Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The gastrointestinal (GI) epithelium is a highly selective barrier that normally prevents the passage of harmful molecules across the mucosa and into the circulation (Bode et al., 1987; Hollander, 1992). An abnormal GI barrier, in contrast, can allow the penetration of normally excluded luminal substances (e.g., endotoxin) across the mucosa and can lead to the initiation and/or perpetuation of inflammatory processes and mucosal damage. This damage, and the ensuing loss of GI barrier integrity, has been implicated in a wide range of inflammatory illnesses, including alcoholic cirrhosis (Bode et al., 1987; Hollander, 1992; Keshavarzian et al., 1994). The underlying difficulty in managing these inflammatory disorders is due in large part to our limited understanding of their pathophysiology.

For example, alcohol [ethanol (EtOH)] intake injures the functional and structural integrity of the intestinal mucosa (Bjorkman and Jessop, 1994) and causes loss of intestinal barrier function (Talbot et al., 1984; Keshavarzian et al., 1994, 1999). This is thought to be important in the development of alcoholic cirrhosis (Bode et al., 1987; Keshavarzian et al., 1999). Little is known, however, as to the underlying mechanisms. While investigating this mechanism, we showed (Banan et al., 1998b, 1999a,b,c, 2000), using monolayers of intestinal cells as a model, that EtOH-induced disruption of barrier integrity requires damage to and disruption of the microtubule cytoskeleton. Oxidative damage appeared to be key because microtubules became oxidized and because several agents, including antioxidants, prevented these effects of EtOH. One of these drugs was L-N⁶-1-iminoethyl-lysine (L-NIL), a selective inhibitor of inducible nitric-oxide synthase (iNOS). iNOS was further implicated by the observations that EtOH increased iNOS activity and that L-NIL prevented not only the iNOS up-regulation but also the EtOH-induced microtubule damage and barrier disruption.

iNOS up-regulation is predicted to lead to NO overproduction (Chen et al., 1996; Salzman et al., 1996; Unno et al., 1997), and many of the toxic effects of NO overproduction are mediated by the peroxynitrite (ONOO), a product of the reaction of NO with superoxide anions (Radi et al., 1991; Ischiropoulos et al., 1992, 1995; Rachmilewitz et al., 1995; Haddad et al., 1994; Muijsers et al., 1997). Indeed, overproduction and uncontrolled generation of ONOO have been proposed in several recent studies to be an important factor in tissue damage during inflammation (Radi et al., 1991; Rachmilewitz et al., 1993, 1995; Ischiropoulos et al., 1995). Nevertheless, the precise pathogenic mechanism by which oxidative stress leads to mucosal abnormalities in the intestine, especially after EtOH insult, is not known. Accordingly, in the present study, we investigated the possibility that NO overproduction and ONOO generation cause the oxidative damage to microtubules that mediates EtOH-induced intestinal barrier dysfunction.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture. Caco-2 cells (from a human colonic cell line) were obtained from American Type Culture Collection (Rockville, MD) at passage 15. Although of colonic origin, these widely studied cells resemble small intestinal cells in that they have defined apical brush borders, form highly tight junctions, and exhibit a highly organized microtubule network on differentiation (Gilbert et al., 1991). These cells also express markers of mature enterocytes such as small intestinal hydrolases (sucrase-isomaltase and alkaline phosphatase) and nutrient transporters. In addition, these cells resemble small bowel epithelium in having receptors for prostaglandins; growth factors [e.g., epidermal growth factor (EGF), insulin-like growth factor-I] and insulin; receptors for vasoactive intestinal peptide and low-density lipoprotein, and transporters such as dipeptides, fructose, glucose, other hexoses, and vitamin B₁₂ (Gilbert et al., 1991). Accordingly, Caco-2 cell model provides a suitable in vitro model for our barrier function studies. Cells were maintained at 37°C in Dulbecco's modified Eagle's medium in an atmosphere of 5% CO₂ and 100% relative humidity. Cells were split at a ratio of 1:6 on reaching confluency every 6 days and set up in either 6-, 24-, or 48-well plates for experiments or in T-175 flasks for the maintenance of stocks. Cells grown for barrier integrity work were split at a ratio of 1:2 and seeded at a density of 200,000 cells/cm² into 0.4-µm Biocoat Collagen I Cell Culture Inserts (0.3-cm² growth surface; Becton Dickinson Labware, Bedford, MA), and experiments were performed at least 7 days postconfluence. The utility, maintenance, and characterization of this cell line have been previously published (Gilbert et al., 1991).

Experimental Design. In the first series of experiments, we evaluated the effect of 30-min exposure of cells to injurious (2.5 and 15%) and noninjurious (1%) concentrations of EtOH (v/v) or vehicle (isotonic saline/Dulbecco's modified Eagle's medium) on cell monolayer barrier integrity, iNOS activity, NO production, ONOO generation (i.e., nitration and oxidation), oxidative stress, and microtubule disassembly and instability as described later. The concentrations of EtOH used in the present investigation are clinically relevant (Dinda et al., 1996; Bjorkman and Jessop, 1994) and have previously been shown by us to cause loss of monolayer barrier function without cell death in this cell line (Banan et al., 1998a,b).

1

1

Determination of Cell Integrity. Live/Dead kits (Molecular Probes, Eugene, OR) were used. This assay measures parameters of cell death: nuclear membrane integrity and chromatin condensation by ethidium homodimer-1 probe, as we described previously (Banan et al., 1999a).

Determination of Epithelial Barrier Function by Fluorometry. Barrier integrity was determined by measuring apical-to-basolateral flux of a fluorescent marker [fluorescein sulfonic acid (FSA), 200 µg/ml, 478 Da); Molecular Probes] as previously described (Unno et al., 1996; Banan et al., 1999a). After pharmacological treatments, fluorescent signals from samples were quantified using a fluorescence multiplate reader. The excitation and emission spectra for FSA were excitation = 485 nm and emission = 530 nm. Clearance (CL) was calculated using the following formula: CL (nl/h/cm²) = Fab/([FSA]_a × S), where Fab is the apical to basolateral flux of FSA (light units/h), [FSA]_a is the concentration at baseline (light units/nl), and S is the surface area (0.3 cm²) (Unno et al., 1996). Simultaneous controls were performed with each experiment.

Assay of iNOS Activity. Cells grown to confluence were removed by scraping, centrifuging, and homogenizing on ice in a buffer containing 50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM EGTA, 12 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4). The homogenates were incubated with a cation-exchange resin (AG 50W-X8, Na⁺ form; Sigma Chemical Co.) for 5 min on ice to deplete endogenous L-Arg. Conversion of L-[³H]Arg (Amersham Corp., Arlington Heights, IL) to L-[³H]citrulline was measured in the homogenates by scintillation counting, as previously described (Salzman et al., 1996; Banan et al., 1999a). Experiments in the absence of NADPH or in the presence of NOS inhibitor L-NMMA (1 mM) determined the extent of L-[³H]citrulline formation independent of NOS activity. Experiments in the presence of NADPH, without Ca²⁺ and with 5 mM EGTA, determined Ca²⁺-independent NOS (iNOS) activity. In selected experiments, the isoform-selective iNOS inhibitor L-NIL was also present (see earlier). Protein concentrations were determined according to the Bradford method (Bradford, 1976).

Western Blot Analysis of Level of iNOS Protein. After treatment with EtOH or vehicle, the cells were washed once with cold PBS, scraped in 1 ml of cold PBS, and harvested in an antiprotease cocktail (2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml phenylmethylsulfonyl fluoride). Protein content was determined according to the Bradford method (Bradford, 1976). For immunoblotting, samples (25 µg of protein/lane) were added to SDS buffer (250 mM Tris-HCl, pH 6.8, 2% glycerol, 5% mercaptoethanol), boiled for 5 min, and then separated on 7.5% SDS-polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to nitrocellulose membranes and then blocked in 3% BSA for 1 h followed by several washes (Tris-buffered saline). The immunoblotted proteins were incubated for 2 h in Tween 20 and Tris-buffered saline and 1% BSA with the primary antibody (mouse monoclonal anti-human iNOS, 1:3000 dilution; Santa Cruz Biotech, Santa Cruz, CA). A horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody at 1:3000 dilution. Membranes were visualized by enhanced chemiluminescence (ECL; Amersham) and autoradiography (Singer et al., 1996).

Chemiluminescence Analysis of NO Concentration in Cultures. NO production was assessed by a novel and sensitive chemiluminescence procedure (Al-Mufti et al., 1998). Briefly, cells were homogenized by sonication, and the endogenous nitrate (NO₃) and nitrite (NO₂), the metabolic degradation products of NO, were then reduced to NO using vanadium(III) (Sigma Chemical Co.) and HCl at 90°C before the measurement of NO concentration by chemiluminescence analysis. Chemiluminescence was measured using a Seivers NOA 280 analyzer (Sievers, Boulder, CO). NO was expressed in micromolar and calculated by comparison with the chemiluminescence of a standard solution of NaNO₂. The absolute NO values were reported as micromoles per 1 × 10⁶ cells.

Determination of Cell Oxidative Stress and Superoxide Anion Production. Oxidative stress and superoxide anion (O₂) production were assessed by measuring the conversion of a nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFD; Molecular Probes) into a fluorescent dye, dichlorofluorescein (DCF) as previously described (Wakulich and Tepperman, 1997; Banan et al., 1999a). The dependence of the assay on monolayer O₂ generation was shown by adding an active superoxide radical scavenger, SOD (300 U/ml), or an inactive superoxide radical scavenger, iSOD. Briefly, monolayers grown in 96-well plates were preincubated with the membrane-permeable DCFD (10 µg/ml for 30 min) before the subsequent treatments (see experimental series 1 and 3). After treatments, fluorescent signals (i.e., DCF fluorescence) from samples were quantified using a fluorescence multiplate reader set at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Immunofluorescent Staining and High-Resolution LSCM of Microtubule Cytoskeleton. Cells from monolayers were fixed in cytoskeletal stabilization buffer and then postfixed in 95% EtOH as previously described (Allen, 1985; Banan et al., 1998a,b). Cell monolayers were subsequently processed for incubation with primary monoclonal mouse anti--tubulin antibody (IgG₁, rat/human reactive; Sigma Chemical Co.) at 1:200 dilution for 1 h at 37°C. Slides were washed three times in Dulbecco's PBS (D-PBS) and then incubated with a secondary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse; Sigma Chemical Co.) at 1:50 dilution for 1 h at room temperature, washed three times in D-PBS and once with deionized H₂O, and subsequently mounted in Aquamount. All antibodies were diluted with D-PBS containing 0.1% BSA. Samples were stored in the dark at 20°C and were examined by both standard fluorescent and LSCM (Zeiss, Munich, Germany). Cell monolayers on slides were observed in a blinded fashion with LSCM using a 63× oil immersion plan-Apochromat objective, NA 1.4 (Zeiss). An argon laser ( = 488 nm) was used to examine fluorescein isothiocyanate-labeled cells, and the cytoskeletal elements were examined for their overall morphology, orientation, and disruption as previously described (Banan et al., 1998b).

Microtubule (Tubulin) Fractionation and Quantitative Immunoblotting of Tubulin. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated as we previously described (Banan et al., 1998a,b). Fractionated S1 and S2 samples were flash frozen in liquid N₂ and then stored at 70°C until immunoblotting. For immunoblotting, samples (5 µg) were placed in SDS sample buffer (250 mM Tris-HCl, pH 6.8, 2% glycerol, 5% mercaptoethanol), boiled for 5 min, and then subjected to electrophoresis on 7.5% polyacrylamide gels. Procedures for Western blotting were performed at room temperature (Banan et al., 1998b). To quantify the relative levels of tubulin, the absorbance of the bands corresponding to immunoradiolabeled tubulin was measured with a laser densitometer. We ensured equal loading of proteins in all Western blots by always loading 5 µg of protein/lane assessed according to the Bradford method (Bradford, 1976). Experimental variations were further minimized by the loading of 5 µg of standard tubulin, which was tested concurrently with each gel. To further ensure reproducibility, each treatment group was run in duplicate and/or triplicate on different days.

Immunoblotting Determination of Tubulin Oxidation and Tubulin Nitration. Oxidation and nitration of the tubulin backbone of microtubules were assessed by measuring protein carbonyl and nitrotyrosine formation, respectively (Banan et al., 1999c, 2000a; Ferro et al., 1997). Carbonylation and nitrotyrosination of tubulin were determined in a similar manner as the quantitative blotting of tubulin (Banan et al., 1998b) except for differences in primary antibodies and buffers. To avoid unwanted oxidation of tubulin samples, all buffers contained 0.5 mM dithiothreitol and 20 mM 4,5-dihydroxy-1,3-benzene sulfonic acid (Sigma Chemical Co.). To determine the carbonyl content, samples were blotted onto a polyvinylidene difluoride membrane, followed by successive incubations in 2 N HCl and 2,4-dinitrophenylhydrazine (DNP; 100 µg/ml in 2 N HCl; Sigma Chemical Co.) for 5 min each. Membranes were then washed three times in 2 N HCl and subsequently washed seven times in 100% methanol for 5 min each, followed by blocking for 1 h in 5% BSA in 10× PBS/Tween 20 (PBS-T). Immunologic evaluation of carbonyl formation was performed for 1 h in 1% BSA/PBS-T buffer containing anti-DNP (1:25,000 dilution; Molecular Probes). Membranes were then incubated with an HRP-conjugated secondary antibody (1:4000 dilution, 1 h; Molecular Probes, Eugene, OR). To determine nitrotyrosine content, after the blocking step earlier (i.e., BSA/PBS-T buffer), membranes were probed for nitrotyrosine by incubation with 2 µg/ml monoclonal anti-nitrotyrosine antibody for 1 h (Upstate Biotechnology, Lake Placid, NY) followed by the HRP-conjugated secondary antibody (as earlier). Wash steps and film exposure were as previously described (Banan et al., 1998b). The relative levels of oxidized or nitrated tubulin were then quantified by measuring, with a laser densitometer, with the absorbance (A) of the bands corresponding to anti-DNP or anti-nitrotyrosine immunoreactivity. Immunoreactivity was expressed as the fraction (ratio) of carbonyl or nitrotyrosine formation in the treatment group to that in the oxidized or nitrated tubulin standard run concurrently.

Statistical Analysis. Data are presented as mean ± S.E. All experiments were carried out with a sample size of at least four to six observations per group. Statistical analysis between or among groups was carried out using ANOVA followed by Dunnett's multiple range test (Harter, 1960). Correlational analyses were performed using the Pearson test for parametric analysis or, when applicable, the Spearman test for nonparametric analysis. For all analyses, a value of P < .05 was deemed to represent statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Injurious Effects of EtOH on Monolayer Barrier Integrity and Its Prevention by a Selective iNOS Inhibitor. Caco-2 monolayers exposed to a range of concentrations of EtOH (1, 2.5, and 15%) for 30 min showed a dose-dependent loss of epithelial barrier function as demonstrated by increased clearance of FSA (Fig. 1). The lowest dose of EtOH that significantly increased FSA clearance was 2.5%. Under these condition, EtOH does not cause cell death as determined by ethidium homodimer-1 probe (Banan et al., 1999a). Pretreatment with a selective iNOS inhibitor (L-NIL) significantly attenuated the EtOH-induced disruption of barrier integrity by up to 65%. Preincubation with an NOS substrate, L-Arg (48 h), synergized with 1% EtOH to create an injurious effect and potentiated the monolayer barrier dysfunction induced by 2.5 and 15% EtOH. L-Arg by itself did not significantly affect monolayer barrier function. Pretreatment with L-NIL completely abolished the "potentiating" interaction between L-Arg and EtOH (Fig. 1). Similar to L-NIL, 1-h preincubation with nonselective NOS inhibitors (L-NMMA or L-NNA) significantly protected against barrier dysfunction by EtOH (FSA clearance = 1223 ± 53 nl/h/cm² for L-NMMA or 1309 ± 93 for L-NNA versus 2548 ± 105 for 15% EtOH). One-hour incubation with L-NMMA or L-NNA by itself did not significantly affect FSA clearance compared with vehicle (not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Deleterious effects of EtOH (30-min exposure) and protective actions of pretreatment (1 h) with an iNOS inhibitor (L-NIL, 1 mM) on barrier integrity of Caco-2 monolayers assessed by FSA clearance. In selected experiments, monolayers were preincubated with L-Arg (48 h) before the aforementioned treatments. FSA clearance was calculated as apical-to-basolateral flux of FSA divided by the concentration of probe in the apical chamber. *P < .05 compared with vehicle, 1% EtOH, or corresponding L-NIL plus EtOH. +P < .05 compared with corresponding 2.5 or 15% EtOH alone. ++P < .05 compared with corresponding L-Arg + EtOH. &P < .05 compared with L-NIL + EtOH (n = 6/group in all figures).

NO-Dependent Oxidative Mechanisms in Injurious Effects of EtOH. Similar to L-NIL, the ONOO scavengers urate and L-cysteine and the O₂ scavenger SOD significantly attenuated the loss of barrier integrity induced by EtOH (Table 1; cysteine and SOD shown). The analogs D-cysteine and iSOD were ineffective.

View this table: [in this window] [in a new window]   TABLE 1 Protective effect of antioxidants on barrier integrity of Caco-2 cells Values are mean ± S.E. after treatments. Apical chambers were loaded with FSA before the exposure of monolayers to EtOH alone for 30 min or preincubation with ONOO scavengers 1) L-cysteine (3 mM) and as control analog D-cysteine or 2) SOD (300 U/ml) and as control the heat-inactivated iSOD for 30 min before EtOH. Controls were incubated in isotonic saline/vehicle. FSA clearance was calculated as apical-to-basolateral flux of FSA divided by the concentration of probe in the apical chamber. n = 6 per group.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   A, EtOH-induced up-regulation of iNOS activity in Caco-2 monolayers. Monolayers were preincubated with an iNOS inhibitor (L-NIL, 1 mM) or vehicle and then exposed to EtOH (30 min). iNOS activity is given in pmol/min/mg of protein. *P < .05 compared with vehicle or L-NIL pretreated. +P < .05 compared with EtOH. B, representative Western blot for the effects of EtOH on iNOS protein levels in Caco-2 cell monolayers. Monolayers were exposed to damaging (2.5%) and noninjurious (1%) EtOH or control (vehicle) for 30 min. Control and 1% EtOH exhibit a low/basal level of iNOS protein, whereas 2.5% EtOH causes a large increase in iNOS protein (~130 kDa). Cell fractions were analyzed by Western immunoblots using monoclonal anti-human iNOS antibody followed by HRP-conjugated secondary antibody. The region of gel shown was between the Mr 126,000 and 218,000 prestained molecular weights run in adjacent lanes. ~, approximate molecular weight for iNOS.

View larger version (60K): [in this window] [in a new window]   Fig. 3.   NO concentration in the intestinal monolayers after exposure to EtOH as determined by chemiluminescence assay. Conditions as in the legend for Fig. 2A. *P < .05 compared with vehicle or L-NIL pretreatment. +P < .05 compared with EtOH.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   A and B, prevention of EtOH-induced oxidative stress by either L-NIL (A) or superoxide anion scavenger (SOD, B) in Caco-2 cell monolayers assessed by the DCF fluorescence assay. Monolayers were exposed to EtOH (2.5 or 15%) in the presence or absence of L-NIL (1 mM) or SOD (300 U/ml or the inactive form iSOD). *P < .05 compared with vehicle, L-NIL, or SOD pretreatment. +P < .05 compared with EtOH.

Role of NO-Dependent Mechanisms in Deleterious Effects of EtOH on Microtubule Cytoskeleton. EtOH dose-dependently decreased microtubule stability (to 27% of control) as determined by laser confocal microscopy and as indicated by the reduced percentage of cells displaying normal microtubules (see Table 2). Similar to effects on barrier function, the lowest EtOH dose that significantly induced instability of microtubules was 2.5%. Additionally, there was a significant (P < .05) positive correlation (r = 0.98) between EtOH doses and percentage of cells with abnormal microtubules. Preincubation with the selective iNOS inhibitor L-NIL or with antioxidants (urate, L-cysteine, or SOD) markedly and significantly blunted the EtOH-induced microtubule instability (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Percentage of Caco-2 cells of monolayers with normal microtubules Values are mean ± S.E. after treatments. Monolayers were exposed to EtOH for 30 min or were preincubated with 1) a selective iNOS inhibitor [L-NIL (1 mM)] or 2) ONOO scavengers urate (0.5 mM) or L-cysteine (3 mM and, as control, D-cysteine) or SOD 300 U/ml and the heat-inactivated iSOD) for 30 min (except 1 h for L-NIL studies) before the exposure to EtOH. Controls were incubated in isotonic saline/vehicle. From 100 to 150 cells from monolayers per slide (well) were examined by high-resolution laser confocal and the percentage of cells displaying normal microtubule cytoskeleton was determined. n = 6 per group.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   The intracellular distribution of the microtubules by high-resolution laser scanning confocal in intestinal cell monolayers incubated with (a) isotonic saline (control), (b) 2.5% EtOH, or (c) L-NIL (1 mM) and then EtOH. Microtubules in control cells appear as an intact filamentous network that courses radially throughout the cytosol. In cells exposed to 2.5% EtOH alone, the microtubules appear disrupted, collapsed, and disorganized. In cells pretreated with L-NIL, normal microtubule architecture is highly preserved and resembles the controls. Bar, 25 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   A, immunoblotting analysis of anti-DNP (carbonylation or oxidation) and antinitrotyrosine (nitration) immunoreactivity of the cytoskeletal tubulin (S2 pool) in Caco-2 monolayers exposed to EtOH. Oxidation or nitration was expressed, respectively, as the fraction (ratio) of carbonyl or nitrotyrosine formation (i.e., absorbance) in treatment group divided by oxidized (or nitrated) tubulin standard. *P < .05 compared with vehicle. B, representation of immunoblotting of the protective effects of L-NIL or antioxidants against nitration immunoreactivity of polymerized tubulin (S2) in Caco-2 cells. Monolayers were pretreated with 1 mM L-NIL, 0.5 mM urate, 1 mM L-cysteine, or 300 U/ml SOD (or their control conditions, D-cysteine and iSOD) before exposure to 2.5% EtOH. Nitration was expressed as described in A. *P < .05 compared with vehicle or urate, L-cysteine, SOD, or L-NIL pretreatment. +P < .05 compared with corresponding D-cysteine or iSOD pretreatment or 2.5% EtOH alone. ++P < .05 compared with corresponding L-cysteine or SOD pretreatment.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   A and B, Western immunoblot of anticarbonyl (A) and antinitrotyrosine (B) immunoreactivity of S2 tubulin in Caco-2 cells after treatments. Conditions as in legend to Fig. 6B. Tubulin fractions were analyzed by Western blots using monoclonal anti-DNP or antinitrotyrosine antibody followed by HRP-conjugated secondary antibody.

View larger version (94K): [in this window] [in a new window]   Fig. 8.   Immunoblotting analysis of the distribution of polymerized tubulin (S2, index of stability) and monomeric tubulin (S1, index of disassembly) in Caco-2 monolayers pretreated with L-NIL or antioxidants before incubation with 2.5% EtOH (conditions identical to those of Fig. 6B). Percentage of polymerization of tubulin pool = [(S2)/total tubulin pool (S2 + S1)]. *P < .05 compared with vehicle, L-NIL, or antioxidant pretreatment. +P < .05 compared with 2.5% EtOH alone, D-cysteine, or iSOD pretreatment. ++P < .05 compared with corresponding L-cysteine or SOD pretreatment.

Cytoskeletal and Barrier Disruption Induced by ONOO Compounds. Our hypothesis predicts that authentic ONOO- or ONOO-generating systems will, similar to EtOH, induce cytoskeleton and monolayer injury by themselves. Indeed, the disruption of monolayer barrier function (increased FSA clearance; Fig. 9A) was caused by incubation 1) with a range of concentrations of authentic added ONOO or 2) with known ONOO donors [e.g., SIN-1 (an NO and O anion donor) or SNAP (an NO donor) in combination with xanthine (X) plus xanthine oxidase (XO) (an O anion donor)]. These effects were prevented by the antioxidants urate, L-cysteine, or SOD but not by D-cysteine or iSOD (Fig. 9B, data for ONOO and SIN-1 shown). At the same concentration (3 mM), D-cysteine was much less protective than L-cysteine (4 versus 94%, respectively). To confirm that this difference in the protective capabilities of cysteine isomers is related to their ability to scavenge ONOO, we measured their scavenging ability in vitro in the presence of added ONOO (absorbance change at 302 normalized to percentages). L-Cysteine (3 mM) removed more than 95% of exogenously added ONOO, whereas D-cysteine (3 mM) removed less than 15% of added ONOO. Furthermore, ONOO compounds did not significantly affect cell viability assessed by ethidium homodimer-1 (not shown). SNAP by itself did not induce barrier dysfunction (not shown).

View larger version (48K): [in this window] [in a new window]   Fig. 9.   A, injurious effects of exogenous authentic ONOO or ONOO donor compounds (SIN-1 and SNAP + X + XO) on the barrier integrity of Caco-2 monolayers determined by FSA clearance. Apical chambers were loaded with FSA before incubation with ONOO (30 min), SIN-1 (1 h), or SNAP/X/XO (6 h). FSA clearance was calculated as described in legend to Fig. 1. *P < .05 compared with vehicle. B, protective effects of antioxidants against ONOO compound-induced intestinal monolayer barrier dysfunction. Monolayers were preincubated with antioxidants or control conditions (for doses, see legend to Fig. 6B) before to exposure to ONOO (1 mM, 30 min) or SIN-1 (5 mM, 1 h). FSA clearance was determined as described in the legend to Fig. 1. *P < .05 compared with vehicle. +P < .05 compared with ONOO or SIN-1 alone. ++P < .05 compared with corresponding L-cysteine or SOD preincubation.

View this table: [in this window] [in a new window]   TABLE 3 ONOO-induced tubulin oxidation/nitration and its prevention by antioxidants in Caco-2 monolayers Values are mean ± S.E. Monolayers were exposed to ONOO or donors or were preincubated (30 min) with ONOO scavengers 1) L-cysteine (3 mM) and as control D-cysteine, 2) urate (0.5 mM), or 3) SOD (300 U/ml) and as control the heat-inactivated iSOD. Controls were incubated in isotonic saline/vehicle. Cells were then processed for immunoblots to assess tubulin oxidation (carbonylation) and nitration (nitrotyrosination). Oxidation or nitration was expressed as the fraction (ratio) of carbonyl or nitrotyrosine immunoreactivity (absorbance) in the treatment group divided by oxidized or nitrated tubulin standards.

View larger version (87K): [in this window] [in a new window]   Fig. 10.   A, injurious effects of chemically synthesized ONOO and ONOO donors on microtubule assembly/polymerization in Caco-2 monolayers and its prevention by antioxidants as determined by the immunoblotting analysis of both polymerized S2 and monomeric S1 tubulin. Cell monolayers were pretreated with antioxidants and then exposed to ONOO or ONOO generators as described in the legend to Fig. 9B. Percentage of polymerization of tubulin pool = [(S2)/total tubulin pool (S2 + S1)]. *P < .05 compared with vehicle. +P < .05 compared with ONOO or SIN-1 alone. ++P < .05 compared with corresponding L-cysteine or SOD preincubation. B, immunofluorescent staining of the microtubules assessed by high-resolution LSCM from intestinal cell monolayers incubated with isotonic saline (a, control), 0.1 mM ONOO (b), or L-cysteine and then ONOO (c). ONOO-exposed cells exhibit collapse and disorganization of the cytoskeleton. In contrast, microtubule structure in antioxidant (L-cysteine)-pretreated cells appears intact and resembles the normal architecture observed in controls. Bar, 25 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Characterizing the pathophysiological mechanism for ethanol-induced barrier dysfunction, as we tried to do in this study, is clinically important because the leaky gut has been proposed to be one of the underlying mechanisms of alcohol-mediated endotoxemia in patients with alcoholic liver disease (Bode et al., 1987; Hollander, 1992; Keshavarzian et al., 1994, 1999). We hypothesized that the cytoskeletal disruption (and barrier dysfunction) that is induced by EtOH requires iNOS activation, NO overproduction, and ONOO formation and that it is these free radical reactions that lead to the oxidative injury to the tubulin-based backbone of the microtubule cytoskeleton. We conclude that all three separate lines of our investigation confirm the hypothesis: 1) measurement of oxidative reactions and cytoskeletal injury after EtOH administration, 2) mimicking EtOH-induced damage using ONOO-generating systems, and 3) preventing EtOH-induced cytoskeletal instability using NOS selective inhibitors and antioxidants. The following section elaborates on this conclusion.

First, we found that under conditions where EtOH oxidizes tubulin, disrupts the microtubule cytoskeleton, and diminishes barrier integrity in cell monolayers, it also creates oxidative stress, including increased levels of O₂ (SOD quenchable DCF fluorescence), NO, and ONOO. These associations strongly suggest that the underlying cause of the injury to the cytoskeletal network (and loss of barrier function) is the oxidation and nitration of its structural tubulin subunits. This mechanism is further supported by a highly significant correlation between increases in NO and nitrotyrosine levels (r = 0.985); increases in nitrotyrosine levels, and either abnormal microtubules (r = 0.95) or microtubule depolymerization (r = 0.94). Also, oxidation in general (as measured by DCF or carbonyl moieties) predicts increases in abnormal microtubules (r = 0.91 and 0.98, respectively).

These data suggest that the reaction between O₂ and NO to form ONOO is important in EtOH-induced damage. This is consistent with previous studies that have documented that O₂ reacts rapidly with NO to form ONOO (Hue and Padmaja, 1993). In our studies, evidence of ONOO generation by EtOH was confirmed by using a O₂ scavenger, SOD, to prevent the interaction of O₂ and NO. This conclusion was further supported by the fact that ONOO scavengers (urate and L-cysteine) also inhibited EtOH-induced cytoskeletal damage (and barrier dysfunction). The demonstration that EtOH administration increases NO and O₂ and at the same time leads to increases in stable ONOO footprints (tubulin-associated nitrotyrosine and carbonyl) further corroborates our interpretation.

Our findings are consistent with earlier reports. For example, previous in vivo studies also implicated oxidative stress and generation of free radicals as key to the pathogenesis of a variety of GI disorders, including EtOH-mediated injury (Kvietys et al., 1990; Keshavarzian et al., 1992; Dinda et al., 1996; McKenzie et al., 1996). ONOO-mediated damage is not limited to the GI tract and has been proposed for other organs. For example, alterations in lung function mediated by tumor necrosis factor (TNF)- were proposed to stem from nitrated proteins such as SOD, antiproteases, and glutathione (Phelps et al., 1995).

Also, studies of systemic diseases and inflammatory GI disorders have shown that oxidation and nitration of proteins occur in vivo and that they can serve as markers of oxidative injury (Haddad et al., 1994; Ischiropoulos et al., 1995; McKenzie et al., 1996; Singer et al., 1996; Ferro et al., 1997). For example, recent studies have shown increased nitrotyrosine and iNOS up-regulation in the inflamed intestinal epithelium in vivo (Salzman et al., 1996; Singer et al., 1996; Kimura et al., 1998). Moreover, it appears that ONOO can oxidize a variety of essential molecules (e.g., sulfhydryls, thiols, ascorbate) and trigger injurious processes, including lipid peroxidation (Muijsers et al., 1997). Indeed, a major product of the reaction of ONOO with proteins (e.g., in macrophages, in lung tissue) is the addition of a nitro group in the ortho position of tyrosine to form nitrotyrosine (Ischiropoulos et al., 1992, 1995).

Second, we showed using ONOO or two different ONOO-generating systems that ONOO mimics the ability of EtOH to disrupt barrier function and to oxidize and damage microtubules. These findings are in accord with previous pharmacological studies in endothelial cells, in test-tube models, and in inflammatory processes (Ischiropoulos et al., 1992, 1995; Radi et al., 1993; Kooy and Royall, 1994; Haddad et al., 1994; Rachmilewitz et al., 1995). For example, ONOO not only can cause chemical oxidation of luminol in the test-tube but also oxidatively injures endothelial cells (Radi et al., 1993; Phelps et al., 1995). Our finding that ONOO can disrupt intestinal barrier function is in accord with finding by Ferro et al. (1997) for endothelial barrier dysfunction. They showed that p42 oxidation and barrier dysfunction induced by TNF- were both mediated by NO and ONOO.

Third, we showed that agents that scavenge ONOO or diminish the formation of ONOO from NO and O₂ attenuate the deleterious effects of both EtOH- and ONOO-generating systems. Our findings parallel a previous study (Phelps et al., 1995) in which urate and SOD protected against TNF--induced, ONOO-mediated lung endothelial injury. Interestingly, some of these same antioxidant enzymes (e.g., SOD) and free radical scavengers (e.g., uric acid) are normal constituents of all tissues (Muijsers et al., 1997). Similarly, a previous study in vivo in rat lung showed that inhibition of NO synthesis abolished the increases in protein nitrotyrosine and protein carbonyl levels (Ischiropoulos et al., 1995).

In our studies, we checked the specificity of the protective effects of antioxidants. First, we showed that analogs that lack the biological activity of these protective agents (iSOD and D-cysteine) were neither capable of protecting against EtOH-induced loss of epithelial barrier function nor had any stabilizing effects on tubulin or on microtubules. iSOD lacks the ability to scavenge O₂ and thus cannot inhibit the reaction of O₂ + NO  ONOO. We surmise that D-cysteine does not protect against EtOH-induced damage because it is much more poorly transported into the cell interior than L-cysteine, which is the natural substrate for the amino acid carrier (Hopfer, 1987). We presume that intracellularly, L-cysteine interacts directly with intracellular ONOO, such as is generated during EtOH exposure. However, it is also possible that L-cysteine enhances the antioxidant defenses of the cell because it is a precursor of the natural antioxidant glutathione (Van Klaveren et al., 1997).

Second, we directly measured ONOO footprints (nitrotyrosine and carbonyl). Not only does this oxidation indicate that added ONOO reaches the interior of our cells but also attenuation of this oxidation indicates that urate and L-cysteine are effective ONOO scavengers. This is consistent with the conclusions of a previous study (Radi et al., 1991).

The protective superiority of 3 mM L-cysteine over 3 mM D-cysteine against added ONOO obviously cannot be attributed to differential transport. It can be attributed to our finding that D-cysteine (15% scavenging) is not equally effective as L-cysteine (95%) in scavenging ONOO in solution.

Other observations we have made (Banan et al., 1999a, 2000a,c) showing that growth factors protect against the injurious mechanisms induced by EtOH or oxidants are consistent with the findings of this study. The long-term goal of our laboratory is to clarify mechanisms of EtOH-induced intestinal barrier dysfunction and then to find a means of preventing this injury. Our current working hypothesis is that chemical agents that are injurious to the intestinal tract (e.g., EtOH, H₂O₂) cause iNOS up-regulation. The activity of the iNOS enzyme leads to intracellular increases in NO and ONOO that directly damage (oxidize) cellular proteins such as tubulin. These effects on tubulin disrupt the microtubule cytoskeleton, causing a loss in integrity of the intestinal barrier, and predispose the organism to inflammation. Clearly, other factors are involved in intestinal injury due to these agents such as increases in intracellular calcium (Banan et al., 1999b) and damage to the actin cytoskeleton (Banan et al., 2000b,c), but microtubule damage appears to be responsible for a significant part of this injury. The effect of growth factors (e.g., EGF) is to counteract these deleterious effects (Banan et al., 1999a, 2000a,c) and to prevent or reverse iNOS induction and its sequelae. In this sense, chemically induced intestinal injury, on the one hand, and its prevention by endogenous defense and repair mechanisms, on the other hand, appear to modulate a single (iNOS-driven) pathway, in opposite directions. Aspects of this model have recently received support from studies using non-GI cell models in which EGF apparently inhibited iNOS up-regulation (Heck et al., 1992; Schini et al., 1992; Asano et al., 1994). Further studies are needed to elucidate the underlying mechanism through which EtOH up-regulates iNOS and protective agents such as EGF may prevent this up-regulation in the GI tract.

In conclusion, our present findings strongly suggest that the underlying mechanism of intestinal epithelial barrier dysfunction induced by EtOH is due to oxidative injury to the cytoskeleton. Our findings also provide avenues for development of novel therapies for alcohol-induced GI disorders such as ONOO scavengers, iNOS inhibitors, or antioxidants.