1 Centre for Investigation, Hospital La Fe, Valencia, 2 Department of Cellular Biology and Anatomical Pathology, Faculty of Medicine, Institute for Biomedical Investigation August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona and 3 Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain
* Author to whom correspondence should be addressed at: Sect. Cell Biology and Pathology, Ctr Invest., Hospital La Fe, Av. Campanar 21, E-46009 Valencia, Spain. Tel.: +34 963 862700 (ext. 50411); Fax: +34 961 973018; E-mail: renau_jai{at}gva.es
(Received 27 October 2003; first review notified 9 December 2003; in revised form 3 January 2004; accepted 12 January 2004)
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ABSTRACT |
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INTRODUCTION |
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PEA produces a wide spectrum of alterations in offspring (fetal alcohol syndrome) including hepatic damage (Habbick et al., 1979; Lefkowitch et al., 1983
) and the fetotoxic effects of ethanol on the liver have been also demonstrated in animals (Renau-Piqueras et al., 1989a
). These alterations include a decrease in body weight and an increase in liver weight and total liver protein (Renau-Piqueras et al., 1997
). One of the most important effects induced by PEA on liver is the alteration of the GC in about 30% of hepatocytes (Renau-Piqueras et al., 1987
). PEA also results in a decrease in protein synthesis, a retention of newly synthesized glycoproteins, a decrease in galactosyltransferase activity and changes in the content of several carbohydrates in the hepatocyte (Renau-Piqueras et al., 1987
; Renau-Piqueras et al., 1989a
,b
).
However, the mechanisms behind PEA-induced glycoconjugate retention in hepatocytes remain unclear. As the biosynthesis of glycans is a process that occurs along the secretory pathway, the effect of ethanol could have several targets. Although there is a lack of evidence that the ethanol-induced alterations of the glycosylation process hinder protein trafficking, modifications of the protein glycosylation state sometimes affect the folding of a nascent protein and compromise its transport (Rasmussen, 1992).
These PEA-induced effects on liver could be the synergetic result of alterations in the glycosylation biosynthetic process and in components of the cytoskeleton involved in intracellular traffic. However, as far as we know there is no information that in fetal liver PEA perturbs the cytoskeleton, which in turn undergoes changes during the differentiation of hepatocytes (Pagan et al., 1996; Vassy et al., 1996
). Therefore, the present study aimed to examine the effect of ethanol intake on several components of the cytoskeleton and on the saccharide composition of glycoproteins in rat hepatocytes during development.
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MATERIALS AND METHODS |
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Chemicals for electron microscopy were from Electron Microscopy Science (Fort Washington, USA), and all the remaining chemicals from Sigma.
Animal treatment
Female Wistar rats weighing 200250 g were fed the Lieber-DeCarli (1976) diet. Female animals received the diet (control or ethanol) for a minimum of 40 days. After mating, the female rats were placed in cages with the same diet during gestation. Newborn rats were decapitated at 09.00 hours, and livers were removed and processed according to the techniques described (Renau-Piqueras et al., 1997
).
All experiments using rats were performed in compliance with the European Community Guide for the Care and Use of Laboratory Animals.
Sodium dodecyl sulphatepolyacrylamide gel electrophoresis and Western blot
Liver proteins were separated in sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) (Laemmli et al., 1970) with 10% polyacrylamide in the separate gel. After electrophoresis, the proteins were transferred to nitrocellulose paper. After blocking with 1% bovine serum albumin (BSA) in Tris buffer solution (TBS) 0.01%, membranes were incubated for 90 min with a primary antibody in the same buffer. Thereafter, the paper was incubated for 60 min with an acid phosphatase-conjugated second antibody. Gel quantification was performed using the Scion Image Beta 4.02 program (Scion, MD).
Purification of adult and newborn fetal rat liver tubulin
Microtubules were prepared by a modification of the temperature-dependent microtubule assembly and disassembly procedure (Gaskin and Cantor, 1974; Yoon et al., 1998
). Livers from pair-fed and ethanol-exposed adult animals, as well as from control and PEA newborn rats, were homogenized with an ultraturrax (2 x 30 s, maximum speed) in ice-cold PEM buffer (100 mmol/l K-PIPES, pH 6.9, 1 mmol/l EGTA and 1 mmol/l MgSO4) containing protease inhibitors (1 mmol/l phenylmethyl sulphonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml soybean trypsin inhibitors, and 1 µg/ml pepstatin A) and 0.1 mmol/l GTP. Homogenates were centrifuged at 4°C sequentially at 10 000 g for 30 min, at 80 000 g for 30 min and at 80 000 g for 60 min to obtain a clear supernatant, which was diluted in PEM up to 4 mg/ml. For microtubule polymerization this solution was incubated at 37°C for 15 min in 1 mmol/l GTP, 1 mmol/l dithioerithritol (DTTO) and 10 µmol/l paclitaxel (taxol; Sigma T-1791). After incubation the purified microtubules were obtained by centrifugation at 30 000 g for 30 min at 20°C in a solution containing 10% sucrose in PEM supplemented with 1 mmol/l GTP, 1 mmol/l DTTO, 10 µmol/l paclitaxel. The resulting pellet (MT and paclitaxel) was resuspended in 1/10 of the initial volume with PEM supplemented with 1 mmol/l GTP, 1 mmol/l DTTO and 10 µmol/l paclitaxel. The analysis of polymerized tubulin was carried out by Western blots.
Immunofluorescence
To analyse actin and intermediate filaments (IF) distribution, cytoskeleton preparations were obtained as described (Otha et al., 1988). To stain microtubules, frozen liver sections were transferred to glass slides, fixed and extracted with methanol at 20°C for 5 min, and stored at 80°C until used (Larkin et al., 1996
). Transferrin and albumin were analysed on frozen unextracted sections fixed in methanol.
Sections were incubated for 60 min with the primary antibody diluted in PBS containing 1% BSA, washed in the same buffer solution and then incubated with the FITC-conjugated second antibody for 60 min. Actin was visualized by incubating cells for 45 min with 0.1 µg/ml of TRITC-labelled phalloidin.
Pre-embedding immunogold of cytoskeleton proteins
Pre-embedding immunolocalization of -tubulin was performed as described (Langanger et al., 1984
). Cryostat sections (10 µm) were washed in a microtubule stabilizing buffer (MTBS) (Yoon et al., 1998
), fixed and extracted for 10 min in a 0.5% Triton X-100/0.3% glutaraldehyde mixture in MTBS, and washed in the same buffer. For the localization of cytokeratin and actin, sections were fixed for 5 min in 0.3% glutaraldehyde in PIPES. Both preparations were subsequently processed for immunocytochemistry (Renau-Piqueras et al., 1989c
)
Electron microscopy and immunogold staining
Rats were perfused with a solution of 0.5% glutaraldehyde plus 4% formaldehyde in 0.1 mol/l cacodylate buffer, pH 7.4. Livers were removed and fragments were immersed in the fixative for 120 min at 4°C, washed in the buffer, incubated for 45 min in NH4Cl, cryoprotected in sucrose, and cryofixed by plunging them into melting nitrogen. Cryosubstitution was carried out using a Leica EM AFS cryosubstitution system (Miñana et al., 2001) and samples were embedded in either Lowicryl K4M or Epon 812.
Transferrin and albumin distribution was carried out using the immunogold technique in both Lowicryl and Epon embedded samples (Miñana et al., 2001; Climent et al., 2002
). As it has been claimed that the main site of alcohol-induced glycoprotein retention in adult liver is the GC, we have quantitatively analysed the gold staining of transferrin and albumin in the GC. This analysis was performed as described (Renau-Piqueras et al., 1989c
, 1997
; Durán et al., 2003
). To assess the distribution of gold particles on microtubules, intermediate and actin filaments, micrographs of equivalent cell portions (the cell cortex, in the case of the actin) were taken following the systematic quadrat subsampling method (Cruz-Orive and Weibel, 1981
) and analysed using a test for clumping (Williams, 1977
; Renau-Piqueras et al., 1989c
). For this, a simple quadratic test system was superimposed onto micrographs and the mean gold particle number per quadrat was estimated; their distribution over the quadrats was evaluated using the variance/mean ratio (R). If this ratio is greater than one, a clumped pattern is indicated, whereas if it is less than unity the distribution would be regular. Significance was determined using the Student's t-test or by calculating the index of dispersion (I). The number of micrographs to be analysed (confidence limit ± 5%) was determined by the progressive mean technique (Williams, 1977
).
Lectin blot analysis
Lectin blotting was used both to differentiate between glycoproteins and non-glycosylated proteins as well as for the characterization of carbohydrate chains of glycoproteins (Tomás et al., 2002). In both cases, a labelling kit from Roche Diagnostics (Barcelona) was used (DIG Glycan Detection Kit, and DIG Glycan Differentiation Kit) following the manufacturer's instructions.
For glycan differentiation, we used the lectins Galantus nivalis (GNA), which labels terminal Man, (13),
(16) or
(12) linked to Man, and Maackia amurensis (MAA), which labels sialic acid
(23) linked to Gal.
Statistical analysis
Data are presented as means ± SD. All experiments were carried out with a sample size of at least four to six observations/group (four to six newborn animals from different ethanol-fed or control rats). Significant differences between group means were determined using the Student's t-test (P 0.05).
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RESULTS |
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Immunogold electron microscopy of cytoskeleton proteins
Analysis of micrographs of cytoskeleton (Fig. 6) using a test for clumping indicated that ethanol changed the distribution of cytokeratins and tubulin (Fig. 7). In contrast, no differences in the distribution of actin were observed. The analysis of cytokeratins indicates that the clumping was greater in cytoskeleton preparations of controls (Fig. 7), whereas an ethanol-induced increase in the R (variance/mean) value was observed in tubulin preparations.
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DISCUSSION |
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Carbohydrate composition of glycoproteins is altered by ethanol
Alteration of the protein glycosylation can modify the folding of a newly synthesized protein and thus compromise its transport (Rasmussen, 1992). Therefore, the reduced rate of secretion of several glycoproteins by several cell types, including hepatocytes, chronically exposed to ethanol may be attributed to ethanol-mediated defective glycosylation of these glycoproteins. We found that the carbohydrate moiety of glycoproteins is also modified by PEA, and thus we show that ethanol affects the levels of several GNA- or MAA-reactive glycoproteins. This effect was different in adult and neonatal rat hepatocytes, suggesting that the carbohydrate moiety of some glycoproteins in neonatal hepatocytes differs from that in adult livers, a finding in agreement with previous studies showing that pre-, postnatal and adult hepatocytes showed different sugar composition of the glycoproteins as well as significant variations in the glucosamine and galactosamine incorporation into proteins. Moreover, postnatal changes in the activity of sialyltranferase and galactosyltransferase in liver Golgi complex membranes have been also described (Leoni et al., 1990
; Oda-Tamai et al., 1991
). Thus, the different effects of ethanol exposure on carbohydrate composition in newborn and adult liver may be a result of the differences in both alcohol metabolism and glycoprotein biosynthesis in newborn compared to adult rats. However, although the mechanisms involved in these effects remain unclear, previous data demonstrate that ethanol-induced changes in the glycoconjugate moieties may well be due to alterations of several glycosyltransferases and/or glycosidases (Guasch et al., 1992
; Rosenberg and Noble, 1994
; Renau-Piqueras et al., 1997
; Lakshman et al., 1999
; Tomás et al., 2002
).
Prenatal exposure to ethanol alters tubulin content, microtubule polymerization and cytokeratins
The retention of protein in the liver of PEA animals could be due to an effect of ethanol on cytoskeleton. Therefore, we analysed the effect of this exposure on some of the main cytoskeleton proteins. In the present work and in previous studies on the effects of ethanol on microtubules in adult hepatocytes it has been demonstrated that tubulin levels are not reduced by ethanol, while a marked portion of this tubulin is polymerization incompetent (Yoon et al., 1998). In contrast, our results also indicate an ethanol-induced increase in the levels of tubulin in neonatal liver. We also tested the ability of isolated tubulin to polymerize and we observe that it is reduced in ethanol-exposed neonatal livers. This suggests that, as occurs in adult liver (Yoon et al., 1998
), a significant amount of tubulin is sensitive to ethanol exposure and assembly incompetent. This alteration could also be a consequence of the affinity of acetaldehyde to
-tubulin, particularly since acetaldehyde-conjugated
-tubulin inhibits tubulin assembly into microtubules (Hamm-Alvarez and Sheetz, 1998
). Our observations in liver using immunofluorescence agree with the results in hepatocytes in control and ethanol-exposed adult livers, wherein the subcellular distribution of microtubules was virtually the same (Yoon et al., 1998
). Conversely, the immunogold analysis, which enables better resolution than immunofluorescence, indicates that PEA induces a significant increase in the clustering of anti-tubulin binding sites, suggesting that ethanol modifies the organization of microtubules. These results are in contrast with studies using electron microscopy in that no differences in the structure of microtubules were observed between control and ethanol-exposed adult liver tissue (Berman et al., 1983
). In addition, we have also found that the amount of dynein and kinesin was similar in ethanol-exposed and control livers, this being consistent with results in adults (Torok et al., 1997
).
Using immunofluorescence we found that the distribution of cytokeratins in control and ethanol-exposed hepatocytes was similar to that described (Otha et al., 1988), and no differences between control and ethanol-exposed cells were observed. In contrast, PEA increases the level of cytokeratins and induces a redistribution of anti-cytokeratin binding sites on IF, which is in accordance to the reduced clustering of gold particles. Although the pathology of IF in PEA hepatocytes has yet to be studied, there is information on the effect of ethanol on cytokeratins in adult liver. Thus, in ethanolic hepatitis, hepatocytes form Mallory bodies (Denk et al., 2000
). In addition, ethanol alters the organization of liver cytokeratins, probably as a result of changes in cytokeratin phosphorylation (Sanhai et al., 1999
). However, it remains unclear whether this mechanism also occurs during development (Tan et al., 1993
; Perrone-Bizzozero et al., 1998
). As the content and distribution of cytokeratins in IF changes throughout liver development (Vassy et al., 1996
), it is possible that the effect of PEA consists of a delay in these changes. Indeed, we have demonstrated that PEA affects glial fibrillary acidic protein, the major IF protein in astrocytes, and delays the morphologic changes that these filaments suffer during the differentiation process (Renau-Piqueras et al., 1989c
). Previous studies have also demonstrated that ethanol exposure affects actin in several cell types (Renau-Piqueras et al., 1989c
; Banan et al., 2000
; Guasch et al., 2003
; Tomás et al., 2003
). In addition, acetaldehyde also forms interactions with muscle actin (Xu et al., 1989
). However, no modifications in the actin cytoskeleton distribution of hepatocytes from alcoholic fatty liver in rats have been observed (Otha et al., 1988
). Similarly, we have been unable to find effects of PEA either on the actin content or on the microfilament morphology.
In conclusion, our results show that in the liver ethanol exposure selectively affects the secretion of certain proteins, induces microheterogeneity in several glycoproteins, and alters the amount and/or distribution of tubulin and cytokeratins; this suggests that the ethanol-induced effects on developing liver could be the synergetic result of alterations in the glycosylation process and in the cytoskeletal components involved in intracellular traffic.
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ACKNOWLEDGEMENTS |
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