Nitrosation by peroxynitrite: use of phenol as a probe.

Nitrosation is an important pathway in the metabolism of nitric oxide, producing S-nitrosothiols that may be critical signal transduction species. The reaction of peroxynitrite with aromatic compounds in the pH range of 5 to 8 has long been known to produce hydroxylated and nitrated products. However, we here present evidence that peroxynitrite also can promote the nitrosation of nucleophiles. We chose phenol as a substrate because the nitrosation reaction was first recognized during a study of the CO2-modulation of the patterns of hydroxylation and nitration of phenol by peroxynitrite (Lemercier et al., Arch. Biochem. Biophys. 345, 160-170, 1997). 4-Nitrosophenol, the principal nitrosation product, is detected at pH 7.0, along with 2- and 4-nitrophenols; 4-nitrosophenol becomes the dominant product at pH >/= 8.0. The yield of 4-nitrosophenol continues to increase even after pH 11.1, 1. 2 units above the pKa of phenol, suggesting that the phenolate ion, and not phenol, is involved in the reaction. Hydrogen peroxide is not formed as a by-product. The nitrosation reaction is zero-order in phenol and first-order in peroxynitrite, suggesting the phenolate ion reacts with an activated nitrosating species derived from peroxynitrite, and not with peroxynitrite itself. Under optimal conditions, the yields of 4-nitrosophenol are comparable to those of 2- and 4-nitrophenols, indicating that the nitrosation reaction is as significant as the nitration of phenolic compounds by peroxynitrite. Low concentrations of CO2 facilitate the nitrosation reaction, but excess CO2 dramatically reduces the yield of 4-nitrosophenol. The dual effects of CO2 can be rationalized if O=N-OO- reacts with the peroxynitrite anion-CO2 adduct (O=N-OOCO-2) or secondary intermediates derived from it, including the nitrocarbonate anion (O2N-OCO-2), the carbonate radical (CO*-3), and *NO2. The product resulting from these reactions can be envisioned as an activated intermediate X-N=O (where X is -OONO2, -NO2, or -CO-3) that could transfer a nitrosyl cation (NO+) to the phenolate ion. An alternative mechanism for the nitrosation of phenol involves the one-electron oxidation of the phenolate ion by CO*-3 to give the phenoxyl radical and the oxidation of O=N-OO- by CO*-3 to give a nitrosyldioxyl radical (O=N-OO*), which decomposes to give *NO and O2; the *NO then reacts with the phenoxyl radical giving nitrosophenol. Both mechanisms are consistent with the high yields of NO-2 and O2 during the alkaline decomposition of peroxynitrite and the potent inhibitory effect of N-3 on the nitrosation of phenol by peroxynitrite and peroxynitrite/CO2 adducts. The biological significance of the peroxynitrite-mediated nitrosations is discussed.

Carbon dioxide modulation of hydroxylation and nitration of phenol by peroxynitrite.

We have examined the formation of hydroxyphenols, nitrophenols, and the minor products 4-nitrosophenol, benzoquinone, 2,2'-biphenol, and 4,4'-biphenol from the reaction of peroxynitrite with phenol in the presence and absence of added carbonate. In the absence of added carbonate, the product yields of nitrophenols and hydroxyphenols have different pH profiles. The rates of nitration and hydroxylation also have different pH profiles and match the trends observed for the product yields. At a given pH, the sum of the rate constants for nitration and hydroxylation is nearly identical to the rate constant for the spontaneous decomposition of peroxynitrite. The reaction of peroxynitrite with phenol is zero-order in phenol, both in the presence and absence of added carbonate. In the presence of added carbonate, hydroxylation is inhibited, whereas the rate of formation and yield of nitrophenols increase. The combined maximum yield of o- and p-nitrophenols is 20 mol% (based on the initial concentration of peroxynitrite) and is about fourfold higher than the maximal yield obtained in the absence of added carbonate. The o/p ratio of nitrophenols is the same in the presence and absence of added carbonate. These results demonstrate that hydroxylation and nitration occur via two different intermediates. We suggest that the activated intermediate formed in the isomerization of peroxynitrous acid to nitrate, ONOOH*, is the hydroxylating species. We propose that intermediate 1, O=N-OO-CO2-, or secondary products derived from it, is (are) responsible for the nitration of phenol. The possible mechanisms responsible for nitration are discussed.

Inhibition of peroxynitrite-mediated oxidation of glutathione by carbon dioxide.

Peroxynitrite reacts with CO2 to from an adduct containing a weak O--O bond that can undergo homolytic and/or heterolytic cleavage to give other reactive intermediates. Because the peroxynitrite/CO2 reaction is fast and physiological concentrations of CO2 are relatively high, peroxynitrite-mediated oxidations of biological species probably involve the peroxynitrite-CO2 adduct and its subsequent reactive intermediates. We have examined the reaction of glutathione with peroxynitrite in the presence and absence of added bicarbonate. In the presence of added bicarbonate, CO2 competes with glutathione for peroxynitrite, resulting in a markedly decreased consumption of glutathione compared with that observed in the absence of added bicarbonate. However, the consumption of glutathione still is much higher than predicted from the assumption that the glutathione-peroxynitrite reaction is the only reaction that can consume glutathione in this system. These results suggest that glutathione partially, but not completely, traps intermediate(s) derived from the peroxynitrite and CO2 reaction. Some rate constants for the trapping of the intermediates are estimated by simulating the reactions, and possible mechanisms for the reaction of peroxynitrite with glutathione in the presence of added bicarbonate are discussed.

The catalytic role of carbon dioxide in the decomposition of peroxynitrite.

The fast reaction of peroxynitrite with CO2 and the high concentration of dissolved CO2 in vivo (ca. 1 mM) suggest that CO2 modulates most of the reactions of peroxynitrite in biological systems. The addition of peroxynitrite to CO2 produces of the adduct ONOO-CO2- (1). The production of 1 greatly accelerates the decomposition of peroxynitrite to give nitrate. We now show that the formation of 1 is followed by reformation of CO2 (rather than another carbonate species such as CO3 = or HCO3-). To show this, it is necessary to study systems with limiting concentrations of CO2. (When CO2 is present in excess, its concentration remains nearly constant during the decomposition of peroxynitrite, and the recycling of CO2, although it occurs, can not be detected kinetically). We find that CO2 is a true catalyst of the decomposition of peroxynitrite, and this fundamental insight into its action must be rationalized by any in vivo or in vitro reaction mechanism that is proposed. When the concentration of CO2 is lower than that of peroxynitrite, the reformation of CO2 amplifies the fraction of peroxynitrite that reacts with CO2. Even low concentrations of CO2 that result from the dissolution of ambient CO2 can have pronounced catalytic effects. These effects can cause deviations from predicted kinetic behavior in studies of peroxynitrite in noncarbonate buffers in vitro, and since 1 and other intermediates derived from it are oxidants and/or nitrating agents, some of the reactions attributed to peroxynitrite may depend on the availability of CO2.

Peroxynitrite-mediated oxidation of D,L-selenomethionine: kinetics, mechanism and the role of carbon dioxide.

Peroxynitrite oxidizes D,L-selenomethionine (MetSe) by two competing mechanisms, a one-electron oxidation that leads to ethylene and a two-electron oxidation that gives methionine selenoxide (MetSeO). Kinetic modeling of the experimental data suggests that both peroxynitrous acid and the peroxynitrite anion react with MetSe to form MetSeO with rate constants of 20,460 +/- 440 M-1 s-1 and 200 +/- 170 M-1 s-1, respectively at 25 degrees C. The enthalpy (delta H++) and entropy (delta S++) of activation for the reaction of peroxynitrous acid with MetSe at pH 4.6 are 2.55 +/- 0.08 kcal mol-1 and -30.5 +/- 0.3 cal mol-1 K-1, respectively. With increasing concentrations of MetSe at pH 7.4, the yield of ethylene decreases and that of MetSeO increases, suggesting, as with methionine, the reactions leading to ethylene and MetSeO have different kinetic orders. We propose that the activated form of peroxynitrous acid, HOONO*, is the one-electron oxidant and ground-state peroxynitrite is the two-electron oxidant in the reaction of peroxynitrite with MetSe. The peroxynitrite anion rapidly adds to CO2 to form an adduct, O = N-OO-CO2- (1), capable of generating potent reactive species, and we therefore examined the role of CO2 in the peroxynitrite/MetSe system. In presence of added bicarbonate, the yield of ethylene obtained from the reaction of 0.4 mM peroxynitrite with 1.0 mM MetSe increases slightly with an increase in the concentration of bicarbonate from 0 to 5.0 mM and remains constant with a further increase of bicarbonate up to 20 mM. The yield of MetSeO, from the reaction of 10 mM peroxynitrite with 10 mM MetSe, decreases by 35% with an increase in the concentration of bicarbonate from 0 to 25 mM. Kinetic simulations show that the decrease in the yield of MetSeO is due to reaction of the peroxynitrite anion with CO2. These results suggest that CO2 partially protects MetSe from peroxynitrite-mediated oxidation and that 1 or its derivatives do not mediate the oxidation of MetSe to MetSeO.

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.

Spin trap studies on the decomposition of peroxynitrite.

The observation of the hydroxyl radical spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) is evaluated as a probe for the production of hydroxyl radicals from the decomposition of peroxynitrite. Although a weak signal corresponding to the DMPO-hydroxyl radical spin adduct (.DMPO-OH) is observed when peroxynitrite is allowed to decompose in the presence of DMPO, it is concluded that this does not constitute proof of the presence of free hydroxyl radicals. The observed rate constant for the decay of peroxynitrite increases from 0.35 to 0.51 s-1 (46% increase) when the concentration of DMPO is increased from 0 to 75 mM. This strongly suggests there is a reaction between DMPO and HOONO, or between DMPO and an activated intermediate of HOONO, to produce the hydroxyl radical spin adduct. The addition of glutathione or cysteine produces a large increase in the intensity of the .DMPO-OH spin adduct signal; experiments employing superoxide dismutase suggest that the increases in the amounts of .DMPO-OH adduct are produced from the decomposition of the spin adduct of the superoxide radical (.DMPO-OOH). The superoxide adduct arises as a result of the autoxidation of thiols, a process known to produce superoxide. The results presented here are incompatible with the formation of free hydroxyl radicals but can be explained in terms of an intermediate of HOONO that is less reactive and more selective than the free hydroxyl radical.

[Peripheral bronchopulmonary cancers. Diagnostic efficiency of fibroscopic samples. Prospective study on 561 patients]. / Cancers bronchopulmonaires périphériques. Rendement diagnostique des prélèvements fibroscopiques. Etude prospective sur 561 malades.

The purpose of this french multicentric study was to evaluate the diagnostic accuracy of samples collected by fibroscopy in peripheral lung cancer with normal endoscopy. Five hundred and sixty-one patients entered the study; a tumour had been diagnosed in 350 of them (62 p. 100). Among these 350 patients, 147 were examined with the help of a light-amplifier screen, and a positive diagnosis was made in 97 (66 p. 100). Guided sample collection in the pathological area (biopsy, brushing, transcatheter aspiration) proved much more rewarding than unguided sample collection (biopsy of the bronchus, simple aspiration or cytology of 3-day sputum): 45 p. 100 positive results versus 18 p. 100 (P less than 0.001). However, sputum cytology still had good diagnostic value since it provided by itself the diagnosis in 14 p. 100 of the cases. Results were significantly inferior in tumours less than 3 cm in diameter.

[Bronchial resection-anastomosis for cicatricial stenosis. Physiopathologic and therapeutic considerations]. / Résection-anastomose bronchique pour sténose cicatritielle. Considérations physiopathologiques et thérapeutiques.

The exploration of a stenosis of an intermediate bronchus in a 50 year-old woman showed it to be severe, limited, and non-malignant. Pre-operative angiography showed hypovascularization of the right lower and middle pulmonary lobes. A short resection of the stenosis, with termino-terminal bronchial anastomosis was performed. Post-operative evaluation showed normal bronchography and angiography. This suggests that bronchial stenosis plays a role in the hypovascularization observed before its surgical correction, the most likely hypothesis being vasoconstriction.

[Testicular function during prolonged corticotherapy]. / Fonction testiculaire au cours de la corticothérapie prolongée.

Prolonged corticosteroid therapy produces a major degree of protein catabolism. Short-term treatment with ACTH or glucocorticoids results, in males, in a fall in plasma testosterone levels. In order to determine the origin of this hypogonadism, the testicular function was investigated in 18 men under prolonged glucocorticoid treatment. The mean plasma testosterone level in men treated for less than 3 months (5.9 +/- 0.8 ng/ml) was not statistically different from that obtained in controls (7.5 +/- 0.5 ng/ml; P greater than 0.05). However, the mean plasma luteinizing hormone level was significantly higher in these patients than in controls (8.2 +/- 1.9 mUl/ml vs 3.6 +/- mUl/ml respectively; P less than 0.001). In patients treated for more than 2 years, the mean plasma testosterone level (3.8 +/- 0.6 ng/ml) was significantly lower than in controls (P less than 0.001), but the mean plasma luteinizing hormone level was normal (5 +/- 1 mUl/ml). Plasma testosterone levels are negatively correlated with the daily dose of glucocorticoid (r = -0.81; P less than 0.001). The free testosterone index was within normal range in 5 of the 9 men under prolonged steroid therapy, near the lower limit of normality in 2 men and definitely low in 2 other men. The gonadotrophin response to the luteinizing hormone releasing hormone was normal, whereas the testicular response to human chorionic gonadotrophin was reduced. These data suggest that prolonged treatment with steroidal anti-inflammatory agents influences both gonadotrophic and testicular function and may be associated with true hypoandrogenism.

Nifedipine in chronic bronchial asthma: a randomized double-blind crossover trial against placebo.

Calcium-channel blockers such as verapamil and nifedipine have been shown to inhibit exercise-induced asthma as well as acutely induced bronchoconstriction, but little is known of their chronic effects, if any, on bronchial asthma. Nifedipine, 60 mg/day for 3 weeks, was compared to placebo in a double-blind randomized crossover study, as an addition to the usual treatment of 11 patients with severe chronic bronchial asthma. Nifedipine decreased the weekly duration of the attacks (102 +/- 34 vs 193 +/- 49 min, p less than 0.05), the number of betamimetic puffs inhaled per week (13 +/- 3 vs 18 +/- 4, p less than 0.05), and the duration of intercritical dyspnoea (7.9 +/- 3.9 vs 15.9 +/- 2.3 h, p less than 0.05) without significantly changing the number of asthma attacks (4.8 +/- 1.2 vs 4.7 +/- 1.7, NS). Nifedipine did not significantly change basal respiratory function, nor did it change heart rate or blood pressure. Side effects were noted in 5 patients taking nifedipine, leading to a decrease in the dosage to 30 mg per day in 3, and in one patient taking placebo. In this study nifedipine had essentially subjective effects, but these warrant a longer-term study of nifedipine or other calcium antagonists in the treatment of bronchial asthma.

[Meteorological conditions and spontaneous pneumothorax. Retrospective study of 165 cases in the Rouen area]. / Conditions météorologiques et pneumothorax spontanés. Etude rétrospective sur 165 cas de la région rouennaise.

The occurrence of a spontaneous "idiopathic" pneumothorax in 165 patients admitted to hospital could be precisely dated over a 5-year period. An analysis of the meteorological conditions recorded at these dates (mean atmospheric pressure, mean temperature, relative and absolute humidity) and of their seasonal distribution demonstrated two peaks of frequency, one in the summer, the other in the winter, and a significant correlation between the occurrence of pneumothorax and a low relative humidity. These results, particularly clear during the exceptionally dry summer of 1976, confirm some data in the literature and suggest that bronchoconstriction induced by moistening of air in the airways might play a role in the pathophysiology of pneumothorax.

[Study of adult morbidity and mortality from pneumococcus in hospital (author's transl)]. / Etude de la morbidité et de la mortalité par pneumococcie, chez l'adulte, en milieu hospitalier.

The authors report the retrospective study of 333 cases of pneumococcal infection recorded in in-patients at the Rouen CHU (University Hospital Centre) from january 1974 to december 1978. Pneumococcal infection involves mainly the lungs (210 cases) according to different features. Pleural involvement is found in 70 cases only. Extra-respiratory localizations are less frequently encountered; they are represented mainly by meningitis (43 cases). Prognosis depends more on diathesis factors such as age and associated pathology than on the site of infection (cases with isolated respiratory involvement being usually less severe). The authors insist on the necessity of prevention especially by vaccination, of which indication is discussed.

[Respiratory function tests in epidermoid cancers of the bronchi]. / A propos de l'exploration fonctionnelle respiratoire au cours du cancer épidermoïde des bronches

The results of functional tests are analyzed in 50 cases of epidermoid bronchial cancers. Severe disorders take place in almost every patient. In one patient out of two, there is an old distension already existing prior to the cancer; in other patients gas exchange disorders can be improved by the intervention. Moreover, from some results a spreading of cancer towards the mediastinum is to be feared.