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.

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.

Cytochemical studies on the fibroblast-preadipocyte relationships in cultured fibroblast cell lines.

The theoretical basis of adipogenesis has always been a matter of debate. One concept suggests that all types of adipocytes are derived from undifferentiated connective tissue cells, whereas another concept suggests that adipocytes develop from specialized cells only that are able to accumulate fat. Many conflicting data have been published with respect to the transition of fibroblasts into preadipocytes. For example, this transition has been declared as impossible for dermal and perimysial fibroblasts. The present study analysed spontaneous accumulation of fat in various types of fibroblasts from different origin (retroocular, skin, NIH/3T3, and L929). It was found that intense Oil Red O-positive triglyceride-containing droplets accumulated in practically all types of fibroblasts provided that the cells were cultured on glass surface. When the cells were cultured on plastic surfaces, lipid staining was inhibited in a variable manner: inhibition was virtually complete in skin fibroblasts, whereas in other types of fibroblasts, inhibition was only partial. It is concluded that all types of fibroblasts can accumulate fat spontaneously, and thus can be considered as preadipocytes. Therefore, interpretations of data obtained with cultures of fibroblasts with respect to adipogenesis have to be reconsidered.

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.

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.

Effect of age on the activity of ceruloplasmin of human blood.

Aminoxidase activity of ceruloplasmin was measured in the serum of 120 people (between 45 and 102 years of age) using the p-phenylenediamine method. A negative linear age-correlation (-30% for the whole lifespan; P < 0.01) was established in this activity with increasing age, nevertheless the total copper content of blood did not change in the same age-range as measured by an atomic absorption spectrophotometric method. Increasing ionic strength, in vitro, caused an exponential decline in blood ceruloplasmin aminoxidase activity of both middle aged and elderly subjects. The age-dependent decrease in ceruloplasmin activity could have a negative effect on the antioxidant functions of blood, and finally on the aging process itself.

The effect of idebenone on the passive K+ and Rb+ permeability of the brain cell membranes of CFY rats as revealed by the Rb+-discrimination ratio.

Rubidium (Rb(+)) uptake and release of brain cortical neurons of adult (12-15 month old), normal, female CFY rats were studied in placebo- and idebenone-treated animals. Treatment period was up to 5 weeks, with 50 mg/kg bw/day idebenone (oxidized form) suspended in 5% gum arabic (verum-group), or only with the latter solvent (placebo-group). Rb(+) can replace up to 60% of the intracellular K(+), and can be used as a tracer of the K(+) movement across the cell membrane. Loading with RbCI was performed from the 3rd week of the idebenone treatment by daily intraperitoneal injections of a dose of 300 mg/kg bw, for 14 days. During the subsequent Rb(+)-release period, the so-called Rb(+)/K(+) discrimination ratio (DR) (Relman et al., 1957, J. Clin. Invest., 36, 1249) was determined on the 3rd and 8th days. Rb(+) and K(+) contents were measured by means of bulk specimen X-ray microanalysis in the intracellular water of brain cells and by atomic absorption spectrophotometry in the serum, in 3-4 animals per group, whereas these concentrations in the cerebrospinal fluid were calculated on the basis of known serum/liquor distribution factors. Normal aging causes a marked increase of DR in brain and liver cells. The values of DR obtained in both placebo and verum groups were identical with those of the age-matched, completely untreated controls. It is important to stress that the subacute idebenone treatment did not cause any deterioration of this parameter, i.e., under the given conditions idebenone does not affect the cell membrane passive Rb(+) and K(+) permeability characteristics in the neurons of adult, normal, female CFY rats.

Effect of sodium-ascorbate and vanadate on the Rb+-uptake of human red blood cells.

To follow the Rb+-uptake of human red blood cells (rbc-s) under different circumstances, a micro-method was developed. According to our experiments the Rb+-uptake of rbc-s in a healthy person was about 3.5-4.0 mumoles Rb/mg Fe at 37 degrees C during 120 minutes. When red cells were incubated with solutions containing different concentrations, between 15 and 120 mmole/l, of Na-ascorbate [Na-ascorbate was applied at the expense of Na-isethionate (sodium salt of 2-hydroxyethan-1-sulfonic acid)] the Rb+-uptake of red cells increased at 37 degrees C with 37 to 70% respectively. In other experiments it was established that the ouabain-sensitive Rb+-uptake of rbc-s decreased with 50% in the presence of 0.1 mmole/l vanadate, while if Na-ascorbate was applied simultaneously with different concentrations of vanadate, or after a preincubation with 1 mmole/l vanadate, the Rb+-uptake of red cells, which had been reduced by vanadate, returned close to that of control. This effect can be explained by the reductive property of Na-ascorbate, i.e. by the transformation of vanadate to vanadil.

The effect of OH(.) radicals generated by Fenton reaction on the growth and cartilage differentiation in limb bud cell culture.

A consistent chondrogenesis takes place in micro high-density cultures of chick limb bud mesenchyme cells stage 22-24. The effect of an increased generation of OH(.) free radicals by Fenton reaction was tested in these cultures. Components of Fenton reaction (i.e., ferrous iron in form of ADP-Fe2+ complex in 0.1 mM concentration, or 0.2 mM H2O2, or the combination of these components with each other, as well as with 0.2 mM ascorbate) were supplemented to the culture medium after the first 24 h. ADP-Fe2+ complex resulted in a drastic decrease of the frequency and confluence of the cartilage nodules seen in light microscope, accompanied electron microscopically by a strikingly increased frequency of occurrence of lipofuscin-like, residual bodies in the cells. Biochemical methods revealed a significant decrease of both the DNA and glycosaminoglycan contents (to 48.6 and 20.7% of the controls, respectively), in day 6 cultures. H2O2 alone caused similar alterations of the cultures, whereas the combination of it with ADP-Fe2+ complex proved to be lethal for the cells. Ascorbate when added to the ADP-Fe2+-treated cultures displayed a slight protective effect for the glycosaminoglycan content but not for DNA. The results are interpreted in terms of free radical theory of aging.

Isolation and physicochemical and functional properties of a calcium binding gluten fraction.

Each milligram gluten protein isolated from bread contains 0.03-0.06 mumol calcium. On theoretical grounds we have concluded that this calcium quantity is bound to the free carboxyl groups not participating in peptide bonds of dicarbonic aminoacids, especially glutaminic acid, making up a large proportion within the aminoacids of gluten. After treatment with EGTA, a well-known calcium complex forming compound, two gluten fractions can be distinguished: water-soluble gluten-ES, and gluten-EP soluble in acetic acid. The aminoacid composition of gluten-ES is similar to that of unfractionated gluten. It is rich in aminodicarbonic acid (glu), aminodicarbonic acid amide (gln) and proline. Further properties of gluten-ES are: immunological similarity to gluten; a molecular mass of 36 000 dalton; an absorption maximum at 275.6 nm; a Ca2+-binding capacity of 0.72 mumol Ca2+/mg protein as measured by atomic absorption spectrophotometry and by Ca2+ ion selective electrode; inhibitory effect of a small quantity (25-30 micrograms) of the compound on the Ca2+-Mg2+ dependent ATPase and Ca2+-uptake of fragmented sarcoplasmatic reticulum. Preliminary experiments have demonstrated that gluten-ES has an influence on other calcium ion mediated systems like actomyosin superprecipitation. We put forward the hypothesis that by its Ca2+-binding capacity, gluten-ES is capable of influencing the level of free calcium and may thus play a part in the pathomechanism of coeliac disease.

Age-dependent decrease of the passive Rb+ and K+ permeability of the nerve cell membranes in rat brain cortex as revealed by in vivo measurement of the Rb+ discrimination ratio.

Young, adult and old male CFY rats (2, 12 and 24 mth of age, respectively) were treated with a daily dose of 30 mg RbCl/100 g body weight, in form of aqueous solution injected intraperitoneally for 14 days. A considerable part of the intracellular K+-content of the body was replaced by Rb+ during this treatment. After cessation of the RbCl injections, a relative steady state came into being in each age group, called Rb+-release period. During this period Rb+ and K+ contents of the blood serum and the cisternal CSF were measured by atomic absorption spectrophotometry, and of the intracellular space of brain cortical cells by energy-dispersive X-ray microanalysis. Ultrastructural features of the brain cortex were also checked by transmission electron microscopy. For X-ray microanalysis, the L-line of Rb at 1.694 keV energy was used at 10 kV accelerating voltage in a scanning electron microscope equipped with an EDAX System F. Rb+ and K+ concentrations were obtained for the cellular dry mass and converted into wet concentrations on the basis of intracellular water contents known from former experiments. Rb+-replacement of K+ did not cause any ultrastructural alteration in the brain cortex. However, the Rb+ accumulation displayed a very significant age-dependent increase: at the beginning of release, adult and old rats had 32.6 and 44.7 mM Rb+ in their intracellular water as against the 8.6 mM found in the young group, and similar proportional difference persisted during 20 days of the release. Rb+ discrimination ratios (DR) calculated either for the blood or the CSF displayed very considerable age-dependent increase: the values of the adult and old groups were 191 and 242% of the young one, indicating that the passive Rb+ (and K+) permeability of the nerve cell membrane decreases throughout the life span of rats. These results give further support to the membrane hypothesis of aging.