYNITRITE

Peroxynitrite, the reaction product of nitric oxide and superoxide, rapidly oxidizes DL-selenomethionine (MetSe) with overall second-order kinetics, first-order in peroxynitrite and first-order in MetSe. The oxidation of MetSe by peroxynitrite goes by two competing mechanism. The first produces ethylene by what we propose to be a one-electron oxidation of MetSe. In the second mechanism, MetSe undergoes a two-electron oxidation that gives methionine selenoxide (MetSe = O); the apparent second-order rate constant, k2(app), for this process is (2.4 +/- 0.1) x 10(3) M-1s-1 at pH 7.4 and 25 degrees C. The kinetic modeling of the experimental data suggests that both peroxynitrous acid (k2 = 20,460 +/- 440 M-1s-1 at 25 degrees C) and the peroxynitrite anion (k3 = 200 +/- 170 M-1s-1 at 25 degrees C) are involved in the second-order reaction leading to selenoxide. These rate constants are 10- to 1,000-fold higher than those for the reactions of methionine (Met) with peroxynitrite. With increasing concentrations of MetSe at pH 7.4, the yield of ethylene decreases, while that of MetSe = O increases, suggesting that the reactions leading to ethylene and selenoxide have different kinetic orders. These results are analogous to those we previously reported for methionine and 2-keto-4-thiomethylbutanoic acid (KTBA),where ethylene is produced in a first-order reaction and sulfoxide in a second-order reaction. Therefore, we suggest that the reaction of peryoxynitrite with MetSe involves a mechanism similar to that we proposed for Met, in which an activated intermediate of peroxynitrous acid (HOONO) is the one-electron oxidant and reacts with first-order kinetics and ground-state peroxynitrite is the two-electron oxidant and reacts with second-order kinetics.

Cigarette smoke chemistry: conversion of nitric oxide to nitrogen dioxide and reactions of nitrogen oxides with other smoke components as studied by Fourier transform infrared spectroscopy

Abstract Nitric oxide is both a critical biological toxin and an important messenger molecule, signalling events as diverse as nerve transmission and smooth muscle relaxation. Fresh cigarette smoke contains from 300 to 500 ppm nitric oxide. While there have been reports of the rate of oxidation of nitric oxide to nitrogen dioxide in cigarette smoke, none have utilized a real-time method and provided detailed kinetic data and modelling. In this paper we present a Fourier transform infrared spectroscopy (FT-IR) method for the simultaneous determination of nitric oxide and nitrogen dioxide in gas phase cigarette smoke and in a number of gaseous mixtures that model smoke. The method uses multivariate least-squares regression analysis, which allows simultaneous quantitation of several components even in the presence of overlapping peaks, and fast data acquisition for kinetic analysis. Model systems containing mixtures of nitric oxide and isoprene, methanol, and/or acrolein and acetaldehyde in air were studied. The concentrations of nitric oxide, isoprene, etc., in the model systems were chosen to duplicate those in authentic cigarette smoke. In our best model (a mixture of nitric oxide, methanol, and isoprene in air) the disappearance of nitric oxide and the appearance of nitrogen dioxide follow time courses that closely duplicate those for cigarette smoke. Furthermore, the production of nitrogen dioxide follows a time course that agrees with our previously published rate of the development of organic free radicals in cigarette smoke. The present work, therefore, substantiates the steady-state mechanism we previously proposed for the production of free radicals in gas phase cigarette smoke.

Synthesis and Rapid Characterization of Amine-Functionalized Silica

Amine-functionalized colloidal silica finds use in a variety of applications and fundamental investigations. To explore convenient methods of synthesis and characterization of research-grade materials in relatively large quantities, nearly monodisperse colloidal silica particles were prepared by base-catalyzed hydrolysis of reagent-grade tetraethyl orthosilicate (TEOS) without the traditional time- and energy-consuming distillation step. Radius was varied reliably from 30 to 125 nm by changing the water/TEOS ratio. Asymmetric flow field flow fractionation (AF4) methods with online light scattering detection proved effective in assessing the uniformity of the various preparations. Even highly uniform commercial standards were resolved by AF4. The surface of the colloidal silica was decorated with amino groups using (3-aminopropyl) trimethoxysilane and spacer methyl groups from methyl-trimethoxysilane. The surface density of amino groups was quantified spectrophotometrically after reaction with ninhydrin; the nature of this analysis avoids interference from sample turbidity. As an alternative to the ninhydrin test, an empirical relationship between surface density of amino groups and zeta potential at low pH was found. The size of the colloidal silica was predictably decreased by etching with HF; this method will be effective for some preparations, despite a modest reduction in size uniformity.

SYNTHESIS OF PEROXYNITRITE BY AZIDE-OZONE REACTION

Publisher Summary This chapter discusses the synthesis of peroxynitrite by azide-ozone reaction. A simple method for preparing stable, concentrated solutions of peroxynitrite that are low in ionic strength, low in alkali, and free of H 2 O 2 . The method is based on the reaction of ozone with azide ions in water at pH 12. There are advantages to the use of these peroxynitrite solutions in chemical investigations, especially where alkali and H 2 O 2 contamination pose a problem. These solutions also are useful in biological studies, if the contamination by the unreacted residual azide is kept to a minimum. A simplification and improvement of the original azide–ozone method is described by using a Sander model 200 ozonator. The Sander ozonator is designed for use with aquariums, and is readily available, inexpensive, and easily used in any laboratory setup. It is possible to prepare peroxynitrite solutions containing an unreacted residual azide of ≤5μM. The residual azide in these peroxynitrite solutions does not interfere with the assay of several hemoprotein and nonhemoprotein enzymes.

SELECTING THE MOST APPROPRIATE SYNTHESIS OF PEROXYNITRITE

Publisher Summary This chapter discusses various methods and the selection criteria used for the synthesis of Peroxynitrite. Ozonation of azide ions is performed at 0–4° in a mildly alkaline solution. This method gives peroxynitrite solutions up to 80 mM that are low in alkali and free of H 2 O 2 , however may contain some unreacted azide. These peroxynitrite preparations are used in several chemical and biochemical reactions, including those mediated by nonhemoprotein and hemoprotein enzymes. The autooxidation reactions of hydroxylamine lead to the formation of peroxynitrite. The reaction of hydrogen peroxide with nitrous acid involves a rapid reaction of an acidified solution of H 2 O 2 with a solution of sodium nitrite followed by stabilization of the product, peroxynitrous acid, with strong alkali. The reaction of alkaline H 2 O 2 with an alkyl nitrite (RONO) can also produce peroxynitrite. Among the various solution-based methods, peroxynitrite prepared by the azide–ozone reaction contains low levels of alkali. All preparations of peroxynitrite contain nitrite and nitrate to a greater or lesser degree. Nitrite is produced when peroxynitrite undergoes decomposition in a transition metal ion-assisted reaction, and nitrate is produced in the acid-catalyzed decomposition of peroxynitrite.

O3-induced formation of bioactive lipids : estimated surface concentrations and lining layer effects

Recent evidence suggests that inhaled ozone (O3) does not induce toxicity via direct epithelial interactions. Reactions with epithelial lining fluid (ELF) constituents limit cellular contact and generate products, including lipid ozonation products, postulated to initiate pathophysiological cascades. To delineate specific aspects of lipid ozonation product formation and to estimate in situ surface concentrations, we studied the O3absorption characteristics of ELF constituent mixtures and measured hexanal, heptanal, and nonanal yields as a function of ascorbic acid (AH2) concentration. Exposures of isolated rat lungs, bronchoalveolar lavage fluid (BALF) and egg phosphatidylcholine (PC) liposomes were conducted. 1) O3 absorption by AH2, uric acid, and albumin exceeded that by egg PC and glutathione. O3 reaction with egg PC occurred when AH2 concentrations were reduced. 2) Aldehydes were produced in low yield during lung and BALF exposures in a time- and O3 concentration-dependent manner. 3) Diminishing BALF AH2 content lowered O3 uptake but increased aldehyde yields. Conversely, AH2 addition to egg PC increased O3 uptake but reduced aldehyde yields. Estimations of bioactive ozonation and autoxidation product accumulation within the ELF suggested possible nanomolar to low micromolar concentrations. The use of reaction products as metrics of O3 exposure may have intrinsic sensitivity and specificity limitations. Moreover, due to the heterogenous nature of O3 reactions within the ELF, dose-response relationships may not be linear with respect to O3 absorption.

Identification of heptanal and nonanal in bronchoalveolar lavage from rats exposed to low levels of ozone

Heptanal and nonanal are identified from in vitro studies as potential biomarkers of exposure to ozone, the former resulting from ozonation of palmitoleic acid and the latter from oleic acid. An analytical method is developed based on derivatization using O-pentafluorobenzylhydroxylamine HCl and gas chromatography. These molecules also are present in the lung lavage of Sprague-Dawley rats exposed to 1.3 ppm ozone for 10 hr. These results suggest aldehydes may be useful dosimeters for ozone and indicate that unsaturated fatty acids in the lung lining fluid layer undergo ozonation in vivo.