Human SLPI inactivation after cigarette smoke exposure in a new in vivo model of pulmonary oxidative stress.

The role of oxidative stress in inactivating antiproteases is the object of debate. To address this question, we developed an in vivo model of pulmonary oxidative stress induced by cigarette smoke (CS) in mice. The major mouse trypsin inhibitor contrapsin is not sensitive to oxidation, and the mouse secretory leukoprotease inhibitor (SLPI) does not inhibit trypsin. Instead, human recombinant (hr) SLPI inhibits trypsin and is sensitive to oxidation. Thus we determined the effect of CS in vivo on hrSLPI antiproteolytic function in the airways of mice. CS caused a significant decrease in total antioxidant capacity in bronchoalveolar lavage fluid (BALF) and significant changes in oxidized glutathione, ascorbic acid, protein thiols, and 8-epi-PGF(2alpha). Intratracheal hrSLPI significantly increased BALF antitryptic activity. CS induced a 50% drop in the inhibitory activity of hrSLPI. Pretreatment with N-acetylcysteine prevented the CS-induced loss of hrSLPI activity, the decrease in antioxidant defenses, and the elevation of 8-epi-PGF-(2alpha). Thus an inactivation of hrSLPI was demonstrated in this model. This is a novel model for studying in vivo the effects of CS oxidative stress on human protease inhibitors with antitrypsin activity.

Responses of thiols to an oxidant challenge: differences between blood and tissues in the rat.

Treatment of rats with diamide (100 mg/kg i.p.) altered the thiol components of the blood to a very different extent than in tissues (liver, kidney, lung, spleen, heart and testis). A total consumption (10 min) and regeneration (120 min) of blood glutathione (GSH), matched by a parallel increase and decrease in glutathione-protein mixed disulfides (GS-SP) was observed. In contrast, no modification of non-protein SH groups (NPSH) and protein SH groups (PSH), GS-SP and malondialdehyde (MDA) was observed in liver, kidney, lung, testis spleen and heart within same time range. In particular, only glutathione disulfide (GSSG) levels and some activities of antioxidant enzymes were modified to a small extent and in an opposite direction in some organs. For example, GSSG, and glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase (CAT) activities appeared up-regulated in one tissue and down-regulated in another. The least modified organ was the liver, whereas lung and spleen were the most affected (lung, GSSG, significantly increased whereas G-6-PDH, glutaredoxin (GRX), GPX, superoxide dimutase (SOD) levels were significantly lowered; spleen, GSSG and the activity of glutathione reductase (GR), G-6-PDH and glutathione transferase (GST) were significantly decreased). The different responses of erythrocytes and organs to diamide were explained by the high affinity of hemoglobin and by the relatively high potential of thiol regeneration in organs. The rapid reversibility of the process of protein S-thiolation in blood and the small effects in organs leads us to propose the existence of an inter-organ cooperation in the rat that regulates protein S-thiolation in blood. Plasma thiols may well play a role in this process.

Altered glutathione anti-oxidant metabolism during tumor progression in human renal-cell carcinoma.

It has been proposed that oxidative stress develops in tumors, with important consequences for growth and progression. To investigate this hypothesis, we measured low m.w. thiols, disulfides, protein-mixed disulfides and a pool of major anti-oxidant enzymes in renal-cortex as well as renal-cell carcinoma (RCC) specimens at stages I-II and III. Our data showed (i) a significant increase in the levels of total intracellular glutathione at both tumor stages (levels were 2.6-2.8 fold higher than those in the normal renal cortex), (ii) a marked lowering of the GSH/GSSG ratio in stage I-II accompanied by a significant decrease of many GSH-dependent enzymes (i.e., GPX, GST, GGT, GR) and (iii) unchanged GSH/GSSG ratio and GSH-dependent enzyme activity in stage III with respect to normal renal cortex. These results indicate that relevant variations exist in the glutathione antioxidant system in the different stages of RCC and support the hypothesis that oxidative stress plays an important role in RCC growth and progression.

Different metabolizing ability of thiol reactants in human and rat blood: biochemical and pharmacological implications.

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position beta125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.

Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress.

Changes in the concentrations of protein-mixed disulfides (XS-SP) of glutathione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in human platelets treated with diamide and t-BOOH in timecourse experiments (time range, 1-30 min) in order to understand the contribution of minor thiols CSH and CGSH to the regulation of glutathione-protein mixed disulfides (GS-SP). Diamide was much more potent than t-BOOH in altering the platelet thiol composition of XS-SP (threshold dose: diamide, 0.03 mM; t-BOOH, 0.5 mM) and caused reversible XS-SP peaks whose magnitude was related to the concentration of free thiols in untreated cells. Thus maximum levels of GS-SP (8 min after 0.4 mM diamide) were about 16-fold higher than those of controls (untreated platelets, GS-SP = 0.374 nmol/10(9) platelets), whereas those of CS-SP and CGS-SP were only 4-fold increased (untreated platelets, CS-SP = 0.112 nmol/10(9) platelets; CGS-SP = 0.024 nmol/10(9) platelets). The greater effects of diamide with respect to t-BOOH were explained on the basis of the activities of fast reactive protein SH groups for diamide and glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t-BOOH. The addition of cysteine (0.3 mM, at 4 min) after treatment of platelets with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to normal values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased gamma-glutamyltranspeptidase activity was found in platelets treated with diamide. Moreover, untreated platelets were found to release and hydrolyze GSH to CGSH and CSH. Ratios of thiols/disulfides (XSH/XSSX) and activities of GR and G-6PDH were also related to a high reducing potential exerted by GSH but not by minor thiols. The lower mass and charge of minor thiols is a likely requisite of the regulation of GS-SP levels in platelets.

Changes in hepatic and renal glutathione-dependent enzyme activities in rabbits and lambs subchronically treated with triphenyltin acetate.

To gain insight into the biochemical mechanisms of organotin toxicity, the effects of oral subchronic exposure (70 d) to triphenyltin acetate (TPTA) on hepatic and renal enzymes involved in glutathione metabolism were investigated in rabbits and lambs. Rabbits were offered a diet fortified with 15, 75 or 150 ppm TPTA, whereas lambs were daily given 1 or 7.5 mg/kg TPTA On the whole, rabbits were more susceptible than lambs and in both species hepatic enzymes were affected to a greater extent than renal enzymes. In rabbit liver, glutathione S-transferase activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was enhanced at 15 ppm and depressed at 150 ppm TPTA, whereas selenium-dependent glutathione peroxidase (Se-GPX) decreased in a dose-related manner; glyoxalase II (GII) activity increased to the same extent at 15 or 75 ppm TPTA but was unaffected at 150 ppm TPTA. For renal enzyme activities in rabbits, only GPX activity was significantly inhibited at 150 ppm TPTA. The only statistically significant changes in lambs were a fall in both hepatic GST accepting DCNB as substrate at 7.5 mg/kg and Se-GPX at 1 or 7.5 mg/kg TPTA, and an increase in renal GII activity at 7.5 mg/kg TPTA. These results suggest that depression of important antioxidant enzymes such as GST and GPX are part of the complex mechanism of organotin toxicity.

Ozonation of blood during extracorporeal circulation. I. Rationale, methodology and preliminary studies.

We investigated whether exposure of blood ex-vivo to oxygen-ozone (O2-O3) through a gas exchanger is feasible and practical. We first evaluated the classical dialysis-type technique but we soon realized that semipermeable membranes are unsuitable because they are hydrophilic and vulnerable to O3. We therefore adopted a system with hydrophobic O3-resistant hollow fibers enclosed in a polycarbonate housing with a membrane area of about 0.5 m2. First we tested the system with normal saline, determining the production of hydrogen peroxide (H2O2) at O3 concentrations from 5 to 40 microg/ml. We then evaluated critical parameters by circulating swine blood in vitro; this revealed that heparin is not an ideal anticoagulant for this system. Finally, we performed several experiments in sheep and defined optimal anticoagulant dose (sodium citrate, ACD), priming solution, volume of blood flow per min, volume and concentration of O2-O3 mixture flowing countercurrent with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 microg/ml is effective; this means that gas exchange and O3 reactivity are rapid and capable of inducing biological effects. The sheep showed no adverse effects even after 50 min of extracorporeal circulation at higher O3 concentrations (20 to 40 microg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.

Role of protein -SH groups in redox homeostasis--the erythrocyte as a model system.

The reactivities of the sulfhydryl groups of rat, turkey, human, and calf hemoglobin were studied together with the enzyme activities of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutaredoxin in lysed erythrocytes to evaluate their roles in regulating redox homeostasis. The results of -SH reactivity showed rate constants spanning four orders of magnitude (k2, calf, 6.67 M-1 s-1; rat -SH fast reacting, 2.8 x 10(4) M-1 s-1). Enzyme activities of glucose-6-phosphate dehydrogenase ranged from 0.402 U/ml (calf) to 0.900 U/ml (rat), glutathione reductase from 0. 162 U/ml (rat) to 0.381 U/ml (human), glutaredoxin from 0.778 U/ml (rat) to 2.28 U/ml (turkey), and glutathione peroxidase from 2.07 U/ml (human) to 27.3 U/ml (rat). Blood samples of the four species were also treated with 0.5-1.5 mM tert-butyl hydroperoxide (t-BOOH) or diamide, and levels of glutathione-derived species [GSH, GSSG, and glutathione-protein mixed disulfides (GS-SP)] were determined within 120 min and related to the corresponding protein -SH group (PSH) reactivities and enzyme repertoires. In all cases t-BOOH rapidly transformed GSH into GSSG by the action of glutathione peroxidase; GSSG was in turn transformed into GS-SP, according to the reaction GSSG + PSH --> GS-SP + GSH, or reduced back to GSH by glutathione reductase. The GSSG reduction was more efficient in rat and human blood, due to the contribution of the fast-reacting -SH of hemoglobin, in the rat, and to the efficiency of the enzyme repertoire of human blood. Calf blood showed a relatively low capacity to restore normal values after oxidative stress, due to its low PSH reactivity and the weak contribution of its enzymes. Diamide treatment, which is known to react nonenzymatically with thiols, gave increased GS-SP levels in rat and turkey, but not in human and calf blood, as expected from the different corresponding PSH reactivities. Species with relatively high PSH reactivity and glucose 6-phosphate dehydrogenase activity, such as the rat, therefore had a higher antioxidant capacity than species (calf) in which these parameters were relatively low.

The role of cysteine in the regulation of blood glutathione-protein mixed disulfides in rats treated with diamide.

The kinetics of GSH, GSSG, and thiol-protein mixed disulfides (RS-SP) of GSH (GS-SP) and cysteine (CYS-SP) were studied in rat blood and liver in the time range 0-120 min after treatment with 100 and 200 mg/kg i.p. of diamide. Total consumption (10 min) and regeneration (120 min) of blood GSH, matched by parallel increases and decreases in RS-SP, were observed. GSSG did not change appreciably. No dose-effect relationship was obtained with either treatment. On the contrary, in vitro treatment of blood with 0.75 mM diamide provoked the same trends of GSH and RS-SP as in vivo (e.g., reversible modifications), whereas treatment with 1.5 mM caused drops and rises in GSH and RS-SP, respectively, without any subsequent return to control values. The presence of a hematic factor responsible for RS-SP regulation is hypothesized in the in vivo experiment. Successive experiments involving in vitro pretreatment with 2 mM diamide and treatment with 0.5 mM of various thiols indicated that cysteine (CYS), but not GSH or N-acetylcysteine, rapidly restored erythrocyte GSH and RS-SP to their basal levels. No evident sign of hemolysis was observed in these experiments. These results indicate that CYS is a diffusible thiol important for RS-SP regulation. Analysis of whole blood of rats treated with 100 mg/kg i.p. diamide and the presence of two reversible peaks (about 10 times the corresponding control level) of CYS-SP and free CYS confirmed the plausible role of CYS in maintaining the reversibility of the process. Preliminary results in liver of rats treated with 100 mg/kg diamide indicated that CYS may act by metabolic cooperation between organs. We suggest that CYS may have a role in the regulation of the intracellular redox state of rat erythrocytes during oxidative stress.

A method to study kinetics of transnitrosation with nitrosoglutathione: reactions with hemoglobin and other thiols.

The rate of protein S-nitrosylation, a reversible process by which S-nitroso thiol (RS-NO) compounds exchange the NO+ moiety with protein SH groups, is essentially governed by two factors, the pK alpha and the accessibility of the protein sulfhydryl. A useful method of following transnitrosation kinetics of various protein and nonprotein SH compounds with GS-NO is described. When the reaction is carried out in the presence of 1-chloro-2,4-dinitrobenzene and glutathione transferases, the rate of RS-NO formation (RSH + GS-NO-->RS-NO + GSH) can be monitored by spectrophotometry at 340 nm in terms of the enzymatic conversion of GSH to a GS conjugate. Unlike methods based on NO release from the S-NO bond, this procedure is rapid and accurate and requires relatively small amounts of thiols. The second order rate constants of S-nitrosylation of human and rat oxy- and deoxyhemoglobin of BSA and other thiols were calculated by this method which confirmed previous results reported in the literature.