Mitogenic and functional responses by nicotine and hydrogen peroxide in AR42J cells: a comparative study
© Walker et al; licensee BioMed Central Ltd. 2008
Received: 13 June 2008
Accepted: 31 July 2008
Published: 31 July 2008
The aim of the current study was to investigate the oxidative effects of nicotine by examining the mitogenic and functional responses in AR42J cells. As a control and for comparison, hydrogen peroxide (H2O2) was used as a source of known oxidative biomarker. Responses were examined by determining cell proliferation through the activation of ERK signaling, basal and CCK-stimulated cell function and measuring lipid peroxidation. AR42J cells have been exposed to either a non-cytotoxic dose of 20 μM H2O2 for 15 min or to 100 μM of nicotine for 3 min respectively. Nicotine and H2O2 at these dose and time intervals produced similar levels of malondialdyde (MDA) production and p-ERK1/2 activation. Immunofluorescence studies employing specific antibody to p-ERK1/2 confirmed the latter. Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells. CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2. These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production. The data suggests that nicotine-induced mitogenic response in AR42J cells closely identifies the response induced by an oxidative biomarker.
Nicotine, one of the main chemicals in tobacco, has been known as a primary psychoactive ingredient that is responsible for the reinforced behavior in smokers. Each year in the United States, 435,000, or 1 in every 5 deaths, are attributed to cigarette smoking . About half of the young adult smokers today who continue to smoke throughout their life will die of a smoke related diseases . Further, it has been shown that smoking is an independent risk factor in the development of chronic pancreatitis and pancreatic cancer [3, 4]. In animal studies it has been shown that nicotine plays a role in the induction of pathophysiology of pancreas [5, 6].
Evidence shows that lipid peroxidation occurs in pancreatic tissues when exposed to nicotine  and that the mitochondrial respiratory chain is affected by nicotine leading to an increased generation of superoxide anions and hydrogen peroxide . Clinical studies have indicated that patients with acute pancreatitis have a higher plasma levels of lipid peroxide than that observed in patients with mild pancreatitis . This suggests that multiple etiological factors other than the release of enzymes may be responsible in this mechanism. As of to-date, however, there have been no reported studies investigating the role of oxyradicals induced by nicotine in the pancreas, and to determine whether oxyradical formation by nicotine contribute to the pathophysiological mechanisms associated with pancreatic injury encountered in smokers We have shown earlier that nicotine induces functional alterations and MAP kinase signaling pathways in pancreatic acinar cells [10, 11]; however, the underlying mechanisms responsible for these observed effects by nicotine are still not completely understood. We surmise in this study that nicotine induces the oxidative stress in pancreatic acinar cells and thus contributes to this mechanism.
Oxidative stress arises when there is an imbalance between the formation of reactive oxygen species (ROS) and removal of oxyradicals by scavenging antioxidants. Increase in ROS production has been directly linked to the oxidation of cellular macromolecules, which may cause direct cellular injury or induce a variety of cellular responses through the generation of secondary metabolic reactive species . Clinical studies have shown that oxidative stress leading to lipid peroxidation appears to be linked to the pathogenesis of chronic pancreatitis . Other evidence showing the production of large amounts of oxygen radicals in lymphocytes due to cigarette smoking  suggests that nicotine derived from cigarette smoking may play a role, in pathophysiological process.
The current study was designed to examine whether exposure of AR42J cells to nicotine causes the production of reactive oxygen species. Thus we have reexamined the mitogenic and functional responses of this cell line to nicotine and compared its effects with hydrogen peroxide (H2O2), a known oxidative biomarker, in the same cellular system in order to evaluate the direct effect of oxidative stress in this cell line. The AR42J cell line was used because of its stability as an immortal tumor cell line and its known similarity in physiological characteristics to primary acinar cells .
Materials and methods
AR42J cells, a rat pancreatic tumor cell line, were obtained from ATCC (Rockville, MD). These cells were grown in 75 cm2 flasks with 12 ml of Ham's F12 nutrient media with 2 mM L-glutamine and 1.5% NaHCO3 (F12K, obtained from Hyclone, Logan, UT), to which 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin were added. The flasks were kept in an incubator maintained at 37°C with a 5% CO2/95% air atmosphere until they reached over 80% confluency.
Assay of Cell Cytotoxicity in the presence of H2O2
The cytotoxic effect of H2O2 on the cells was measured using an LDH-Cytotoxicity Assay Kit from Biovision Research Products (Mountain View, CA). The kit consisted of a lyophilized catalyst and a dye reagent. The test samples were prepared with various doses of H2O2 ranging from 10–100 μM and cell cytotoxicity measurements were performed following the recommendation of the manufacturer. The cytotoxicity study with nicotine varying doses has been reported previously in this cell system . A dose of 100 μM nicotine was found to be non-toxic. The percent of cytotoxic cells increased significantly beyond this dose level of nicotine. Thus this dose of nicotine was selected for this comparative study.
Measurement of cellular lipid peroxidation products induced by H2O2 and nicotine
Lipid peroxidation assay was conducted using MDA-586 method (Oxis Research, Portland, OR) with whole cell lysates obtained after treatment with nicotine or H2O2. Malondialdehyde (MDA, Sigma-Aldrich, St Louis, MO) was used as a standard. Both the standards and whole cell lysates were incubated for 1 h in a 45°C water bath with N-methyl-2-phenylindole (NM2P, dissolved in acetonitrile) and diluted with methanol together with concentrated HCl. The ratio of cell lysate to the volume of NM2P solution was 1:5. Methods of measurement of MDA from tissue homogenates and blood have been reported recently from our laboratory [16, 17].
MAPK Signaling assay by Western Blot Analysis
Whole cells lysates were prepared from flasks containing more than 80% confluent cells that were tyrpsinized. About 1–2 × 106 cells were plated per flask. The cells were allowed to attach. The cells were incubated overnight in serum free media. Cells were treated with 100 μM nicotine or 20 μM H2O2, washed with cold PBS and placed on ice. Two hundred fifty microliters of RIPA buffer containing PMSF/protease III cocktail inhibitor was added. The cells were lysed, sonicated and incubated on ice for 40 min. The cell protein mixture was then spun down at 12,000 rpm for 10 min; supernatant removed and kept on ice. Protein concentration was determined by Bradford assay with bovine serum-albumin as the standard .
For Western Blot analysis, a total of 40 μg of cellular protein was loaded onto 12% SDS-polyacrylamide gels and electrophoresed for 1/12 h at a steady voltage of 120 V. The separated protein bands were then transferred to nitrocellulose membranes (BioRad Laboratories, Hercules, CA). The primary antibodies used for probing the nitrocellulose membrane overnight were obtained from Cell Signaling (Danvers, MA). The antibodies used were: anti-ERK1/2, anti-pERK1/2. Subsequently membranes were probed with horseradish peroxidase-conjugated secondary antibody (Pierce Biotechnology Inc, Rockford, IL). Enhanced chemiluminescence (ECL+, Amersham BioSciences, Piscatway, NJ) was used to visualize the bands. The band intensity was quantified using a STORM 860 Imager (Molecular Dynamics, Inc, Sunnyvale CA).
Cell Proliferation Assay
Cell proliferation studies were conducted after treatments with 100 μM nicotine, or 20 μM H2O2 using commercially available Cell Viability and Cytotoxicity Assay Kit (Cell Counting Kit, CCK-8, Dojindo Molecular Technologies Inc. Gaithersburg, MD). Ninety six-well microplates were used and 2 × 104 cells per well were plated. The cells were allowed to attach for 24 h in media containing 10% FBS. Following this, the cells were kept in 0.05% FBS containing media overnight before being treated with 100 μM nicotine or 20 μM H2O2. Twenty μl of CCK-8 dye was added to each well at specified time interval and incubated further for 3 h at 37°C. The absorbance was measured at 450 nm.
Localization of MAPK signals measured by Immunofluorescence Imaging
For these studies, 4 × 104 cells per well were plated in 4-well Lab-Tek chamber slides (Becton Dickinson Labware, Franklin Lakes, NJ). The cells were allowed to attach for 24 h in 10% FBS media before transferring to serum free media overnight. The cells were then exposed to 100 μM nicotine for 3 min or 20 μM H2O2 for 15 min. After washing briefly with cold PBS, the cells were fixed with 2% paraformaldehyde for 20 min at room temperature, permeablized with 1% Triton X-100 in PBS for 5 min followed by extensive washing with PBS. Blocking was done using 1% bovine serum-albumin and 5% goat serum in PBS. Incubation with primary antibody to p-ERK (1:100 dil) in 1% bovine serum-albumin was continued for 24 h at 4°C. Following incubation the slides were washed 3 times, 10 min each with PBS. After washing, the cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (1:50 dilution, Sigma, St. Louis MO), at room temperature for 45 min. Slides were then washed extensively (3 times for 10 min) in PBS. Mounting media from Invitrogen Technologies (Carlsbad, CA) was used to mount the samples. The slides were then viewed under confocal microscope and images were analyzed using Fluorescan 2 software (Fluorescan Labsystems OY, Helsinki, Finland). The negative controls for immunostaining were cells that were unexposed and incubated with secondary antibody alone.
Assay of basal and stimulated cell function by bioassay
For cell function studies, the cells were grown to 80% and above in confluency. The flasks containing an average 4–6 × 106 cells, were washed with Hepes-Ringer Buffer (HRB) before treating with 100 μM nicotine in HRB for 3 mins or 20 μM H2O2 in HRB for 15 min. After incubation the cells were washed with HRB. The cells were then trypsinized using 1 × Trypsin EDTA (Mediatech Inc, Herndon, VA). 5 mls of HRB were then added and the cells were centrifuged for 5 min at 1000 rpm. The supernatant was discarded and the cells were resuspended in HRB. The resuspended cells were incubated with and without CCK (10-10 M) for 30 min at 37°C. After the incubation, the media was removed following centrifugation. Amylase activity was measured employing procion yellow starch as substrate (PRO Chemical & Dye; Somerset, MA) using the method of Jung . The cell pellets were washed with ice cold PBS, lysed with water by sonication and centrifuged. The cell lysate was analyzed for both amylase and protein content.
Experimental values are calculated as mean ± SEM of the number of experiments indicated in the legends. Data were evaluated for statistical significance with one-way ANOVA. A p-value of 0.05 or less was considered as statistically significant.
Effects of H2O2 on Cell Toxicity
Induction of lipid peroxidation by H2O2 and nicotine in AR42J cells
Activation of ERK signaling by H2O2
Response of H2O2 and nicotine in activation of ERK signaling in AR42J cells
Cytoplasmic localization of activated p-ERK1/2 by H2O2 and nicotine as determined by immunofluorescence
Effects of H2O2 or nicotine on AR42J cell Proliferation
Influence of H2O2 and nicotine Cell Function
The effects of nicotine on cell proliferation and secretion has been reported in this cell line . The current study re-examined the effects of nicotine in the same cell system with the added exposure to hydrogen peroxide to determine whether nicotine-induced effects on these cells are regulated via oxidative stress pathway. Thus hydrogen peroxide was selected as a well known marker for oxidative damage. Previous studies have shown that exposure of human osteosarcoma cell line to 1–10 mM H2O2 induced reactive oxygen species (ROS) formation, DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in this cell line . Since the presence of excessive ROS is known to cause cellular damage by hydroxyl radical attack  it was imperative to perform dose response studies with H2O2.
Cell cytotoxicity experiments have been first performed to determine the effects of H2O2 at dose-range from 10 – 100 μM. The results show that for concentrations beyond 20 μM, the percentage cytotoxicity was significantly higher than the control and as high as greater than 20%. Other studies have also confirmed an increase in cell toxicity beyond 20 μM . Thus, in order to avoid any cell injury and to maintain the cells within the physiological range, this concentration of H2O2 was selected for the current study.
Lipid peroxidation occurs through free radical attacks of poly-unsaturated fatty acids leading to formation of lipid hydroperoxides as well as conjugated dienes and aldehydes such as malondialdehyde (MDA). In order to investigate whether nicotine induces the generation of oxygen free radicals within the cell, the lipid peroxidation was measured in response to nicotine and H2O2. The data from our experiments showed that both nicotine and H2O2 had significant increases in the concentrations of MDA formation as compared to the control untreated cells suggesting that both nicotine and H2O2 induced ROS formation in AR42J cells. It has been shown that ROS formation above a critical level in oligodendrocytes is followed by an increase in anti-oxidant enzymes possibly to scavenge oxidative by products such as H2O2 [26–28]. This mechanism is important for cell survival . In this study we show that the concentration of hydrogen peroxide that induced MDA formation also induced the activation of p-ERK1/2 signaling (Figure 3). These results are consistent with the data on p-ERK1/2 activation as reported in cultured endothelial cells in which peak responses of p-ERK activation is shown to occur after 15 min of exposure followed by its return to baseline at 60 min . In our study we have also observed that the activation of pERK-1/2 by H2O2. These observations have been confirmed further by co-localization of p-ERK1/2 within the cytosol by immunoflorescence study.
Since ERK is known as a signal for mitogen-activated protein kinase (MAPK) pathway and has been shown to be involved in growth, differentiation and development in mammalian system , we sought to investigate its role following its induction by nicotine or hydrogen peroxide in AR42J cell proliferation employing MTT assay as reported earlier . Our data show that while nicotine does promote significant growth within the first 48 hours of incubation in low serum media, H2O2 treated cells, on the other hand, do not show such an increase in proliferation and its effects on cell proliferation is similar to that of untreated cells (Figure 5). It has been suggested that the induction of p-ERK1/2 by H2O2 is a cell specific response , where H2O2 may be able to utilize multiple pathways to produce mitogenic effects depending on the cell type. Thus we surmise that even though the cells do not proliferate at a faster rate with H2O2 as observed for the cells exposed to nicotine, the rate of proliferation by H2O2 is similar to that of the control untreated cells. These complex regulatory mechanisms have been described previously by Watanabe et al , and others  Thus it is possible that induction of ERK signaling by H2O2 may be critical in the regulation of cellular protection in the early stage of cell response to oxidative stress. One other interpretation may be that H2O2 induced p-ERK activation and MDA formation are not involved in cellular proliferation.
Cell function studies have been conducted by stimulating the cells with a previously determined maximal dose of cholecystokinin (CCK). These experiments are aimed to determine whether there are any differences in cell function when they are exposed to nicotine or H2O2 at their optimum concentrations. While the CCK-induced amylase secretion with nicotine exposure is significantly higher than the unexposed cells, there is no increase in amylase secretion by H2O2 over and above that of control. It has been shown that CCK can evoke marked changes in pancreatic acinar cell mitochondrial activity and that CCK-8 evoked responses are blocked by H2O2 . Impairment of mitochondrial activity in the presence of H2O2 (1 mM) may represent a mechanism by which cellular damage can occur leading to its dysfunction and pathology. The dose of 20 μM of H2O2 used in this study is nontoxic and therefore, the data from our studies showing normal cell function appear consistent with those observations.
H2O2 is a known oxidative agent and is used here as a biomarker by which its effect on AR42J cells can be directly compared to the effects of nicotine following its exposure. Functional and cell proliferation studies show a significant difference in the effects between nicotine and H2O2 on AR42J cells. This indicates that while nicotine exposure does result in the production of ROS within the cells, there are certain other key factors induced by nicotine that differentiates its effects from that of H2O2. In the current study, we have aimed to show that one of these key factors for cell injury is in the production or ROS. Since ROS has been shown to cause DNA single strand breakdown , it is reasonable to consider further investigation of the role of nicotine in signal transduction pathways and its oxidative role in pancreatic cell injury. In in-vivo studies, Wittel et al  investigated the effect of cigarette smoke inhalation in rats. Their studies showed that morphological damage to pancreas induced by inhalation of cigarette smoke may likely be mediated by alteration of acinar cell function. Our studies in in-vitro cell culture using nicotine as marker supports the observation made by Wittel et al .
This study was supported, in part, by funds from Central Arkansas Veterans Healthcare System and the University of Arkansas for Medical sciences. The authors thank Dr. Chhanda Bose for her valuable discussion and technical suggestions. A part of this study was presented at the Annual Meeting of The American Gastroenterological Association, Digestive Disease Week, held in Los Angeles, California, May 20–25, 2006.
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