EFFECT OF ETHANOL ON GASTROINTESTINAL TIGHT JUNCTIONS AND P-GLYCOPROTEIN EXPRESSION AND FUNCTIONALITY

Alcoholism, alcohol abuse or alcohol use disorder (AUD) is considered as a major untreated epidemic health concerns in modern societies. According to the 2014 report of World Health Organization (WHO), alcoholism causes approximately 6% of all cases of death every year. It has been evident that chronic alcohol consumption leads to organ damage, and in some cases, it progresses to cirrhosis and carcinoma. Some pharmacotherapy strategies have been proposed during decades to treat AUD; however, the efficacy of these medications to reduce drinking or alcohol abstinence remained controversial. Naltrexon, acamprosate, desulfiram, and nalmefen have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for alcohol abstinence. However, clinical studies show that the efficiency of these medications in AUD treatment is limited. As a result, finding a targetable biomarker in alcoholic patients to decrease alcohol craving and prevent organ damage remained as a major health challenge. According to the literatures, paracellular tight junction proteins are highly affected by ethanol (EtOH). In this work, the effect of EtOH on the expression of paracellular proteins as well as efflux transporters (mainly P-glycoprotein) was investigated. The effects of EtOH on paracellular route of drug permeation as well as P-glycoprotein-mediated drug efflux provide more insights onto how pharmacokinetic characteristics are impacted in alcoholic individuals. Understanding of the pharmacokinetic changes will lead to dose-adjustment for drugs that are administered to alcoholic patients. Moreover, finding novel biomarkers to treat different stages of alcoholism would be another beneficial outcome of this research. Gastrointestinal (GI) tract is considered as the first organ to be affected by alcohol. The highest EtOH concentration reaches to GI tract right after each alcohol drink. Due to propensity of GI tract epithelium with the highest EtOH concentration entering the body, more harmful effects would be predicted in GI tract after alcohol ingestion. Hence, estimating the actual concentration of EtOH in GI lumen after each alcohol intake and subsequently other organs seems to be beneficial. To address this question, a full physiologically based pharmacokinetic (PBPK) model was developed for EtOH as described in Manuscript I (to be submitted to the European Journal of Pharmaceutics and Biopharmaceutics). This manuscript describes development of an advanced dissolution, absorption, and metabolism (ADAM) model integrated into the Simcyp Simulator® 15 for alcohol. In this work, three common alcoholic beverages including beer (325 mg/kg body weight), wine (300 mg/kg body weight), and whisky (400 mg/kg body weight) were selected to be investigated. After simulation of beverages ingestion, the concentration-time profile of EtOH in stomach and duodenum as well as plasma and peripheral tissues was predicted. According to the results, the highest EtOH concentration was observed in stomach right after beverage ingestion and the concentration significantly decline during a 3-hour period. Eventually after stomach, duodenal concentration was the highest. The theoretical concentrations of EtOH in our model were validated according to the experimentally reported results. Moreover, EtOH concentration-time profile in welland poorly-perfused tissues was estimated. Results present that liver and muscle showed the highest and the lowest rate of EtOH absorption, respectively. Manuscript II (to be submitted to the Journal of Molecular Pharmaceutics) outlines the effect of clinically relevant EtOH concentrations on the expression and functionality of P-glycoprotein (P-gp) in Caco-2 cell monolayer. EtOH did not show significant alteration in cell viability at concentrations found in GI tract. To investigate the EtOH effect on P-gp, expression of P-gp was induced by treating normal cells with vinblastine (10 nM). Immunofluorescent (IF) images of normal and P-gp induced Caco-2 showed that the abundance of P-gp decreased by increasing EtOH concentration and treatment time. Moreover, the effect of EtOH on the abundance of xenobiotic transporters in normal and P-gp induced Caco-2 cells was analyzed. Sequential Windowed data independent Acquisition of the Total High-resolution Mass Spectra (SWATH-MS) proteomics approach showed that the abundance of P-gp polypeptides was decreased after treatment of normal and P-gp induced Caco-2 cells with EtOH for 4 and 24 h. Moreover, Calcein-AM assay showed that by increasing concentration of EtOH to 25 mM, the efflux activity of P-gp was reduced in P-gp induced Caco-2 cells. Increasing EtOH concentration more than 25 mM did not show significant effect on P-gp functionality. Furthermore, EtOH effect on transport parameters of talinolol (Tal, a P-gp substrate) and PF-5190457 (PF-57), an alcohol craving treatment currently undergoing clinical trials) was investigated in the presence and absence of verapamil (P-gp inhibitor). According to the results, EtOH showed significant decrease in efflux ratio (ER) of Tal in Caco-2 cells treated with 50 mM EtOH for 24 h in the presence of verapamil (200 μM). However, EtOH did not show significant effect on ER of PF-57 in the presence or absence of verapamil. Manuscript III (to be submitted to Biochimica et Biophysica Acta (BBA) Biomembranes) outlines the effect of clinically relevant EtOH concentrations on the organization of paracellular membrane proteins in Caco-2 cell monolayer. Neither EtOH, nor its metabolite, acetaldehyde (AA), showed significant alteration in cell viability at concentrations found in GI tract. Transepithelial electrical resistance (TEER) assay showed that the paracellular hyper permeability of Caco-2 cells induced by EtOH and AA was reversible. Fluorescent Lucifer yellow (LY) permeation showed that paracellular transport of LY was enhanced after treatment of Caco-2 cells with EtOH. Transmission electron microscopy (TEM) images of EtOH-treated Caco2 cells showed the disintegration of membrane proteins including tight junctions (TJs), adherent junctions (AJs), and desmosomes (DS). Moreover, the Sequential Windowed data independent Acquisition of the Total High-resolution Mass Spectra (SWATHMS) proteomics was used to analyze the EtOH effects on paracellular proteins. SWATH-MS showed that the abundance of TJs, AJs, and DS were diminished after treatment of Caco-2 cells with EtOH for 4 and 24 h. Manuscript IV (to be submitted to the Journal of Proteomics–clinical applications) outlines the proteome of sigmoid colon obtained from human subjects using labelfree quantification proteomics. The sigmoid colon of healthy human subjects was compared to alcoholic patients with and without liver disease. Moreover, the proteome of GI tract in chronic-binge rat model was investigated and compared to that in control rats. SWATH-MS proteomics exhibits as a prominent technique in quantitative analysis of proteins from limited biopsy samples. In this work, the proteome of human sigmoid colon biopsies as well as alcoholic rat GI tracts were studied. Results show that the expression level of some proteins in sigmoidal colon samples of alcoholic patients was altered compared to the healthy subjects. Significant differences were observed in expression of proteins in AWLDLQ subjects compare to the HC. No significant difference was observed in the expression of any investigated protein in ALD patients. Moreover, the effect of chronic EtOH consumption on proteins of different parts of GI tracts was examined in rat models. Vimentin and desmin showed a significant induction in pCol of binge-chronic rat models compare to the control group. The power of SWATH-MS proteomics in analysis of clinical biopsies might be helpful in identification of biomarkers to cure different stages of alcoholism. In summary, analyzing the proteome of Caco-2 cell monolayer treated by EtOH revealed alteration in efflux transporters (e.g., P-gp) as well as paracellular barrier proteins (i.e., TJs, AJs, and DS). Mainly, EtOH-treatment showed decrease in the expression level of paracellular barriers and efflux transporters. The proteomics evidence were confirmed with immunofluorescent assay, transmission electron microscopy (TEM) images, Calcein-AM assay, and transport behavior of talinolol (a P-gp probe). Furthermore, analyzing the proteome of binge-chronic rat models as well as human alcoholic patients with or without liver disease were accomplished. We anticipate that proteomic analyses of GI tract from alcoholic patients would be beneficial to explore protein biomarkers for early detection and treatment of alcoholrelated diseases in liver and GI tract. In this way, the power of mass spectrometry proteomics (i.e., SWATH-MS) in analysis of clinical biopsies might be helpful in identification of novel biomarkers to cure different stages of alcoholism.

however, the efficacy of these medications to reduce drinking or alcohol abstinence remained controversial. Naltrexon, acamprosate, desulfiram, and nalmefen have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for alcohol abstinence. However, clinical studies show that the efficiency of these medications in AUD treatment is limited. As a result, finding a targetable biomarker in alcoholic patients to decrease alcohol craving and prevent organ damage remained as a major health challenge. According to the literatures, paracellular tight junction proteins are highly affected by ethanol (EtOH).
In this work, the effect of EtOH on the expression of paracellular proteins as well as efflux transporters (mainly P-glycoprotein) was investigated. The effects of EtOH on paracellular route of drug permeation as well as P-glycoprotein-mediated drug efflux provide more insights onto how pharmacokinetic characteristics are impacted in alcoholic individuals. Understanding of the pharmacokinetic changes will lead to dose-adjustment for drugs that are administered to alcoholic patients. Moreover, finding novel biomarkers to treat different stages of alcoholism would be another beneficial outcome of this research.
Gastrointestinal (GI) tract is considered as the first organ to be affected by alcohol.
The highest EtOH concentration reaches to GI tract right after each alcohol drink.
Due to propensity of GI tract epithelium with the highest EtOH concentration entering the body, more harmful effects would be predicted in GI tract after alcohol ingestion.
Hence, estimating the actual concentration of EtOH in GI lumen after each alcohol intake and subsequently other organs seems to be beneficial. To address this question, a full physiologically based pharmacokinetic (PBPK) model was developed for EtOH as described in Manuscript I (to be submitted to the European Journal of Pharmaceutics and Biopharmaceutics). This manuscript describes development of an advanced dissolution, absorption, and metabolism (ADAM) model integrated into the Simcyp Simulator ® 15 for alcohol. In this work, three common alcoholic beverages including beer (325 mg/kg body weight), wine (300 mg/kg body weight), and whisky (400 mg/kg body weight) were selected to be investigated. After simulation of beverages ingestion, the concentration-time profile of EtOH in stomach and duodenum as well as plasma and peripheral tissues was predicted. According to the results, the highest EtOH concentration was observed in stomach right after beverage ingestion and the concentration significantly decline during a 3-hour period. Eventually after stomach, duodenal concentration was the highest. The theoretical concentrations of EtOH in our model were validated according to the experimentally reported results.
Moreover, EtOH concentration-time profile in well-and poorly-perfused tissues was estimated. Results present that liver and muscle showed the highest and the lowest rate of EtOH absorption, respectively.

Manuscript II (to be submitted to the Journal of Molecular Pharmaceutics)
outlines the effect of clinically relevant EtOH concentrations on the expression and functionality of P-glycoprotein (P-gp) in Caco-2 cell monolayer. EtOH did not show significant alteration in cell viability at concentrations found in GI tract. To investigate the EtOH effect on P-gp, expression of P-gp was induced by treating normal cells with vinblastine (10 nM). Immunofluorescent (IF) images of normal and P-gp induced Caco-2 showed that the abundance of P-gp decreased by increasing EtOH concentration and treatment time. Moreover, the effect of EtOH on the abundance of xenobiotic transporters in normal and P-gp induced Caco-2 cells was analyzed.
Sequential Windowed data independent Acquisition of the Total High-resolution Mass Spectra (SWATH-MS) proteomics approach showed that the abundance of P-gp polypeptides was decreased after treatment of normal and P-gp induced Caco-2 cells with EtOH for 4 and 24 h. Moreover, Calcein-AM assay showed that by increasing concentration of EtOH to 25 mM, the efflux activity of P-gp was reduced in P-gp induced Caco-2 cells. Increasing EtOH concentration more than 25 mM did not show significant effect on P-gp functionality. Furthermore, EtOH effect on transport parameters of talinolol (Tal, a P-gp substrate) and PF-5190457 (PF-57), an alcohol craving treatment currently undergoing clinical trials) was investigated in the presence and absence of verapamil (P-gp inhibitor). According to the results, EtOH showed Manuscript IV (to be submitted to the Journal of Proteomics-clinical applications) outlines the proteome of sigmoid colon obtained from human subjects using label-free quantification proteomics. The sigmoid colon of healthy human subjects was compared to alcoholic patients with and without liver disease. Moreover, the proteome of GI tract in chronic-binge rat model was investigated and compared to that in control rats. SWATH-MS proteomics exhibits as a prominent technique in quantitative analysis of proteins from limited biopsy samples. In this work, the proteome of human sigmoid colon biopsies as well as alcoholic rat GI tracts were studied. Results show that the expression level of some proteins in sigmoidal colon samples of alcoholic patients was altered compared to the healthy subjects.
Significant differences were observed in expression of proteins in AWLDLQ subjects compare to the HC. No significant difference was observed in the expression of any investigated protein in ALD patients. Moreover, the effect of chronic EtOH consumption on proteins of different parts of GI tracts was examined in rat models.
Vimentin and desmin showed a significant induction in pCol of binge-chronic rat models compare to the control group. The power of SWATH-MS proteomics in analysis of clinical biopsies might be helpful in identification of biomarkers to cure different stages of alcoholism.
In summary, analyzing the proteome of Caco-2 cell monolayer treated by EtOH revealed alteration in efflux transporters (e.g., P-gp) as well as paracellular barrier proteins (i.e., TJs, AJs, and DS). Mainly, EtOH-treatment showed decrease in the expression level of paracellular barriers and efflux transporters. The proteomics evidence were confirmed with immunofluorescent assay, transmission electron microscopy (TEM) images, Calcein-AM assay, and transport behavior of talinolol (a P-gp probe). Furthermore, analyzing the proteome of binge-chronic rat models as well as human alcoholic patients with or without liver disease were accomplished. We anticipate that proteomic analyses of GI tract from alcoholic patients would be beneficial to explore protein biomarkers for early detection and treatment of alcohol-   Table I

Introduction
The effect of drinking alcohol on drug absorption and bioavailability has received considerable attention during recent years. 1-3 Ethanol (EtOH) alters the body exposure to the extended-release drug formulations through induction of dose dumping. [4][5][6] While, the increased release of the dosage incorporated in prolonged-release formulations has serious safety concerns, FDA has recommended to characterize the effect of alcohol on the release profile of drugs. 5 Apart from the effect of alcohol on drug release and solubility behaviors, many EtOH-drug interactions have been reported. 7,8 For instance, analgesics 9 , antidepressants 10 , antibiotics 11 , anticoagulants 12 , and antidiabetics 13 have been proven to show adverse reactions in acute and chronic alcohol drinkers. Hence, the alcohol destination in human body could reveal its profound role in various metabolic pathways that affects the metabolism and disposition of drug substances.
When alcohol is absorbed, it undergoes two main oxidative and non-oxidative metabolic pathways. Those include (i) reversible oxidative conversion to acetaldehyde catalyzed by alcohol dehydrogenases (ADH1A, ADH1B*1, ADH1C*2, and ADH4) [14][15][16] and cytochrome P450 2E1 (CYP2E1) 17 in liver and intestine followed by irreversible oxidation to acetate and acetyl Co-A by aldehyde dehydrogenase 2 (ALDH2) 18 , and acyl-coenzyme A synthetase short-chain family member 2 (ACSS2) 19 ; (ii) EtOH involvement in sugars, amino and fatty acids through acetyl Co-A; (iii) oxidation via the tricarboxylic acid (TCA) cycle to CO 2 ; and (iv) non-oxidative conversion to fatty acid ethyl esters (FAEE) and phosphatidylethanols 20 . Fig. 1 shows a schematic illustration of EtOH role in different metabolic pathways occurring in human GI tract and liver (hepatocytes). In this scheme, EtOH oxidation to acetaldehyde and acetate (Fig. 1a), conversion of acetate to acetyl CoA (Fig. 1b), and acetyl CoA transformation to citrate and fatty acyl CoA (Figs 1c and d) have been shown.
The actual site and magnitude of EtOH first-pass metabolism in human remained controversial over the years. Some researches confirmed that a significant fraction of administered EtOH is cleared by first-pass metabolism primarily in gastric mucosa.
Julkunen et al., 21 , has shown that significant EtOH first-pass metabolism in rat is taking place in GI tract, mainly due to the presence of ADH4 in stomach. Meanwhile, other works suggested that approximately entire EtOH first-pass metabolism occurs in liver and gastric metabolism accounts for a small fraction of total EtOH clearance. 22 While determination of EtOH concentration in different organs is not easy to be experimentally measured, development of an in silico model to predict the concentration-time profile of EtOH in human tissues seems to be beneficial. One of the most helpful theoretical approaches that widely used in drug discovery and development is physiologically based pharmacokinetic (PBPK) modeling and simulation methodology. 23 The concept of PBPK modeling was first introduced by Teorell 24 in 1937 and developed rapidly during recent years due to emerging commercially available softwares (Simcyp ® , GastroPlus ™ , and PK-Sim ® ) 25 and ease of access to preclinical data. 26 Nowadays, a number of drug labels affirm that PBPK modeling was used to conduct clinical studies. Therefore, PBPK modeling is considered as a powerful tool in prediction of clinical data via evaluation of intrinsic (population properties, and genetics) and extrinsic (drug-drug interaction) factors. It combines the physicochemical data for a compound with the predefined physiological and biological properties of a specific population to obtain a mechanistic approach for that compound in the biological system. 27 In Moreover, the concentration-time profile of EtOH was predicted in liver, gut, brain, kidney, pancreas, spleen, skin, heart, muscle, and lung after ingestion of three common alcoholic beverages (beer, wine, and whisky). Finally, the bioavailability parameters of EtOH including area under the plasma concentration-time curve (AUC), maximum concentration observed (C max ), and time to achieve C max (T max ) were predicted and discussed in various tissues in fasted-and fed-states.

EtOH PBPK model
A whole-body PBPK model was constructed according to the advanced dissolution, absorption, and metabolism (ADAM) model integrated in Simcyp ® (Fig.   2). In this model, GI tract is divided into nine parts including stomach, duodenum, jejunum, ileum, and colon comprising of 1, 1, 2, 4, and 1 segments, respectively. This model shows the body as composed of 13 tissue compartments and 2 blood compartments (arterial and venous pools). The methodology and structure of the Simcyp ® population-based PBPK modeling was described in detail, previously [28][29][30] .
Anatomical and physiological parameters were modified according to the published literatures when it was essential ( Table 1).
The simulation of fasted-and fed-state gastrointestinal concentration of EtOH after ingestion of three common alcoholic beverages was implemented onto a PBPK modeling platform (Simcyp ® Simulator) and was validated based on the in vivo data obtained from literature. Physicochemical properties, physiological/population details, and trial design used for EtOH modeling have been summarized in Table 1.
In all trial designs standard adult body weight was set as 70 kg. 31 Three common alcoholic beverages, including beer (325 mg EtOH/kg body weight, 500 mL), wine (300 mg EtOH/kg body weight, 200 mL), and whisky (400 mg EtOH/kg body weight, 80 mL) were modeled in fasted-and fed-state using healthy human adult population.
In the modeling of fed-state, a volume of 250 mL of liquid meal was applied and initial volume of stomach fluid was modified accordingly in population details. Furthermore, other model input parameters such as, mean gastric emptying time, initial volume of stomach fluid, volume of intake alcohol, and dosage were retrieved from the literature and used for model development ( Table 1).

EtOH absorption, distribution, and elimination
After ingestion of alcoholic beverages orally, EtOH is absorbed rapidly from GI tract via passive diffusion. 32 The EtOH absorption starts from mouth and continues along the GI tract. About 15% of the initial EtOH dose is absorbed into stomach in fasted state, while only 30% of alcohol is passed to the GI tract once EtOH is administered with food. 22 According to the ADAM model EtOH oral absorption from luminal fluid to enterocytes was incorporated into unstirred boundary layer (UBL) (Simcyp ® manual). A permeability-limited membrane (basolateral side of enterocytes), which separates the enterocytes from intestinal interstitial fluid (ISF), is considered as the second step. Finally, a lymphatic route of absorption facilitates the EtOH entering systemic circulation.
Although the predominant site of EtOH metabolism is liver, the presence of ADH isozymes in mucosa of stomach, duodenum and jejunum begins the alcohol metabolism before liver. 33 The presence of ADH4 in stomach and upper sides of GI tract are proposed to be responsible for first pass metabolism of EtOH before reaching to liver. 32 Less than 2% of the ingested dose is metabolizing by gastric ADH4. 22 While, almost the whole EtOH elimination is taking place in liver, less than 10% of alcohol is excreted from lung, kidney, and skin. The breath alcohol clearance is 0.16 L/h, renal clearance is 0.06 L/h, and sweat clearance is 0.02 L/h. 34 Additional sites of EtOH clearance that were reported in the previously published literatures were applied in this modeling to simulate more realistic pharmacokinetic behavior.

The genetics of alcohol metabolism by ADHs
The list of the involved ADHs in EtOH metabolism and their corresponding Michaelis-Menten kinetic parameters has been shown in Table 2. According to the  22 Moreover, the concentration-time profile of EtOH in GI tract, liver, brain, kidney, pancreas, spleen, skin, heart, muscle, and lung after ingestion of three common alcoholic beverages (beer, wine, and whisky) were simulated. Rubbens and co-workers 39,40 are illustrated together. Fig. 3a) shows the observed and predicted EtOH concentration-time profiles in stomach after beer (500 mL) intake.

EtOH concentration-time profile in stomach and duodenum
Based on the results, no significant difference was observed in experimental and theoretical values for stomach EtOH concentration neither in fasted-nor in fed-state ( Fig. 3a and b). Fig. 3c shows approximately two times overestimation of C max after ingestion of wine (200 mL) in predicted EtOH concentration compare to the observed values in fasted-state stomach. After 50 min, predicted and observed EtOH concentrations were aligned with each other. According to the Fig. 3d, predicted and observed EtOH concentration-time profiles in fed-state found to be in good agreement with each other. Fig. 3e illustrates EtOH profile after ingestion of whisky (80 mL) in fasted-state. The initial predicted EtOH concentration was two times higher than that in the observed experiments; however, after 30 min they became aligned. Fig. 3f indicates EtOH profile after consumption of the same amount of whisky in fed-state.
Results show that there is no significant difference in the predicted versus observed profiles of EtOH in stomach.
The EtOH concentration-time profile in duodenum of healthy human volunteers in fasted- (Fig. 4a) and fed-state (Fig. 4b) after beer intake are shown. According to and fed-state, respectively. The predicted C max of EtOH in fasted-and fed-state is approximately two times higher than that in the observed graphs. Fig. 4e shows the overestimation of EtOH concentration in fasted-state after ingestion of whisky.
According to the graphs, the predicted and observed results were the same after 30 min of drink. Fig. 4f illustrates that predicted EtOH concentration at C max is two times greater than that in the observed plot, while the difference between predicted and observed values for EtOH concentration decreased by the time. Table 3 shows key bioavailability parameters of EtOH in fasted-and fed-state after intake of alcoholic beverages. It summarizes C max , T max , and AUC of EtOH after drinking alcoholic beverages.

Key bioavailability parameters of EtOH in stomach and duodenum
Results for beer show that C max of EtOH in fasted-state stomach is higher than that in fed-state. Similar trend was observed in duodenal EtOH C max in fasted-versus fed-state. While, duodenal C max of EtOH was decreased from 29.76 g/L in fasted-state to 16.67 g/L in fed-state, T max was roughly doubled ( Table 3). According to the obtained data for AUC 0-180 , there is a significant increase in AUC 0-180 of EtOH in fedstate stomach compare to fasted-state. However, the increased AUC 0-180 in fed-state duodenum was not as much. In the case of wine, C max of EtOH in fed-state stomach was lower than that in fasted-state. Likewise, duodenal EtOH C max in fed-state was less than that in fasted-state. Duodenal EtOH T max in fed-state was more than two times greater than that in fasted-state ( Table 3).  Table 3, C max of EtOH in fasted-state stomach after ingestion of whisky was 232.62 g/L, which is three times greater than that in fedstate stomach. Similarly, higher duodenal C max was observed in fasted-state than that in fed-state. T max of EtOH after whisky ingestion was comparable with the T max obtained for wine in duodenal fasted-and fed-state. Interestingly, AUC 0-180 of EtOH did not show any significant alteration in stomach and duodenum from fasted-to fedstate. illustrate F abs and F met of EtOH after beer intake. No significant difference in EtOH F abs and F met in different parts of GI tract was detected between fasted-and fed-state.

EtOH fraction dose absorbed and metabolized in GI tract
Similar pattern was observed in EtOH F abs and F met following wine ( Fig. 5c and d) and whisky ( Fig. 5e and f) administration. In all depicted graphs, the highest F abs and F met were corresponded to the upper GI tract sides which decreased by moving toward the lower GI tract areas. According to the Fig. 6a, the predicted EtOH C max after ingestion of whisky was 34% underestimated the observed EtOH plasma concentration in fasted-state. After 50 min of alcohol administration, the predicted and observed EtOH concentration values more aligned with each other. (Fig. 6b) shows that the highest predicted EtOH concentration after ingestion of whisky was ~ 60% underestimated the observed plasma EtOH C max .  Table 5 shows the C max , t max , and AUC achieved for EtOH in different organs of healthy human Caucasian population after ingestion of alcoholic beverages. In this table, the EtOH bioavailability parameters were obtained using the Simcyp ® model for well-perfused organs (e.g., gut, liver, lung, heart, and kidney) and poor-perfused tissues (e.g., skin, muscle, and brain). Based on the results, the highest and lowest EtOH C max were acquired for liver and muscle tissues, respectively.

Fig. 7
shows the concentration-time profile of EtOH in liver-gut-brain axis after ingestion of three alcoholic beverages. Fig. 7a and b) show EtOH concentration-time profile in liver for fast-and fed-state, respectively. Higher C max for EtOH in fastedstate was mentioned compare to that in fed-state. Fed-state EtOH T max was later than that in fasted-state. However, AUC drastically decreased in the case of fed-state.
EtOH concentration-time profile in gut for fast- (Fig. 7c) and fed-state ( Fig. 7d) conditions are illustrated. Higher C max for EtOH in fasted-state was observed compare to that in fed-state. According to the graphs, the elimination phase of EtOH in fedstate was slower than that in fasted-state. AUC of EtOH in gut tissue decreased in fed-state compare to the fasted-state. C max , T max , and AUC of EtOH in brain were depicted in fasted- (Fig. 7e) and fed-state (Fig. 7f). EtOH concentration-time profile in brain found to be close to the profile presented for gut tissue. As indicated in Fig. 7e and f, brains EtOH C max , T max , and AUC in fasted-state were higher than that in fed-state.

Discussion
Physiologic properties of human GI tract greatly affect the intragastric EtOH The present model provided the possibility to prediction EtOH concentration-time profile in organ and tissues. Among all organ and tissues, liver showed the highest C max and the lowest T max and AUC. That means higher rate and lower extent of EtOH absorption into the liver compare to the other organs. A probable justification for these results could be this fact that liver is one of the well-perfused organs and it is the main organ for EtOH metabolism. Conversely, muscle showed the lowest C max and highest T max . The reason is that muscle is one of the poor-perfused organs. The predicted EtOH concentration profile in the liver-gut-brain axis illustrated the significant decrease in EtOH concentrations in fed-state compare to the fasted condition. The lower concentration of EtOH in fed-state tissues concordant with the reduced plasma concentration of EtOH after food intake.

Conclusion
In the present study, a full PBPK model was developed for EtOH using ADAM   39,40 Single dose (mg/kg body weight) 325 (beer), 300 (wine), 400 (whisky) 39,40 Proportion of dose inhaled (%) 15 22 a ADAM: advanced dissolution absorption and metabolism, LogP O:W (octanol:water partition coefficient), α and β are scale and shape parameters in Weibull distribution, respectively. P app : apparent permeability, K D : dissociation constant of the EtOHprotein complex, HAS: human serum albumin.    Maximum concentration of EtOH (C max , mg/L), time to reach C max (t max , h), and area under the concentration-time curve (AUC, mg·h/L) after ingestion of three common alcoholic beverages (beer 500 mL, wine 200 mL, and whisky 80 mL) during 6 h. *C max of EtOH in liver after intake of alcoholic beverages in fasted-state was higher than the other organs. However, it was not statistically significant compared to all other organs (data were not shown).  EtOH is oxidized to acetaldehyde in hepatocytes through alcohol dehydrogenases (ADH1A, ADH1B*1, ADH1C*1, ADH1C*2, and ADH4) and cytochrome P450 2E1 (CYP2E1). Reactive oxygen species (ROS) are generated due to the interference of EtOH with electron transport complexes in mitochondrial membrane. Acetaldehyde is converted to acetate by aldehyde dehydrogenases (ALDH1/2). b) Acetate is metabolized to acetyl CoA, which considered as an important metabolic intermediate of tricarboxylic acid (TCA) and β-oxidation of fatty acids. c) Citrate could be converted to acetyl CoA by ATP citrate lyase (ACL). The generated acetyl CoA may result in DNA acetylating or involve in fatty acyl CoA production. d) Fatty acyl CoA is associated with lipogenesis reactions, which results in triglycerides, very lowdensity lipoproteins (VLDL) and finally alcoholic fatty liver in chronic alcoholic patients. The excess amount of EtOH in chronic alcoholic patients may result in elevated acetate in blood, lactic acidemia, and hyperlipidemia.  The EtOH doses for beer, wine, and whisky were 325, 300, and 400 mg/kg body weight. The standard human body weight was considered as 70 kg. The Simcyp ® data were compared with the experimental data reported by Rubbens et al [38,39], while the numerical data points were visualized using Plot Digitizer (http://plotdigitizer.sourceforge.net). , [22] and visualized using Plot Digitizer (http://plotdigitizer.sourceforge.net). The amount of EtOH in their study, which was administered orally, was 150 mg/kg body weight.

INTRODUCTION
The effect of drinking alcohol on drug absorption and bioavailability has received considerable attention during recent years. 1, 2 Ethanol (EtOH) alters the body exposure to the extended-release drug formulations through induction of dose dumping. [3][4][5] Hence, US Food and Drug Administration (FDA) has recommended to characterize the effect of alcohol on drug release profile. 6 Furthermore, EtOH affects the pharmacokinetic and pharmacodynamic of medication such as, ezogabine, 7 elvitegravir, 8 and opioids 9 in alcoholic patients. Acute EtOH consumption primarily affects the rate and extent of drug absorption 10 and to a lesser extend drug clearance. 11 Hence, estimation of EtOH interaction with the involved absorption mechanisms in gastrointestinal (GI) tract is beneficial to understand how EtOH alters the disposition of oral drugs.
The human colorectal adenocarcinoma cell line Caco-2 is widely used as a standard model for human intestinal epithelium to assess drug permeation and transport mechanisms. 12,13 Caco-2 monolayer has been broadly used to study all four possible drug transport pathways across epithelial cells including passive transcellular, paracellular, carrier mediated routes, and transcytosis. 14,15 One of the most abundant transporters in Caco-2 cell line and human GI tract that plays an important role in drugs absorption and disposition, is permeability-glycoprotein (P-gp). P-gp, also known as multi drug resistant protein-1 (MDR-1), is a 170 kDa transmembrane efflux transporter encoded by ATP-binding cassette (ABC) superfamily. [16][17][18] P-gp (ABCB1) is the most extensively studied transporter in mammalian that was first discovered in colchicine-resistant Chinese hamster ovary cells. 19 In human, P-gp is expressed on the apical side of GI epithelial cells, the apical side of epithelial cells in proximal tubules of kidney, the apical surface of epithelium in placenta, the biliary canalicular membrane of hepatocytes in liver, and endothelial cells in blood brain barrier (BBB). 20 Intestinal P-gp plays a significant role in absorption and disposition of a wide variety of drugs by limiting their cellular uptake from intestinal lumen into the enterocytes. Consequently, the functionality of P-gp and its expression level greatly influence the pharmacokinetic, safety, and efficacy profiles of drugs.
The expression of P-gp has been extensively investigated at mRNA and protein levels. 21,22 While, the mRNA level of P-gp does not necessarily translate into the amount of expressed protein, 23 the quantification of protein seems to be more reliable approach for P-gp expression assessment. 24,25 Enzyme-linked Immunosorbent Assay (ELISA), 26 flow cytometry, 27 and immunoblotting (Western blotting) 28 methods are the most well-known methods for identification and quantification of P-gp and other transporters. However, those methods suffer from some limitations such as, low throughput performance, intensive laboring, applying expensive antibodies, and low specificity. Mass spectrometry (MS), which avoids a vast majority of those flaws, has gained a great demand in protein studies. [29][30][31] Among all MS-based proteomics methods, the Sequential Windowed data independent Acquisition of the Total Highresolution Mass Spectra (SWATH-MS) offers superior advantages, like high accuracy and reproducibility over the other MS approaches. 32 Transport behavior of P-gp substrates across Caco-2 cell monolayer is broadly used by pharmaceutical industries to measure human intestinal P-gp activity. 33,34 Caco-2 cell monolayer transport assay is considered as a well-established method to determine if a compound is substrate, inhibitor or both for P-gp. While, P-gp is expressed in various organs (e.g., GI tract, BBB, liver, and kidney), inhibition of P-gp greatly affect the pharmacokinetic and efficacy of P-gp substrates. Consequently, inhibition of P-gp in GI tract increases the oral absorption of drugs that are P-gp substrate. Elevated plasma concentration of drugs with narrow therapeutic index can cause severe toxicity. Therefore, dose adjustment is needed for P-gp substrates once they are co-administered with a P-gp inhibitor.
In addition to Caco-2 monolayer transport assay, fluorescent probe efflux assay can also be used to evaluate P-gp functionality. Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent P-gp substrate that cleaves by intracellular esterases to form fluorescent impermeable calcein. 35,36 This method is widely used as a highthroughput, facile, and real-time P-gp inhibition assay. 37 In the present study, the effect of EtOH on expression and functionality of P-gp in normal and P-gp induced Caco-2 cell monolayer was analyzed. EtOH cytotoxicity in Caco-2 cells was examined and immunohistochemistry staining was used to visualize the expressed P-gp on the Caco-2 cell membrane. Moreover, SWATH-MS proteomics approach was used to find out if EtOH can alter the expression level of P-gp in Caco-2 cell monolayer. Furthermore, the transport behavior of talinolol (Tal, β 1 -antaginist) and PF-5190457 (PF-57, a ghrelin reverse agonist), as two model drugs, was investigated to find out the effect of EtOH P-gp activity. Finally, the Calcein-AM fluorescent assay was conducted to prove the effect of EtOH on P-gp efflux functionality.
where, R monolayer is the measured resistance of cell monolayer, R blank represents the resistance of filter inserts without cell monolayer, and A is the available filter inserts surface area.   Apparent permeability coefficient (P app ) of Tal was calculated according to the following equation:

Immunohistochemistry
where where, P app (AP-BL) and P app (BL-AP) stand for apparent permeability from AP to BL and BL to AP sides, respectively. Immunohistochemistry. Figure 3 shows the immunofluorescent (IF) microscope images of P-gp protein in normal (Figure 3a, c, and e) and P-gp induced (Figure 3b, d, and f) Caco-2 cells after buffer (Figure 3a and b) and EtOH (Figure   3c-f) treatments for 24 h. The higher expression level of P-gp proteins in the P-gp induced Caco-2 cells are clearly mentioned in IF images. In the absence of EtOH, no clear sign of P-gp was observed in normal Caco-2 cells (Figure 3a), while the localization of P-gp is clearly mentioned in P-gp-induced cells by monoclonal P-gp antibody (Figure 3b). Figure 3c and d show the normal and P-gp-induced cells after 24 h of treatment with EtOH (25 mM). There is no change in the appearance of normal and P-gp-induced cells after treatment with 25 mM EtOH. However, by increasing EtOH concentration to 50 mM, a clear decrease in the number of cells with distinct P-gp localization was observed (Figure 3f).

SWATH-MS.
Two P-gp digested peptides including K.LVTMQTAGNEVELENAADESK.S and K.GTQLSGGQK.Q were recognized and normalized according to the trypsin digested β-galactosidase intensity and total protein content. The cellular position of each peptide has been shown in Figure 4. Table 2 shows the abundance of six efflux transporters in 25, 50, and 100 mM EtOH-treated cells to the control group. According to the results, no significant difference was observed in the abundance of P-gp in control versus EtOH treated normal Caco-2 cells after 4 and 24 h of treatment (Table 2). Similarly, MRP-2, MRP-3, and MRP-4 showed signs of reduced expression in EtOH-treated groups versus control ( Table 2).

Calcein-AM Functional Assay.
To investigate the effect of EtOH on the functionality of P-gp, Calcein-AM assay was conducted to ensure the remained fluorescent yield of calcein inside the cells. in the presence and absence of verapamil (ER <2). According to the Table 1, the ER of PF-57 in P-gp induced Caco-2 cells was 7.1 ± 0.6 (ER>2) that proves PF-57 is a Pgp substrate. The ER of PF-57 was dropped to 1.7 ± 0.1 in the presence of verapamil. show that ER of Tal and PF-57 was significantly (P = 0.002) decreased in the presence of verapamil (Figure 6a and b). Moreover, P-gp induced Caco-2 cells treated with 50 mM EtOH showed significant decrease in ER of Tal in the presence of P-gp inhibitor (P = 0.04) compare to the control group. Further, the ER of PF-57 did not show any significant changes by increasing EtOH concentration (Figure 6b).

DISCUSSION
The mitochondrial toxicity of alcohol was examined after 24 To investigate the effect of drinking alcohol on the expression and functionality of P-gp, vinblastine was used to induce the expression of P-gp in Caco-2 cells. 45 Shirsaka and co-workers had previously explored that the mRNA level of P-gp in P-gp induced Caco-2 cells (vinblastine-induced cells) is approximately five times higher than that in normal Caco-2 cells. 46  Pxr and constitutive androstane activated receptor (car) mRNA expression in binge mice models 55 . According to the current study and the previous findings about EtOH effects on the induction of P-gp, it could be hypothesized that induction of nuclear receptors does not necessarily translate to protein expression.
The functionality of P-gp in transport of Tal was investigated in P-gp induced  Apical to basolateral apparent permeability (P app (AP-BL) ) and basolateral to apical apparent permeability (P app (BL-AP) ) as well as uptake ratio (UR) and efflux ratio (ER) across normal and P-gp induced Caco-2 cell lines are shown for Tal. Based on the reported data, ER of Tal was increased in P-gp induced Caco-2 cells, in which P-gp is overexpressed. All experiments were done in triplicate and data were shown as Mean ± Standard Error of Mean (Mea ± SEM, n = 3).      been investigated in alcohol-mediated liver diseases [5][6][7] . It has been evident that existence of a gut-liver-brain axis is important in EtOH-induced organ damage 8,9 .
Therefore, understanding the effect of alcohol on GI epithelium that plays a pivotal

Chemical and Reagents
Molecular biology grade EtOH and AA >99.5%, iodoacetamide (IAA), dithiothreitol (DTT), ammonium bicarbonate, and sodium deoxycholate were Proteomics grade trypsin and trypsin-predigested β-galactosidase (originated from Escherichia coli) were purchased from SCIEX (Framingham, MA). All other reagents used in this study were of analytical grade.

Cytotoxicity Assay
The cytotoxicity of EtOH and AA treatment on the Caco-2 cells was evaluated according to the colorimetric method using a water-soluble tetrazolium salt (WST-1).
In this method, the cleavage of the tetrazolium salt to formazan by active cellular

Transepithelial Electrical Resistance (TEER)
TEER is widely used as a real-time, non-destructive, and label free method to characterize the quality of the epithelial or endothelial barrier function in cell monolayer. TEER assay was used as a real-time, non-destructive, and label free method to characterize the quality of the cell monolayer integrity. The TEER of the normal and P-gp-induced Caco-2 cell monolayers were evaluated by an EVOM 2 (World Precision Instruments, Sarasota, FL) equipped with STX2 "chopstick" silver/silver chloride (Ag/AgCl) electrodes. TEER comprises from four different resistance on cell membrane that has been mentioned in Eq. 1.
Where, R a stands for apical cell membrane resistance, R b is basolateral cell membrane resistance, R tj represent for tight junction resistance, and R ic expresses the intercellular resistance 28 .
TEER assay was performed 21-days post seeding of normal and vinblastinetreated Caco-2 cells on filter inserts. TEER measurements were done before starting of EtOH treatment as well as 4 and 24 h after alcohol exposure to the Caco-2 monolayers. The relative changes in TEER values before and after EtOH treatments were compared to that for buffer treatment, as the control group. TEER values were calculated according to the following equation (Eq.2): (2) where, R monolayer is the measured resistance of cell monolayer, R blank represents the resistance of filter inserts without cell monolayer, and A is the available filter inserts surface area.

Transmission electron microscopy (TEM)
Caco-2 cell monolayers, grown on Transwell filter membranes, were fixed with 1.5% glutaraldehyde in 0.15 M sodium cacodylate buffer at 4 °C for several days.

LY Permeation through Caco-2 monolayer
Permeation of LY, a fluorescent marker, used to verify tight junction integrity in Caco-2 monolayers. Caco-2 cells were seeded onto semi-permeable PET filter inserts Apparent permeability coefficient (P app ) of LY was calculated according to the following equation: where, A stands for surface area of the cell monolayer (0.33 cm 2 ), C D (0) is the initial concentration of the drug added to the donor compartment, t is the time of transport, M r represents the mass of compound in the receiver side, and dM r /dt accounts for the flux of the drug across the cell monolayer. Uptake ratio (UR) and efflux ratio (ER) were obtained as below; where, P app (AP-BL) and P app (BL-AP) stand for apparent permeability from AP to BL and BL to AP sides, respectively.

Membrane Protein Extraction and Sample Preparation
The expression level of junctional membrane proteins were analyzed in normal Protein to trypsin ratio was 20:1 (w/w). Barocycler repeated cycles of hydrostatic pressure at 35000 psi for 90 cycles (90 min) to improve the protein digestion.
Afterwards, 10 µL of 2.5% formic Acid in 1:1 (v/v) mixture of water/acetonitrile was added to the digest and centrifuged at 10,000 ×g for 5 minutes at 4°C. Finally, 2.5 µL of β-galactosidase (31.25 pmol), as an external control, was added to the digested protein solution before injection to LC-MS/MS.

SWATH-MS Analysis
The SWATH-MS proteomics analysis and data processing were accomplished  to 60 min before the start of next run. The amount of protein per injection on the column was 10 μg. In each batch, trypsin-digested β-galactosidase that is a quality control standard (1.65 pmol/injection) was injected to each sample to monitor mass calibration of the TOF detector and normalization of peptides intensity in SWATH label free quantification (LFQ) approach. The LFQ was performed using Skyline, which is an open source application for targeted proteomics quantitative data analysis.

Protein Quantification
Membrane Proteins and digested peptides concentrations were analyzed using   Cytotoxicity results showed that at EtOH concentration < 500 mM, which is more clinically relevant, no significant cytotoxicity is induced by EtOH compared to the control group (buffer-treated cells). Further, AA did not show significantly reduction in cell viability even after three days of incubation at 1000 µM. Elamin and coworkers have previously shown that exposure of Caco-2 spheroids to EtOH and AA did not show any significant reduction in cell viability 33 .  The reduction in TEER value by EtOH in Caco-2 cells was reported previously. 34,37,38 Fisher and co-workers suggested that EtOH and its metabolite acetaldehyde are able to increase the paracellular permeability of Caco-2 cells without altering viability. 34 The proposed mechanism for EtOH-induced enhancement in paracellular permeability is disruption of TJs proteins. The proposed mechanism is activation of myosin light chain kinas (MLCK) which results in phosphorylation of MLC and occluding and subsequently destabilization of TJs 39, 40 . Table 1 shows the permeability of LY, a fluorescent marker for paracellular pathway, across mature (highly integrate paracellular barrier) and non-mature (loose barrier) Caco-2 monolayers. As shown in the Table 1  Cresci et al have shown that EtOH-exposed mice showed reduction in the expression and co-localization of zonula occludens-1 (ZO-1) and occludin in the ileum and proximal colon 42 . The reduction in occludin and ZO-3 expression level was confirmed in our study using normal and vinblastine-induced Caco-2 cells ( Table 2).

SWATH-MS proteomics
Moreover, Zhao and co-workers have shown that EtOH exposure in mice decreased the expression of occludin leading to intestinal hyper permeability 43 (Table 2). However, normal and vinblastine-induced Caco-2 cells treatment with 50 mM did not show any decrease in expression level.
It has been mentioned that the levels of gut AJs (e.g., β-catenin and E-cadherin) and desmosome plakoglobin were clearly decreased in binge alcohol-exposed rats 46 .  Apical to basolateral (P app (AP-BL) ) and basolateral to apical apparent permeability (P app (BL-AP) ) as well as uptake ratio (UR) and efflux ratio (ER) across mature and nonmature Caco-2 cells monolayers. Data are shown as Mean ± SEM (n = 3).     (Figure 3d). However, Caco-2 treatment with 50 mM EtOH (Figure 3b) did not affect the MV and the structure of MV after 24 h treatment with 50 mM EtOH was comparable to the control groups (Figure 3a and c). The organization of tight junctions (TJs), adherens junctions (ADs), and desmosomes (DS) are disrupted after 24 h treatment of cells with 50 and 100 mM EtOH (Figure 3b and d) compare to the control groups (Figure 3a and c).

This manuscript has been prepared for submission to the "Proteomics-Clinical
Applications"

Introduction
Alcohol abuse or alcohol use disorder (AUD) is considered as an untreated epidemic health concern in modern societies 1  Alcoholic liver disease (ALD) comprises a wide spectrum of liver disorders ranging from alcoholic steatosis to alcoholic steatohepatitis (ASH), alcoholic hepatitis, progressive fibrosis, cirrhosis, and hepatocellular carcinoma. 5,7 Reports show that while a majority portion of heavy alcohol drinkers develop steatosis, only a minority of patients with steatosis progresses to ASH and subsequently cirrhosis and carcinoma 8,9 . There is evidence suggesting that EtOH is not the only reason in progression of liver diseases but also the existence of a gut-liver-brain axis needs to be considered as another player. Consequently, many pathophysiological mechanisms that are affected by EtOH exposure and implicated in disease development remained unknown. Hence, analyzing proteome of alcoholic patients would be beneficial to explore protein biomarkers for early detection and treatment of alcohol-related diseases in liver and GI tract 5 . It has been evident that the paracellular permeability in GI tract could be considered as a practical indicator to determine the severity of EtOH damage.
The artificial sweetener sucralose (a chlorinated derivative of sucrose) is considered as a useful GI tract permeability probe 10 . Resistance of sucralose to bacterial fermentation makes it as a suitable marker for whole GI tract permeability.
The higher urinary excretion of sucralose indicates the higher gut leakiness. 11 The usefulness of sucralose permeation in determining gut leakiness has been investigated in alcoholic steatohepatitis. 12 The alcoholic subjects without liver disease can be In this study, the proteome of sigmoid colon obtained from healthy human subjects was compared with that in ALD patients. Furthermore, alcoholic patients with no liver disease and different intestinal permeability were also examined.
Moreover, the proteome of GI tract of chronic-binge rat model was investigated and compared with that in control rats. analysis. Figure 1 shows a schematic illustration of the experimental procedure from tissue lysis and protein digestion to injection of samples to LC-MS/MS and data analysis.

Triple-TOF MS analysis in SWATH mode
The SWATH-MS proteomics analysis and data processing were accomplished based on the previously published method. 19,20 Briefly The digested P-gp peptides were separated on an Acquity UHPLC Peptide BEH C18 (2.1 × 150 mm 2 , 300 Å, 1.7 μm) equipped with Acquity VanGuard precolumn (2.1 × 5 mm 2 , 300 Å, 1.7 μm). Autosampler temperature was kept at 10 °C and the column temperature was maintained at 40 °C during all injections. The chromatographic separation was performed with a runtime of 120 min at 100 μL/min with a gradient method using mobile phase A (98% water, 2% acetonitrile, 0.1% formic acid) and mobile phase B (98% acetonitrile, 2% water, 0.1% formic acid). A gradient chromatographic elusion method was performed as follows: 98% A from 0 to 3 min, 60% to 90% A from 3 to 48 min, 20% A held from 49 to 52 min to flush the column, 98% A at 53 min. The column was equilibrated at 98% A from 53 to 60 min before the start of next run. The amount of protein per injection on the column was 10 μg. In each batch, trypsin-digested β-galactosidase that is a quality control standard (1.65 pmol/injection) was injected to each sample to monitor mass calibration of the TOF detector and normalization of peptides intensity in SWATH label free quantification (LFQ) approach. The LFQ was performed using Skyline, which is an open source application for targeted proteomics quantitative data analysis.

Statistical Analysis
The normality of data sets in each experiment was checked with Shapiro-Wilk test. One-way analysis of variance (ANOVA) with Dunnett's T3 post hoc test (SPSS 23, SPSS, Inc.) was used for normally distributed observations. Kruskal-Wallis (KW) one-way ANOVA (non-parametric ANOVA) with Dunn's multiple comparison post hoc test was used in non-normally distributed data. In all analyses, probability values <0.05 was considered significant.

EtOH affects the expression of ALDH2 in human sigmoid colon
The expression level of proteins in sigmoid samples of ALD, AWLDLQ, AWLDHQ, and HC subjects has been investigated by SWATH-MS proteomics. The fold changes in proteins expression are mentioned in Table 1. According to the results, the expression of the most proteins was not significantly altered in alcoholic subjects versus HC. However, aldehyde dehydrogenase2 (ALDH2), filamin A, and glutathione S-transferase A1 (GSTA1) showed significant differences across groups.
ALDH2 detoxifies acetaldehyde (major EtOH metabolite) into acetate. The role of ALDH2 in metabolism of EtOH inside the cells has been illustrated in Figure 2. 21 Many studies on mice models have shown that ALDH2 overexpression in liver cells ameliorates the pathological damages induced by chronic EtOH intake. 22 Moreover, Chaudhry and co-workers have shown that ALDH2 deficiency enhances EtOHinduced disruption of paracellular barriers in alcohol-fed mice models. 23 In our proteomics results, expression of ALDH2 in ALD and AWLDHQ was insignificantly lower than that in HC group ( Table 1). Progression of alcoholism to liver disease in ALD subjects as well as higher paracellular damage in AWLDHQ group might be induced due to the lower ALDH2 expression. In contrast, AWLDLQ subjects showed significantly higher expression of ALDH2 compared to the HC. More expression of ALDH2 in AWLDLQ patients might be the reason for retaining paracellular integrity in these subjects.

Figure 3a
shows a Box-plot illustrating the relative abundance of ALDH2 across four groups of subjects. KW with Dunn's post hoc test showed that there is statistically significant difference in expression of ALDH2 across AWLDHQ versus AWLDLQ subjects (P-value = 0.005). Higher expression of ALDH2 in AWLDLQ subjects may explain why paracellular barrier in these subjects is more integrated than that in AWLDHQ. Figure 3b shows a Box-plot illustrating the relative abundance of GSATA1 across four groups of subjects. KW with Dunn's post hoc test showed that there is statistically significant difference in expression of GSTA1 across ALD versus AWLDLQ subjects (P-value = 0.02). According to the Table 1, the GSTA1 expression in ALD subjects was insignificantly lower than that in HC (P = 0.23). It was previously indicated by Ma and co-workers that GSTA1 is downregulated in EtOH-induced in mice model. 24 In that study, the protective role of GSTA1 in scavenging the free radicals generated by EtOH was suggested to justify the GSTA1 release from liver. The lower expression of GSTA1 in sigmoid samples of ALD subjects was confirmed in our proteomics study. However, the expression level of GSTA1 in AWLDLQ was higher than that in HC ( Table 1). Sigmoid colon in AWLDLQ subjects is possibly less affected by reactive oxygen species generated by EtOH consumption. Enhanced expression of GSTA1 in AWLDLQ may suggest the different role of this enzyme in EtOH-induced injury in GI tract compared to liver. Figure 4 shows the role of filamin A in relation to other cytoskeletal components.

EtOH affects the expression of filamins in human sigmoid colon
The expression of filamin A is decreased in ALD, AWLDLQ, and AWLDHQ subjects compares to the HC (Table 1). However, the reduction of filamin A expression was statistically significant in AWLDLQ subjects (P-value = 0.05). Tobin and co-workers have hypothesized that clinically relevant EtOH concentration (20-40 mM) disrupts the mu-opioid receptor (MOP)-filamin A interaction 25 . Our results showed alteration in the expression level of filamin A in alcoholic subjects compared to the HC group.
According to our results, it could be suggested that diminished expression of filamin A in alcoholic patients is probably the reason for disrupting MOP-filamin A interaction.
No significant difference was observed in the expression of any investigated protein in ALD patients compared to the HC. The reason is the organ of analysis.
While, the site of EtOH-induced damage in these subjects is liver, proteomics analysis of liver samples from ALD patients might be more relevant.

EtOH affects the expression of proteins in rat GI tract
The fold changes in proteins expression in stomach, ileum, pcolon, and d-Col of EtOH-treated versus control rat groups are mentioned in Table 2. Results showed the significant increase of vimentin in pCol region of alcoholic rat models (P-value = 0.05). Vimentin is an intermediate filament protein involved in cellular structure and integrity. Induction of vimentin was not statistically significant in dCol ( Table 2).
Results show expression of vimentin was not observed in stomach samples of control and binge rats. Moreover, the vimentin expression was slightly decreased in ileum but not statistically significant (P-value = 0.8).
Kelso et al have shown that vimentin is upregulated in binge rat models with neurodegeration. 26 They found that a prominent increase in vimentin expression happened 4 and 7 days after the last EtOH dose. Duly and co-workers has shown that binge alcohol rat models induced liver fibrogenesis by increasing the expression of vimentin. 27 in another study on alcohol-fed mice, Ambade et al have shown that chronic EtOH exposure leads to vimentin upregulation. 28 Although, these works had been done in brain and liver tissues, our pCol proteomics result is in agreement with their findings.  Non-parametric Mann-Whitney U test was used to determine the significance of difference between ALD, AWLDLQ, and AWLDHQ groups versus healthy control (HC) group, respectively. *Statistically significant decrease was observed in expression of FLNA in AWLDLQ versus HC group. The expression of GSTA1, and ALDH2 were significantly higher in AWLDLQ subjects compared to the HC group. Abbreviations: E-stom: stomach of EtOH-treated rats, C-Stom: stomach of control rats, E-Ile: ileum of EtOH-treated rats, C-Ile: ileum of control rats, E-dCol: distal colon of EtOH-treated rats, C-dColon: distal colon of control rats, E-pCol: proximal colon of EtOH-treated rats, C-pColon: proximal colon of control rats. E-stom/C-Stom shows the fold change observed in the proteins abundance in stomach of EtOH-treated versus stomach of control rat group. Likewise, E-Ile/C-Ile, E-dCol/C-dCol, and E-pCol/C-pCol show the fold change observed in the protein abundance of Ile, dCol, and pCol from EtOH-treated versus control rat groups. Non-parametric Mann-Whitney U test was used to determine the significance of difference between EtOH-treated groups versus control group in each part of GI tract. *Statistically significant difference was observed in expression of Vim in alcohol-treated rats versus control groups.   Protein abundance across control (HC), alcoholic with liver disease (ALD), alcoholic without liver disease low quartile sucralose permeation (AWLDLQ), and alcoholic without liver disease high quartile sucralose permeation (AWLDHQ) groups was illustrated. Kruskal-Wallis H-test with post hoc Dunn's was conducted to find out the significance level among groups. The P-value of difference between AWLDLQ and AWLDHQ subjects was 0.005. Furthermore, the P-value between AWLDLQ and ALD was 0.02 for GSTA1.