Open Access

Mitogenic and functional responses by nicotine and hydrogen peroxide in AR42J cells: a comparative study

Tobacco Induced Diseases20084:5

https://doi.org/10.1186/1617-9625-4-5

Received: 13 June 2008

Accepted: 31 July 2008

Published: 31 July 2008

Abstract

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.

Background

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 [1]. About half of the young adult smokers today who continue to smoke throughout their life will die of a smoke related diseases [2]. 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 [7] and that the mitochondrial respiratory chain is affected by nicotine leading to an increased generation of superoxide anions and hydrogen peroxide [8]. 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 [9]. 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 [12]. Clinical studies have shown that oxidative stress leading to lipid peroxidation appears to be linked to the pathogenesis of chronic pancreatitis [13]. Other evidence showing the production of large amounts of oxygen radicals in lymphocytes due to cigarette smoking [14] 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 [15].

Materials and methods

Cell Culture

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 [10]. 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 [18].

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 [19]. 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.

Statistical Analysis

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.

Results

Effects of H2O2 on Cell Toxicity

In order to determine the cytotoxic dose of H2O2 on AR42J cells, cells were exposed for 24 h to a graded dose of H2O2. The percent cytotoxicity as determined by LDH release is shown in Figure 1. In untreated control cells the percent of LDH release was 5.4 ± 1.1% while cells exposed to 20 μM H2O2 it was 6.7 ± 3.9%. This value was not significantly different from the control. As the concentration of H2O2 increased beyond 20 μM, the percentage of cytotoxic cells was increased. With 100 μM H2O2, it was 28.0 ± 0.3%. This is not surprising since it has been reported that EL-4 murine lymphoma cells exposed to H2O2 doses beyond 20 μM, increased number of cytotoxic cells was found by Zhou et al [20]. Thus we have used this non-cytotoxic concentration of 20 μM of H2O2 in all the subsequent studies.
Figure 1

Measurement of cytotoxic effect of H 2 O 2 . The cells were exposed to H2O2 in the dose range of 10–100 μM. A BioVision LDH-Cytotoxiciy Assay Kit (Mountain View, CA) was used, and the absorbance was measured after 24 hr incubation in 96-well plates at a wavelength of 495 nm. The percentage cytotoxicity was calculated as the ratio of absorptions of wells treated with H2O2 and untreated wells; N = 8, *, P < 0.05, significantly different from the uncxposed control.

Induction of lipid peroxidation by H2O2 and nicotine in AR42J cells

To determine whether H2O2 and nicotine would affect the cell's ability to accumulate reactive oxygen species as measured by the production of malondialaldehyde (MDA) within the cells, cells were treated with either H2O2 (20 μM) for 15 min or with nicotine (100 μM) for 3 min, a non cytotoxic dose determined earlier [10]. Cell lysates were used for the measurement of MDA production as described in the Materials and Methods section. As shown in Figure 2, the concentration of MDA measured in cells exposed to H2O2 or nicotine were 0.28 ± 0.02 μmol/mg and 0.26 ± 0.02 μmol/mg, respectively. These values were significantly higher than the value of 0.11 ± 0.01 μmol/mg in control cells (p < 0.01).
Figure 2

Lipid peroxidation represented by MDA concentration in AR42J cells. Control untreated cells, cells exposed to 20 μM H2O2 for 15 min or to 100 μM nicotine for 3 min were lysed after treatments and the lysates were used in an MDA-586 Assay (Oxis Research, Portland, OR). The values are expressed as μM/mg protein in y-axis. The vertical bar represents treatments with H2O2 or nicotine, N = 5, *, P < 0.05, significantly different from the uncxposed control.

Activation of ERK signaling by H2O2

It has been shown that in other cells, ERK1/2 is activated by H2O2 treatment [20, 21]. To determine the effects of H2O2 on ERK1/2 activation in AR42J cells, the cells were exposed to H2O2 (20 μM) for various times. Cell lysates were prepared and measured for total and p-ERK1/2 activation employing the specific antibodies to total and p-ERK1/2 and analyzed by Western blots. With 20 μM H2O2, a 3 fold increase in band intensity was observed at 15 min which was significantly higher (p < 0.02) when compared to the control cells (Figure 3A). Expressing band intensity as the fold increase above the control, a steady increase in p-ERK activation was observed with increasing concentrations of H2O2 at 15 min of incubation, attaining a 7-fold increase with 100 μM H2O2 (Fig. 3B). Total ERK1/2 in all instances showed no alteration with H2O2 incubation and indicated a uniformity of loading of the wells with samples.
Figure 3

Dose and time dependent induction of ERK1/2 in AR42J cell s. Cell lysates were loaded onto an SDS gel, separated by electrophoresis, blocked in 1% fat free milk and probed with antibodies to total and phosphorylated (p) ERK1/2. Horseradish peroxidase-coupled anti IgG was used as a secondary antibody. Bands were visualized with ECL-plus and quantified using a STORM Imager. The data shown as means ± SEM of n = 5 experiments. A: Induction of ERK1/2 in cells exposed to 20 μM H2O2 for 10–60 min compared to control untreated cells. B: Induction of ERK in cells exposed to 10–20 μM H2O2 for 15 min. C: Band intensity showing the fold increase in the time dependent induction of ERK1/2. D: Band intensity showing the fold increase in the dose dependent induction of ERK1/2.

Response of H2O2 and nicotine in activation of ERK signaling in AR42J cells

The non cytotoxic dose of hydrogen peroxide as determined from the study described above was used in this study along with the non-cytotoxic dose of nicotine reported earlier from this laboratory [10]. The concentration of nicotine used in this study was 100 μM with an incubation time of 3 min. A comparison of p-ERK1/2 activation of cells exposed to nicotine or H2O2 is shown in Figure 4. The data show that there were increases in p-ERK1/2 activation in cells exposed to 100 μM nicotine for 3 min or 20 μM H2O2 for 15 min. The band intensities expressed as the fold increase were 2 fold higher with both nicotine and H2O2 and were significantly higher when compared to the unexposed cells (p < 0.05).
Figure 4

Induction of ERK1/2 in AR42J cells exposed to nicotine or H 2 O 2 . Control untreated cells, cells exposed to 20 μM H2O2 for 15 min or 100 μM nicotine 3 min were lysed and the lysates were used for Western blotting. A: Western blot visualization with ECL-plus using STORM Imaging software. B: Band intensity showing the fold increase as mean ± SEM of n = 5 experiments. *, P < 0.05, significantly different from the uncxposed control.

Cytoplasmic localization of activated p-ERK1/2 by H2O2 and nicotine as determined by immunofluorescence

Activation of pERK1/2 in AR42J cells by nicotine and H2O2 were onfirmed by immunofluorescence study after exposing the cells to nicotine or H2O2, and probing the fixed cells with antibody to p-ERK1/2. As shown in Figure 5, immunostaining revealed that the p-ERK1/2 signals were distributed throughout the cell cytoplasm with considerably higher fluorescent intensities of p-ERK1/2 observed when cells were treated with nicotine or H2O2. Cells not exposed to nicotine or H2O2, on the other hand, showed less fluorescent intensities compared with treated cells. The immunofluorescence data supported the observation of increased induction of pERK1/2 by nicotine and H2O2 as analyzed by Western Blot.
Figure 5

Induction of p-ERK1/2 in AR42J cells as indicated by immunohistochemistry. AR42J cells were grown and treated with 100 μM nicotine for 3 min; 20 μM H2O2 for 15 min. The cells were fixed with paraformaldehyde and treated with antibody to p-ERK1/2 for 1 h. After washing, the cells were treated with secondary antibody labeled FITC. Slides were observed using a confocal microscope A: Control untreated cells probed with primary antibody to p-ERK1/2 B: Cells exposed to nicotine for 3 min probed with primary antibody to p-ERK1/2 C: Cells exposed to H2O2 for 15 min probed with primary antibody to p-ERK1/2. Negative controls were done by incubating in FITC labeled secondary antibody only. (D – F). D: Control untreated cells. E: Cells exposed to nicotine for 3 min. F: Cells exposed to H2O2 for 15 min. The experiment was repeated in a set of four under each treatment.

Effects of H2O2 or nicotine on AR42J cell Proliferation

In order to compare the nicotine-induced ERK1/2 activation in the growth of AR42J cells with that of H2O2-induced ERK1/2, cell proliferation experiments were performed. The details of the experiments were described in the Materials and Methods section. The cell proliferation was evaluated from 24–96 h of incubation in media containing 0.05% serum and using an MTT assay kit. The absorbance was measured at 450 nm. As shown in Figure 6, there was a significant difference in proliferation pattern between nicotine exposed cells; the cells treated with H2O2 or unexposed control cells. The maximum proliferation occurred at 48 h interval and then declined at later periods. The absorbance for nicotine exposed cells at 48 h interval was 0.3 ± 0 arbitrary units while that for controls it was 0.2 ± 0.0 arbitrary units (p < 0.05). There was no significant difference between the proliferation observed for cells exposed to H2O2 and the control cells.
Figure 6

The effect of nicotine or hydrogen peroxide on the proliferation of AR42J cells. The cells were plated in 96-well plates and allowed to attach overnight before transferring to 0.05% serum media for 10–12 h before beginning of the study. The cells were then treated with 100 μM nicotine or 20 μM H2O2 and the proliferation measured at 24–96 h using a cell counting kit from Dojindo Molecular Technologies according to manufacturer's instructions. The data points represent mean ± SEM of 5 experiments. ,-- control unexposed cells; ■, --- Nicotine treated; ▲ --, H2O2 treated. *, P < 0.05, significantly different from the uncxposed control.

Influence of H2O2 and nicotine Cell Function

The effects H2O2 and nicotine on cell function were assessed by determining their ability to stimulate the secretagogue induced enzyme release. The concentrations of nicotine and H2O2 used in the studies were 100 μM and 20 μM for a time of exposure of 3 and 15 min respectively. The washed cells were incubated with or without cholecystokinin (CCK) at their maximal stimulating concentration of 10-10 M as reported earlier [22]. The release of amylase in response to CCK was measured as percent of initial content and is shown in Figure 7. The percent of amylase released from the cells treated with nicotine was (18.2 ± 1.8% of initial content) and it was significantly higher from the percent amylase released from either the control cells (9.5 ± 1.0.% of initial content, or from the H2O2 treated cells (8.2 ± 1.1% of initial content, p < 0.05).
Figure 7

Comparison of cell function of AR42J cells when exposed to nicotine or H2O2. The cells were treated with 100 μM nicotine for 3 min or 20 μM H2O2 15 min. Cells were subsequently washed with Hepes-Ringer and incubated at 37°C with or without CCK-8 (10 nM) for 30 min. Amylase released into the incubation medium was measured with procion yellow starch as a substrate. The data are expressed as percent initial content and represent mean ± SEM of 5 experiments. open bar---control unexposed; hatched bar---- CCK-8 stimulated; *, p < 0.05, significantly different from the control; **, p < 0.05, significantly different from H2O2 exposed cells.

Discussion

The effects of nicotine on cell proliferation and secretion has been reported in this cell line [10]. 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 [23]. Since the presence of excessive ROS is known to cause cellular damage by hydroxyl radical attack [24] 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 [25]. 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 [2628]. This mechanism is important for cell survival [28]. 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 [29]. 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 [30], we sought to investigate its role following its induction by nicotine or hydrogen peroxide in AR42J cell proliferation employing MTT assay as reported earlier [10]. 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 [31], 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 [21], and others [32] 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 [33]. 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 [34], 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 [35] 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 [35].

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences
(2)
Department of Geriatrics, University of Arkansas for Medical Sciences
(3)
Medical Research, Central Arkansas Veterans Healthcare System

References

  1. Mokdad AH, Marks JS, Stroup DF, Gerberding JL: Actual causes of death in the United States, 2000. JAMA. 2004, 291: 1238-1245. 10.1001/jama.291.10.1238.View ArticlePubMedGoogle Scholar
  2. Morbidity and Mortality Weekly Report, Tobacco use–United States. 1900–1999. MMWR Morb Mortal Wkly Rep. 1999, 48: 986-993.Google Scholar
  3. Talamini G, Bassi C, Falconi M, Sartori N, Salvia R, Rigo L, Castagnini A, Di FV, Frulloni L, Bovo P, Vaona B, Angelini G, Vantini I, Cavallini G, Pederzoli P: Alcohol and smoking as risk factors in chronic pancreatitis and pancreatic cancer. Dig Dis Sci. 1999, 44: 1303-1311. 10.1023/A:1026670911955.View ArticlePubMedGoogle Scholar
  4. Lin Y, Tamakoshi A, Hayakawa T, Ogawa M, Ohno Y: Cigarette smoking as a risk factor for chronic pancreatitis: a case-control study in Japan. Research Committee on Intractable Pancreatic Diseases. Pancreas. 2000, 21: 109-114. 10.1097/00006676-200008000-00001.View ArticlePubMedGoogle Scholar
  5. Rayford PL, Chowdhury P: Mecamylamine, a nicotinic receptor channel antagonist, affects amylase secretion by isolated pancreatic acinar cells. J Assoc Acad Minor Phys. 2001, 12: 105-108.PubMedGoogle Scholar
  6. Chowdhury P, MacLeod S, Udupa KB, Rayford PL: Pathophysiological effects of nicotine on the pancreas: an update. Exp Biol Med (Maywood). 2002, 227: 445-454.Google Scholar
  7. Wetscher GJ, Bagchi M, Bagchi D, Perdikis G, Hinder PR, Glaser K, Hinder RA: Free radical production in nicotine treated pancreatic tissue. Free Radic Biol Med. 1995, 18: 877-882. 10.1016/0891-5849(94)00221-5.View ArticlePubMedGoogle Scholar
  8. Yildiz D, Liu YS, Ercal N, Armstrong DW: Comparison of pure nicotine- and smokeless tobacco extract-induced toxicities and oxidative stress. Arch Environ Contam Toxicol. 1999, 37: 434-439. 10.1007/s002449900537.View ArticlePubMedGoogle Scholar
  9. Park BK, Chung JB, Lee JH, Suh JH, Park SW, Song SY, Kim H, Kim KH, Kang JK: Role of oxygen free radicals in patients with acute pancreatitis. World J Gastroenterol. 2003, 9: 2266-2269.PubMedGoogle Scholar
  10. Bose C, Zhang H, Udupa KB, Chowdhury P: Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion. Am J Physiol Gastrointest Liver Physiol. 2005, 289: G926-G934. 10.1152/ajpgi.00138.2005.View ArticlePubMedGoogle Scholar
  11. Chowdhury P, Bose C, Udupa K: Nicotine induced proliferation of isolated rat pancreatic acinar cells: Effect of cell signaling and function. Cell Proliferation. 2007, 40: 125-141. 10.1111/j.1365-2184.2007.00418.x.View ArticlePubMedGoogle Scholar
  12. Chiarugi P: Reactive oxygen species as mediators of cell adhesion. Ital J Biochem. 2003, 52: 28-32.PubMedGoogle Scholar
  13. Ganesh PC, Rao SMN: Evidence for oxidant stress in chronic pancreatitis. Indian J Gastroenterol. 1999, 18: 156-157.Google Scholar
  14. Piperakis SM, Visvardis EE, Sagnou M, Tassiou AM: Effects of smoking and aging on oxidative DNA damage of human lymphocytes. Carcinogenesis. 1998, 19: 695-698. 10.1093/carcin/19.4.695.View ArticlePubMedGoogle Scholar
  15. Christophe J: Pancreatic tumoral cell line AR42J: an amphicrine model. Am J Physiol. 1994, 266: G963-G971.PubMedGoogle Scholar
  16. Soulsby ME, Phillips B, Chowdhury P: The effects of a soy-protein diet o elevated brain lipid peroxide levels induced by simulated weightlessness. Annals of Clin & Lab Sci. 2004, 34: 103-106.Google Scholar
  17. Chowdhury P, Soulsby M, Kim K: L-carnitine influence on oxidative stress induced by hind-limb unloading in Adult Rats. Aviat Space Environ Med. 2007, 78: 554-556.PubMedGoogle Scholar
  18. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3.View ArticlePubMedGoogle Scholar
  19. Jung DH: Preparation and application of Procion Yellow starch for amylase assay. Clin Chim Acta. 1980, 100: 7-11. 10.1016/0009-8981(80)90179-5.View ArticlePubMedGoogle Scholar
  20. Zhou Y, Wang Q, Evers BM, Chung DH: Signal transduction pathways involved in oxidative stress-induced intestinal epithelial cell apoptosis. Pediatr Res. 2005, 58: 1192-1197. 10.1203/01.pdr.0000185133.65966.4e.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Watanabe N, Zmijewski JW, Takabe W, Umezu-Goto M, Le Goffe C, Sekine A, Landar A, Watanabe A, Aoki J, Arai H, Kodama T, Murphy MP, Kalyanaraman R, Darley-Usmar VM, Noguchi N: Activation of mitogen-activated protein kinases by lysophosphatidylcholine-induced mitochondrial reactive oxygen species generation in endothelial cells. Am J Pathol. 2006, 168: 737-1748. 10.2353/ajpath.2006.050648.View ArticleGoogle Scholar
  22. Chowdhury P, Hosotani R, Rayford PL: Inhibition of CCK or carbachol stimulated amylase release by nicotine. Life Sciences. 1989, 45: 2163-2168. 10.1016/0024-3205(89)90083-0.View ArticlePubMedGoogle Scholar
  23. Ogawa Y, Kobayashi T, Nishioka A, Kariya S, Ohnishi T, Hamasato S, Seguchi H, Yoshida S: Reactive oxygen species-producing site in radiation-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization. Int J Mol Med. 2004, 13: 69-73.PubMedGoogle Scholar
  24. Riley PA: Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int J Radiat Biol. 1994, 65: 27-33. 10.1080/09553009414550041.View ArticlePubMedGoogle Scholar
  25. Iijima R, Takahashi H, Namme R, Ikegami S, Yamazaki M: Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger. FEBS Lett. 2004, 561: 163-166. 10.1016/S0014-5793(04)00164-4.View ArticlePubMedGoogle Scholar
  26. Baud O, Greene AE, Li J, Wang H, Volpe JJ, Rosenberg PA: Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes. J Neurosci. 2004, 24: 1531-1540. 10.1523/JNEUROSCI.3989-03.2004.View ArticlePubMedGoogle Scholar
  27. Meiners S, Ludwig A, Lorenz M, Dreger H, Baumann G, Stangl V, Stangl K: Nontoxic proteasome inhibition activates a protective antioxidant defense response in endothelial cells. Free Radic Biol Med. 2006, 40: 2232-2241. 10.1016/j.freeradbiomed.2006.03.003.View ArticlePubMedGoogle Scholar
  28. Martindale JL, Holbrook NJ: Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol. 2002, 192: 1-15. 10.1002/jcp.10119.View ArticlePubMedGoogle Scholar
  29. Yang B, Oo TN, Rizzo V: Lipid rafts mediate H2O2 prosurvival effects in cultured endothelial cells. FASEB J. 2006, 20: 1501-1503. 10.1096/fj.05-5359fje.View ArticlePubMedGoogle Scholar
  30. Cobb MH: MAP kinase pathways. Prog Biophys Mol Biol. 1999, 71: 479-500. 10.1016/S0079-6107(98)00056-X.View ArticlePubMedGoogle Scholar
  31. Zhang J, Jin N, Liu Y, Rhoades RA: Hydrogen peroxide stimulates extracellular signal-regulated protein kinases in pulmonary arterial smooth muscle cells. Am J Respir Cell Mol Biol. 1998, 19: 324-332.View ArticlePubMedGoogle Scholar
  32. Song HJ, Lee TS, Jeong JH, Min YS, Shin C, Sohn UD: Hydrogen peroxide-induced extracellular signal-regulated kinase activation in cultured feline ileal smooth muscle cells. J Pharmacol Exp Ther. 2005, 312: 391-398. 10.1124/jpet.104.074401.View ArticlePubMedGoogle Scholar
  33. Granados MP, Salido GM, Pariente JA, Gonzales A: Effect of H2O2 on CCK-8-evoked changes in mitochondial activity in isolated mouse pancreatic acinar cells. Biology of the Cell. 2005, 97: 847-856. 10.1042/BC20040513.View ArticlePubMedGoogle Scholar
  34. Weitberg AB, Corvese D: Oxygen radicals potentiate the genetic toxicity of tobacco-specific nitrosamines. Clin Genet. 1993, 43: 88-91.View ArticlePubMedGoogle Scholar
  35. Wittel UA, Pandey KK, Andranifahanana M, Johansson SL, Cullen DM, Akhter MP, Brand RE, Prokopczyk B, Batra SK: Chronic pancreatic inflammation induced by environmental tobacco smoke inhalation in rats. Am J Gastroenterol. 2006, 101: 148-159. 10.1111/j.1572-0241.2006.00405.x.View ArticlePubMedGoogle Scholar

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© Walker et al; licensee BioMed Central Ltd. 2008

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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