We have been using polarized hepatic WIF-B cells to examine ethanol-induced liver injury. Previously, we determined microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells. We proposed that the ethanol-induced alterations in microtubule dynamics may explain the ethanol-induced defects in membrane trafficking that have been previously documented. To test this, we compared the trafficking of selected proteins in control cells and cells treated with ethanol or with the histone deacetylase 6 inhibitor trichostatin A (TSA). We determined that exposure to 50 nM TSA for 30 minutes induced microtubule acetylation (ϳ3-fold increase) and stability to the same extent as did ethanol. As shown previously in situ, the endocytic trafficking of the asialoglycoprotein receptor (ASGP-R) was impaired in ethanol-treated WIF-B cells. This impairment required ethanol metabolism and was likely mediated by acetaldehyde. TSA also impaired ASGP-R endocytic trafficking, but to a lesser extent. Similarly, both ethanol and TSA impaired transcytosis of the single-spanning apical resident aminopeptidase N (APN). For both ASGP-R and APN and for both treatments, the block in trafficking was internalization from the basolateral membrane. Interestingly, no changes in transcytosis of the glycophosphatidylinositol-anchored protein, 5 -nucleotidase, were observed, suggesting that increased microtubule acetylation and stability differentially regulate internalization. We further determined that albumin secretion was impaired in both ethanol-treated and TSA-treated cells, indicating that increased microtubule acetylation and stability also disrupted this transport step. Conclusion: These results indicate that altered microtubule dynamics explain in part alcoholinduced defects in membrane trafficking. (HEPATOLOGY 2008;47:1745-1753 A lcoholic liver disease is a major biomedical health concern in the United States. Although the progression of this disease has been well described clinically, little is known about the molecular basis for liver injury. Research aimed at identifying the molecules and pathways that promote alcohol-induced hepatotoxicity has been hampered by the lack of good in vitro models. Our recent studies have been performed in WIF-B cells, an emerging model system for the study of alcohol-induced liver injury. These hepatic cells are highly differentiated and form discrete apical and basolateral membrane domains in culture. Domain-specific membrane proteins are localized in WIF-B cells as they are in hepatocytes in situ, and many liver-specific functions are maintained. 1 Importantly, WIF-B cells metabolize ethanol using alcohol dehydrogenase (ADH) and cytochrome P4502E 1 . 2 Like hepatocytes, ethanol-treated WIF-B cells display a reduced redox state and increased triglycerides. 2 More recently, we discovered that microtubule repolymerization was impaired in ethanol-treated WIF-B cells, 3 consistent with reports performed in vitro and in isolated hepatocytes. [4][5][6] We also found that steady-state micro...