Prenatal exposure to ethanol alters the synthesis and glycosylation of proteins in fetal hepatocytes.

In the present work we have studied the effect of prenatal exposure to alcohol on the synthesis, glycosylation, and transport of proteins in fetal hepatocytes isolated from 21-day-old fetuses derived from control and chronic alcoholic rats. Protein synthesis was evaluated both in a cell-free system and in hepatocytes after (35S)methionine and (3H)leucine incorporation, respectively. Glycosylation was assessed using (3H)mannose and (3H)galactose as precursors. Protein synthesis was significantly decreased in treated hepatocytes. In control hepatocytes, quantitative electron microscope autoradiography showed that both (3H)leucine and (3H)mannose incorporation occur first in the rough endoplasmic reticulum (rER). Later the silver grains appeared over the Golgi apparatus, and, finally there was a transport towards the cell periphery. After pulse, silver grains corresponding to (3H)galactose incorporation appeared over the Golgi apparatus. The label then moved to the hepatocyte periphery. Alcohol treated hepatocytes showed a retention of grains over the Golgi apparatus with a diminution in the label at the cell periphery. These results indicate that prenatal exposure to alcohol induces a decrease in the synthesis of proteins in the hepatocyte as well as an alteration in the process of glycosylation and/or transport of secretory proteins.

Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.

We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.

Long-term high-protein diet induces biochemical and ultrastructural changes in rat liver mitochondria.

We have shown in previous work that long-term high-protein diet treatment (420 days) induces important biochemical and stereological changes in rat liver mitochondria. In this paper we have studied the time course for these changes in rats fed a high-protein diet for 30, 90, 180, and 420 days. The liver carbamoyl-phosphate synthetase I (ammonia), which represents 15-20% of the mitochondrial protein, increased ca. 2.5-fold in 30 days, with no further significant changes during the treatment. This increase was accompanied by an increment in the serum urea levels and a diminution in the half-life of blood urea, which could be interpreted as compensatory mechanisms for detoxification of blood and for maintaining osmotic pressure. The stereological study indicates that there is an enlargement of individual mitochondria in rats fed the high-protein diet, and that the maximum enlargement occurred at 90 days of treatment. The analysis of data shows, however, that the increase in mitochondrial volume density was due mainly to proliferation of normal mitochondria. These mitochondria were functionally normal as demonstrated by the unaltered P:O ratio during treatment. The total content of liver amino acids was increased, and the taurine/glycine ratio (which has been related to metabolic stress) was greatly increased. The possible correlation between the increases of both liver taurine levels and cell volume is discussed.

Chronic ethanol consumption affects filipin-cholesterol complexes and intramembranous particles of synaptosomes of rat brain cortex.

To assess the effect of ethanol on the planar distribution of cholesterol as well as on the surface architecture of presynaptic terminals of rats, synaptosomes isolated from cerebral cortex of rats chronically exposed to alcohol were incubated with filipin, a cytochemical marker for beta-hydroxycholesterol, and analyzed using both conventional (qualitative and quantitative) and freeze-fracture electron microscopy. Synaptosomes incubated in the absence of filipin were used as cytochemical controls. Biochemical determination indicates a 12% increase of cholesterol in synaptosomal membranes from alcohol treated rats. This increase was confirmed by a significant increment in the number of filipin-cholesterol complexes. Synaptosomes of treated rats showed a reduction in the total number of synaptic vesicles (SV) as well as a decrease in the density and total number of intramembranous particles (IMP) per synaptosome. In control rats, most synaptosomal IMP were distributed in clusters whereas in those of rats exposed to alcohol they were distributed at random. These changes in distribution of IMP were also observed in presynaptic terminals analyzed "in situ." These findings indicate that ethanol acts on the presynaptic terminals. The variations in cholesterol content as well as in the density and distribution of IMP appear to be related to alcohol-induced changes in the physicochemical properties of components of the synaptosomal membrane.

Prenatal exposure to ethanol alters lateral plasma membranes and gap junctions of newborn rat hepatocytes as revealed by freeze-fracture.

The effects of prenatal exposure to ethanol on the lateral (contiguous) plasma membrane of newborn rat hepatocytes was investigated using qualitative and quantitative freeze-fracture. The number of free intramembranous particles decreased by 11% and 17% in P- and E-faces, respectively. Alcohol also induced the appearance of particle-free areas within the gap junctions, in addition to a three-fold increase in the mean area and an increment in the percent of plasma membrane occupied by these elements. However, no differences in either the size of gap junction particles or in the mean interparticle distance were found between control and alcohol-treated animals. On the other hand, prenatal exposure to ethanol did not alter the structure of tight junctions.

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study.

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.

Effects of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma cells. A flow-cytometry and cytochemical study.

The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.

Ammonium chloride-induced alterations in growth kinetics and ultrastructure of murine neuroblastoma cells. A flow cytometric and stereologic study.

Neuro-2a and L-132 cells have been used as a model in the study of ammonia toxicity. Incubation of neuro-2a cells for 48 h in the presence of 2 mM NH4Cl caused inhibition of their growth and accumulation of cells in the G2M phase of the cell cycle as demonstrated by fluorocytometric methods. Mitotic figures were absent in the treated cell preparations. On the other hand, ammonia had no effect on L-132 cells treated in the same way. Electron microscopy of neuro-2a cells incubated with 2 mM NH4Cl for 5 days showed striking qualitative and quantitative ultrastructural changes compared with control cells. Treated cells doubled in absolute volume and showed marked alterations in the shape and organization of mitochondria. The absolute volume of mitochondria was also increased which, together with a decrease in their total number, suggests that ammonia induces fusion between adjacent mitochondria. Increases in the total number of lysosomes, multivesicular bodies and lipid droplets were also found in treated cells.

The effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules.

The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.

Alterations in the cytochemical activity of several phosphatases in hepatocytes from rats exposed prenatally to ethanol.

The activities of acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, uridine diphosphatase, inosine diphosphatase, thiamine pyrophosphatase and 5'-nucleotidase have been investigated cytochemically in hepatocytes of the offspring of alcohol-fed rats, using cerium ions as a capturing agent and qualitative and quantitative electron microscopy. All these enzyme activities were decreased in the experimental animals compared with controls not exposed to ethanol. The pattern of deposition of the product of glucose-6-phosphatase activity in the cisternae of the endoplasmic reticulum was also different in the two groups. The phosphatases analyzed are functional markers of different cell components, and the results suggest that prenatal exposure of rats to ethanol causes functional alterations in the endoplasmic reticulum, Golgi apparatus, lysosomes and plasma membrane of hepatocytes.