Barbara Botti

Decreased susceptibility to oxidative stress-induced apoptosis of peripheral blood mononuclear cells from healthy elderly and centenarians

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma-glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells.

No Direct Evidence of Increased Lipid Peroxidation in Hemodialysis Patients

Lipid peroxidation, as measured by the thiobarbituric acid test, has been reported to have increased in hemodialysis (HD) patients, even though the test has low specificity in vivo. Conjugated diene fatty acid (CDFA) hydroperoxides are formed during lipid peroxidation, but not all conjugated dienes (CD) detected in humans originate from lipid peroxidation: octadeca-9,11-dienoic acid, a nonhydroperoxide CD derivative of linoleic acid (CDLA), has a dietary origin. We evaluated CDFA hydroperoxides, CDLA and linoleic acid, using high-performance liquid chromatography, in lipids extracted from plasma, adipose tissue and RBC membranes obtained from 25 patients treated with HD, 16 patients treated with hemodiafiltration (HDF) and 29 controls. No differences in the levels of CDFA hydroperoxides and linoleic acid were seen in any of the groups. Concentrations of CDLA were found to be significantly high in the adipose tissue and low in the RBC membranes of HD patients. HDF-treated patients showed the same results as HD patients. No direct evidence of increased lipid peroxidation was found in HD patients. This does not exclude the possibility that lipid peroxidation is increased and escapes direct detection due to the bodys homeostatic control eliminating the increased production of hydroperoxides. Both HD- and HDF-treated patients showed a significant change in CDLA concentrations, either in the adipose tissue, or in the RBC membranes. These dietary CD may be mistaken for markers of lipid peroxidation by conventional methodologies.

Conjugated Diene Fatty Acids in Patients with Chronic Renal Failure: Evidence of Increased Lipid Peroxidation?

Conjugated diene fatty acids (CDFA) were evaluated by second derivative spectrophotometry in the plasma and adipose tissue of 42 chronic renal failure (CFR) patients in conservative treatment, 40 patients treated by hemodialysis (HD) with cuprophane, cellulose acetate or hemophan, 29 treated by hemodiafiltration (HDF) with polysulfone, polyacrylonitrile or polyamide, and 28 healthy controls. Plasma CDFA were also evaluated at the beginning, at 30 min and at the end of the dialytic session. CDFA were unchanged in CRF patients with creatinine clearance (Ccr) > 10 ml/min respect to the controls, CRF patients with Ccr < 10 ml/min showed a higher level of CDFA both in plasma and adipose tissue (p < 0.02). HD patients showed values similar to those of the control group. The lowest level of CDFA was found in HDF patients (p < 0.01 for plasma, p < 0.05 for adipose tissue versus both control and any other group). A significant relationship between plasma and adipose tissue CDFA was found in all groups. In the group of CRF patients with Ccr < 10 ml/min, females exhibited a higher level of CDFA both in plasma and adipose tissue. No significant change was found during dialytic session, independently from the membrane used. CDFA are not only primary products of lipid peroxidation, but also have a dietary origin, primarily from dairy products. Taking into account the reduced dietary intake, the increase in end-stage CRF may be due to an enhanced oxidative stress and/or to abnormalities in CDFA metabolism. Uremic patients, particularly in the predialytic stage, should be considered at risk for increased oxidative stress. HDF treatment better corrects the abnormality compared to conventional HD.

Detection of Free Radical Intermediates in the Oxidative Metabolism of Carcinogenic Hydrazine Derivatives

Hydrazine derivatives are widely used in agriculture, in industry, as rocket propellants, and in medicine. Hydrazines also occur naturally in tobacco and mushrooms. Many hydrazines tested in animal studies appear to be carcinogenic and induce tumors in various target tissues in mice, hamsters, and rats. The use of hydrazine derivatives in humans is of ten complicated by adverse side-effects such as liver injury and rheumatoid arthritis. A number of studies have demonstrated that hydrazine derivatives are activated to reactive intermediates, such as free radicals, through a variety of cellular oxidative metabolic pathways. The aim of this work is to demonstrate the occurrence of free radical intermediates during the metabolic activation of various hydrazine derivatives and to characterize the enzymatic system(s) responsible for the activation to free radical species. The hydrazines studied are acetylhydrazine, isoniazid, isopropylhydrazine, iproniazid, methylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine. The model systems chosen are those of rat liver microsomes and isolated hepatocytes. Free radical intermediates have been demonstrated by the electron spin resonance spectroscopy coupled to spin trapping technique. The activation mechanism has been characterized using inhibitors of the mixed function oxidase system and of the FAD-dependent oxygenase system. Glutathione was able to scavenge, with high efficiency, the free radicals produced.

Dietary iron deficiency in the rat. I. Abnormalities in energy metabolism of the hepatic tissue

Severe iron deficiency was induced in rats by rearing nursing dams and their offspring on a diet comprising all the requisite nutrients and trace metals except iron. The iron deficient 5-week-old rats exhibited a severe anemia and a drastic decrease in iron content of the hepatic tissue and of the mitochondrial fraction. Cytochromes c + c1 and b were moderately but significantly reduced. A large increase in liver concentration was observed in iron-deficient animals; whereas there was no modification in total lipid, cholesterol, phospholipid and fatty acid composition of the mitochondrial membrane. Mitochondria from iron-deficient rats presented a partial uncoupling of the oxidative phosphorylation process. This functional derangement was completely reversed by the presence of either bovine serum albumin or L-carnitine plus ATP. This behaviour suggested that endogenous long-chain fatty acids could be primarily involved in the onset of mitochondrial dysfunction. The hepatic energy state of the liver appeared dramatically decreased under the pathological condition of severe iron-deficiency anemia. The possibility of a direct link between the partial loss of coupled functions observed in isolated mitochondria and the heavy energy deficit detected in the liver is discussed.

Biochemical mechanism of GSH depletion induced by 1,2-dibromoethane in isolated rat liver mitochondria. Evidence of a GSH conjugation process

HPLC measurements of GSH and GSSG levels in isolated rat liver mitochondria, on addition of 1,2-dibromoethane (DBE), revealed the presence of a glutathione (GSH)-conjugating pathway of DBE. This process required the structural integrity of the mitochondrial matrix and inner membrane complex and was inhibited by the uncouplers of oxidative phosphorylation, particularly 2,4-dinitrophenol. On the other hand it was not affected by the energetic state of the mitochondria, since other mitochondrial inhibitors like KCN and oligomycin did not have any effect on it. This process also did not require the involvement of mitochondrial inner membrane transport systems, based on the measurement of the mitochondrial transmembrane potential. The involvement of mitochondrial GSH-S-transferases, located either in the matrix or in the intermembrane space, is discussed.

Lipid Peroxidation and Bioactivation of Halogenated Hydrocarbons in Rat Liver Mitochondria During Experimental Siderosis

It is firmly extablished that increased amount of iron accumulated in hepatic parenchymal cells is associated with tissue injury, fibrosis and ultimately, cirrhosis1. However, the pathogenetic mechanism of iron in determining the liver injury has not been experimentally proven2. Currently two hypothesis have been put forward in order to explain the hepatocellular injury in chronic iron overload. The first implies that the excess iron largely occurring in lysosomes, physically disrupts these organelles with the release of cell damaging hydrolytic enzymes3. The second one presupposes that the pathological accumulation of iron elicits membrane lipid peroxidation in cellular organelles resulting in structural and functional alterations of cell integrity4. Indeed, iron could promote the production of reactive oxygen species such as Superoxide anion (O·− 2), hydroxy radicals (OH·− 4), and hydrogen peroxide (H2O2)5. The experimental evidence, gathered up to now, indicates that chronic iron overload may induce in vivo lipid peroxidation of mitochcndrial membranes6–8. Furthermore, the in vivo occurrence of lipid peroxidation in the mitochondrial membranes has been suggested to be responsible for some anomalies in liver mitochondria isolated from rats made siderotic either by dietary iron9,10 or by intraperitoneal injection of iron-(III)- gluconate complex11–13.