Alcoholic liver disease and apoptosis.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol A. Casey and Amin Nanji. The presentations were (1) Mechanisms of apoptosis in alcoholic liver disease, by Amin A. Nanji; (2) Impaired receptor-mediated endocytosis: Its role in alcoholic apoptosis, by Carol A. Casey; (3) Toxicity of ethanol in HepG2 cells that express CYP2E1, by Arthur I. Cederbaum; (4) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis, by M. Adachi; and (5) Apoptosis in alcoholic hepatitis, by T. Takahashi.

Hyperphosphorylation of the asialoglycoprotein receptor in isolated rat hepatocytes following ethanol administration.

Ethanol administration leads to altered function and impaired receptor-mediated endocytosis of the hepatocyte asialoglycoprotein receptor (ASGP-R). The purpose of the present study was to examine the effects of ethanol on the phosphorylation of the ASGP-R to determine whether this post-translational modification could contribute mechanistically to the observed ethanol-induced alterations. The methodological approach of this work involved the measurement of the phosphorylation state of the receptor obtained from isolated rat hepatocytes, using a combination of experimental designs from the biosynthetic incorporation of phosphate to the determination of steady-state phosphotyrosine levels. We report here that both short-term (1- to 2-week) and chronic (5- to 7-week) periods of ethanol administration resulted in a significant increase in the steady-state phosphotyrosine protein in the ASGP-R. In addition, in vitro incorporation of [gamma-(32)P]ATP using a permeabilized cell assay system similarly showed an increase in tyrosine-phosphorylated receptors. Furthermore, metabolic radiolabeling of hepatocytes with [(32)P]orthophosphate demonstrated hyperphosphorylation of the ASGP-R in cells obtained from chronically ethanol-fed animals. Finally, our results revealed that dephosphorylation of the ASGP-R was unaffected by ethanol administration, indicating that kinase activity rather than impaired phosphatase action contributes to the increased phosphorylation state of the receptor. Overall, the results presented in this study demonstrated that the extent of tyrosine phosphorylation of the receptor is significantly higher in hepatocytes obtained from ethanol-fed animals. We conclude that hyperphosphorylation of the ASGP-R may be a contributing factor to the impaired function of the receptor elicited by ethanol administration.

Ethanol-impaired hepatic protein trafficking: concepts from the asialoglycoprotein receptor system.

OBJECTIVES: Alcohol abuse with its resulting liver injury is a major health problem worldwide. Recent studies have shown that the process of receptor-mediated endocytosis (RME) is especially susceptible to the deleterious effects of ethanol. DESIGN AND METHODS: In our laboratory, we have shown that after as early as 1 week of ethanol administration, binding, internalization and degradation of asialoorosomucoid, a ligand for the asialoglycoprotein receptor (ASGP-R), is significantly impaired. We have also demonstrated that ethanol administration impairs ATP-dependent acidification of prelysosomal endosomes. RESULTS: These impairments are seen using ligands internalized by the non-specific process of fluid phase endocytosis as well as those internalized by coated pit endocytosis. In addition, we have identified ethanol-induced alterations in post-translational modifications of the receptor including phosphorylation and fatty acid modification (palmitoylation). CONCLUSIONS: Impaired function of this receptor could lead to alterations of membrane internalization events after ethanol administration and contribute to ethanol-induced alterations in protein trafficking and signaling in the liver.

Effects of ethanol on receptor-mediated endocytosis in the liver.

Ethanol administration impairs multiple aspects in the process of receptor-mediated endocytosis (RME) in the liver. Studies from our laboratory over the last 10 years have carefully examined RME by the hepatocyte-specific asialoglycoprotein receptor (ASGP-R). We have identified a time course for ethanol-induced defects in RME and established that many of the impairments occur initially in the centrilobular region of the liver and as early as one week after ethanol administration. Impaired intravesicular acidification in ethanol-fed animals has been identified, and these defects in acidification could alter multiple protein trafficking pathways including RME. In addition to altered acidification, altered receptor function (including receptor inactivation) could also contribute to impaired trafficking. Current studies in our laboratory are aimed at an examination of posttranslational modifications in the receptor (acylation and phosphorylation) that are known to affect its function. A role for the ASGP-R in the process of alcoholic apoptosis is also being examined because proper functioning of the ASGP-R is thought to be important in clearance of apoptotic cells.

Differential effects of monensin on asialoglycoprotein receptor function after short-term ethanol administration.

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) by the hepatic asialoglycoprotein receptor (ASGP-R). Previous work on this receptor has shown that its activity can be perturbed by the carboxylic ionophore monensin. This agent has been shown to preferentially affect receptor-ligand dissociation and receptor redistribution of one subset (State 2) of ASGP-R, while receptor function in a second subset (State 1 receptors) is unaffected. In the present study, we examined the effect of monensin on ASGP-R activity and intracellular receptor-ligand dissociation after 7-10 days of ethanol feeding, a time when we have shown altered ASGP-R function in ethanol-fed animals. Hepatocytes from male Wistar rats (fed an ethanol-containing or control diet) were utilized. Ethanol administration decreased total ligand binding by 35-40% (P < 0.01) without a change in receptor protein content. After monensin treatment, surface receptors on cells from control animals were inactivated and redistributed to the cell interior. In cells from ethanol-fed animals, a similar pattern of monensin-induced inactivation was shown, but no redistribution occurred. Intracellular receptor-ligand dissociation was impaired in both cell types, although the monensin-induced effect on dissociation was significantly less dramatic (two-fold) in the hepatocytes from ethanol-fed animals as compared with controls. Thus, although receptors on both cell types were susceptible to monensin, cells from the ethanol-fed animals were less vulnerable to the added effects of this agent. Since monensin affects functioning of State 2, but not State 1 receptors, a very early effect of ethanol may be a preferential impairment in the State 2 receptor population.

Chronic ethanol ingestion impairs TGF-alpha-stimulated receptor autophosphorylation.

The effects of chronic ethanol feeding on the binding of transforming growth factor-alpha (TGF-alpha) and TGF-alpha-stimulated receptor autophosphorylation were investigated in isolated rat hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 6-8 weeks, these cells exhibited a marked impairment of TGF-alpha-stimulated autophosphorylation of the receptor that binds this growth factor compared with hepatocytes from the pair-fed controls. This impaired autophosphorylation of receptor tyrosine residues was accompanied by significant decreases in the amount of surface-bound TGF-alpha. Immunoanalysis indicated no changes in receptor number, indicating that decreased receptor content was not responsible for decreased TGF-alpha binding in the hepatocytes from the ethanol-fed rats. In conclusion, chronic ethanol feeding reduced TGF-alpha binding to hepatocytes with a concomitant decrease in the ability of the receptor tyrosine kinase to autophosphorylate its tyrosine residues. These changes were not accompanied by decreased receptor protein content. These defects could lead to altered signal transduction and to impaired reparative and regenerative processes in the liver.

Effects of monensin on ethanol-induced alterations in function of hepatocellular asialoglycoprotein receptor subpopulations.

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) in hepatocytes. RME mediated by the asialoglycoprotein receptor seems to be especially impaired by ethanol. In the present study, we determined susceptibility of RME to alterations in ethanol-fed and pair-fed control animals by the addition of a carboxylic ionophore, monensin. Monensin inhibits acidification of prelysosomal vesicular compartments, which results in a decrease in the rate of receptor-ligand dissociation within the cell. Low levels (25 microM) of monensin have been shown to preferentially affect receptor-ligand dissociation of one subset (state 2) of asialoglycoprotein receptor, whereas dissociation in a second subset (state 1 receptors) seems to be relatively unaffected. We examined whether ethanol treatment preferentially affected one or another of these receptor subpopulations. Male Wistar rats were fed a standard ethanol-containing (36% of calories) or control diet for 10 to 14 weeks, and hepatocytes were prepared from the animals. Similar to previous results from our laboratory, surface and total ligand and antibody binding were decreased by 30 to 45% (p < 0.01) in ethanol animals, compared with controls. An ethanol-induced impairment of receptor-ligand dissociation was also apparent in these cells, as shown by increased ratios of bound-to-free ligand during continuous endocytosis. After monensin treatment, surface receptors on both ethanol and control cells showed a similar pattern of redistribution to the cell interior and intracellular inactivation. When kinetics of intracellular receptor-ligand dissociation were examined upon addition of monensin, the bound-to-free ligand ratios in both control and ethanol cells increased dramatically and to an equal extent. These results indicate that, in the ethanol animals, the pattern of sensitivity to monensin is unchanged relative to controls. Thus, the relative proportion of state 1 and state 2 receptor populations do not seem to be affected after long-term feeding, and ethanol may be a perturbant that affects both state 1 and state 2 receptor function.

Ethanol feeding causes inactivation of both state 1 and state 2 rat hepatic asialoglycoprotein receptors.

Previous studies have shown that ethanol feeding in rats causes inactivation and redistribution of approximately 50% of the total asialoglycoprotein receptors (ASGPRs) in hepatocytes (Tworek et al., J. Biol. Chem. 271:2531, 1996), and that two equal populations of hepatic ASGPRs mediate ligand uptake and processing via two functionally different pathways (Weigel in Glycoconjugates: Composition, Structure and Function, Marcel Dekker, 1992, p. 421). The purpose of this study was to determine if ethanol feeding causes preferential inactivation of only one of these two ASGPR populations, which have been designated state 1 and state 2 ASGPRs. The state 2, but not state 1, ASGPRs are inactivated in isolated hepatocytes by a variety of drugs and inhibitors. State 2 ASGPRs can also be inactivated in permeable cells by ATP treatment and then reactivated by treatment with fatty acyl coenzyme As. In the present study, permeable cell assays for state 2 ASGPR inactivation and reactivation were used to assess whether hepatocytes from ethanol-fed rats contain inactive state 2 ASGPRs. The results show that preferential inactivation of one ASGPR population does not occur after ethanol feeding. That inactive ASGPRs could not be reactivated by treatment with palmitoyl-coenzyme A to a greater extent in ethanol-fed versus control cells indicates there is not a larger pool of inactivated state 2 ASGPRs in treated cells. We conclude that ethanol feeding causes equal inactivation of both state 1 and state 2 ASGPRs. Ethanol feeding may represent the first treatment found to inactivate state 1 ASGPRs.

Decreased binding and autophosphorylation of the epidermal growth factor receptor in ethanol-fed rats.

We have shown previously that binding and processing of epidermal growth factor are impaired in livers of ethanol-fed rats. In the current study, we examined these ethanol-induced alterations in greater detail by studying both high and low affinity epidermal growth factor binding as well as the ability of added ligand to stimulate receptor autophosphorylation. We also measured the binding of anti-receptor antibody to intact and permeabilized cells in order to determine more accurately the levels of receptor protein. Hepatocytes were isolated from ethanol-fed and pair-fed control rats. Ligand binding, antibody binding, and ligand-induced receptor autrophosphorylation were measured in the respective cell populations. In ethanol-fed animals, binding to both high and low affinity states of the hepatic epidermal growth factor receptor was decreased by 40-50% (P < 0.01). This ethanol-induced decrease in ligand binding was accompanied by a reduced ability of epidermal growth factor to stimulate receptor autophosphorylation (32% decrease, P < 0.01). In contrast, binding of anti-receptor antibody was not altered in ethanol-fed animals. In conclusion, chronic ethanol feeding decreased epidermal growth factor binding with a concomitant decrease in the ability of the receptor tyrosine kinase to phosphorylate tyrosine residues. These changes were not accompanied by an actual decrease in receptor protein content. These findings could be relevant to modified responses to this growth factor in the livers of chronic ethanol-fed animals.

Zonal differences in ethanol-induced impairments in fluid-phase endocytosis in rat hepatocytes.

We have previously shown that ethanol administration impairs the processes of fluid-phase endocytosis (FPE) and receptor-mediated endocytosis (RME) in isolated rat hepatocytes after as early as 1 week of ethanol administration. The defects in RME were most prominent in the perivenule (PV) region of the liver lobule, the area wherein alcoholic liver disease has been shown to start and predominate. We undertook the present study to see if changes in FPE were likewise more apparent in the PV versus the periportal (PP) region of the liver. For these studies, we fed male Sprague-Dawley rats with an ethanol-supplemented liquid diet or an isocaloric control diet for 1 or 5 weeks. PV and PP hepatocytes were isolated using a digitonin-collagenase perfusion method. Internalization and efflux of the marker dye, Lucifer Yellow, was then examined in the cell populations. After as early as 1 week of feeding, cells from the PV region in ethanol-fed animals showed dramatic impairments in the net internalization of dye, compared with PV cells from the pair-fed controls, and these changes persisted throughout the 5-week feeding period. In contrast, internalization of Lucifer Yellow in cells from the PP region of the liver were not different between control and ethanol animals. Because net internalization represents the balance between uptake into the cells versus efflux from the cells, we examined these components individually. Early uptake of the dye into the cell was not altered by ethanol treatment. The decreased net internalization seemed to be caused by enhanced efflux of the dye, which was significantly increased in PV cells, compared with the same cell type in control animals. Cells from the PP region of the ethanol-fed animals did not exhibit altered efflux after either 1 or 5 weeks of feeding. These results indicate that ethanol-induced impairments in FPE are more dramatic in the PV region of the liver, and these impairments seem to result from an ethanol-induced enhancement of efflux.

Decreased binding of asialoglycoproteins to hepatocytes from ethanol-fed rats. Consequence of both impaired synthesis and inactivation of the asialoglycoprotein receptor.

Chronic ethanol administration alters the process of receptor-mediated endocytosis in isolated rat hepatocytes. Using the asialoglycoprotein receptor (ASGP-R) as a model, we have previously shown decreased binding of asialoglycoproteins to this receptor after as early as 1 week of ethanol administration. In the present study, we further analyzed the mechanism(s) responsible for this impairment by determining the ligand and antibody binding characteristics of the ASGP-R in rats fed ethanol over a 5-week time course. The results presented here demonstrate that ethanol treatment for 4 days significantly impaired total ligand binding without affecting antibody binding. Ethanol administration for a longer period of 1-2 weeks resulted in intermediate impairments in both ligand and antibody binding. After 5 weeks of ethanol exposure, ligand and antibody binding were equally lowered. In contrast to total cellular receptor binding, surface binding of both ligand and antibody were decreased over the entire time course of ethanol administration. Our data indicate that the ASGP-R is initially inactivated during the time course of ethanol exposure and that a redistribution of surface receptors to intracellular compartments occurs. Northern blot analysis showed that there was a significant decrease in receptor mRNA content in the 5-week chronically fed animals but not in the animals fed for 1 week. In addition, after 5 weeks of ethanol feeding, biosynthetic labeling of the ASGP-R was decreased in the ethanol cells, indicating impaired synthesis of the ASGP-R. In summary, an early inactivation of the ASGP-R occurs during ethanol exposure followed by an actual decrease in protein and mRNA content for the receptor.

Effect of hyperosmolarity on both receptor-mediated and fluid-phase endocytosis in ethanol-fed animals.

We have shown previously that chronic ethanol administration impairs hepatic receptor-mediated endocytosis (RME) of asialoorosomucoid (ASOR), epidermal growth factor and insulin, whereas early uptake by fluid-phase endocytosis (FPE) of a fluorescent dye, Lucifer Yellow (LY), is not altered. Results of these studies suggested that ethanol-induced injury was primarily affecting endocytosis in coated pit areas of the plasma membrane while internalization in noncoated membrane areas was unaffected. In the present study, we investigated the effects of blocking clathrin-coated pit mediated endocytosis by hyperosmolarity on FPE of LY and on RME of ASOR. We also examined the effects of hyperosmolarity on the binding and internalization of insulin, a ligand endocytosed by both RME and FPE. Uptake of LY by noncoated regions of the membrane was not altered in control animals, whereas in hepatocytes from ethanol-fed animals uptake of LY was decreased by 35-40% in the presence of 0.12 M sucrose (P < 0.05). These hyperosmolar conditions almost completely inhibited (> 85%) the endocytosis of 125I-ASOR by RME in both ethanol and control cells. Results with insulin showed slight effects (20-30% impairment) on uptake of the ligand in the presence of sucrose. These results are consistent with previous reports that in normal cells the coated pit pathway is impaired by hyperosmolarity, whereas endocytosis in noncoated regions is unaltered. It appears, however, that both FPE and RME in hepatocytes from ethanol-fed animals are susceptible to perturbation by hyperosmolarity. These results indicate that the noncoated pit pathway may be sensitive to stressful conditions such as hyperosmolarity after ethanol treatment.

The Semmes-Weinstein monofilament as a potential predictor of foot ulceration in patients with noninsulin-dependent diabetes.

The objective was to evaluate the Semmes-Weinstein monofilament as a potential predictor of foot ulceration. A case-control study was carried out in a teaching hospital clinic specializing in diabetes and hyperlipidemic disorders. Two groups of patients with noninsulin-dependent diabetes mellitus were studied: 168 with no history of foot lesions (No-Ulcer group, aged 58.1 +/- 9.7 years, duration 11.5 +/- 7.8 years) and 14 who had at least one foot ulceration during the last year (foot ulcer group, aged 60.8 +/- 7.8 years', duration 17.1 +/- 10.5 years). The authors defined and measured a "monofilament index" using a size 5.07 Semmes-Weinstein monofilament on three locations on each foot and then measured peroneal nerve current perception thresholds using the Neurometer at three frequencies: 5 Hz, 250 Hz, and 2,000 Hz. The monofilament index score was lower in patients in the foot ulcer group than in patients in the No-Ulcer group (1.71 +/- 2.36 vs 5.21 +/- 1.81, P < 0.001). A cutoff point at an index of 5 out of 6 yielded a sensitivity rate of 85.71% and a false-positive rate of 16.07%. The peroneal nerve current perception threshold was higher (P < 0.005) in patients in the foot ulcer group than in patients in the No-Ulcer group for all frequencies (605.8 +/- 414.6 vs 181.5 +/- 272.3 for 5 Hz; 743.2 +/- 361.7 vs 251.6 +/- 283.7 for 250 Hz; 859.4 +/- 220.2 vs 423.3 +/- 252.6 for 2,000 Hz). (ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ethanol feeding on the interaction of rat hepatocytes with laminin peptides.

Laminin, a complex glycoprotein of the extracellular matrix, contains a number of biologically active sites. These sites are involved in cell growth, attachment, differentiation, and gene expression. Our previous studies have shown that chronic ethanol consumption by rats impairs hepatocyte attachment to various components of the extracellular matrix including laminin. In this study, three synthetic peptides (PA22-2, YIGSR, and RGD) that correspond to three distinct functional sites on the laminin molecule were used to investigate the effect of ethanol consumption on their cognate receptors. Initially, varying concentrations of each peptide were incubated with isolated hepatocytes from ethanol-fed and pair-fed control rats. These hepatocytes were then assayed for the ability to attach to laminin. The results indicated that all three peptides effectively inhibited laminin-mediated cell adhesion: the degree of inhibition appeared similar between pair-fed controls and ethanol-fed animals. Of the three peptides, PA22-2 showed the most dramatic inhibition of attachment. Therefore, we investigated the ability of hepatocytes to attach directly to PA22-2 itself. Attachment of hepatocytes from ethanol-fed animals to PA22-2 was impaired by 30% after 4 days and 90% by 14 days. Conversely, no significant difference in attachment to the entire laminin molecule was observed in ethanol-fed animals at these early time points. These results indicated that the ethanol-induced impairment of hepatocyte attachment to laminin may be caused by the decreased interaction of hepatocytes with specific functional sites on the laminin molecule and that specific receptors on the hepatocyte may be affected differently.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term ethanol feeding selectively impairs the attachment of rat perivenous hepatocytes to extracellular matrix substrates.

BACKGROUND/AIMS: We have previously shown that long-term ethanol consumption by rats results in a profound decrease in hepatocyte attachment to various extracellular matrix substrates. The present study investigated whether differences in attachment exist between cells isolated from either the periportal or perivenous regions of the liver. METHODS: Rats received long-term ethanol, and hepatocytes were selectively isolated by the digitonin-collagenase perfusion method. The ability of periportal and perivenous cells isolated from ethanol-fed and pair-fed control rats to attach to plates coated with either laminin, fibronectin, or type I collagen was then assayed. RESULTS: With all substrates, the attachment of perivenous hepatocytes isolated from ethanol-fed animals was significantly impaired. Time-course studies showed that although the rate of attachment of perivenous cells from ethanol-fed animals was only slightly reduced, a dramatic decrease in absolute attachment was observed. Furthermore, the perivenous cells isolated from ethanol-fed animals detached more readily from the substrate-coated plates than the corresponding periportal cells or either periportal or perivenous cells from pair-fed controls. CONCLUSIONS: Long-term ethanol consumption impairs hepatocyte-extracellular matrix interactions more severely in the perivenous region of the liver. This finding could be relevant to the pathological changes observed in alcoholic liver injury.

Chronic ethanol administration impairs degradation of formaldehyde-treated albumin by the perfused rat liver.

Nonparenchymal cells of the liver appear to be important in the pathogenesis of various liver diseases, including that caused by ethanol. It is known that chronic ethanol administration impairs the process of receptor-mediated endocytosis in hepatocytes. Liver endothelial cells are also actively endocytic cells, playing a prominent role in the clearance from the circulation of a variety of macromolecules. In this study, we assessed the effect of ethanol administration on this "scavenger" function of liver endothelial cells by measuring the degradation of formaldehyde-treated albumin in isolated, perfused livers of ethanol-fed rats. Rats were pair-fed for 1 or 4 weeks with a liquid diet containing either ethanol as 36% of total calories or an isocaloric amount of carbohydrate. Chronic ethanol administration in this manner for 1 or 4 weeks significantly impaired the degradation of this endothelial cell ligand (by 60 +/- 9% and 37 +/- 9%, respectively). Liver perfusions were also performed on rats that had been administered ethanol acutely or in which ethanol was added to the perfusate. No acute effect of ethanol on the degradation of this ligand was seen. These results demonstrate that chronic ethanol ingestion impairs receptor-mediated endocytosis of formaldehyde-treated albumin by liver endothelial cells, indicating that the adverse effects of ethanol on protein trafficking within the liver are not limited to the hepatocytes.

Zonal differences in ethanol-induced impairments in hepatic receptor binding.

We have previously shown ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid (ASOR), epidermal growth factor (EGF), and insulin in isolated rat hepatocytes. The present study was undertaken to compare the binding of these three ligands in both Zone 1 (periportal [PP] region) and Zone 3 (perivenule [PV] region) of rat liver. Cells from the PV region of ethanol-fed animals bound significantly less EGF (40% decrease) than did cells from the same area in control rats. EGF binding was decreased to a lesser extent (15-25%) in PP cells from ethanol-fed animals compared to controls. When binding of ASOR was examined, ethanol feeding significantly impaired binding in both PP cells (30-35% decrease) and PV cells (50-55% decrease), again showing a greater ethanol-induced impairment in the PV region. Insulin binding in ethanol animals was decreased by 20-25% in both regions compared to controls. In addition, we found that ASOR receptor recycling was impaired to a greater extent in the PV than in the PP region of liver after ethanol feeding, indicating selective impairment of receptor function in the centrilobular region of the liver.

Chronic ethanol administration impairs ATP-dependent acidification of endosomes in rat liver.

Ethanol administration has been shown to alter various protein trafficking pathways in the liver, including protein secretion, plasma membrane assembly and receptor-mediated endocytosis. Molecules such as proteins are transported into and within the cells via endosomal vesicles, and acidification of these endosomes appears to be critical for efficient sorting and trafficking of proteins. In the present study we examined whether ethanol administration impaired the ATP-dependent acidification of endosomes. Liver endosomes were prepared from rats after intravenous labeling with fluorescently labeled ligands and the initial pH and extent of ATP-dependent acidification was determined. Our results showed that the intrinsic pH was significantly higher and that the ATP-dependent acidification of the interior of the endosomes was inhibited in ethanol-fed versus control animals. This impairment of endosomal acidification may play a role in the impaired protein trafficking which occurs as a result of ethanol administration.

Time course of ethanol-induced impairment in fluid-phase endocytosis in isolated rat hepatocytes.

The time-course effects of long-term ethanol administration on fluid-phase endocytosis were studied in isolated rat hepatocytes. Rats were pair-fed an ethanol-supplemented liquid diet or an isocaloric control diet for 3 days, 1 wk, 2 wk or 5 wk. Hepatocytes were isolated and incubated at 37 degrees C with various concentrations of the fluid-phase marker Lucifer yellow. Net internalization of the marker dye was determined. After as little as 1 wk, ethanol-fed rats demonstrated marked decreases in the net internalization of dye compared with pair-fed controls; these changes persisted throughout 5 wk of feeding. Because net internalization is the balance between uptake into the cells vs. efflux from the cells, these components were examined individually. Early uptake was not significantly decreased by ethanol feeding; however, efflux of preloaded Lucifer yellow from cells from the ethanol-fed animals was markedly faster than efflux from pair-fed controls. This increased efflux was more prominent in the longer preload time (90 min) compared with a shorter preload time (15 min), indicating an alteration in dye distribution among various intracellular pools. These ethanol-induced changes in fluid-phase endocytosis were apparent for 1 wk through 5 wk of feeding and were similar for all Lucifer yellow concentrations examined. These results indicate that the decreased net internalization of Lucifer yellow through fluid-phase endocytosis is mainly a result of an ethanol-induced increase in efflux possibly caused by altered intracellular trafficking rather than by reduction in uptake.