Aging and red blood cell membrane: a study of centenarians.

Successful aging, characterized by little or no loss in physiological functions, should be the usual aging process in centenarians. It is known that well-preserved physiological functions depend on the proper functioning of cell systems. In this article we focus on cell membrane integrity and study the red blood cell membrane to evaluate the effect of physiological aging in centenarians. Fifteen healthy, self-sufficient centenarians, mean age 103 years, were examined by assessing hemocytometric values and some relevant characteristics of the erythrocyte membrane, i.e., the cholesterol/phospholipid molar ratio, the distribution of phospholipid classes and their fatty acid composition, the integral and skeletal protein profiles. The centenarians showed a significant decrease in the red blood cell count (p < 0.0002), hemoglobin (p < 0.0002), and hematocrit (p < 0.0005). The red blood cell membrane showed a significantly increased cholesterol/phospholipid molar ratio (p < 0.01), with a concomitant increase in polyunsaturated fatty acids in phosphatidylcholine (p < 0.001) and, to a lesser extent, in phosphatidylethanolamine. The electrophoretic pattern of membrane proteins was qualitatively normal compared to controls but the densitometric analysis showed a significant increase in the integral protein band 4.2 (p < 0.05) and in the skeletal protein actin (p < 0.001). Extreme longevity seems to be associated with a substantial integrity of the erythrocyte membrane. Moreover, the evident increase in polyunsaturated fatty acids and in actin are likely to improve the membrane fluidity and to strengthen the membrane structure.

Antioxidant activity of 3,4-DHPEA-EA and protocatechuic acid: a comparative assessment with other olive oil biophenols.

Olive oil contains several phenolic compounds with antioxidant activity, whose levels depend strongly on the kind of cultivar grown, fruit ripening effects and the oil extraction process. Therefore, the beneficial effects exerted by olive oil consumption on the resistance of low density lipoproteins (LDLs) to oxidation depend not only on an increased intake of mono-unsaturated fatty acids (e.g. oleate) which are less prone to oxidation, but also phenolic antioxidants. The aim of this study was to analyze in vitro effects exerted on the oxidative modification of Cu-stimulated human LDL by two olive oil biophenols, i.e. 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and protocatecuic acid. These compounds have not been investigated in as much detail as the better-known olive oil biophenols - such as tyrosol (p-HPEA), o-coumaric acid, vanillic acid, caffeic acid, oleuropein and 3,4-dihydroxyphenylethanol (3,4-DHPEA). Modification of LDL was tested by measuring the formation of intermediate and end products of lipid peroxidation such as conjugated dienes, lipid hydroperoxides, cholesterol and cholesteryl ester oxides, as well as studying the decrease in oxidizable substrates like polyunsaturated fatty acids. In addition, the increase in LDL negative charges was evaluated. The results demonstrate the two-tested olive oil biophenols show high antioxidant activities. In particular, protocatecuic acid and 3,4-DHPEA-EA show an antioxidant activity comparable with that of caffeic acid, oleuropein and 3,4-DHPEA. They are not only able to retard lipid peroxidation, but also to reduce the extent of its activity.

Genotoxic activity of nitrilotriacetic acid in Chinese hamster cells.

Nitrilotriacetic acid (NTA), a chelating agent, was tested for its ability to induce chromosomal damage in Chinese hamster cells. The chemical was shown to exert a weak genotoxic activity increasing the frequency of micronuclei after prolonged treatments. The analysis of kinetochore containing-micronuclei showed that NTA prevailingly induces chromosomal aberrations as compared to chromosome loss in hamster cells. Furthermore, immunostaining with an alpha-tubulin antibody showed clear alterations in the interphase microtubule network of cells treated for 24 h with 3 mM NTA. The microtubule effects of the chemical may be partly responsible for its cytotoxic effects.

Cytokinesis-block micronucleus assay with kinetochore detection in colchicine-treated human fibroblasts.

A modified micronucleus assay using antikinetochore antibody has been developed in cytokinesis-blocked human fibroblasts as a simple method to identify aneuploidy-inducing agents. Different protocols for inducing binucleated cells by cytochalasin B in colchicine-treated human fibroblasts were investigated. A dose-related increase in kinetochore-positive micronuclei was obtained when cytochalasin B was given subsequent to colchicine treatment. No induction of micronuclei was observed in combined treatments of the two substances. These results indicate that the detection of kinetochores in micronucleated cytokinesis-blocked human fibroblasts can be effectively applied to the identification of environmental agents with aneuploidy-inducing potential. However, in testing such compounds particular attention should be paid to the protocol used for inducing cytokinesis-blocked cells.

Age-related variations in plasma and liver lipids of Yoshida rats: a comparison with Wistar rats.

Lipoprotein and liver lipids of spontaneously hyperlipidemic Yoshida rats were compared with those of normolipidemic Wistar animals for studying their age- and strain-related differences. Both strains showed an age-related increase in the total plasma cholesterol concentration. However, the Yoshida strain had a higher content of cholesteryl esters and triglycerides than the Wistar strain in both young and adult animals (2- and 8-month-old animals, respectively). The free cholesterol content was also higher, but only in the 8-month-old animals. Both strains showed an age-related increase in the proportion of HDL1 and a symmetrical decrease in both the HDL2 and HDL3 subfractions, but the variations were more evident in the Yoshida strain. The study of strain-related differences suggested that the spontaneous hypertriglyceridemia of the Yoshida strain was not only related to the higher amount and proportion of the VLDL fraction, but also to the higher content of triglycerides in the LDL fraction. The livers of Yoshida rats accumulated more triglycerides (with an age-related progression) than those of Wistar rats. The major lipid classes in the liver of Yoshida rats contained a significantly higher proportion of monounsaturated fatty acyls. Furthermore, this proportion showed an age-related increase in all the lipid classes, but in cholesteryl esters. This suggested that liver desaturases had a relevant role in the development of hyperlipidemia, and of its age-related variations, in the Yoshida strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effect of oleuropein, an olive oil biophenol, on low density lipoprotein oxidizability in rabbits.

On the basis of the results obtained with pilot studies conducted in vitro on human low density lipoprotein (LDL) and on cell cultures (Caco-2), which had indicated the ability of certain molecules present in olive oil to inhibit prooxidative processes, an in vivo study was made of laboratory rabbits fed special diets. Three different diets were prepared: a standard diet for rabbits (diet A), a standard diet for rabbits modified by the addition of 10% (w/w) extra virgin olive oil (diet B), a modified standard diet for rabbits (diet C) differing from diet B only in the addition of 7 mg kg(-1) of oleuropein. A series of biochemical parameters was therefore identified, both in the rabbit plasma and the related isolated LDL, before and after Cu-induced oxidation. The following, in particular, were selected: (i) biophenols, vitamins E and C, uric acid, and total, free, and ester cholesterol in the plasma; (ii) proteins, triglycerides, phospholipids, and total, free, and ester cholesterol in the native LDL (for the latter, the dimensions were also measured); (iii) lipid hydroperoxides, aldehydes, conjugated dienes, and relative electrophoretic mobility (REM) in the oxidized LDL (ox-LDL). In an attempt to summarize the results obtained, it can be said that this investigation has not only verified the antioxidant efficacy of extra virgin olive oil biophenols and, in particular, of oleuropein, but has also revealed a series of thus far unknown effects of the latter on the plasmatic lipid situation. In fact, the addition of oleuropein in diet C increased the ability of LDL to resist oxidation (less conjugated diene formation) and, at the same time, reduced the plasmatic levels of total, free, and ester cholesterol (-15, -12, and -17%, respectively), giving rise to a redistribution of the lipidic components of LDL (greater phospholipid and cholesterol amounts) with an indirect effect on their dimensions (bigger by about 12%).

Mitotic indirect non-disjunction in phytohemagglutinin stimulated human lymphocytes.

In a previous publication we demonstrated that in cells of Vicia faba micronuclei derived from whole lagging chromosomes or chromatids may perform DNA synthesis and mitotic condensation in synchrony with main nuclei and be regained by main nuclei at the next mitosis, giving rise to trisomic cells together with diploids. This process was called 'mitotic indirect non-disjunction' (MIND). In the present work the occurrence of MIND was studied in human lymphocytes cultivated in vitro. Human lymphocytes were treated with low colcemid concentrations until fixation; BrUdR was supplied together with colcemid to distinguish the number of mitoses performed by the cells (M1, M2 and M3 cells). The frequencies of M1 ana-telophases with single lagging chromosomes/chromatids and of M2+ prophases with single micronuclei in synchronous motitic condensation with main nuclei were evaluated. On this basis the expected frequencies of both monosomic and trisomic M2 cells were calculated, according to the hypothesis of MIND. Their observed frequencies were very close to those expected. These results support the hypothesis of the occurrence of MIND in human lymphocytes.

Tyrosol, the major olive oil biophenol, protects against oxidized-LDL-induced injury in Caco-2 cells.

Experimental and clinical evidence suggest that oxidative stress causes cellular damage, leading to functional alterations of the tissue. Free radicals may thus play an important role in the pathogenesis of a number of human diseases. Among pro-oxidant agents, oxidized LDL lead to the production of cytotoxic reactive species, e.g., lipoperoxides, causing tissue injury and various subsequent pathologies including intestinal diseases. Thus, to analyze the oxidative damage induced by oxidized LDL to intestinal mucosa, we evaluated morphological and functional changes induced in the human colon adenocarcinoma cell line, Caco-2. In addition, we examined the protective effects exerted by tyrosol, 2-(4-hydroxyphenyl)ethanol, the major phenolic compound present in olive oil. Caco-2 cell treatment (24 and/or 48 h) with oxidized LDL (0.2 g/L) resulted in cytostatic and cytotoxic effects characterized by a series of morphological and functional alterations: membrane damage, modifications of cytoskeleton network, microtubular disorganization, loss of cell-cell and cell-substrate contacts, cell detachment and cell death. The oxidized LDL-induced alterations in Caco-2 cells were almost completely prevented by tyrosol which was added 2 h before and present during the treatments. Our results suggest that some biophenols, such as those contained in olive oil, may counteract the reactive oxygen metabolite-mediated cellular damage and related diseases, by improving in vivo antioxidant defenses.