M. Ledig

Ethanol-induced cell death in cultured rat astroglia

Because of the important role of glial cells in brain maturation and reports on their delayed proliferation following ethanol exposure, it was considered of interest to investigate the mechanism of ethanol action on these cells. Biochemical parameters related to the apoptotic and necrotic processes in astroglial cells exposed for 1 week to 50 and 100 mM ethanol were examined. Ethanol increased intracellular calcium levels without changing transglutaminase activity and nitrite levels. Moreover, DNA fragmentation was noted with flow cytometry and with the random oligonucleotide primed synthesis assay but neither following agar gel electrophoresis nor in UV microscopy of cell nuclei. The DNA patterns obtained were different from these seen in programmed cell death. Additionally, immunocytochemical analysis showed greater fragility of astrocytes than oligodendrocytes to ethanol. These results support the hypothesis that astroglial cells in vitro exposed to ethanol die due to necrotic but not apoptotic mechanisms.

Superoxide dismutase activity in rat brain during acute and chronic alcohol intoxication

The effect of acute and chronic ethanol administration on rat brain superoxide dismutase (SOD) activity was studied. Intraperitoneal injections of ethanol led to an inhibition of SOD activity. When ethanol was fed as the sole fluid, the SOD activity decreased progressively, reaching a plateau after 6 weeks of treatment. Withdrawal of ethanol produced a recovery of control values within 48 hr. SOD activity was also decreased in rats born from ethanol-drinking mothers. Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O2− radicals; this may account for some nervous system disorders during alcohol intoxication.

Concentrations of physiologically important metal ions in glial cells cultured from chick cerebral cortex

Energy dispersive x-ray fluorescence and atomic absorption spectroscopy were used to determine the concentrations of Mg, Ca, Mn, Fe, Zn, and Cu in primary cultures of astroglial cells from chick embryo cortex in chemically defined serum-free growth medium. The intracellular volume of cultured glia was determined to be 8.34 μl/mg protein. Intracellular Mn, Fe, Zn, and Cu in these cells were ca. 10–200 μM, or 20–200 times the concentrations in the growth medium. Mg2+ was 7 mM in glial cells, only four-fold higher than in growth medium. Glutamine synthetase (GS), compartmentalized in glia, catalyzes a key step in the metabolism of neurotransmitterl-glutamate as part of the glutamate/glutamine cycle between neurons and glia. Hormones (insulin, hydrocortisone, and cAMP) added to growth medium differentially altered the activity of GS and the intracellular level of Mn(II), but not Mg(II). These findings suggest the possibility that glutamine synthetase activity could be regulated in brain by the intracellular levels of Mn(II) or the ratio of Mn(II)/Mg(II), which may in turn be controlled indirectly by means of transport processes that respond to hormones or secondary metabolic signals.

Modulation of Oxygen-Radical-Scavenging Enzymes by Oxidative Stress in Primary Cultures of Rat Astroglial Cells

Cytosolic and mitochondrial alterations induced by exposure of rat astroglial primary cultures to reactive oxygen species (ROS) generated by a xanthine/xanthine oxidase (X/XO) mixture or by lipopolysaccharide (LPS) have been investigated biochemically and immunochemically. In the presence of ROS generated by X/XO, a significant decrease in Cu,Zn superoxide dismutase (Cu,Zn-SOD) and in glutamine synthetase (GS) activity was observed whereas mitochondrial Mn-SOD activity and enzyme protein levels were significantly enhanced. Similar effects on GS, Cu,Zn- and Mn-SOD activities were observed by glucose/glucose oxidase treatment of the cells. Addition of LPS to the cell growth medium also specifically induces Mn-SOD synthesis but was without effect on Cu,Zn-SOD. It is suggested that in all these tested situations, hydrogen peroxide could represent a specific inducer of the observed phenomenon and it may therefore be considered as an intracellular messenger involved in the regulation of some aspects of astroglial oxidative metabolism, particularly the defence against ROS.

Influence of light and dark on the free amino acid pattern of the developing chick retina.

Summary Changes in free amino acids of the developing chicken retina were studied. All amino acid levels except GABA, glutamine, glycine and lysine decreased between the 17th and the 20th incubation days, then increased between the 20th incubation day and the 1st day after hatching. Adult levels were attained within 30 days of hatching. Taurine concentrations increased in the retina after hatching, contrasting with the post-natal decrease of this amino acid in brain and other organs. The influence of light and darkness on the developing free amino acid pattern in retina was examined. Incubation of eggs in total darkness or in continuous illumination did not affect the normal changes in amino acid levels. During the first days after hatching a significant increase in GABA content was observed in the retina of light-exposed chicks; taurine levels were increased by more than 90% in the dark-reared animals. A possible role of taurine as a synaptic transmitter or modulator in retina is discussed.

Free radical scavenging systems of rat astroglial cells in primary culture : effects of anoxia and drug treatment

Hypoxic injury of rat astroglial cells in primary culture initiates several modifications of their functional integrity. A significant decrease of the cellular oxygen consumption was observed in astrocytes submitted to a 15 h low oxygen pressure. The addition of almitrine (dialylamino-4′,6′-triazinyl 2′)-1-(bis-parafluorobenzydryl)-4-piperazine, a chemoreceptor agonist, restored almost completely the respiratory activity of the hypoxia treated cells. In order to test the hypothesis that oxygen free radical formation may contribute to the cellular damage resulting from ischemia, the activities of the following antioxidant enzymatic systems have been determined in the cultured astrocytes: Cu,Zn-and Mn-superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), glutathione reductase (GSH-RED), and catalase (CAT). Only a significant and specific decrease of the Mn-SOD activity was observed after the hypoxia-normoxia exposure. The other oxygen radical scavenging systems were not modified. The addition of almitrine antagonized the decrease of the Mn-SOD activity observed in the low oxygen pressure treated cells, but results clearly point-out the importance of oxygen radical production in the astroglial response after hypoxic injury. A beneficial effect of almitrine toward the observed alteration has been underlined. It is suggested that some mitochondrial alterations could be related to some aspects of theastroglial hypoxic stress.

Combined effects of ethanol and manganese on cultured neurons and glia

Manganese is essential for normal development and activity of the nervous tissue. Mn2+ ions are involved in protein synthesis and may prevent free radical damage. Since it is now established that alcohol degradation may produce free radicals, we studied the effect of Mn2+ on ethanol induced alterations using cultured nerve cells as an experimental model of the central nervous system. Neurons and glial cells were cultured from rat brain cortex; a tumoral rat glial cell line (C6) was also examined. We measured enzymatic markers of nerve cell maturation (enolase, glutamine synthetase) and superoxide dismutase, a scavenger of free radicals; all these enzymes being activated by Mn2+ ions. Only for the glial cell types an alcohol antagonizing effect was found when Mn2+ was combined with ethanol. Neurons were not sensitive to that Mn2+ effect.

Chick brain glutamine synthetase and Mn2+−Mg2+ interactions

Glutamine synthetase (GS) from the chick brain was purified to apparent homogeneity by ammonium sulfate fractionation followed by affinity chromatography, electrofocusing and Sephadex G-150 chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate analysis in polyacrylamide gel. By sedimentation equilibrium analysis and gel electrophoresis analysis, it was shown that the enzyme has a subunit molecular weight of 45,000 and a native molecular weight of 364,000, which is consistent with an octameric structure. Sedimentation analysis in the presence of Mg2+ revealed three different forms of macromolecules corresponding respectively to a monomer, a tetramer and an octamer. Among eight cations tested (Ca2+, Co2+, Fe2+, Li+, Mg2+, Mn2+, Ni2+, Zn2+) only Co2+, Mg2+ and Mn2+ supported GS activity; the order of activatory ability was Mg2+>Co2+>Mn2+. The maximum activating effect of Mn2+ occurs only within a very narrow range of concentration: with an excess of cation causing strong inhibition of GS activity. For each cation, maximal GS activity occurs at a defined cation/ATP ratio. A regulatory system in which Mn2+, modulates the Mg2+ dependent GS activity, is proposed; such cation interactions may be of significance in the intracellular control of glutamine synthesis.

Regional distribution of superoxide dismutase in rat brain during postnatal development

Superoxide dismutase in nervous system protects readily oxidizable compounds such as catecholamines against toxic effects of oxygen. We investigated superoxide dismutase activity during development in 5 brain regions selected for a wide range of catecholamine concentration and turnover: cerebellum, neocortex, striatum, hypothalamus and medulla-pons. The cytosolic and the particulate enzyme were measured from birth to 6 months of age. In cerebellum the cytosolic enzyme shows considerable activity on the first postnatal days; the particulate enzyme is less active, both reach a maximum at 3 months. In cortex and striatum both activities were low during the postnatal days and reach a plateau at 3 months. In hypothalamus both activities are higher during the postnatal days and reach a maximum at 3 months. In medulla-pons the values are 2 times higher than in all other regions; the cytosolic enzyme reaches a maximum at 2 months whereas the particulate enzyme reaches a plateau at 3 months. Thus our results show an increase of superoxide dismutase activity during development in all brain regions; the highest activities were found in regions with high catecholamine content.

Paternal alcohol exposure : developmental and behavioral effects on the offspring of rats

The effect of paternal alcohol exposure on neurochemical and behavioral parameters was investigated using as a model system glial cells derived from newborn rat brain and cultured for 4 weeks. The total brain neurochemical parameters from rats born to mothers sired by an alcohol treated father were also investigated. Enzymatic markers of nerve cell development (enolase isoenzymes and glutamine synthetase) and the defense system (superoxide dismutase) against free radicals formed during alcohol degradation were measured in order to evaluate nerve cell damage. Behavioral locomotor tests (open-field, novelty-seeking, light/dark) were carried out to show long-lasting effects of paternal alcoholization on the offspring. Behavioral and developmental alterations were found until 1 year of age in the offspring and a significant growth retardation was observed in the males. Our results suggest that paternal alcohol exposure produces developmental and behavioral effects in the offspring. The consequence of either alcohol withdrawal during stage one spermatogenesis, or maternal diet supplementation with manganese during pregnancy were investigated. It was observed that some of the effects of paternal alcohol exposure on the offspring may be reversed by these treatments.