Fabiola Giannerini

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 β125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.

Influence of amalgam fillings on Hg levels and total antioxidant activity in plasma of healthy donors.

In order to evaluate the influence of specific factors on mercury (P-Hg) levels and antioxidant power (P-FRAP) in human plasma, 26 healthy donors were examined by a dentist, their plasma analyzed for Hg by atomic absorption spectrometry and for total antioxidant activity by FRAP method. Hg plasma concentration was found to be correlated with the number of amalgam fillings, suggesting that Hg released from fillings is a source of Hg in non-occupational exposed subjects. P-FRAP correlated negatively with P-Hg suggesting a pro-oxidant role of the Hg released from amalgam fillings. Though age by itself was not significantly correlated with P-FRAP, when considered together with P-Hg in multivariate analysis, it was found to be a major related cofactor. Multivariate analysis showed no influence of fish consumption or cigarette smoking on P-FRAP.

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.

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 μg/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 counter-current with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 μg/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 μg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.