Lipid composition of liver peroxisomes isolated from untreated and clofibrate-treated mice and rats.

Peroxisomes were isolated from liver tissue of control and clofibrate-treated adult male NMRI mice and Sprague-Dawley rats. Phospholipids, cholesterol, triglycerides and free fatty acids were measured in the peroxisomes. The fatty acid profiles of the phosphatidylethanolamine, the phosphatidylcholine, the triglyceride and the free fatty acid fractions were also analyzed. Phosphatidylethanolamine was the dominating phospholipid in peroxisomes from untreated animals. The fatty acid profiles of phosphatidylethanolamine, free fatty acids and triglycerides were similar for untreated mice and rats but differences between the species were observed in the pattern derived from phosphatidylcholine. Phosphatidylcholine was the most abundant phospholipid after clofibrate treatment. Clofibrate treatment caused an increase in the concentrations of phospholipids and unsaturated long-chain fatty acids and a decrease in the concentrations of triglycerides, free fatty acids, cholesterol and shorter saturated fatty acids.

Perchloroethylene-induced reduction in glial and neuronal cell marker proteins in rat brain.

Rats were exposed to continuous inhalation of 300 and 600 p.p.m. of perchloroethylene for 4 and 12 weeks. Exposure to 600 p.p.m. for 4 to 12 weeks resulted in a slower increase in brain weight. Brain region weights, total proteins and DNA were decreased in frontal cerebral cortex and brain stem but not in hippocampus after exposure to 600 p.p.m. for 12 weeks. Four marker proteins were measured to monitor the specific neurotoxic effects of perchloroethylene: S-100 protein and glial fibrillary acidic protein as glial cell markers and neurone specific enolase and neurofilament 68 kD polypeptide as neuronal markers. The concentrations of glial and neuronal cytoskeletal proteins (glial fibrillary acidic protein and neurofilament 68 kD polypeptide) were reduced in frontal cerebral cortex. The total tissue contents of glial proteins (S-100 protein and glial fibrillary acidic protein) were decreased in all 3 brain regions investigated (frontal cerebral cortex, hippocampus and brain stem). Neurone specific enolase was unchanged by perchloroethylene exposure. These results indicate that exposure to perchloroethylene reduces the number of brain cells, possibly glial cells, and interferes with the metabolism of cytoskeletal elements in both glial and neuronal cells.

The use of experimental studies to reveal suspected neurotoxic chemicals as occupational hazards: acute and chronic exposures to organic solvents.

The nervous system differs from many other body organs by its central control of vital functions and its low regeneration capacity. Organic solvents have, as a group, been suspected to have neurotoxic effects. Because of their similar physical properties and the fact that in industrial uses, they are often present in various mixtures, organic solvents have also been regarded, unfortunately, to induce common neurotoxic effects. However, it is evident from experimental studies using specified exposure conditions that different organic solvents have very diverse neurotoxic effects and also that the toxic mechanism may differ between acute and chronic exposure. No specific method used to describe a neurotoxic effect or single toxic response can be used for the overall occupational risk assessment of all organic solvents. Each solvent has to be considered as having its own unique toxic effects.

Exposure of rats to high concentrations of 1,1,1-trichloroethane and its effects on brain lipid and fatty acid composition.

Exposure of rats to 1,1,1-trichloroethane (TRI) (1200 p.p.m.) for 30 days resulted in changes in the fatty acid pattern of the brain ethanolamine phosphoglyceride. A decrease was observed in stearic acid (18:0) and arachidonic acid (20:4), while the 22-carbon (n-3) fatty acids were increased. These changes in the fatty acid pattern were similar to that observed previously in the rat for another solvent, perchloroethylene, at a lower exposure concentration (320 p.p.m). Both these solvents are little metabolised and it seems that a common mechanism exists whereby these solvents alter the fatty acid pattern of brain phospholipid upon exposure. The relatively low uptake of TRI makes a high exposure level (1200 p.p.m.) necessary to attain a blood concentration high enough for the changes to appear.

Long-term exposure of rats to perchloroethylene, with and without a post-exposure solvent-free recovery period: effects on brain lipids.

The effects of perchloroethylene were studied in the rat brain after continuous exposure for 90 days at 320 ppm. Animals were also allowed after exposure to recover in a solvent-free atmosphere for 30 days. Lipid and fatty acid compositions were studied in particular. Changes were observed in the fatty acid pattern of ethanolamine phosphoglyceride in the cerebral cortex. Fatty acids 18:0, 20:1 (n-7) and 20:4 (n-6) were decreased while very-long-chain polyunsaturated fatty acids were increased. Most fatty acid changes were normalized during the 30 days post-exposure solvent-free recovery period with the exception of the minor fatty acid 20:1 (n-7). However, the previously unchanged fatty acid 18:1 was found to be decreased after the recovery period. Cholesterol was also decreased at this time and a tendency to reduction of myelin-enriched lipids after exposure to perchloroethylene might indicate a persisting loss of myelin membranes.

Brain lipid changes after organic solvent exposure.

Animals (rats, gerbils and guinea pigs) were exposed to various common solvents. The brain lipids were extracted and analysed. In particular the fatty acid pattern of a major phospholipid, ethanolamine, phosphoglyceride was studied. A major finding was that some chlorinated solvents, trichloroethylene, perchloroethylene and 1,1,1-trichloroethane, had effects on the ethanolamine phosphoglyceride fatty acid pattern, while most other solvents, like toluene, xylene, white spirit and Freon 11 lacked this effect. Relatively small, but significant, changes were observed in the proportions of the polyunsaturated fatty acids of both the n-6 and n-3 series. The selective effect of the chlorinated solvents might indicate that they have a specific effect on the enzymatic regulation of membrane fatty acid composition. However, the lack of effects of other solvents might also be explained by low blood levels, due either to a low uptake (Freon 11, white spirit) or an extensive metabolism (toluene, xylene). Intrauterine exposure of guinea pigs to perchloroethylene (160 ppm) during the last half of gestation had minor effects on the ethanolamine phosphoglyceride fatty acid pattern. Thus it appears that animals during the period of rapid brain growth are equally sensitive to exposure as adult animals.

Effects of exposure to Freon 11, 1,1,1-trichloroethane or perchloroethylene on the lipid and fatty-acid composition of rat cerebral cortex.

Organic solvents are often present as mixtures in various industrial and house-hold products. The adverse effects arising from exposure to these solvents have often been generalized to concern the whole group of solvents. In an examination of the possibility that organic solvents have general effects on experimental animals, rats were continuously exposed to vapors of the halogenated solvents Freon 11, perchloroethylene, and 1,1,1-trichloroethane. The lipid composition and fatty-acid pattern of ethanolamine phosphoglyceride from the cerebral cortex were analyzed. It was observed that only perchloroethylene had effects on the brain lipid composition. Cholesterol and total phospholipids were slightly reduced. Among the fatty acids the proportion of stearic acid was reduced and those of docosapentanoic, 22:5 (N = 6), and of docosahexanoic, 22:6 (N = 3), acids were increased. The changes in the fatty-acid pattern indicate that an alteration occurs in the desaturation of fatty acids. It seems probable that the chloroethylenes have specific effects on the fatty-acid pattern of brain phospholipids not shared by other solvents.

Brain lipid changes in rats exposed to xylene and toluene.

Rats were continuously exposed to vapors of xylene (320 ppm) for 30 or 90 days, other groups were exposed to toluene (320 ppm) for 30 days. After termination of exposure, different brain regions were removed for the determination of their lipid contents and ethanolamine phosphoglyceride fatty acid patterns. The 2 solvents had different effects on the animals. Xylene exposure resulted in limited transient changes. After 30 days exposure an increase in the liver to body weight ratio and a decrease in linoleic acid of ethanolamine phosphoglyceride in the cerebral cortex were observed. These changes were normalized after 90 days exposure. Toluene exposure, on the other hand, resulted in decreased weights of the body, of the brain as a whole and of the cerebral cortex. Liver weights were unchanged. Total phospholipids were found to be reduced in the cerebral cortex where also a slight increase in phosphatidic acid was observed. In this brain region a minor fatty acid of ethanolamine phosphoglyceride, 22:5 (n-3), was decreased. No changes were observed in the brainstem. The data on brain weights and lipid composition after exposure to toluene indicates a breakdown of phospholipids resulting in a loss of gray matter. The mechanism for these changes is uncertain but may involve degradation of phospholipids by phospholipase D. The effective metabolism of xylene and toluene seem to protect from fatty acid changes of brain phospholipids previously observed after exposure to chlorinated ethylenes.

Lipid composition and fatty acid pattern of the gerbil brain after exposure to perchloroethylene.

Continuous inhalation of perchloroethylene (PCE) (320 ppm) for 3 months by Mongolian gerbils resulted in an altered fatty acid pattern of a brain phospholipid. A minor decrease in the brain weight was also observed. In ethanolamine phosphoglyceride of the cerebral cortex and the hippocampus, a decrease was found among the minor fatty acids derived from linolenic acid with a corresponding increase in several fatty acids of the linoleic acid family. Linoleic acid itself was decreased. Stearic acid was also decreased in both the cerebral cortex and the hippocampus. These changes in the fatty acid pattern indicate increased desaturation. PCE might alter the desaturase activity either directly by interfering with the protein moieties of the enzyme system, or indirectly by changing the properties of the lipid matrix. The observed changes in fatty acid composition are also consistent with the current hypothesis that solvents and anesthetics perturb the lipid matrix of membranes, possibly inducing complex compensatory changes in the membrane lipid composition.

Two procedures to remove polar contaminants from a crude brain lipid extract by using prepacked reversed-phase columns.

Two procedures were developed using prepacked, reversed-phase columns (Bond Elut) for the separation of lipids from water-soluble contaminants. A crude lipid extract from brain tissue was diluted stepwise with a methanol/water (method or a methanol/saline (method mixture and, with each step, was passed through the column. As the polarity of the solvent was increased, all lipids became bound to the column, while the water-soluble compounds remained in the eluate. After three subsequent dilutions and column elutions, the eluate containing the more polar contaminants was discarded. The bound lipids were then eluted with a small volume of chloroform/methanol (1:2, v/v). Alternatively two fractions were eluted, the first fraction eluted with methanol/water (12:1, v/v), contained gangliosides, phosphatidylserine, phosphatidylinositol, phosphatidic acid and sulfatides. The second fraction, eluted with chloroform/methanol (1:2, v/v), contained all remaining phospholipids, cerebrosides and cholesterol. For both methods a quantitative recovery of cholesterol and phospholipids was obtained. In method 2, when water was replaced by saline in the dilution solvent mixture, gangliosides were also bound and quantitatively recovered.

Fatty acid changes in rat brain ethanolamine phosphoglycerides during and following chronic exposure to trichloroethylene.

Rats were exposed by continuous inhalation to a moderate level of trichloroethylene (1720 mg/m3). The fatty acid pattern of brain ethanolamine phosphoglycerides was examined during exposure and after an additional exposure-free period. Alterations in the fatty acid pattern were noted after 30 days of exposure. An increased ratio of linoleic acid-derived (n-6) to linolenic acid-derived (n-3) fatty acids was observed in the cerebral cortex, the hippocampus, and the brain stem. Of the major fatty acids, arachidonic acid (20:4(n-6)) was increased in the cerebral cortex and the brain stem, while docosahexenoic acid (22:6(n-3)) was decreased in the cerebral cortex and the hippocampus. A further change in these fatty acids was observed in the cerebral cortex following a longer exposure period of 90 days. The 22-carbon linoleic acid-derived fatty acids were also increased after 90 days of exposure. These findings imply that trichloroethylene affects the metabolism of ethanolamine phosphoglyceride fatty acids in the rat brain by inhibiting desaturation of the linolenic acid family and by increasing desaturation of the linoleic acid family. The effect of trichloroethylene was partially reversible, since a postexposure solvent-free period revealed a rapid partial normalization of 22:6(n-3), which is the most important fatty acid of the linolenic acid family in the rat brain. However, the precursor of this fatty acid, 22:5(n-3), was decreased during the first 10 exposure-free days. This suggests that desaturation over the first steps was still impaired. A complete normalization of the fatty acid pattern was not observed during the 30-day solvent-free period. The decreased number of double bonds and shorter chain lengths detected after solvent exposure is consistent with the idea of a compensatory remodeling of membrane lipid composition based on membrane stability with regard to phase preference.

Membrane lipid changes in organic solvent tolerant neural cells.

Although, organic solvents are known to interact with neural membranes and to possess acute anesthetic properties, very little is known about the chronic effects of organic solvents on neural membranes. In the present study lipid compositional changes were examined after chronic exposure to the chlorinated organic solvents, trichloroethylene (TCE) and perchloroethylene (PCE). Animals were exposed continuously in inhalation chambers. Each experimental group had its own control group exposed simultaneously to air under identical conditions. Exposure to these organic solvents had only minor effects on lipid class composition. A tendency towards a decreased cholesterol to phospholipid ratio was observed, whereas the content of cerebrosides was unaffected. However, the proportions of the polyunsaturated fatty acids of ethanolaminephosphoglyceride (EPG) was consistently changed after exposure to either of these solvents for three months at (320 ppm). After longer exposure periods such alterations were observed at even lower solvent concentrations. This change in fatty acid pattern was characterized by an increase in linolenic acid derived fatty acids, with a concomitant decrease in fatty acids of the linolenic acid family. These changes may be an effort to compensate for the increased hydrophobic volume of membranes induced by dissolved solvent molecules. This compensation could be achieved by insertion of less coned lipid components into the membrane. The observed decrease in cholesterol to phospholipid ratio and the increase in the less saturated linoleic acid derived fatty acids could serve this function. Another possibility could be that chlorinated organic solvents interfere with the protein structure of desaturases, resulting in an alteration in the speed of desaturation and a changed fatty acid composition.

Fatty acid composition of ethanolamine phosphoglycerides in different areas of the gerbil brain after chronic exposure to trichloroethylene.

Exposure of Mongolian gerbils to trichloroethylene (TCE) (320 ppm) in an inhalation chamber continuously for 3 mo resulted in an altered fatty acid pattern of phospholipid in discrete areas of the brain. Lipids were extracted from four brain regions: the cerebral cortex, the hippocampus, the cerebellar vermis posterior, and the brain stem. No changes induced by TCE were found in lipid class distribution among the different regions examined. Whole brain weights and weights of the dissected pieces were also unchanged. In ethanolamine phosphoglycerides from the cerebral cortex and the hippocampus, a decrease was found among long-chain fatty acids derived from linolenic acid with a corresponding increase of the linoleic acid family. The cerebellar vermis and the brain stem were less affected. Since areas rich in gray matter were affected more than those with a high proportion of white matter, it seems reasonable to assume that the fatty acid alterations of ethanolamine phosphoglycerides occur mainly in the gray matter. Furthermore, we suggest that the alterations can be a compensatory mechanism for the membrane fluidizing properties of TCE.

Chronic effects of perchloroethylene on the composition of lipid and acyl groups in cerebral cortex and hippocampus of the gerbil.

Continuous exposure of Mongolian gerbils to perchloroethylene (PCE) (120 ppm) for 12 months in an inhalation chamber caused no changes in body or brain weights. The protein content, the concentration of lipid phosphorus or cholesterol were unaltered in the cerebral cortex and hippocampus. However, a small change in the fatty acid pattern of phospholipid was observed. In the phosphatidylethanolamine of cerebral cortex and hippocampus a decrease was found among the long-chain, linolenic acid-derived, fatty acids. The ratio 22:4 (N-6)/22:5 (N-3) was increased, indicating a shift towards the corresponding linoleic acid-derived 22-carbon fatty acids. The observed changes among poly-unsaturated fatty acids are similar to those appearing after peroxidation and either protein or essential fatty acid malnutrition. However, an attractive explanation for the changes is that they represent a response to the fluidizing properties of PCE.

Chronic effects of trichloroethylene upon S-100 protein content and lipid composition in gerbil cerebellum.

Gerbil rats were exposed to trichloroethylene (TCE) vapors intermittently (8 h/d) at 510 ppm or continuously at 170 ppm for five months. The cerebellar content of S-100 protein and the phospholipid fatty-acid profiles were determined. S-100 protein, a possible marker for astrocytic reactivity, indicated delayed astrocytic reactivity in the anterior cerebellar hemisphere and a decrease of S-100 protein in the posterior cerebellar vermis. Minor lipid changes were observed. The fatty-acid profiles of ethanolamine phosphoglycerides showed a tendency towards alterations among the 22-carbon fatty acids, with a decrease in 22:5 (N-3), similar to those shown earlier for cerebral cortex and hippocampus of the gerbil. Two monoenoic fatty acids were decreased, the 20:1 of phosphatidyl-ethanolamine and the 18:1 of the phosphatidyl-serine. This occurrence could indicate a decrease in myelin in areas where these two fatty acids were found to be enriched.

Chronic exposure to trichloroethylene: lipid and acyl group composition in gerbil cerebral cortex and hippocampus.

Inhalation exposure of Mongolian gerbils to trichloroethylene (50 and 150 ppm) for 12 months caused only small changes in the lipid composition of their cerebral cortex and hippocampus. Total protein content and lipid class distribution were almost unaffected. However, the cholesterol to phospholipid ratio decreased in the cortex of animals exposed to trichloroethylene. This might be due to changes in the relative proportions of different membranes or cellular types. The finding of most interest was among the long-chain polyunsaturated fatty acids where changed ratios were found between long-chain derivatives of the linoleic and the linolenic acid series and a decrease in two minor fatty acids, 20:5 (n-3) and 22:5 (n-3). We think it is likely that such changes occur as a result of peroxidation of polyunsaturated fatty acids or in order to withstand the fluidizing properties of trichloroethylene.