Báo cáo khoa học: Expression, localization and potential physiological significance of alcohol dehydrogenase in the gastrointestinal tract pdf

Expression, localization and potential physiological significance of alcohol dehydrogenase in the gastrointestinal tract Julia Vaglenova 1, *,‡, Susana E. Martı ´ nez 1, †,‡, Sergio Porte ´ 1 , Gregg Duester 2 , Jaume Farre ´ s 1 and Xavier Pare ´ s 1 1 Department of Biochemistry and Molecular Biology, Universitat Auto ` noma de Barcelona, Spain; 2 OncoDevelopmental Biology Program, The Burnham Institute, La Jolla, CA, USA ADH1 and ADH4 are the major alcohol dehydrogenases (ADH) in ethanol and retinol oxidation. ADH activity and protein expression were investigated in rat gastrointestinal tissue homogenates by enzymatic and Western blot analyses. In addition, sections of adult rat gastrointestinal tract were examined by in situ hybridization and immunohistochem- istry. ADH1 and ADH4 were detected along the whole tract, changing their localization and relative content as a function of the area studied. While ADH4 was more abundant in the upper (esophagus and stomach) and lower (colorectal) regions, ADH1 was predominant in the intestine but also present in stomach. Both enzymes were detected in mucosa but, in general, ADH4 was found in outer cell layers, lining the lumen, while ADH1 was detected in the inner cell layers. Of interest were the sharp discontinuities in the expression found in the pyloric region (ADH1) and the gastroduodenal junction (ADH4), reflecting functional changes. The precise localization of ADH in the gut reveals the cell types where active alcohol oxidation occurs during ethanol ingestion, providing a molecular basis for the gastrointestinal alcohol pathology. Localization of ADH, acting as retinol dehydro- genase/retinal reductase, also indicates sites of active retinoid metabolism in the gut, essential for mucosa function and vitamin A absorption. Keywords: ethanol; immunohistochemistry; in situ hybridi- zation; retinol; retinoic acid. The major pathway for the elimination of ethanol is through its oxidation to acetaldehyde that occurs mostly in liver [1], though ethanol metabolism is also significant in other tissues [2]. Alcohol dehydrogenase (ADH) is the main enzyme responsible for the first step in ethanol elimination [3]. ADH is expressed in several molecular forms, grouped in five enzymatic classes [4], and four of them have been well characterized at the protein level in mammals [5,6]. In the rat, ADH1 has a low K m for ethanol and it is responsible for the hepatic ethanol metabolism [7]. ADH2 and ADH3 are not active at moderate concentrations of ethanol [7,8]. ADH4 has high K m and k cat values for ethanol [9], and it is found in gastrointestinal mucosa, blood vessels, central nervous system and many epithelia, but it is absent in normal liver [2,10,11]. Moreover, these ADH forms have retinol dehydrogenase activity [12–17], and recent genetic studies in knockout mice have demonstrated that ADH1, ADH3, and ADH4 participate in the retinoic acid (RA) synthesis pathway [16,18,19]. Previous studies have shown that the rat ADH system is comprised of single isozyme representatives of each class, making it a simpler system to study, compared to the human ADH [5,6]. In spite of several reports on the localization of ADH in rodent [2,7,20–22] and human [23–30] gastrointestinal tissues, these works are only partial. This paper presents a complete analysis of the whole gastrointestinal tract in the rat: ADH activity levels were measured by spectrophotometric assays, ADH expression pattern by electrophoretic and Western blot analyses, and the localization of ADH (at mRNA and protein levels) in the distinct cell layers of each gastrointestinal region by in situ hybridization (ISH) and immunohistochemistry (IHC) 1 . Our results demonstrate that ADH1 and ADH4 coexist throughout the gastrointestinal tract and provide new data to understand the physiological role of ADH classes in the gastrointestinal tract and the etiopathogeny related to alcohol abuse. Experimental procedures Animals Adult Sprague–Dawley rats (n ¼ 5; male, 200–250 g) were used. Animal protocols were approved by the Ethical Committee of the Universitat Auto ` noma de Barcelona. After decapitation, gastrointestinal organs were removed and processed rapidly as described below. Correspondence to X. Pare ´ s, Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universitat Auto ` noma de Barcelona, E-08193 Bellaterra, Barcelona, Spain. Fax: + 34 93 5811264, Tel.: + 34 93 5813026, E-mail: xavier.pares@uab.es Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; IHC, immunohistochemistry; ISH, in situ hybridization; RA, retinoic acid. Note: àThese authors made equal contributions to this study. *Present address: Department of Pharmacal Sciences, 401 Pharmacy Bldg., Auburn University, Auburn, AL 36849, USA. Present address:BiologyDepartment,BostonCollege,321Higgins Hall, 140 Commonwealth Ave., Chesnut Hill, MA 02467, USA. (Received 27 February 2003, revised 21 April 2003, accepted 28 April 2003) Eur. J. Biochem. 270, 2652–2662 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03642.x ADH activity assay and starch gel electrophoresis Tissues from gastrointestinal tract were dissected carefully and subsequently washed in ice-cold homogenization buffer (50 m M sodium phosphate, pH 7.6, 0.5 m M dithio- threitol). The specimens were cut into small fractions and homogenized at 4 °C. Crude homogenates were centrifuged (24 000 g,4°C, 30 min) and supernatants were used for activity assay or analysis by starch gel electrophoresis [2]. After electrophoresis, gels were stained for ADH activity using 100 m M 2-buten-1-ol as a substrate. Also, ADH activity of homogenates was monitored at 340 nm in a UV-VIS spectrophotometer (Cary 400Bio; Varian), in 0.1 M NaCl/P i , pH 7.5, 2.4 m M NAD + ,at25°C, using 10 m M ethanol or 1 M ethanol as a substrate. At 10 m M ethanol, we determined the contribution of ADH1 (K m ¼ 1.4 m M , k cat ¼ 40 min )1 ) [7]. At 1 M ethanol, the observed activity was mainly due to ADH4 (K m ¼ 2.4 M , k cat ¼ 2600 min )1 )[9].Atthis ethanol concentration, ADH1 shows substrate inhibition [31] and the contribution of ADH3 is still negligible because of its extremely low activity at pH 7.5 [7]. One activity unit corresponds to the reduction of 1 lmol NAD per min. Protein concentrations were estimated by the method of Bradford [32] using bovine serum albumin as standard. In situ hybridization analysis (ISH) Generation of ADH1 and ADH4 specific sense and antisense riboprobes was performed as reported previously [11]. The gastrointestinal tract was removed and divided into regions corresponding to the various tissues. After dissection, digestive samples were immediately rinsed in NaCl/P i (0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl) and immersed in 4% (w/v) paraformaldehyde in NaCl/P i for 12 h. The paraffin-embedded tissues were sliced into serial 8-lm sections using a Leica microtome and attached to coated microscope slides. Insituhybrid- ization and subsequent immunochemical chromogenic detection of digoxigenin-labeled hybrids was performed as previously described [11]. The hybridization signal corresponding to each probe appeared highly specific, as demonstrated by the negative controls performed with the sense RNA probes. Protein immunoblotting and Western blot analysis Homogenates were prepared from fresh adult rat tissue as reported previously [11], except that 1 m M phenyl- methanesulfonyl fluoride, 1 lgÆmL )1 leupeptin, and 1 lgÆmL )1 pepstatin were added as protease inhibitors. Protein blots were incubated with affinity-purified rabbit antiserum raised against mouse ADH4 (1 : 500) [21]. Immunodetection was carried out using goat anti-(rabbit IgG)-alkaline phosphatase conjugate (Bio-Rad) for 1 h at room temperature. Alkaline phosphatase activity was then visualized by incubation with 0.1 M Tris/HCl, pH 9.5, containing 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium as substrates according to the instructions of the Alkaline Phosphatase Conjugate Substrate kit (Bio-Rad). Immunohistochemistry Rat gastrointestinal tissues were fixed, processed routinely, and embedded in paraffin as described for ISH. Localiza- tion of ADH4 was investigated using affinity-purified antibodies specific for ADH4 [21] diluted to 1 : 500 on serial 5-lm tissue sections. Slides were treated with xylene and hydrated through a graded series of decreasing ethanol concentrations. Endogenous peroxidase activity was blocked with 1% (v/v) hydrogen peroxide for 15 min. After rinsing in Tris/HCl-buffered saline, slides were blocked with 2% (v/v) of normal serum, and the primary antibody was applied for 1 h. Biotinylated goat anti-(rabbit IgG) Ig (Dakopatts) was used as a secondary antibody and was visualized by avidin–biotin complex (Strept–ABCom- plex–HRP; Dakopatts; dilution 1 : 400 in blocking solu- tion) with peroxidase detection using the Vectastain Universal Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA). 3,3¢-Diaminobenzidine tetra- hydrochloride (DAB; Sigma-Aldrich) was used as a chromogen (50 mg DAB in 100 mL 0.05 M TBS, pH 7.4, with 33.3 lLH 2 O 2 , prepared prior to use). Tissues were then rinsed in Tris/HCl, dehydrated and mounted using a xylene-based medium (ENTELLANÒ neu; Merck). Adja- cent slides were stained with Harris hematoxylin (Vecta- stain), dehydrated through a graded series of increasing ethanol concentrations, followed by two xylene washes, and cover-slipped with ENTELLANÒ neu (Merck). Both the omission of anti-ADH4 IgG and the preadsorption of anti-ADH4 IgG with excess of purified recombinant ADH4 abolished the positive reaction in the control sections, demonstrating the specificity of the staining. Control experiments had showed that anti-ADH4 IgG immunoreacted with recombinant purified rat ADH4 but did not cross-react with any other ADH classes. Image analysis Following ISH and IHC techniques, digestive tract sections were examined under a Leica DMRD fluorescense micro- scope with a Hamamatsu C5310 CCD or a Leica DC200 camera. Image acquisition was carried out with IMAGE PROPLUS software and imported into Adobe PHOTOSHOP v5.5 (Adobe). Color images were transformed into black and white images using a grey-scale function, and brightness and contrast were adjusted. All sections were examined concurrently and compared to published pictures and schemes [33]. Results ADH expression in rat gastrointestinal homogenates Homogenates from gastrointestinal tissues (tongue, eso- phagus, stomach, duodenum, jejunum, ileum, caecum, colon and rectum) were analyzed for the presence of ADH at activity and protein levels by using starch gel electrophoresis, spectrophotometric measurements, and immunoblotting (Fig. 1). Both ADH1 and ADH4 were detected throughout the entire gastrointestinal tract but with a differential tissue distribution. ADH1 was detected mainly in duodenum and the colorectal region, while ADH4 Ó FEBS 2003 Alcohol dehydrogenase in the gastrointestinal tract (Eur. J. Biochem. 270) 2653 was highly expressed in the upper (mainly esophagus and stomach) and colorectal regions. ADH3 was detected in all tissues examined. No large differences were found in activity or in the tissue distribution of the ADH forms between different animals (Fig. 1). Localization of ADH in tongue and esophagus Immunohistochemistry (IHC) of rat tongue showed ADH4 in the mucosa. The signal was detected in the papillae, specifically in the stratified squamous epithelium (Fig. 2B, C,E). ADH4 was also detected in the endothelium of microvessels (Fig. 2C). Insituhybridization (ISH) analysis of esophagus revealed that ADH1 mRNA was only localized in the base line of the stratified squamous epithelium (data not shown). In contrast, ADH4 mRNA was present at high level in all cell layers of stratified squamous epithelium (Fig. 2G). No specific signal was detected in the lamina propia and muscularis mucosae.In good agreement, ADH4 immunostaining was detected in the stratified squamous epithelium (Fig. 2I). Interestingly, a strong ADH4 protein signal was observed in the keratinized layer of epithelium, where the ADH4 mRNA was not detected. Localization of ADH in stomach and the gastroduodenal junction ADH1 and ADH4 mRNAs were both expressed in the gastric mucosa from cardiac to pyloric stomach but each form was confined to distinct layers and cell types. In the stomach body, ADH1 was localized in the medium and basal layers of the mucosa, and muscularis mucosae but not in mucus-secreting cells (Fig. 3A). However, towards the pyloric region, ADH1 gradually appeared in the mucus- secreting epithelium as well (cf. Fig. 3B,C,D). In contrast, ADH4 mRNA was detected in the mucus-secreting cells, in some of the inner cell layers, and in muscularis mucosae Fig. 1. Detection of ADH1 and ADH4 in tis- sue homogenates of the gastrointestinal tract. (A) Starch gel electrophoresis stained for activity using 100 m M 2-buten-1-ol as a sub- strate. (B) Graphic representation of ADH1 (black bars) and ADH4 (grey bars) activity levels. Activity assays were performed with 0.1 M sodium phosphate, pH 7.5, at 25 °C, with 10 m M (ADH1) or 1 M (ADH4) ethanol as a substrate and 2.4 m M NAD + as a coen- zyme. Values are expressed as the arithmetical mean ± SD of measures from four different animals, each determination run in duplicate. (C)Immunoblotanalysisoftissueextracts (30 lg) using affinity-purified rabbit anti- (mouseADH4)IgG.Lanes:T,tongue; E, esophagus, S, stomach; D, duodenum; J,jejunum;I,ileum;C,caecum;Cl,colon, R, rectum. Liver (L) was used as a control. 2654 J. Vaglenova et al. (Eur. J. Biochem. 270) Ó FEBS 2003 throughout the cardiac, fundic, and pyloric regions (Fig. 3E,I,J). A strongly positive and specific signal was found in the epithelial cells lining the surface of gastric pits of the gastric body (Fig. 3F). Detection by IHC confirmed expression of ADH4 in the mucus-secreting cells of pylorus (Fig. 3G). Therefore, in the surface epithelium, ADH1 and ADH4 only overlapped in the gastric region close to the pylorus. The endothelium lining small blood vessels within the gastric mucosa and submucosa also showed ADH4 expression (data not shown). Fig. 2. Localization of ADH4 in tongue and esophagus by ISH and IHC analyses. Hematoxylin-stained section of filiform (A) and fungiform (D) tongue papillae. Immunodetection of ADH4 protein in stratified epithelium of tongue mucosa (B, C and E). Control section incubated with anti- ADH4 IgG preadsorbed with 15 lg recombinant mouse ADH4 (F). Detection of ADH4 mRNA in stratified squamous epithelium in sections of esophagus hybridized with antisense riboprobe (G). Control section of esophagus hybridized with ADH4 sense riboprobe (H). Immunodetection of ADH4 protein in queratinized stratified epithelium of esophageal mucosa (I) and control section of rat esophagus incubated with anti-ADH4 IgG preadsorbed with 15 lg recombinant mouse ADH4 (J). C, circumvallate papilla; E, squamous stratified epithelium; FI, filiform papilla; LP, lamina propia; MM, muscularis mucosae. Calibration bars: A–F (shown in F), G–H (shown in H) and I–J (shown in J), 50 lm. Ó FEBS 2003 Alcohol dehydrogenase in the gastrointestinal tract (Eur. J. Biochem. 270) 2655 Fig. 3. Localization of ADH1 and ADH4 in stomach body, pyloric region and the gastroduodenal junction by ISH and IHC analyses. Detection of ADH1 mRNA in the gastric mucosa of stomach body (A) and region close to pylorus (B–D). ADH4 mRNA (E,F,I,J) and protein (G) in the cells lining gastric pits of mucosa in the stomach body (E,F) and the pyloric region (G,I,J), but absent in duodenal mucosa (I). Control section incubated with anti-ADH4 IgG preadsorbed with 15 lg recombinant mouse ADH4 protein (H). B, Brunner glands; GP, gastric pits; L, lumen; MM, muscularis mucosae; SM, submucosa; V, villi. Calibration bars: A,E (shown in E) and I, 400 lm;B–D(showninD)andJ,200lm;F,G,H(showninG),50lm. 2656 J. Vaglenova et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Localization of ADH in small intestine In duodenum, ISH showed abundance of ADH1 mRNA in the absorptive mucosa and muscularis mucosae layer (data not shown), following the same pattern observed in the lower pyloric region (Fig. 3D). While ADH4 was abundant in stomach mucosa outer cell layers (Fig. 3E), it was absent in the duodenum external mucosa after a sharp transition at the gastroduodenal junction (Fig. 3I,J). ADH4 was only detected in muscularis mucosae. In the jejunum and ileum, both ADH1 and ADH4 mRNAs were detected throughout the epithelium in intestinal villi and crypts of Lieberku ¨ hn (Fig. 4A,B). By IHC, ADH4 was prominent in the epithelial cells lining intestinal villi, in contrast to crypts of Lieberku ¨ hn, which stained weakly (Fig. 4C). Connective tissue, lamina propia, and muscularis mucosae were not stained. Localization of ADH in the colorectal region Analysis of colorectal sections showed that ADH1 mRNA was localized primarily in the cells of the lower part of the crypts of Lieberku ¨ hn (Fig. 5A,B). In contrast, ADH4, at mRNA and protein levels, was detected uniformly along the crypts of Lieberku ¨ hn and in the surface brush-border epithelium (Fig. 5D,E,G,H). ADH4 immunostaining was Fig. 4. Localization of ADH1 and ADH4 in jejunum and ileum. ADH1 (A) and ADH4 (B) mRNA detection in jejunal mucosa. Immunodetection of ADH4 protein in ileal mucosa (C). Omission of anti-ADH4 IgG in an adjacent control section of ileum (D). CL, crypt of Lieberku ¨ hn; LP, lamina propia; MM, muscularis mucosae; SM, submucosa; V, villi; v, vessel. Calibration bars (shown in D): A,B, 200 lm; C,D, 50 lm. Ó FEBS 2003 Alcohol dehydrogenase in the gastrointestinal tract (Eur. J. Biochem. 270) 2657 absent in the submucosa, lamina propia and muscularis mucosae. Discussion Although several works had provided information on the ADH distribution in rodent digestive organs [2,7,20,21,34], the present report represents the most thorough study on the localization of the ethanol-metabolizing ADHs in the digestive tract tissues of adult rat. In previous reports, ADH1 was, in general, undetected in upper digestive organs, including stomach while ADH4 was not found in several intestinal regions [21,34]. Notably, here we demon- strate that ADH1 and ADH4 are expressed throughout the Fig. 5. Localization of ADH1 and ADH4 in the colorectal region. ADH1 (A,B) and ADH4 (D,E) mRNA detection in the longitudinal (A,D) and transversal (B,E) section of crypts of Lieberku ¨ hn. Control sections hybridized with ADH1 (C) and ADH4 (F) sense riboprobe. Immunodetection of ADH4 protein in longitudinal (G) and transversal (H) section of the Lieberku ¨ hn glands of colorectal mucosa. Control section incubated with anti- ADH4 IgG preadsorbed with 15 lg recombinant mouse ADH4 protein (I). CL, crypt of Lieberku ¨ hn; G, goblet cell; LP, lamina propia; MM, muscularis mucosae; SB, striated border of enterocytes. Calibration bars: A–F (shown in F) and G–I (shown in I), 50 lm. 2658 J. Vaglenova et al. (Eur. J. Biochem. 270) Ó FEBS 2003 rat gastrointestinal tract. Each ADH form, however, is confined to specific regions and cell populations. Thus, ADH1 is localized predominantly in the intestinal area whereas ADH4 is prominent in the most external parts (esophagus, stomach and colorectum) of the digestive system. In each tissue, except for the duodenum, ADH1 is confined to the inner cell layers of the mucosa, while ADH4 is localized in the outer cell layers exposed to the lumen. Interestingly, duodenum is the only region where ADH4 is absent from the external cell layers of the mucosa. The precise colocalization of mRNA, protein and activity demonstrates that these enzymes are present in the same regions where their mRNA is found. However, the restric- tion of the ADH1 and ADH4 expression to a relatively small number of cell types in specific regions could explain the previous difficulty of demonstrating their presence in various digestive organs [2,7,20,24,35]. Also, it should be considered that there may exist some rat/mouse species differences in ADH localization along the gastrointestinal tract that account for the slightly different ADH localization reported here for rat as compared to that previously reported for mouse [21]. Although ADH1 and ADH4 are found in all digestive tube organs, discontinuity exists regarding the cellular layers where the enzymes are expressed. Thus, while ADH1 is not expressed in the gastric pits of most of the stomach mucosa, it is of interest the progressive increase in expression in this external area as the mucosa reaches the pyloric region (Fig. 3B,C,D). Even more impressive is the sharp disap- pearance of ADH4 expression from the mucosa outer cell layers in the gastroduodenal junction (Fig. 3J). The sudden change in functional requirements in the transition between stomach and duodenum is therefore also reflected by marked differences in the expression levels of the ADH enzymes. Comparison of the present data from rat with the partial information available from human [24–25,29,30, and S. Porte ´ ,S.E.Martı ´ nez,J.Farre ´ sandX.Pare ´ s, unpublished results 2 ], indicates that the general pattern of ADH distribu- tion in the gastrointestinal tract is similar in the two species. The present results along with previous in vitro studies on the substrate specificity of ADH1 and ADH4 [2,7,9,12–14] provide the basis to hypothesize some physiological functions for these enzymes in the gastro- intestinal tract. However, precaution should be taken when extrapolating conclusions to human because of different ADH4 K m values for ethanol between rat and human (2.4 M vs. 37 m M , respectively) [9] and differences in diet, intestinal flora, etc. Role of gastrointestinal ADH in retinoid metabolism The expression of ADH1 and ADH4 in certain cell layers of gastrointestinal tissues, and its colocalization with the biochemical apparatus associated with RA responsiveness and metabolism [36–42], support the contribution of ADH1 and ADH4 (both exhibiting retinol dehydrogenase activity [12–15,17]) to RA generation in adult gastrointestinal tract. ADH1 and ADH4 displayed some nonoverlapping locali- zation which might reflect distinct roles, as has been suggested by studies with knockout animals [43]. ADH4, located in the most external tissues and cell layers with a high epithelial cell turnover, is well suited to fulfill a function in RA synthesis. In this sense, esophageal, gastric and colorectal mucosa show NAD + -dependent RA formation from all-trans-retinol, that is disturbed by inhibitors of ADH and aldehyde dehydrogenase (ALDH) [44,45]. On the other hand, b-carotene absorbed by intestinal enterocytes is converted to retinal which is subsequently reduced to retinol for transport and storage [46]. Thus, ADH1 and ADH4 (k cat /K m for retinal ¼ 500 m M )1 Æmin )1 [13] and 1750 m M )1 Æmin )1 [14], respectively) could be also involved in the step to generate retinol that would be immediately esterified in vivo. This could shift the reaction equilibrium towards retinal reduction, even in the absence of a favorable NAD/NADH ratio. Interestingly, we have shown that ADH4 is not present in duodenal enterocytes, where most b-carotene cleavage occurs [46]. Therefore, ADH1 would be the main ADH for the physiological retinal reduction in duodenum, although microsomal retinal reductases may also contribute to this function [47,48]. ADH4, specialized in retinal generation from retinol in specific tissues [21], could not be necessary in duodenal enterocytes where retinal is directly formed from b-carotene. Role of gastrointestinal ADH in alcohol metabolism and pathology Substrate specificity predicts that both ADH1 and ADH4 participate in the elimination of ingested alcohols and aldehydes, ethanol generated by intestinal microbial flora, and products of lipid peroxidation [12,13]. ADH4, located in the upper part of the gastrointestinal tract and the luminal part of the mucosa, would be in contact with the highest concentrations of ingested alcohols and aldehydes, and in areas subjected to high levels of oxidative stress. Therefore, ADH4 could act as a first metabolic barrier. Likewise, ADH1, that is positioned more internally along the tract and within the mucosa, could act as a second metabolic barrier. The localization of ADH4 suggests its contribution to the first-pass metabolism [49–53], mostly at high ethanol concentration (K m ¼ 2.4 M ) [9]. In addition, we have demonstrated here that ADH1 is also present in the upper digestive tract and therefore it may have a role as well in the first-pass metabolism, mostly at low ethanol concentrations (K m ¼ 1.4 m M ) [7]. In the lower gastrointestinal tract, colonic flora is the major source of endogenous ethanol in mammals that is produced constantly [54–56]. The main function of the high amount of ADH1 in colon might be the elimination of this endogenous ethanol. The presence of ADH throughout the gut can be related to alcohol pathology. Thus, ethanol and acetaldehyde have been associated with epithelial hyperegeneration of the mucosa and cancer [57–59]. On the other hand, disturbance of RA metabolism may be related to carcinogenesis [58, 60–62], and ethanol is a competitive inhibitor of retinol oxidation by ADH [12,51,63–66]. The esophagus and the colorectal region are especially vulnerable to alcohol injury [58,59], and these are tissues with the highest ADH activity (Fig. 1) where acetaldehyde-metabolizing ALDH2 is virtu- ally absent or scarce [25]. Thus, 50 l M acetaldehyde hampers RA formation [45], suggesting that acetaldehyde produced by ADH could also disturb RA generation catalyzed by retinal-active ALDH1 which has been also Ó FEBS 2003 Alcohol dehydrogenase in the gastrointestinal tract (Eur. J. Biochem. 270) 2659 detected in these gastrointestinal areas [25,30,41,42,67,68]. The impairment of RA formation by ethanol and acetal- dehyde could be an explanation for mucosal damage, increased cell proliferation and the high incidence of esophageal and colorectal neoplasia in alcohol abusers. In conclusion, we have detected ADH1 and ADH4 in distinct cell types of specific regions throughout the gastrointestinal tract, which evidences a local level of ethanol metabolism. Active ethanol oxidation in specific gastrointestinal regions can be related to some deleterious effects of ethanol. The involvement of ADH1 and ADH4 in retinol oxidation makes these enzymes relevant to gastro- intestinal functions that require RA. The impairment of retinol oxidation by inhibition of ADH during ethanol consumption may be an additional mechanism of gastro- intestinal alcohol pathology. 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ADH1 and ADH4 coexist throughout the gastrointestinal tract and provide new data to understand the physiological role of ADH classes in the gastrointestinal