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 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 vinblastin (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 Caco-2 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 (SWATH-MS) 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 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 immunoflourescent 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-related 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