The effect of in vitro exposure to white spirit on [Ca2+i] in synaptosomes from rats exposed prenatally to white spirit.

Female rats were exposed to white spirit (400 and 800 ppm for 6 hr/day) at day 7-20 during pregnancy. Thirty-five days after birth all female offspring were sacrificed, the brains removed, and the synaptosomal fractions prepared for in vitro studies. The cytosolic calcium concentration was measured using the FURA-2 technique. The results show that cytosolic calcium was increased in synaptosomes from rats exposed to white spirit prenatally compared to synaptosomes from unexposed rats. When synaptosomes were exposed to white spirit in vitro, the cytosolic calcium concentration changes were identical in all groups of rats. The membrane leakage measured as FURA-2 leakage from the synaptosomes identical in all three groups of animals. The results suggest that prenatal exposure to white spirit induces long-lasting and possibly irreversible changes in calcium homeostasis in the rat nervous system.

The effect of ageing and in vitro exposure to xylene and KCl on [Ca2+]i in synaptosomes from rats exposed prenatally to xylene.

Female rats (Mol: WIST) were exposed prenatally to 500 p.p.m. of technical xylene on days 7-20. At the age of fourteen months the rats were sacrificed and the synaptosomal fraction prepared for in vitro studies. The cytosolic calcium concentration was measured using the FURA-2 technique. The cytosolic calcium was increased in synaptosomes from old rats compared to those from rats at the age of three months, but no effect of prenatal exposure was seen. When synaptosomes were incubated with xylene, potassium or both, the cytosolic calcium concentration was changed identically in all groups of rats. When synaptosomes were incubated simultaneously to xylene and potassium a dramatical leakage of FURA-2 was observed. The mechanisms behind the membrane leakage are discussed.

Late postnatal growth in the dentate area of the rat hippocampus compensates for volumetric changes caused by early postnatal toluene exposure.

The present report assesses the reversibility of previously described quantitative morphological alterations in the dentate area of the hippocampi of rats exposed to 500 ppm toluene for 12 h daily from postnatal day 1 (P1) to P28. Following the exposure, rats were allowed to survive until P120. The volumes of hippocampal components, as they appear in Timm-stained histological sections, were determined using the Cavalieri estimator. At P120, no differences were apparent in the volumes of the dentate components of control and experimental animals. The differences observed immediately after exposure were eliminated by significantly larger increases in the sizes of early affected components in experimental animals. Different periods of neurogenesis in rodents and primates are briefly discussed as possible confounders of rodent models of permanent toluene-induced morphological alterations in primates.

Effect of organic solvents on nervous cell membrane as measured by changes in the (Ca2+/Mg2+) ATPase activity and fluidity of synaptosomal membrane.

The effect of various solvents on the central nervous system was studied by using rat brain synaptosomal membranes as an in vitro model. The activity of (Ca2+/Mg2+) ATPase and the membrane fluidity was determined. The alteration of the ATPase activity depended on the physio-chemical characteristics of the solvent in question. Incubation with aliphatic alkanes caused a stimulation of the ATPase activity whereas mixed hydrocarbons as kerosene, white spirit and gasoline inhibited the enzyme. Incubation with chlorinated hydrocarbons caused a biphasic response dependent on the concentration. Oxygen-containing hydrocarbons exhibited various effects as found after incubation with hydrocarbons. The different effects of the solvents on the ATPase activity suggest that the lipophilicity of the solvents is one of more parameters affecting the membrane. Furthermore, the biphasic response following the incubation with chlorinated hydrocarbons indicates that more mechanisms are involved in the enzyme effect. The membrane fluidity is increased with higher concentrations of the solvents. From the results it is concluded that the ATPase activity depends not only on the membrane fluidity and volume, but also on the hydrophilic vicinity of the enzyme molecule.

Effects of simultaneous ethanol and toluene exposure on nerve cells measured by changes in synaptosomal calcium uptake and (Ca2+/Mg2+)-ATPase activity.

The effect of simultaneous exposure of rats to toluene and ethanol on synaptosomal calcium uptake and (Ca2+/Mg2+)-ATPase activity was studied. Rats were exposed to 500 p.p.m. toluene by inhalation for 12 hr a day during four weeks. During the exposure period, the rats had access to 5% sucrose solution containing 20% ethanol or to 5% sucrose solution alone. Rats drinking ethanol exhibited a smaller weight gain than rats drinking water alone. Furthermore, rats exposed simultaneously to toluene and ethanol had a higher ethanol intake than unexposed rats. The toluene exposure caused a higher synaptosomal calcium uptake in vitro. Ethanol intake did not change the synaptosomal calcium uptake in vitro. The synaptosomal calcium uptake in rats exposed to toluene and ethanol was nearly identical to that measured in control rats. In vivo exposure to toluene, or ethanol, or toluene/ethanol simultaneously did not affect the (Ca2+/Mg2+)-ATPase activity in vitro. Incubation with toluene in vitro decreased the (Ca2+/Mg2+)-ATPase activity in a concentration dependent manner. Ethanol had only a slight effect on the enzyme. Simultaneous incubation with toluene and ethanol showed an antagonistic effect of ethanol on the toluene inhibition of the ATPase activity.

Mucosal/submucosal blood flow in the gut wall determined by local washout of 133Xenon.

A 133Xe washout technique for measuring the blood flow in the intestinal mucosa is introduced and evaluated. In 11 anaesthetized pigs a laparotomy was performed and the mucosal blood flow rate in the intestine of the pig was determined by a local epimucosal application of 133Xe. In both the colon and the small intestine the 133Xe washout plotted in a semilogarithmic diagram showed a multiexponential configuration. Histological examination and localization studies showed shunting by diffusion of 133Xe in the intestinal mucosa explaining the multiexponential configuration of the washout curve. Therefore the initial slope of the washout was used for measuring blood flow rate. Blood flow rate was simultaneously measured by microsphere entrapment technique. There was an excellent correlation between the blood flow rate determined by the two techniques the correlation coefficient R being 0.89 in the small intestine and 0.996 in the colon.

The effects of alcohols in vitro on the nervous cell membrane measured by changes in the (Ca2+/Mg2+) ATPase activity and fluidity of the synaptosomal membrane.

The effect of various alkanols on the central nervous system was studied by using rat brain synaptosomal membranes as an in vitro model. The activity of (Ca2+/Mg2+)ATPase and the membrane fluidity were determined. The n-alkanols exhibited an increased molar inhibition of the ATPase activity with an increase in the carbon chain length up to 1-octanol. 1-octanol and 1-decanol caused a biphasic effect on the ATPase activity depending on the alkanol concentration, whereas 1-dodecanol caused a stimulation of the ATPase activity. All alkanols studied caused an increased fluidity of the membrane. Our results indicate that the effect of alkanols on the ATPase activity depends on changes in the border layer between the membrane and the surrounding medium and on a binding of the alkanols to the enzyme molecule. Furthermore, the two-way effect of 1-octanol and 1-decanol and the stimulatory effect of 1-dodecanol indicate that more mechanisms are involved. In addition, the results indicate that changes in the membrane fluidity do not seem to be a prerequisite of the ATPase inhibition.

Mucosal/submucosal blood flow in the small intestine in pigs determined by local washout of 133Xe and microsphere techniques.

In 11 anaesthetized pigs a laparotomy was performed and the mucosal and submucosal blood flow rate in the small intestine of the pig was determined by a local application of 133Xe and by 6.5-microns radioactive microspheres. The 133Xe washout plotted in a semilogarithmic diagram showed a multiexponential configuration. As localization studies of 133Xe in the intestinal mucosa showed a constant high concentration of 133Xe in the luminal part of the mucosa due to shunting by diffusion, the initial slope of the 133Xe washout was used for blood flow determination in the mucosa/submucosa. There was a good relationship between blood flow determined by the two techniques. The correlation coefficient, R, between the two techniques was 0.89.

The effect of low-level toluene exposure on the developing hippocampal region of the rat: histological evidence and volumetric findings.

With the intention of investigating possible morphological alterations effected by toluene in the developing CNS, rat pups were exposed to 100 ppm and 500 ppm of atmospheric toluene from postnatal day 1 until sacrifice at postnatal day 28, when the hippocampal region (area dentata, Ammon's horn, subiculum) was examined light microscopically and alterations in the volumes of the layers of the subdivisions were determined. The layers of Ammon's horn and the subiculum were not affected qualitatively or quantitatively by the 500 ppm exposure. Within the area dentata, the volume of the granule cell layer was 6% smaller in animals exposed to 100 ppm and 13% smaller in animals exposed to 500 ppm than they were in controls. The volumes of the hilus, which is a terminal field of granule cell axons, and the commissural-associational zone of the dentate molecular layer, which is the terminal field of the hilar projection to the granule cells, were smaller (12% and 19%) in animals exposed to 500 ppm than they were in controls. Argyrophilic cells were found in the granule cell layer of all animals exposed to 500 ppm. Pronounced granule cell degeneration was found in one animal exposed to 500 ppm. The granule cell layer of animals exposed to 100 ppm appeared qualitatively normal. The alterations reported here support the few earlier reports of morphological alterations in the CNS of adult laboratory animals. Effects of toluene similar to those described, that is alterations in specific neuron populations and their afferent and efferent terminal fields may complement changes in neurophysiology and behavior that have been observed in prenatally and perinatally exposed rodent pups. Causal relationships, however, remain to be elucidated.

The effect of toluene exposure for up to 18 months (78 weeks) on the (Ca2+/Mg2+)ATPase and fluidity of synaptosomal membranes isolated from rat brain.

The effect of toluene on the central nerve system was studied by using rat brain synaptosomal membranes as in vitro and in vivo models. The activity of Ca2+/Mg2+ ATPase and the membrane fluidity were determined. Short-term exposure in vivo to 500 p.p.m. of toluene had an inhibitory effect on the enzyme studied whereas long-term exposure to toluene caused an increased activity. Exposure to toluene had no effect at all on the membrane fluidity. The in vitro experiment showed an effect of toluene on both parameters. The alteration in the enzyme activity and membrane fluidity was parallel in the exposed animals as well as those of control. Our results show that long-term exposure to toluene affects nerve cell membranes by other mechanisms than those observed under in vitro conditions.

Calcium uptake in brain synaptosomes from rats exposed to daily toluene for up to 80 weeks.

Rats were exposed to toluene, 500 p.p.m., for 12 hrs/day for up to 80 weeks. The brains were removed and the synaptosomes were prepared. Potassium-stimulated and unstimulated synaptosomes were incubated with 45Ca2+ for 1/2, 2, 4, 8, and 16 min. Toluene exposure for 4 and 12 weeks caused a significant, approximately 20%, increase in 45Ca2+ uptake into unstimulated synaptosomes. The effect was of moderate quantity and transient, since it was not significant after 30 and 80 weeks of exposure. It seems doubtful whether the demonstrated change in calcium uptake should have any relevance in connection with the "organic solvent neurotoxicity syndrome".

The effect of long-term lithium treatment on the incorporation and distribution of 32P-orthosphosphate into the phospholipids from rat synaptosomes.

Rats were treated with lithium added to their diet for five weeks (40 mmol LiCl/kg diet). Mean plasma concentration was 0.45 mmol Li+/plasma. The investigation was divided into two sections. I) In an in vivo experiment in which the rats were injected with 32P-orthosphosphate for 20 hours, and with carbamoylcholine for 20 min. prior to their death, the distribution of 32P in the synaptosomal phospholipids, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylcholine (PC), was investigated. II) An in vitro experiment which was carried out in order to establish the effect of carbamoylcholine on the incorporation of 32P into total phospholipids from extracted synaptosomes. In I) the incorporation of 32P-orthosphosphate into PI from carbamoylcholine-stimulated rats was significantly lower than from unstimulated rats, whereas there was no difference between the incorporation of 32P into PI from synaptosomes from control animals and lithium-treated animals. In II) the incorporation of 32P-orthosphosphate was significantly lower in unstimulated synaptosomes from lithium-treated rats than from control rats, while the increase in the 32P-incorporation after stimulation followed the same pattern with regard to synaptosomes from control rats and lithium-treated rats. The results support the idea of lithium exerting an effect on basal synaptosomal activity but not on stimulated synaptosomal activity.

The effect of chronic lithium treatment on the sialic acid content in rat brain synaptosomes.

Rats were fed a lithium-containing diet for five weeks (40 mmol LiCl/kg fodder). Plasma lithium concentration was 0.44 mmol Li+/l plasma. The synaptosomes were isolated an incubated with 3H-glucosamine for 60 min. The glycopeptides and the gangliosides were extracted and the sialic acid content and the 3H-sialic acid content were measured. The lithium treatment produced an increase in the synaptosomal glycopeptide content. The sialic acid content was simultaneously increased in glycopeptides as well as gangliosides. The specific activity of sialic acid was reduced to 75% of the control value in the sialoglycopeptide fraction.

The effect of lithium on the incorporation of 3H-glucosamine into glycopeptides and the transformation of 3H-glucosamine into sialic acid in rat brain.

Rats were fed a lithium-containing diet (40 mmol LiCl/kg of diet) for five weeks. The plasma-lithium concentration was 0.48 mmol Li+/l of plasma. 3H-glucosamine (400 nCi/kg body weight was injected intraperitoneally, and 24 hrs later the brain was divided anatomically into hemisphere, cerebellum, pons and thalamus. The brain parts were defatted and the sialic acid-containing glycopeptides were separated by column chromatography before determination of 3H-glucosamine, 3H-sialic acid and total sialic acid. Neither the total amount nor the specific activity of sialic acid were influenced by the lithium treatment. Also the 3H-content of the sialic acid-free glycopeptides remained unchanged.

The effect of lithium on the incorporation of 32P-orthophosphate into synaptosomal phospholipids from rat brain.

Rats were treated with lithium added to the diet for five weeks (40 mmol LiCl/kg diet). The mean plasma lithium concentration was 0.48 mmol/l plasma, and the blood was drawn at 8 a.m. The brains were removed and synaptosomes were prepared according to the method of Gray & Whittaker (1962) and Bradford (1972). The synaptosomes were incubated for 120 min. with 32P-orthophosphate, either in a lithium-containing medium or in a lithium-free medium. The 32P-incorporation was lower in the synaptosomes from lithium-treated rats than the 32P-incorporation in synaptosomes from control rats regardless of the medium chosen. The results indicate that lithium treatment in vivo decreases the 32P-incorporation into synaptosomal phospholipids and that the effect remains after the removal of the lithium ion.

The influence of lithium on water binding ability, consistency and macromolecules in the rat brain.

Wistar male rats were treated with lithium for 50, 80, and 120 days, respectively. Lithium was added to the diet (40 mmol/kg of diet), the plasma lithium concentration being in the range of 0.55-0.70 mmol/1 of plasma. The treatment caused a significantly more rigid consistency of the brain tissue. The water content and water binding ability in various parts of the brain - measured by a drying course - was unchanged. The content of hexosamine and protein in total brains was significantly decreased (about 10 per cent) after the treatment. The content of chondroitin sulphate in various parts of the brain was unchanged, whereas the content of hyaluronic acid was significantly increased in the cerebellum (25 per cent) and in the thalamic area (20 per cent). The results suggest an influence of lithium on the macromolecules from the extracellular space, causing an alteration of the neuronal function.