Luigi Atzori

Sulfur Dioxide-Induced Bronchoconstriction in the Isolated Perfused and Ventilated Guinea-Pig Lung

SO2 exposure (50-500 ppm) of isolated, perfused and ventilated guinea pig lungs, via the air passages, caused a concentration-related reduction in dynamic compliance and conductance. No changes in pulmonary perfusion flow was noted at any SO2 concentration. Formed sulfite was detected in lung lavage fluid as well as in the perfusate. Pretreatment of the lungs with a low concentration of SO2 (10 ppm) for 30 min protected against bronchoconstriction by a high concentration of SO2 (250 ppm). A similar protective effect was noted by pretreatment with sodium sulfite (3 mM) in the lung perfusate.

The mechanism of Hg2+ toxicity in cultured human oral fibroblasts: the involvement of cellular thiols.

To study amalgam-related toxicity in a primary target cell type, human oral fibroblasts were grown in a low-serum medium containing 1.25% fetal bovine serum and exposed to Hg2+, a corrosion product of amalgam. A 1-h exposure to various concentrations of Hg2+ resulted in a dose-dependent loss of colony forming efficiency. Removal of the low-molecular-weight thiol cysteine from the medium increased the toxicity of Hg2+ almost 50-fold in comparison with complete medium or medium without fetal bovine serum. Accordingly, fetal bovine serum was not found to contain detectable levels of low-molecular-weight thiols. The levels of cellular free protein thiols were shown to be depleted Hg2+ at significantly lower concentrations of the metal ion than those required to decrease the levels of the major cellular low-molecular weight thiol glutathione. These decreases were dependent on the exposure conditions, i.e. the presence of serum and thiols, in a manner similar to the effect on colony forming efficiency. Other functions commonly related to cell viability, including the accumulation of the vital dye neutral red, the cytosolic retention of deoxyglucose and the mitochondrial reduction of tetrazolium were also inhibited by Hg2+, albeit at higher concentrations. Finally, the depletion of cellular glutathione, by pre-exposure of the cells to the glutathione synthesis inhibitor buthionine sulfoximine, somewhat increased the toxicity of Hg2+ and potentiated the depletion of protein thiols. Taken together, the toxicity of Hg2+ in human oral fibroblasts was demonstrated in several assays of which colony forming efficiency was the most sensitive, cell killing by this agent was related to its high affinity for protein thiols, whereas glutathione showed a significant, but limited, ability to protect the cells from Hg2+ toxicity.

Sulfur Dioxide-Induced Bronchoconstriction via Ruthenium Red-Sensitive Activation of Sensory Nerves

The mechanism of sulfur dioxide-induced bronchoconstriction was studied using isolated perfused and ventilated guinea-pig lungs. They were exposed to sulfur dioxide after pretreatment with different compounds, either via the pulmonary artery or via the air passages. Neither the cyclooxygenase inhibitor indomethacin (30 microM) nor the H1-receptor antagonist diphenhydramine (15 microM), given via the perfusate, attenuated the sulfur dioxide-induced bronchoconstriction. Furthermore, sulfur dioxide exposure did not cause a release of either thromboxane or histamine into the perfusate. In experiments with atropine equivocal results were obtained with regard to protection against sulfur dioxide-evoked bronchoconstriction. Intratracheal instillation of the local anesthetic agent lidocaine (1 mg/50 microliters) markedly reduced the sulfur dioxide-induced bronchoconstriction. Also, ruthenium red (10 microM), an agent with calcium entry-blocking properties and an inhibitor of capsaicin-induced bronchoconstriction, was able to inhibit the effect of sulfur dioxide. The sulfur dioxide-induced bronchoconstriction was associated with release of calcitonin gene-related peptide, a sensory neuropeptide. The effect of sulfur dioxide was also inhibited by a Ca(2+)-free buffer plus EGTA. These results suggest that sulfur dioxide-induced bronchoconstriction in the guinea-pig lung is the result of a local effect on sensory nerves (C-fiber activation). The mechanism seems to be dependent on the Ca(2+)-dependent release of sensory neuropeptides and to be linked to opening of the cation channel, which is associated with the proposed capsaicin receptor on sensory nerves as revealed by the inhibitory effect of ruthenium red.

Sulfur Dioxide and Sodium Metabisulfite Induce Bronchoconstriction in the Isolated Perfused and Ventilated Guinea Pig Lung via Stimulation of Capsaicin-Sensitive Sensory Nerves

In this study the relationship between sulfur dioxide-induced sensory nerve activation and acute bronchoconstriction was assessed. We also studied the effects of sodium metabisulfite, an agent that is suggested to increase airway resistance via activation of sensory nerves. Sulfur dioxide (250 ppm) induced a characteristic biphasic bronchoconstriction. Concomitantly sulfur dioxide induced the release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive sensory nerves into the pulmonary circulation. In lungs of guinea pigs pretreated with a neurotoxic dose of capsaicin, the first phase of bronchoconstriction was reduced and the overflow of CGRP was not detectable. Tetrodotoxin abolished the initial phase of the bronchoconstriction induced by sulfur dioxide, indicating that a local neural reflex depending on sodium channels was operant. Inhibition of the vanilloid receptor with capsazepine slightly, although not significantly, reduced the contractile responses to sulfur dioxide. Sodium metabisulfite, when infused via the pulmonary circulation (3 mM), induced bronchoconstriction which was abolished by capsaicin pretreatment, but not significantly reduced by capsazepine. The results indicate that in the isolated guinea pig lung inhaled sulfur dioxide induces initial bronchoconstriction in part via sensory nerve activation, while other mechanisms are involved in the late effect. Sensory nerve activation appears to be the only mechanism for bronchoconstriction induced by infused sodium metabisulfite. A role for sensory nerve-mediated bronchoconstriction by sulfur dioxide or sodium metabisulfite via activation of the vanilloid receptor could not be conclusively demonstrated by this study using capsazepine.

Hydroperoxide-Induced Broncho- and Vasoconstriction in the Isolated Rat Lung

The effects of different hydroperoxides on lung mechanics and perfusate flow rate and their mechanisms of action were studied in isolated perfused rat lungs. The administration of hydrogen peroxide, t-butyl hydroperoxide, cumene hydroperoxide, linoleic acid hydroperoxide, and linoleic acid ethylester hydroperoxide (0.1-2 mM) to the perfusate caused a marked decrease in lung compliance, conductance, and perfusate flow rate, with constriction strength of t-butyl hydroperoxide greater than hydrogen peroxide greater than cumene hydroperoxide greater than linoleic acid ethylester hydroperoxide greater than linoleic acid hydroperoxide. Although the hydroperoxides probably had to enter lung cells to exert their effects, no relationship was found between constriction strength and amount of hydroperoxide taken up by the lung. Reduced sensitivity was apparent after repeated dosing, depending on the length of time between dosing. The addition of the iron chelator Desferal (1 mM) had no effect on the hydroperoxide-induced broncho- and vasoconstriction, although free iron was reduced by 50% in the lungs. The administration of the antioxidants diphenyl-p-phenylenediamine (50 microM) or butylated hydroxyanisole (200 microM) to the perfusate 20 min prior to the hydroperoxide attenuated the hydroperoxide-induced effects as well as arachidonic acid-induced broncho- and vasoconstriction. Our findings have shown that hydroperoxides that can enter the lung cells will also induce both vaso- and bronchoconstriction in the isolated perfused rat lung.

Vasoconstriction and bronchoconstriction induced by 2,5-Di(tert-butyl)1,4-benzohydroquinone, an endoplasmic reticular Ca2+-ATPase inhibitor in isolated and perfused rat lung

The microsomal Ca2+-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) induced bronchoconstriction and vasoconstriction in the isolated perfused and ventilated rat lung. These effects were accompanied by increased levels of thromboxane and prostacyclin in the effluent perfusate. The effect of tBuBHQ was inhibited by L-655,240, a thromboxane receptor antagonist, indicating thromboxane-A2-mediated bronchoconstriction and vasoconstriction. Accordingly, the cyclooxygenase inhibitor indomethacin largely blocked the effects of tBuBHQ. The involvement of a phospholipase in the generation of thromboxane A2 (TXA2) was supported by dibucaine protection on tBuBHQ effects. The results from this study indicate that tBuBHQ, probably by inhibiting the microsomal Ca2+-ATPase, can trigger the arachidonic acid cascade leading to the formation of TXA2, which in turn causes bronchoconstriction and vasoconstriction in rat lung.

Sodium Metabisulfite and Citric Acid Induce Bronchoconstriction via a Sulfite-Sensitive Pathway in the Isolated Guinea Pig Lung

Inhalation of sodium metabisulfite (MBS; 80 mM; pH 2.9 +/- 0.1) or citric acid (CA; 0.4 M; pH 2.0 +/- 0.1) aerosols induced a reduction in compliance and conductance in the isolated perfused and ventilated guinea pig lung without affecting perfusion flow. The effect was dependent on the pH of the nebulized solution since inhalation of 80 mM MBS aerosols at pH 7.4 did not induce any effect on bronchial tone. Concomitantly to the bronchoconstriction induced by MBS or CA an increased level of calcitonin gene-related peptide (CGRP-LI) in the effluent perfusate was observed, indicating activation of sensory nerves. Sodium sulfite, a dissolution product of MBS, has previously been shown by our studies to reduce bronchoconstriction induced by inhalation of sulfur dioxide, in the isolated perfused and ventilated guinea pig lung. In the present study perfusion of the lung with sodium sulfite (3 mM) before and during exposure to aerosols with either MBS or CA attenuated the bronchoconstriction induced by the acidic solutions. The release of CGRP-LI induced by MBS or CA was not affected by sodium sulfite. Sulfite treatment did not modify perfused guinea pig lung reactivity towards acetylcholine (4 nmol), bradykinin (100 pmol), histamine (10 nmol), serotonin (500 pmol) and substance P fragment 5-11, a substance P analogue resistant to degrading enzyme (500 pmol). However, an inhibitory effect by sodium sulfite was observed on bronchoconstriction induced by the NK-2 agonist neurokinin A fragment 4-10 (NKA 4-10, 25 pmol). These results indicate that MBS- or CA-induced bronchoconstriction was dependent on the low pH of the aerosol solution and coincided with activation of sensory nerves. Sulfite modulation of the bronchoconstricting action of inhaled MBS and CA is suggested to be related to a sulfite-sensitive step in the signal transduction of the neuropeptide NKA.

Critical Events in the Toxicity of Redox Active Drugs

The mechanism of toxicity of redox active compounds such as quinones, bipyridylherbicides and nitrocompounds varies with the compound1. Common to these types of redox active compounds is that they may be enzymatically reduced by one electron reduction catalyzed by the microsomal enzymes NADPH, cyt-P450 reductase and NADH cyt-b5 reductase and the mitochondrial NADH ubiquinone oxidoreductase. The free radical intermediates formed may be reactive by themselves or, depending on their one electron redox potentials, react with molecular oxygen with the resulting formation of superoxide anion radical (\( {O_2}\overline \bullet \)). This process is termed redox cycling. The so formed (\( {O_2}\overline \bullet \)) readily dismutates either enzymatically or nonenzymatically to hydrogen peroxide (H2O2) which in turn, if not metabolized, undergoes heterolytic cleavage in a Fenton-type reaction with the formation of the very reactive hydroxyl radical (·OH). In addition to redox cycling some redox active compounds may react directly with cellular constituents. They may for instance oxidize both pyridine nucleotides and soluble and protein-bound thiols, as well as arylate and alkylate protein and DNA. Many quinones have for instance been shown to readily react with GSH to form GSH-quinone conjugates and with protein thiols to form alkylated proteins2–4.