Hydroxyl free radicals induce cell differentiation in SK-N-MC neuroblastoma cells.

The SK-N-MC cell line is frequently used as a model of neuronal differentiation induced by 5-bromodeoxyuridine (BrdU). In this study, the differentiation properties of this cell line were investigated under hydroxyl free radical generation, and compared to BrdU treatment. Hydroxyl free radicals were generated in the cultures by the Fenton reaction, i.e. by simultaneous addition of ADP-Fe2+ complex and H2O2. Microscopic morphological signs, as well as the acetylcholinesterase and ganglioside sialidase activities were considered as markers of neuronal differentiation of this cholinergic neuroblastoma cell line. Apart from the altered morphological appearance, the marker enzymes displayed significant increases after both types of intervention. We suggest that hydroxyl free radicals can induce in vitro cell differentiation. They apparently play a more complex role in cell physiology than simply causing oxidative damage.

Experimental gerontology in Hungary.

Gerontological research has some past and sporadically also some highlights in Hungary, but its present state can be easily deduced from the following data. During the last 12 years and more, well over 10,000 Hungarian scientific papers have been published in well-recognized national or international journals. Altogether approximately 1% of them have been classified as gerontological publications from Hungary. This low figure shows that gerontology has low priority and--unfortunately low support--in Hungary. This statement does not intend to downgrade Hungarian gerontologists, however points out that the Hungarian trends are not far from those of European or world wide interest in aging. Despite the recognition that we have to accept the inevitable fact that industrial societies will have (they already have) an aging population with all the social and medical problems arising, the focus of interest is wide from this significant and interesting (sub)population, which is neglected (sometimes even despised); yet everybody is absolutely eager to join this club. The average of the Hungarian research achievements and publication activities are among the better European achievements. There are some highlights and new trends even initiated by some outstanding Hungarian scientists, yet the overall weight of gerontology research is still an orphan in the Hungarian scientific life. We deal in this short and far from complete summary almost exclusively with experimental gerontology. We have to apologize if we have not included everybody, who also contributed even significantly to this field because the time for the preparation of this overview was short.

The involvement of hydroxyl free radicals in differentiation of the PC-12 rat pheochromocytoma cell line.

These experiments tested the differentiation properties of the PC-12 cell line under conditions of in vitro generation of OH&z.rad; free radicals by Fenton reaction. This involves the simultaneous addition of the following reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.025 mM), final concentrations. Superoxide dismutase activity, the increase of which is considered as a marker of differentiation, catalase and glutathione peroxidase enzyme activities were investigated, which all displayed significant increases after single and repeated interventions with hydroxyl free radicals, while the cell number remained nearly at the starting-value. It is known that the differentiation takes place when the cell number has reached a plateau. These data, therefore, suggest that hydroxyl free radicals can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damages. It is interesting that the cells can maintain high levels of these enzyme activities for a relatively long time (2 or 4 days) after a very short flux of hydroxyl free radicals.

Enzyme activities in the light of the membrane hypothesis of aging. [An answer to K. Kitani, Mech. Ageing Dev. 107 (1999), 299-322].

The paper of Kitani cited in the title has raised an apparent contradiction regarding the validity of certain aspects of the membrane hypothesis of aging (MHA). He collected data showing that a number of detoxifying liver enzyme activities, although decline with age in male Fischer 344 rats, remain at an unchanged level in females of the same strain. He concluded that the main assumption of the MHA, according to which intracellular enzyme activities generally decline with age, cannot be maintained, and invoked me (p. 312) 'ellipsis to provide in the future ample (and convincing) evidence' in this respect. The present paper answers this criticism by showing that the apparent contradiction mentioned above is based on a misunderstanding on behalf of Kitani. Namely, MHA speaks about the general, density-dependent decline of the catalytic rate constant of any enzyme (k(cat)), i.e., activity per mole of enzyme, being the true specific activity of the enzymes. This parameter inevitably decreases at the increased physical density of the intracellular colloids during aging. This statement derives from the molecular enzyme kinetic models, and has extensively been proven experimentally, too. On the other hand, Kitani speaks about enzyme activities per mg total protein content of certain tissue extracts, which is a very illdefined parameter, since the concentration of the measured enzyme remains unknown. Therefore, this latter parameter is irrelevant from the point of view of MHA in any aspect.

Comparison of the lateral diffusion coefficient of hepatocyte plasma membrane proteins in three strains of sensescence accelerated mouse (SAM).

The lateral diffusion coefficients of proteins (D(p)) were measured in hepatocyte plasma membrane in freshly prepared liver smears by means of the fluorescence recovery after photobleaching (FRAP) method. D(p) was measured after development of peroxide-induced autofluorescence (PIAF) in a total of 115 senescence accelerated mice (SAM), distributed in three strains, at least five age-groups in each, as follows: (i) SAMR1TA (25 males and 22 females), medium life span (MLS) in months, under specific pathogen free (SPF) conditions, MLS(spf) 20.1 and 20.0, respectively, while under conventional conditions, MLS(Deltapf)=18.9 in average for both sexes; (ii) SAMP6/Ta (18 males and 17 females), MLS(spf)=17.1 and 15.3, respectively, and MLS(cc)=8.1 for both sexes; (iii) SAMP6/Ta (17 males and 16 females), MLS(spf)=15.6 and 14.7, respectively, and MLS(cc)=10.0 for both sexes. A highly significant negative linear age-correlation of D(p) (R=0.975 or higher) was found in each strain, being roughly proportional with the MLS(cc) values. Since the studied mice kept under SPF conditions survived longer, than under conventional conditions, the actual age-dependent decay rates of D(p) values did not differ significantly in two pairs of comparisons (female R1/P6 and female R1/P8), whereas they did in all other possible pairs, including also the normal C57BL/6 mice. The main conclusion can be drawn that the D(p) of hepatocyte membranes is a good biomarker of aging and survival also in SAM, as in all other inbred and outbred rodents, studied so far.

On the useful role of OH&z.rad; free radicals in differentiation of cultured human fibroblasts.

The working hypothesis assuming that oxygen free radicals cannot be considered only as harmful by-products of the oxidative metabolism has been experimentally tested. Human fibroblasts were grown in culture from the following five types of tissues: (1) normal orbital fat; (2) orbital fat of patients with endocrine ophtalmopathy (EOP); (3) normal orbital muscle; (4) orbital muscle of EOP patients; (5) skin. These fibroblasts (second to 12th passages) were treated for 2x72 h with the Fenton reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.055 mM), final concentrations. This treatment caused a slowing down of the cell proliferation, induced various morphological signs of differentiation, and significantly increased (40-150%) the total superoxide dismutase (SOD) and catalase (CAT) activities of the fibroblasts. Authors suggest that the increased expression of these enzymes may play a general role in the cell differentiation mechanisms, meaning that the generation of oxygen free radicals is an essential, useful factor even during the early phases of development, and may not be taken only as a harmful process during aging.

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.

Age-dependence of the lateral diffusion coefficient of concanavalin-A receptors in the plasma membrane of ex vivo prepared brain cortical nerve cells of BN/BiRijHsd rats.

A new method has been developed for ex vivo preparation of brain cortical cells of BN/BiRijHsd rats to make them suitable for the measurement of the lateral diffusion coefficient of the membrane components by means of fluorescence recovery after photobleaching (FRAP). The method involves chopping the brain cortex into pieces of less than 1 mm. These parts are stained with a fluorescent label (e.g., concanavalin-A-fluorescein, Con-A-FL conjugate) and then gently pressed onto a microscope slide using the coverslip. In the resulting specimen, the largest cells of the cortex can be recognized in phase-contrast image, sufficiently stained by the label and ready for the FRAP measurement. The lateral diffusion coefficient of Con-A-receptor proteins (Dp) was measured in such brain cell preparations of 15 female rats in four age groups (5.6-31.8 months) and 11 males in three age groups (13.8-31.8 months). Highly significant negative, linear age correlation of Dp (R=-0.9958 in females, and -0.9956 in males) were found, the regression equations being D(p female) =(8.8311-0.1425 X)(-10) and D(p male)=(9.3240-0.1630 X)(-10) cm2/s, respectively, where X is age in months. The data confirm that the lateral mobility of plasma membrane proteins represents an important biomarker of cellular aging in the brain cortical cells of BN/BiRijHsd rats.

Alpha lipoic acid (ALA) protects proteins against the hydroxyl free radical-induced alterations: rationale for its geriatric topical application.

The well known OH* free radical scavenging properties of alpha-lipoic acid (ALA) cannot be easily utilized for biological experiments, because the compound is practically insoluble in water. We elaborated a simple method of preparing its Na-salt (Na-ALA) which proved to be water soluble. It has been demonstrated by ESR spin trapping experiments with DMPO, using the Fenton reaction as the source of OH* free radicals that Na-ALA maintains its OH* free radical scavenging ability: it reacts nearly an order of magnitude faster with these radicals than the spin trap itself. It was tested in two different systems to determine whether Na-ALA was able to protect bovine serum albumin (BSA) against the OH* free radical-induced polymerization and protein oxidation. (i) OH* free radicals were generated by Fenton reaction in the presence of BSA. This protein is polymerized by these radicals shown by the loss of its water solubility; Na-ALA exerted a considerable protective effect against this type of protein damage. (ii) BSA oxidation was induced by Co-gamma irradiation of 80 krad, resulting in a strong increase in the protein carbonyl content. Na-ALA inhibited this carbonyl formation very efficiently. The data suggest that the interaction of the OH radical with Na-ALA takes place on the disulfide group, yielding thiosulfinate or thiosulfonate. The results indicate that the geriatric topical application of Na-ALA may have an established rationale.

Age-dependence of the lateral diffusion coefficient of Con-A receptor protein in the skeletal muscle membrane of C57BL/6J mice.

The lateral diffusion coefficient (Dp) of the Con-A receptor protein was measured in the sarcolemma of the quadriceps femoris (QF) muscle of male and female C57BL/6JNia mice in four age groups between 2 and 26 months. Freshly prepared, ex vivo taken muscle strips were stained with Con-A-FL conjugate for 10 min, and fluorescence recovery after photobleaching (FRAP) measurements were carried out on 20-30 cells per animal, at 37 degrees C. Using this technique, Dp, and the fractional recovery (mobile fraction = FR%) of these proteins can be measured. In the youngest male and female age groups, Dp values of 5.72E-10 and 5.43E-10 cm2/s, and FR% values of 43.3 and 36.3%, were found, respectively. Dp displayed a characteristic, significant, negative, linear correlation with age in both sexes. The slope of the linear regression line calculated per month of age was 1.06E-11 and 0.96E-11 cm2/s for males and females, respectively; both of them differ from zero highly significantly. FR% values tended to increase slightly with age, yet the estimated average Dp = D(FR), calculated for the total Con-A receptor pool, maintained its significant, negative, linear age-correlation. The physiological significance of these changes needs to be clarified in the future.

Age-dependence of the lateral diffusion coefficient of lipids and proteins in the hepatocyte plasma membrane of BN/BiRijHsd rats as revealed by the smear-FRAP technique.

The lateral diffusion coefficients of lipids (D(l)) and proteins (D(p)) were measured in hepatocyte plasma membranes in freshly prepared liver smears of Brown Norway (BN/BiRijHsd) rats by means of the fluorescence recovery after photobleaching (FRAP) method. D(l) was measured after staining with N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE) in 14 males in four age groups (4.3-30.4 months), and 12 females (5.0-31.0 months). D(p) was measured after the development of peroxide-induced autofluorescence (PIAF) in 16 males in four age groups (4.7-30.8 months) and 15 females in four age groups (4.0-28.1 months). Highly significant negative, linear age-correlations of D(l) (R=-0.972 in males, and -0.840 in females, respectively) were found, the average data being 1.39x10(-9) cm(2)/s in the young male rats, and only 9.26x10(-10) cm(2)/s in the oldest ones, whereas in females, these two extremes were 1.44x10(-9) and 8.97x10(-10) cm(2)/s, respectively. Protein lateral mobility also declined linearly with age; in the male rats it decreased from 2.7x10(-10) to 1.8.x10(-10) cm(2)/s (R=-0.903), while in the females it decreased from 2.64x10(-10) to 2.21x10(-10) cm(2)/s (R=-0.955) between the youngest and oldest age groups. There were also some characteristic differences in the fractional recoveries (FR%), too. These trends are comparable with those of F344 rats studied previously, supporting the conclusion that the changes in the lateral mobility of plasma membrane proteins and lipids represent important biomarkers of cellular aging also in the BN/BiRijHsd rats.

The membrane hypothesis of aging: its relevance to recent progress in genetic research.

This review outlines the main concepts of the membrane hypothesis of aging (MHA) developed over the past two decades. MHA offers a general cell biological mechanism to explain the main trends of age-dependent changes in terms of intracellular physicochemistry. An essential point in MHA is that the inevitable plasma membrane alterations result in the accumulation of dry mass in the intracellular space, a process that is essential to cellular development and organismal maturation, but which becomes a rate limiting factor above a certain physical density of the cell colloids. The main statements of the MHA are supported by recent developments in molecular genetics. Specifically, the great majority of the products of oncogenes and antioncogenes are localized to the plasma membrane, indicating a central role of the plasma membrane in mitotic regulation, cell differentiation and senescence.

Age-dependence of the lateral mobility of lipids in hepatocyte plasma membrane of male rats and the effect of life-long dietary restriction.

The lateral diffusion constant of lipids (D(1)) in hepatocyte plasma membranes was measured in liver smears by means of the fluorescence recovery after photobleaching (FRAP) method, applying the label, N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE). Nineteen ad libitum fed, male Fischer-344 rats in four age groups (2.1-29.8 months of age) were studied. A highly significant negative linear age-correlation of D(1) (cc = 0.958) was found. D(1) values were 1.39 x 10(-9) cm2/s in the young rats, and only 6.77 x 10(-10) cm2/s in the oldest rats. Lipid lateral mobility is changing in parallel with that of proteins, having been measured previously also with the FRAP method by the authors. Fractional recovery values (FR%) of the lipids were lower than those of proteins even in the young ages, but also decreased linearly with age, therefore, the parameter, D, x FR decreased even steeper with age than D(1) itself. D(1) was also measured in a group of six male Fischer 344 rats having been kept on dietary restriction (DR) since their age of 1 month until 30 months of age (applying the every-other-day (EOD) feeding). DR caused an increase of D(1), compared with the age-matched ad libitum fed animals: the mean was 9.24 x 10(-10) cm(2)/s. FR% and D(I), x FR again increased considerably under DR. The results are interpreted in terms of the increased protein and lipid turnover under DR.

Relative intranuclear magnesium and phosphorus contents in normal and tumor cells of the human thyroid gland as revealed by energy-dispersive X-ray microanalysis.

Energy dispersive X-ray microanalysis was performed on altogether 42 surgically removed tissue specimens of 32 patients, which were taken either from intact thyroid parts or various histopathologically verified tumors of the thyroid gland. The tissue specimens were processed with the freeze-fracture-freeze-drying technique and then analyzed in the so-called bulk specimen form. The studies were carried out during the years 1980-81, when intranuclear monovalent ionic composition was studied in detail. From the retained total elemental peak list, it was possible to calculate retrospectively the relative intranuclear Mg and P contents. The data processed by nested (hierarchical) analysis of variance show that the intranuclear Mg content of the 5 diagnostic groups (normal thyroid tissue, thyroiditis, benign adenomas, differentiated carcinomas and undifferentiated thyroid tumors) increases significantly, in parallel with the increasing malignancy, but the P content remains unchanged. One can conclude that the elevated intranuclear Mg content in the tumors of high malignancy may be of diagnostic importance, and a warning signal for the therapeutic approaches based on Mg-supplementations.

The influence of dystrophin on lateral diffusion of proteins in sarcolemma of L-185 and C2 myoblasts and mature striated muscle cells of rats and mice, as measured by FRAP technique.

The expression of dystrophin can be suppressed in cultured skeletal muscle cells (coded L-185 from rat, and C2 from mouse) after a proper genetic manipulation. The influence of presence or absence of dystrophin on the lateral diffusion constant of Con-A-receptors was studied in the cell membrane of such cells (and also of mature skeletal muscle fibres of rat and mouse) by means of the fluorescence recovery after photobleaching (FRAP) technique, applying a novel fluorescent label called Con-A-BODIPY-FI conjugate. It has been established that the normal maturation, of myoblasts into skeletal muscle fibres involves a significant decrease of the mobility of Con-A receptors in the sarcolemma. In the absence of dystrophin, this maturation process cannot take place; the membrane proteins display an increasing mobility during the culture time, which is of lethal effect for these cells.

An in vitro model of aging: the influence of increasing physical density on enzyme activities of trypsin, xanthine oxidase and superoxide dismutase.

The enzyme activities of trypsin (using an artificial substrate, Nalpha-benzoyl-L-arginine-ethylester = BAEE), xanthine oxidase (XOD) and superoxide dismutase (SOD) were measured in the absence and presence of various concentrations of the following inert, water-soluble polymer viscogens: polyvinylpyrrolidone (PVP-40), polyethyleneglycol (PEG-6000) and bovine serum albumin (BSA). Enzyme activities measured in the absence of viscogens were taken as 100%. In the presence of the viscogens, enzyme activities decreased considerably as follows: (i) Trypsin: to 2 or 12% in reaction mixtures containing 64 mg/ml PVP-40 or 481 mg/ml PEG-6000, respectively. (ii) XOD: to 29.3% in a reaction mixture containing 116 mg/ml PVP-40, to 68.9% in a medium containing 266 mg/ml PEG-6000, and 38.1% in the presence of 138 mg/ml BSA. (iii) SOD: to 40.0, 19.9 and 16.6% in the same media as listed for XOD, respectively. The observations are consistent with the predictions of the molecular enzyme kinetic model (MEKM), and are also of importance for the membrane hypothesis of aging, since the latter explains the loss of cell functions by an age-dependent increase of intracellular density which may cause serious enzyme inhibitions.

Induction of age pigment accumulation in the brain cells of young male rats through iron-injection into the cerebrospinal fluid.

The OH free radical formation can be increased in the brain by intralumbar iron injections into the cerebrospinal fluid (CSF) through the Fenton reaction. Ferrous ammonium sulphate was injected to male CFY rats of 3 months of age. The animals survived a single dose of 4 mumoles, and this treatment was repeated daily for 3 or 6 days. The total iron contents of the large brain and the cerebellum were measured by atomic absorption spectroscopy; 3 days of iron administration resulted in significant increases of iron contents only in the cerebellum, whereas after 6 days, both parts of the brain showed considerably higher iron contents. Electron microscopic point-counting morphometric analysis revealed a 65% increase of volume density of the lipofuscin in the large cells of parietal cortex after 6 days of iron-treatment. The Purkinje cells displayed a particularly frequent occurrence of an "apoptosis"-like structural alteration under the effect of iron. This kind of experimental approach is complicated by the facts that (i) the increase of OH radical flux above a certain level is lethal, and (ii) the young animals have a very active elimination mechanism of the waste products. Nevertheless, the enhancement of OH radical yield by an increased availability of iron for Fenton reaction resulted in an accumulation of lipofuscin even in young rats.