Ethanol-induced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: Evidence for oxidative and cytoskeletal mechanisms

Exposure of intestinal mucosa to ethanol (EtOH) disrupts barrier function a nd growth factors [epidermal growth factor (EGF) and transforming growth fa ctor-alpha (TGF-alpha)] are protective, but the mechanisms remain obscure. Accordingly, we sought to determine whether the molecular mechanism of EtOH -induced intestinal barrier dysfunction involves oxidative stress and disas sembly of microtubules and whether the mechanism of protection by EGF or TG F-alpha involves prevention of these alterations. To this end, human coloni c (Caco-2) monolayers were exposed to 0 to 15% EtOH with or without pretrea tment with EGF or TGF-alpha (10 ng/ml) or with oxidative or cytoskeletal mo dulators. Effects on cell viability, barrier function, tubulin (microtubule s), and oxidative stress were then determined. Cells were also processed fo r immunoblots of polymerized tubulin (S2; index of stability) and the monom eric tubulin (S1; index of disruption). EtOH dose-dependently decreased the stable S2 polymerized tubulin and concomitantly increased measures of oxid ative stress, including oxidation and nitration of tubulin, fluorescence of dichlorofluorescein, and inducible nitric oxide synthase activity. EtOH al so dose-dependently disrupted barrier function and extensively damaged micr otubules, and these effects were prevented by pretreatment with antioxidant scavengers: L-cysteine, superoxide dismutase, and L-N-6-1-iminoethyl-lysin e (an inducible nitric oxide synthase inhibitor). In monolayers exposed to EtOH, pretreatment with EGF or TGF-alpha prevented the oxidation and nitrat ion of tubulin, increases in the levels of the unstable S1 tubulin, disrupt ion of microtubules, and barrier dysfunction. A microtubule stabilizer (pac litaxel, Taxol) mimicked, in part, the effects of EGF and TGF-alpha, wherea s a microtubule disruptive drug (colchicine) prevented the protective effec ts of these growth factors. We concluded that mucosal barrier dysfunction i nduced by EtOH involves oxidative stress, which causes the disassembly of t he microtubule cytoskeleton. Protection by EGF and TGF-alpha involves the p revention of these EtOH-induced alterations in microtubules.