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Ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells: Role of fatty acid ethyl esters

Hai Wu, Kamlesh K. Bhopale, G. A. S. Ansari, Bhupendra S. Kaphalia
DOI: http://dx.doi.org/10.1093/alcalc/agm044 1-8 First published online: 17 October 2007

Abstract

Aims: To understand the mechanism(s) of alcoholic pancreatitis and role of fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol) in ethanol-induced pancreatic injury. Methods: A time- and concentration-dependent synthesis of FAEEs and the cytotoxicity of ethanol and its predominant fatty acid esters were studied in rat pancreatic tumour (AR42J) cells in cultures. Role of FAEEs in ethanol-induced cytotoxicity was investigated by measuring the synthesis of FAEEs, injury markers and apoptosis in cells incubated simultaneously with ethanol and FAEE synthase inhibitor, 3-benzyl-6-chloro-2-pyrone. The cells were pre-incubated with caspase-3 inhibitor (N-acetyl-DEVD-CHO) to measure the effect of caspase-3 inhibition on ethanol-induced apoptosis. Results: The levels of FAEEs synthesized in cell cultures incubated with 800 mg% ethanol for 6 h were ∼10-fold higher (60 nmol/25 × 106 cells) than those in cells incubated with 100 mg% ethanol (5.4 nmol/25 × 106 cells). Ethanol exposure resulted in a concentration-dependent apoptosis (10, 12 and 13% at 200, 400 and 800 mg% ethanol, respectively, vs 5% in controls). A similar concentration-dependent apoptosis was also found in the cells incubated with ethyl oleate (one of the predominant FAEEs reported in alcoholic patients). Inhibition of FAEE synthesis and resultant apoptosis was found in the cells incubated simultaneously with pancreatic FAEE synthase inhibitor and ethanol. Ethanol-induced apoptosis was significantly inhibited in cells pre-incubated with caspase-3 inhibitor. Conclusions: These results support our hypothesis that ethanol-induced cytotoxicity in AR42J cells is mediated by the non-oxidative metabolite(s) of ethanol, and caspase-3 mediated apoptosis could be one of the mechanisms involved in ethanol-induced pancreatic injury.

Introduction

Chronic alcohol abuse is the second major cause of chronic pancreatitis after the biliary duct diseases and many alcohol abusers die before reaching to the clinical stage of the disease (Pitchumoni et al., 1984; Singh and Simsek, 1990; Lankisch and Banks, 1998). The clinical picture of acute pancreatitis is a continuing inflammation due to tissue autolysis by activated proteolytic enzymes, typically causing pain and/or permanent loss of pancreatic function. Chronic pancreatitis may result from recurrent episodes of acute pancreatitis and is described as functionally and morphologically different from acute pancreatitis (Lankisch and Banks, 1998). The mechanism and etiology of alcoholic pancreatitis is currently not well understood.

Oxidative metabolism of ethanol to acetaldehyde, catalysed by alcohol dehydrogenase (ADH), is the major pathway for its disposition in the liver (Rognstead and Grunnet, 1979). Such oxidative metabolism is reported negligible in the extrahepatic organs such as pancreas, frequently damaged in chronic alcoholism (Laposata and Lange, 1986). An acute large bout or subchronic ingestion of alcohol as well as chronic alcohol abuse are known to inhibit/impair hepatic ADH (Nuutinen et al., 1983; Sharkawi, 1984; Palmer and Jenkins, 1985; Panes et al., 1989, 1993; Kaphalia et al., 1996). Similar inhibition was also observed in vitro following incubation of ADH with ethanol (Shore and Theorell, 1966; Zahlten et al., 1980). Inhibition of hepatic ADH by 4-methyl pyrazole is reported to increase non-oxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) by several folds in the pancreas and cause pancreatitis-like injury in rats (Manautou and Carlson, 1991; Werner et al., 2002). The FAEE synthase is reported to be highest in the pancreas (Laposata and Lange, 1986; Bhat and Ansari, 1990; Kaphalia and Ansari, 2003) and can be up-regulated by subchronic exposure to ethanol (Pfutzer et al., 2002). Therefore, non-oxidative metabolism of ethanol to FAEEs could be a prevalent and favoured pathway for ethanol disposition in the pancreas during chronic alcohol abuse.

The metabolism of ethanol in the pancreas has been reviewed earlier in detail by Wilson and Apte (2003). Detectable activities of ADH and cytochrome P4502E1 (CYP2E1) have been reported in the pancreata of humans and rats (Foster et al., 1993; Kessova et al., 1998; Norton et al., 1998; Chrostek et al., 2003). Irrespective of over expression of CYP2E1, hepatic ADH-deficiency facilitates synthesis of FAEEs (Bhopale et al., 2006; Wu et al., 2006). Therefore, hepatocellular ADH activity appears to be a dominant factor in ethanol metabolism.

Although several animal models used to study the acute and chronic alcoholic pancreatitis have provided insights in several factors contributing to the alcoholic pancreatitis (Schneider et al., 2002), a suitable animal model for the chronic alcoholic pancreatitis is still not available. Increased synthesis of FAEEs in the pancreas is shown to be associated with pancreatitis-like injury in rat and deer mouse models (Werner et al., 2002; Bhopale et al., 2006). These fatty acid esters are reported to cause several-fold greater cytotoxicity than ethanol or its oxidative metabolite, acetaldehyde, in freshly isolated rat pancreatic acinar cells (Criddle et al., 2004). Because of short life span of dispersed freshly isolated pancreatic acinar cells, rat pancreatic tumour (AR42J) cell line has been used for investigating the biology of isolated pancreatic cells (Christophe, 1994). Earlier, we reported a significant synthesis of FAEEs in AR42J cells (Kaphalia et al., 1999). However, metabolic basis of ethanol-induced cytotoxicity has not been investigated in AR42J cells, so far. In this study, we examined time- and concentration-dependent synthesis of FAEEs and evaluated their cytotoxicity in AR42J cells.

Materials and Methods

Materials

FAEE standards, ethyl palmitate, ethyl oleate and p-nitrophenyl acetate (PNPA) were obtained from Sigma Chemical Co. (St Louis, MO). Cell lysis buffer, protein assay kit and leupeptin from Pierce Biotechnology, Inc. (Rockford, IL) and HPLC grade solvents and SentiVerse (scintillation flour) from Fisher Scientific (Fairlawn, NJ) were used. [1-14C] Ethanol (specific activity 56 mCi/mmol) was purchased from American Radiolabelled Chemicals, Inc. (St Louis, MO).

Polyclonal antibodies against human CYP2E1 and ADH (yeast) were obtained from (Oxford Biomedical Research, Oxford, MI) and Rockland (Gilbertsville, PA), respectively. Antibodies to rat pancreatic cholesterol esterase (ChE) were generously provided by Dr Gallo (Gallo et al., 1978). 3-Benzyl-6-chloro-2-pyrone (3-BCP, an inhibitor of pancreatic ChE) was synthesized according to Bailey et al. (1995) and characterized as described earlier (Kaphalia et al., 2004a). Cell permeable N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO, caspase-3 inhibitor) was procured from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA).

Cell culture

AR42J cells obtained from ATCC (Rockville, MD) were cultured at 37 °C at sub confluent densities (80%) in F-12K medium supplemented with 2 mM L-glutamine, 20% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified air atmosphere containing 5% CO2. Cell cultures were regularly monitored for the mycoplasma using Mycoplasma Detection Kit (Boehringer Mannheim, Mannheim, Germany). Cells were dispersed in 5 ml 0.25% trypsin solution containing 2.2 mM EDTA for cell counting (Kaphalia et al., 1999; Wu et al., 2006). Frozen rat pancreata (PelFreez Biologicals, Arkansas) were homogenized in 0.05 M phosphate buffer (pH 7.4). FAEE synthase, ADH and CYP2E1 were measured in the cell lysates and rat pancreatic homogenate by Western blot analysis using respective antibodies (Kaphalia and Ansari, 2003; Wu et al., 2006). Viability, apoptosis and necrosis of the cells were determined by TACS Annexin V-FITC Apoptosis Detection kit (R&D Systems, Minneapolis, MN).

For the metabolism and cytotoxicity studies, 100, 200, 400 and/or 800 mg% ethanol in the culture medium was incubated with ∼80% confluent cells in T75 polystyrene tissue culture flasks for 6 h as described previously (Wu et al., 2006). Studies with 3-BCP (FAEE synthase inhibitor) and Ac-DEVD-CHO (caspase-3 inhibitor) were conducted in culture medium and/or in lysate of cells incubated with 800 mg% ethanol.

Metabolism of ethanol

Residual ethanol and acetaldehyde levels in culture medium

Concentration of ethanol and its oxidative metabolite, acetaldehyde, were determined in the culture medium of cells incubated with ethanol by head space analysis using a Hewlett Packard 5890 GC equipped with flame ionization detector (Strassnig and Lankmayr, 1999; Wu et al., 2006). Recoveries for ethanol and acetaldehyde from the culture medium were >95%.

Time- and concentration-dependent formation of FAEEs

Time-dependent formation of FAEEs was studied using sub confluent cells incubated with 800 mg% [1-14C] ethanol (specific activity 0.125 μCi/mM) at 37 °C for 1, 3 and 6 h. For concentration-dependent formation of FAEEs, sub confluent cells were incubated with 100, 200, 400 or 800 mg% [1-14C] ethanol (specific activities 1.0, 0.5, 0.25 or 0.125 μCi/mM, respectively). After incubation, cells were scraped along with culture media from the tissue culture flasks, lipids extracted with chloroform: methanol (2: 1, by volume) and separated by thin layer chromatography (Kaphalia et al., 1999; Wu et al., 2006). The total 14C-label in the ester region was measured and expressed as nmol FAEEs/25 ×106 cells.

Markers of pancreatic injury

Amylase, lipase and PNPA-hydrolyzing activity in cell culture media

Both amylase and lipase are synthesized and stored in acinar cells of the pancreata. Elevated activities of these enzymes in the plasma are generally considered markers of acute pancreatic injury (Lankisch and Banks, 1998). Therefore, amylase and lipase activity were determined in the culture medium of AR42J cells incubated with ethanol using Biotron Diagnostic kits (Biotron Diagnostics Inc. Hemet, CA, Product # 017-C and 049, respectively) and expressed as U/l.

FAEE synthase, a membrane-bound as well as cytosolic enzyme found abundant in the mammalian livers and pancreata, is released in the serum of patients with pancreatic disease (Aleryani et al., 1996). Rat pancreatic FAEE synthase has been purified and characterized to be triacylglycerol lipase and ChE (Riley et al., 1990; Kaphalia and Ansari, 2003), and found to be structurally and functionally different than that in the livers (Kaphalia et al., 1997; Kaphalia and Ansari, 2001a). Pancreatic as well as hepatic FAEE synthase are known to co-express PNPA-hydrolyzing (Kaphalia et al., 1997; Kaphalia and Ansari, 2001a, 2003). Therefore, PNPA-hydrolyzing activity was measured in the culture medium as a surrogate of FAEE synthase activity according to Erlanson (1970) and expressed as nmoles PNPA hydrolysed/ml/min.

Trypsinogen activation peptide assay

Activation of trypsinogen to trypsin releases trypsinogen activation peptide (TAP). This peptide is attached at the amino-terminus of trypsinogen in vertebrates and contains a sequence tetra-L-aspartyl-L-lysine (D4K) (Hurley et al., 1988). The activation process occurs in the proximal small intestine during digestion following cleavage at lysine carbonyl by a gut hormone, enterokinase. However, premature activation of trypsinogen within the acinar cells in the pancreas may cause pancreatitis and release TAP in the pancreas and circulation. Antibodies to tyrosine-D4K (YD4K) conjugated with keyhole limphet hemocyanin (KLH) were raised in white New Zealand rabbits in Protein Expression and Purification Laboratory, Biomolecular Resource Facility, The University of Texas Medical Branch, Galveston, TX.

TAP was measured in the cell lysate by an enzyme-linked immunosorbent assay (ELISA). The 96-well microplate (Fisher, Pittsburgh, PA) was coated with 100 μl 0.38 mM peptide (2, 4, 8, 16 and 32 times diluted) using coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) as standard or 80 μl coat buffer and 20 μl cell lysate at 4 °C overnight. After washing thrice with washing buffer (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0), 200 μl of blocking buffer (50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0) was added to each well and the plate was incubated at room temperature for 30 min. 100 μL rabbit anti-TAP antibody (1 : 2000 diluted) was added to each well and incubated at room temperature for 60 min. Supernatant was removed and each well washed five times as described above. 100 μL goat anti-rabbit IgG-HRP conjugate (Southern Biotech, Alabama, 1 : 2000 diluted) was transferred to each well and again incubated at room temperature for 60 min. 3,3,5,5′-Tetramethylbenzidine (100 μl) was added to each well and the reaction stopped after 10 min by adding 100 μl H2SO4 (2 M). The absorbance was read at 450 nm using a microtiter plate reader (Bio-Rad, Hercules, CA) and data is expressed as nmol/mg protein.

Ethanol- and FAEE-induced cytotoxicity

For apoptosis studies, 5 × 105 trypsinized AR42J cells/well were seeded in 24 well plates and incubated overnight at 37 °C. The cells were treated with ethanol (100–800 mg% ethanol), ethyl oleate or ethyl palmitate (62.5–500 μM) and incubated for 6 h. Stock solutions of ethyl oleate or ethyl palmitate were prepared in ethanol and 10 μl of the stock solutions were mixed slowly with culture media to give final concentration of 62.5–500 μM. Cells incubated with or without 10 μl ethanol served as control. Cytotoxicity (apoptosis and necrosis) was measured by TACS Annexin V-FITC Apoptosis Detection kit as per manufacturer's instructions. In brief, after the incubation, cells were trypsinized and collected by centrifugation at 500 × g for 10 min at room temperature. The cells were washed by resuspending in 500 μl ice-cold phosphate buffer and centrifuged as described above. The cell pellet was gently re-suspended in Annexin V incubation reagent (10× binding buffer, propidium iodide, Annexin V-FITC) and incubated for 15 min at room temperature. The cell suspension was re-centrifuged as described above. The cell pellet was re-suspended in 400 μl binding buffer and subjected to flow cytometry (BD FACSCanto benchtop flow cytometry system, BD Biosciences, Franklin Lakes, NJ) within 1 h for maximal signal. The apoptosis is expressed as percent apoptotic cells.

FAEE synthesis and caspase-3 inhibition studies

Inhibition of FAEE synthesis and apoptosis by 3-BCP

In preliminary studies, PAPA-hydrolyzing activity was inhibited ∼50% at 6 h in the culture medium by 10 μM 3-BCP. Therefore, sub confluent AR42J cells in culture were simultaneously exposed to 100–800 mg% [1-14C] ethanol as described earlier in the presence or absence of 10 μM 3-BCP (pancreatic FAEE synthase inhibitor) for 6 h. The cells along with media were extracted with chloroform: methanol (2: 1, by volume) thrice. The pooled extract was dried under nitrogen and 14C-label in the ester fraction was measured and expressed as nmol FAEEs/25 × 106 cells (Kaphalia et al., 1999; Wu et al., 2006). Effect of FAEE synthase inhibitor on ethanol-induced apoptosis was studied in cells incubated with 800 mg% ethanol in the presence of 10 μM 3-BCP. The assay procedure for PNPA-hydrolyzing activity was essentially according to Erlanson (1970) as described earlier (Kaphalia et al., 1999). Apoptosis was measured using TACS Annexin V-FITC Apoptosis Detection kit as described earlier.

Inhibition of ethanol-induced apoptosis by caspase-3 inhibitor

About 80% confluence AR42J cells in T25 polystyrene tissue culture flasks were incubated with caspase-3 inhibitor, Ac-DEVD-CHO (0.1 μM), for 2 h followed by incubation with 800 mg% ethanol for 6 h. Cells were scraped and centrifuged at 500 × g for 10 min. The pellet was washed with Dulbecco's phosphate buffered saline and the cell pellet was re-suspended in the chilled cell lysis buffer (provided with the kit) for 30 min followed by centrifugation at 10 000 × g for 10 min. Since necrosis was not altered in cells incubated with ethanol or ethyl oleate as compared to the controls, apoptosis was measured by Cell Death Detection ELISA kit (Roche Applied Science, Indianapolis, IN; Cat. No. 1774425) for the quantitative measurement of cytoplasmic histones-associated-DNA-fragments using mouse monoclonal antibodies directed against DNA and histones. The absorbance was measured by ELISA reader (Bio-Rad) at 405 nm against 2, 2′-azino-bis [3-ethylbenzothiozoline-6-sulfonic acid] solution as a blank. Caspase-3 activity was assayed using Calbiochem Kit (La Jolla, CA; Cat #235418) in the cell lysate and expressed as U/μg protein.

Statistical analysis

All data are presented as mean ± standard error of the mean (S.E.M.). Analysis of Variance (ANOVA) followed by Student-Newman Kuel's post-hoc test was used to analyse the data using the GraphPad/instat program. P values <0.05 were considered statistically significant.

Results

The formation of FAEEs was found to be time- and concentration-dependent. Ethanol or ethyl oleate produced concentration-dependent increases in apoptosis. Ethanol-induced apoptosis was significantly inhibited by the inhibitors of FAEE synthase or caspase-3. These results suggest role of FAEEs in ethanol-induced cytotoxicity.

Western blot analysis

Western blot analysis of FAEE synthase, CYP2E1 and ADH in the lysates of AR42J cells and homogenate of rat pancreas is shown in Fig. 1. Contrary to the higher CYP2E1 levels in rat pancreas, negligible ADH was found in AR42J cells as well as in rat pancreas. Based on densitometric analysis of the blots, ChE-related FAEE synthase was ∼25% more in AR42J cells than in rat pancreatic homogenate.

Fig. 1

Western blot analysis of AR42J cell lysate and rat pancreatic homogenate using antibodies to ADH (yeast), CYP2E1 (human) and pancreatic cholesterol esterase (rat). Forty μg protein was loaded into each well and β-actin used as loading control.

Metabolism of ethanol and formation of FAEEs

Oxidative versus non-oxidative metabolism of ethanol

Approximately 72, 79, 62 and 67% residual ethanol was estimated in the cell culture medium at 6 h after incubation with 100, 200, 400 or 800 mg% ethanol, respectively. The acetaldehyde concentration (∼0.1%) was detectable only in the culture media of the cells incubated with 800 mg% ethanol (Table 1). The total FAEEs formed in the cell culture was found to be 19, 22 and 56 nmoles/25 × 106 cells at 1, 3 and 6 h after incubation with 800 mg% ethanol, respectively [Fig. 2(A)] and 5, 12, 20 and 60 nmoles FAEEs/25 × 106 cells incubated with 100, 200, 400 or 800 mg% ethanol, respectively [Fig. 2(B)]. These results suggest that the formation of FAEEs in AR42J cells is time- and concentration-dependent.

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    Fig. 2

    Time-dependent (A) and concentration-dependent (B) synthesis of FAEEs by the AR42J cells incubated with ethanol for 6 h at 37 °C. For time-dependent studies, cells were incubated with 800 mg% ethanol. Values are mean ± S.E.M. (N = 4).

    Inhibition of FAEE formation by FAEE synthase inhibitor

    Simultaneous exposure of AR42J cells to 3-BCP and ethanol inhibited FAEE synthesis at all concentrations of ethanol used in the present study (Fig. 3). However, a significant inhibition was found only in the cells incubated with 800 mg% ethanol (27.6 nmoles/25 × 106 cells incubated with 3-BCP vs 47.3 nmoles/25 × 106 cells without 3-BCP).

    Fig. 3
    Fig. 3

    Inhibition of FAEE synthesis by 3-BCP in AR42J cells incubated with different concentrations of ethanol for 6 h at 37 °C. Values are mean ± S.E.M. (N = 4). **P < 0.01.

    Markers of ethanol-induced pancreatic injury

    Amylase, lipase and PNPA-hydrolyzing activity

    Decreases in amylase and lipase activity were found in the culture medium of cells incubated with 400 and 800 mg% ethanol as compared to the controls. However, only lipase activity was significantly decreased in the culture medium of the cells incubated with 800 mg% ethanol. Amylase and lipase activities were 168 and 15 U/l, respectively, in the culture medium of cells incubated with 800 mg% ethanol versus 181 and 27 U/l, respectively, in controls (Table 2). PNPA-hydrolyzing activity was not altered significantly in the culture medium after incubation with ethanol at all concentrations used in the present study (Table 2).

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    Table 2

    Amylase, lipase and PNPA-hydrolyzing activity in the culture medium and TAP levels in lysate of AR42J cells incubated with different concentrations of ethanol for 6 h at 37 °C

    Ethanol in cell culture medium (mg%)
    Control100200400800
    Amylase (U/l)180.9 ± 6.2180.1 ± 11.3180.1 ± 18.0167.5 ± 12.7168.1 ± 10.1
    Lipase (U/l)27.2 ± 2.322.8 ± 2.527.6 ± 1.721.2 ± 3.015.0 ± 1.3*
    PNPA-hydrolyzing activity (nmol/min/ml)15.9 ± 0.215.9 ± 0.716.8 ± 0.315.7 ± 0.716.0 ± 0.3
    TAP (nmol/mg protein)10.4 ± 2.620.0 ± 1.0*20.9 ± 0.9**20.9 ± 1.1**19.1 ± 0.8*
    • Values are mean ± S.E.M. (N = 5).

    • * P < 0.05,

    • ** P < 0.01 versus control.

    TAP

    Concentration of TAP was not detectable in the culture medium of cells incubated with ethanol. Despite a significant increases in TAP levels in the lysates of cells incubated with ethanol (100–800 mg%) as compared to the controls, a dose-dependent response was not observed in the present study (Table 2).

    Ethanol- and FAEE-induced cytotoxicity in AR42J cells

    Ethanol- and FAEE-induced apoptosis

    Apoptosis was found to be ∼9, 10, 12 and 13% in the cells incubated with 100, 200, 400 and 800 mg% ethanol, respectively, as compared to 5% in the control (Fig. 4). A similar concentration-dependent apoptosis was also observed for ethyl oleate (62.5–500 μM, Fig. 5). No significant change was found in the necrotic cells in ethanol- or ethyl oleate-treated cells as compared to that in control cells (data not shown). Surprisingly, no significant apoptosis was observed in the cells incubated with ethyl palmitate (data not shown), suggesting that ethyl oleate could be one of the potential FAEEs involved in ethanol-induced pancreatic injury.

    Fig. 4

    Apoptosis in AR42J cells incubated with different concentrations of ethanol for 6 h at 37 °C. Values are mean ± S.E.M. (N = 6). *P < 0.05, **P < 0.01.

    Fig. 5

    Apoptosis in AR42J cells incubated with different concentrations of ethyl oleate for 6 h at 37 °C. Values are mean ± S.E.M. (N = 4). *P < 0.05, **P < 0.01.

    Inhibition of ethanol-induced cytotoxicity by 3-BCP

    Simultaneous exposure of AR42J cells to ethanol and 3-BCP significantly decreased ethanol-induced apoptosis and reversed the inhibition of lipase activity from 13 to 16 U/l in the culture medium of cells incubated with 800 mg% ethanol (Table 3). However, a similar reversal was not observed for amylase activity (data not shown). Similarly, 3-BCP could also not inhibit ethanol-induced TAP levels significantly (Table 3).

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    Table 3

    Effect of 3-BCP on ethanol-induced apoptosis, lipase secretion and TAP levels in AR42J cells

    Treatment groups
    ControlEthanolEthanol + 3-BCP
    % apoptosis4.4 ± 0.510.4 ± 0.9**7.63 ± 8.8*
    Lipase (U/l)22.9 ± 1.912.6 ± 1.1*15.9 ± 1.0
    TAP (nmol/mg protein)14.8 ± 2.621.7 ± 1.7**18.3 ± 0.9
    • Values are mean ± S.E.M. (N = 5).

    • * P < 0.05,

    • ** P < 0.01 versus control or ethanol group.

    Inhibition of ethanol-induced apoptosis by caspase-3 inhibitor

    Cells incubated with 800 mg% ethanol in the presence of caspase-3 inhibitor, Ac-DEVD-CHO, significantly decreased apoptosis [Fig. 6(A)] as well as caspase-3 activity [Fig. 6(B)] as compared to those treated with ethanol only.

    Fig. 6

    Effect of caspase-3 inhibitor (N-acetyl-DEVD-CHO) on ethanol-induced apoptosis (A) and caspase-3 activity (B) in AR42J cells incubated with 800 mg% ethanol for 6 h at 37 °C. Values are mean ± S.E.M. (N = 5). *P < 0.05 versus ethanol group.

    Discussion

    Pancreas is one of the major target organs of injury during chronic alcohol abuse and FAEEs are implicated in alcoholic pancreatitis (Laposata and Lange, 1986). ADH-deficiency could be one of the major reasons for the greater synthesis of FAEEs in AR42J cells as supported by previous findings in the pancreata of rats pre-treated with 4-methyl pyrazole and in ADH-deficient deer mice (Manautou and Carlson, 1991; Werner et al., 2002; Bhopale et al., 2006). Therefore, ADH deficiency appears to be an important determinant involved in disposition of ethanol via non-oxidative metabolism. These findings support our hypothesis that hepatic ADH deficiency facilitates non-oxidative metabolism such as formation of FAEEs. Due to its highest FAEE synthase activity and greater FAEE synthesis than other organs/tissues, the pancreas is considered a major target of ethanol toxicity or disease during chronic alcohol abuse (Laposata and Lange, 1986; Kaphalia and Ansari, 2001b; Kaphalia and Ansari, 2003). Apart from its own FAEE biosynthesis, pancreas can also accumulate a large quantity of FAEEs from the circulation (Wilson and Apte, 2003).

    Ethanol-containing diet is known to increase the sensitivity of cholecystokinin octapeptide (CCK)-induced pancreatitis in rats (Pandol et al., 1999). Such sensitivity to pancreatitis could also be due to the increased formation of FAEEs as CCK is known to induce FAEE synthase activity (Huang and Hui, 1994). Most of the glandular functions of acinar cells are mimicked by AR42J cells (Christophe, 1994). Formation of FAEEs and associated cytotoxicity reported in the present study support the utility of AR42J cells as a cell culture model to study the mechanism of ethanol-induced pancreatic acinar cell injury. These cells also have low capacity to oxidize ethanol as revealed by the presence of relatively low levels of acetaldehyde in the media of cells incubated with ethanol and low ADH (Fig. 1); a metabolic condition similar to that observed in chronic alcohol abusers (Nuutinen et al., 1983; Sharkawi, 1984; Palmer and Jenkins, 1985; Panes et al., 1989, 1993).

    A residual 532 mg% ethanol concentration found in the media of cells after 6 h incubation with 800 mg% ethanol is comparable to the blood alcohol concentration generally found in chronic alcoholic cases (Lindblad and Olsson, 1951; Christopoulos et al., 1973; Hammond et al., 1973; Berild and Hasselbalch, 1981). However, a much lower level of FAEEs synthesized in AR42J cells incubated with 800 mg% ethanol than the levels reported in the plasma of chronic alcoholic patients explains relatively low cytotoxicity observed in the present study (Kaphalia et al., 2004b). The FAEE levels equivalent to that reported in chronic alcohol abusers should be generated for the cytotoxicity studies to understand the role of FAEEs in ethanol-induced pancreatic injury.

    In view of the reported pancreatic toxicity of FAEEs (Wilson et al., 1990; Kaphalia and Ansari, 2001b; Criddle et al., 2004), ethanol-induced cytotoxicity in AR42J cells as observed in this study could be associated with formation of FAEEs. A significant inhibition of ethanol-induced apoptosis by 3-BCP, an inhibitor of FAEE synthase, further supports our hypothesis that FAEEs are involved in the ethanol-induced cytotoxicity.

    Elevated serum amylase and lipase are widely used as diagnostic markers in acute pancreatitis (Lankisch and Banks, 1998). However, the decreased lipase and amylase activity observed in the culture medium of AR42J cells incubated with ethanol could be related to the formation of FAEEs as found for plasma markers of hepatic injury in rats administered ethanol, halogenated alcohols or 2-chloroethyl linoleate (Kaphalia et al., 1992, 1996). Such changes may be associated with impaired membrane transport properties and/or decreased ATP synthesis due to the uncoupling effect of FAEEs on mitochondrial oxidative phosphorylation (Lange and Sobel, 1983).

    Plasma TAP has been used as a marker of pancreatic injury (Schmidt et al., 1992; Werner et al., 2002). However, lack of ethanol-induced concentration-dependent response of TAP levels as observed in the present study is surprising and needs further studies. Pancreatitis-like injury (pancreatic edema, TAP activation and vacuolization of acinar cells) by ethyl palmitate is reported in a rat model (Werner et al., 1997). Saturated as well as unsaturated FAEEs are reported to elicit elevation of free ionized cytosolic Ca2+ in the pancreatic acinar cells in culture (Criddle et al., 2004). However, a differential toxic response of ethyl oleate and ethyl palmitate in AR42J cells observed in the present studies could not be explained. The findings reported in this study suggest that ethanol-induced cytotoxicity in AR42J cells is related to the formation of FAEEs based on concentration-dependent formation of FAEEs and associated cytotoxicity. This conclusion is further supported by the observed results of reduced ethanol-induced apoptosis and toxicity by 3-BCP (FAEE synthase inhibitor) and inhibition of ethanol-induced apoptosis and caspase-3 by caspase-3 inhibitor. As reported earlier (Wu et al., 2006), ethanol-induced apoptosis appears to be mediated by the non-oxidative metabolism of ethanol to FAEEs. Therefore, a detailed evaluation of cytotoxicity of individual FAEEs detected in the human plasma and tissues could be important in understanding the mechanism of alcoholic pancreatic injury. The results of this study also suggest that pancreatic acinar cells are the major site for the synthesis and accumulation of FAEEs, and FAEEs contribute significantly to ethanol-induced injury.

    Acknowledgments

    This work was supported by NIH grant AA13171 from the National Institute on Alcohol Abuse and Alcoholism. The authors also acknowledge the assistance of Synthetic Organic Chemistry Core for the synthesis of 3-BCP supported through NIEHS Center grant P30ES06676.

    References

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