Inhalation Toxicology, 2009; 21(12): 1007–1012

O R G I N A L A R T I C L E

Evidence for cigarette smoke-induced oxidative stress in the rat pancreas

Jianyu-Hao, Guang-Li, and Baosen-pang

Department of Gastroenterology, Beijing Institute of Respiratory Diseases, Beijing Chao Yang Hospital, Captial Medical University, Beijing, P.R. China

Address for Correspondence: Jian-Yu Hao, MD, Department of Gastroenterology, Beijing Chao Yang Hospital, Capital Medical University, 8 Baijiazhuang Road, Beijing 100020, P.R. China. E-mail: haojianyu@medmail.com.cn

(Received 14 September 2008; revised 03 December 2008; accepted 04 December 2008)

Introduction

A wealth of epidemiological findings clearly indicates that cigarette smoking is associated with the induction of pancrea-titis, with the progression of chronic pancreatitis independent of etiology, and with pancreatic cancer (Talamini et al., 2007; Hart et al., 2008; Lindkvist et al., 2008; McKay et al., 2008). Cigarette smoking is currently thought to promote pancrea-titis and carcinogenesis through the actions of multiple con-stituents present in tobacco smoke and tar (Malfertheiner & Schutte, 2006). Despite strong correlations, the molecular mechanisms underlying these associations have remained elusive. Experimental studies have been limited by the pau-city of animal models of human smoking and by the thou-sands of chemicals present in cigarette smoke. Recently, however, a rodent model of controlled, chronic exposure to cigarette smoke inhalation was designed which yielded important new findings regarding smoking and pancreatitis. Chronic pancreatic inflammation with fibrosis and scarring of

acinar structures was observed within 12 weeks of exposure to cigarette smoke (Wittel et al., 2006a, 2008). The predominant lesions were characterized by focal increases in extracellular matrix and subsequently decreased acinar structures, but areas distinguished by infiltration of transforming growth factor- (TGF-)-positive immune cells expressing inflam-matory mediators were also observed. The findings with this rodent model strongly favor a role for cigarette smoke-induced alterations in acinar cell function in the development of pancreatitis in smokers. Furthermore, in animals exhibiting focal pancreatic acinar lesions in response to environmental tobacco smoke, gene expression in pancreatic ductal cells was increased (Wittel et al., 2006b, 2008), consistent with the involvement of such lesions in pancreatic carcinogenesis.

Reactive oxygen species (ROS) are heavily implicated in the development and progression of the inflammatory process (Ma et al., 2008). Both the smoke and tar of tobacco contain high concentrations of ROS, and serum ROS

ISSN 0895-8378 print/ISSN 1091-7691 online © 2009 Informa UK LtdDOI: 10.1080/08958370802665937

AbstractBackground/aims: Recent findings with a rodent model of cigarette smoke inhalation revealed a causal relation-ship between chronic exposure to cigarette smoke and the development of pancreatitis. The present study was conducted to ascertain whether cigarette smoke induces oxidative stress in the rat pancreas concurrently with inflammation.Methodology: Rats (six per treatment group) were treated for 0, 3, 6, 9, or 12 weeks with cigarette smoke (0.7 mg/L). Pancreatic tissues were examined for histological and pathological alterations and serum for changes in inter-leukin-6 concentration. Pancreatic expression and localization of α-smooth muscle actin, transforming growth factor-β1, and collagen-1 were determined as measures of progressive inflammation/fibrosis. Pancreatic super-oxide dismutase and glutathione peroxidase activities and malondialdehyde content were measured as indices of oxidative stress.Results: Inflammatory cell infiltration and ductal hyperplasia were detected in pancreata after 12 weeks of treat-ment with cigarette smoke. The serum interleukin-6 concentration increased significantly and pancreatic glu-tathione peroxidase activity declined significantly after 12 weeks of treatment. No other significant changes were observed.Conclusions: Pancreata of rats exposed chronically to cigarette smoke exhibit inflammation concurrently with suppression of glutathione peroxidase activity. These observations favor a role for oxidative stress in the induc-tion of pancreatitis associated with chronic cigarette smoke inhalation.

Keywords: chronic pancreatitis; pancreatic duct hyperplasia; environmental cigarette smoke; oxidative stress; interleukin-6; glutathione peroxidase

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1008 J.-Y. Hao and G. Li

concentrations among smokers increase with the number of cigarettes smoked (Hayashi et al., 2007). Furthermore, anti-oxidant enzyme systems of healthy subjects are disturbed by cigarette smoking (Aycicek et al., 2005; Chavez et al., 2007; Orosz et al., 2007; Sidel et al., 2007). Oxidative stress in response to cigarette smoking is also linked with the genera-tion of circulating proinflammatory cytokines, such as inter-leukin-6 (IL-6) (Orosz et al., 2007). Although oxidative stress is believed to play an important role in the pathophysiology of acute pancreatitis (Formela et al., 1995; Schulz et al., 1999; Gomez-Cambronero et al., 2002), it is unclear whether such stress is associated with the chronic pancreatitis of smokers. The present study was therefore undertaken to ascertain whether pancreata of rats exposed to cigarette smoke dis-play increased susceptibility to oxidative stress concurrently with inflammatory histopathology.

Materials and methods

Experimental animals and groupingThirty clean male Sprague–Dawley rats (200–300 g) were purchased from the Experimental Animal Center, National Institute for the Control of Pharmaceutical and Biological Products (China). Rats were maintained in the animal labo-ratory of the Beijing Chao Yang Hospital for 1 week prior to initiating experiments. The day/night interval was 12 h, and suitable temperature and humidity were provided. The envi-ronment was rated as clean by the Beijing Administration Office of Laboratory Animals.

Animals were then randomly divided into two groups: a cigarette inhalation group (n = 24) and a control group (n = 6).

MaterialsZongnanhai cigarettes were purchased from Beijing Tobacco. Each cigarette contained 14 mg of tar, 1.2 mg of nicotine, and 14 mg of carbon monoxide. Kits for determination of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) were obtained from the Nanjing Jiancheng Bioengineering Institute. An enzyme linked immunosorbent assay (ELISA) kit for the measure-ment of IL-6 was purchased from North Biotech, Beijing. Monoclonal antibodies to -smooth muscle actin (-SMA) and to collagen type 1 were obtained from Sigma, USA. Transforming growth factor-1 (TGF-1), goat anti-mouse antibodies, goat anti-rabbit secondary antibodies, and trypsin were purchased from Beijing Zhong Shan-Golden Bridge Biological Technology.

Rat model of exposure to environmental cigarette smokeThe animal model for this study was based on that described by Wittel et al. (2006a, 2006b, 2008) for the induction of chronic pancreatic inflammation in response to prolonged exposure to cigarette smoke, and has been previously uti-lized to establish a mouse model of chronic obstructive pulmonary disease (Pang et al., 2003). Rats were placed in

glass boxes (80 × 60 × 58 cm) with a hole of 1 cm diameter in the upper right corner. Smoke was collected in 100-ml syringes before injection into the boxes. Rats were exposed to cigarette smoke for 30 min twice daily, with an interval of 4 h between exposures. Each animal received smoke from 14 cigarettes over each 30-min treatment period (total of 28 cigarettes each day). The smoke concentration in the box was approximately 7.6% or 0.7 mg/L. Animals in the control group received a normal diet and water supply but no addi-tional treatment.

SamplingSix animals each were sacrificed at the 3rd, 6th, 9th, and 12th week of treatment with cigarette smoke. The six rats in the control group were sacrificed concurrently with the treat-ment group at the 12th week. Animals in the treatment groups received cigarette smoke on the day prior to, but not on the day of, sacrifice. After sacrifice, blood samples were taken immediately from the abdominal aorta to prepare serum for measurements of IL-6 concentration. Meanwhile, sections of pancreatic tissue were fixed in neutral formaldehyde for hematoxylin and eosin (H&E) staining, with the remaining portion of the tissue stored at –80°C for subsequent determi-nations of SOD and GSH-PX activities and MDA content.

H&E stainingA standard H&E staining protocol was followed. Briefly, slices were stained with hematoxylin for 15 min, treated with 1% HCl/ethanol for several seconds, and then stained with eosin for 2 min. After dehydration, sealing with neutral resin was performed and slices were photographed.

Determination of SOD and GSH-PX activities and of MDA contentFollowing standard homogenization of tissues, SOD and GSH-PX activities and MDA contents were determined according to the instructions provided by manufacturers of the kits. Absorption was measured with a Beckman spectro-photometer. For normalization, SOD and GSH-PX activities and MDA contents are expressed per mg of tissue protein.

Immunohistochemical analysesPancreatic tissue slices were prepared as described above. No further preparation was required for -SMA or TGF-1 staining. For collagen-1 staining, slices were first subjected to digestion with 0.1% trypsin at 37°C for 15 min. Samples were incubated with primary antibody or phosphate buff-ered saline (PBS) (negative control) at 4°C overnight before treatment with secondary antibody at room temperature for 40 min. The slides were then developed with 3,39- diaminobenzidine tetrahydrochloride, double-stained with hematoxylin, dehydrated, sealed with neutral resin, and photographed.

Statistical analysesAll data are presented as mean ± standard deviation. To test for differences between the control and experimental

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Smoking and oxidative stress in the pancreas 1009

groups, the two-sample t test was used. All statistics were performed using SPSS software (version 15.0; SPSS Inc., Chicago, IL). A p value of 0.05 was considered significant. When statistical comparisons were performed as a function of weeks of exposure to cigarette smoke, the adjusted alpha was considered as 0.013 (9 = 0.05/4).

Results

Histological and pathological alterationsNo histological or pathological alterations were observed in pancreatic tissues of rats in the control group (Figure 1A). Examination of pancreatic tissues from animals treated for 3, 6, or 9 weeks with cigarette smoke also revealed no obvi-ous histological or pathological changes (not shown). In contrast, pancreatic duct hyperplasia was evident in 50% (3/6) of animals that were treated for 12 weeks with cigarette smoke (Figure 1B). Alterations consistent with the presence of inflammation were also observed in tissues from these rats. Specifically, modest infiltration of inflammatory cells,

including plasma cell infiltration into the interstitial matrix in some pancreatic leaflets and around pancreatic ducts, and lymphocytic infiltration were observed in tissues from all animals in this group (Figure 1C).

Markers of inflammation and fibrosisThe concentration of IL-6, an established mediator of inflammation, present in the serum of rats exposed to cigarette smoke was found to increase with the weeks of exposure. A statistically significant difference in IL-6 con-centration was observed for animals treated with cigarette smoke for 12 weeks as compared to non-treated controls (20.44 vs. 17.2 pg/ml; p = 0.007; Figure 2D). Evidence for the presence of progressive inflammation in pancreatic tissues of rats subjected to treatment with cigarette smoke was also sought. Three markers of inflammation and fibrosis were therefore measured in pancreatic tissues of treated and non-treated animals: -SMA, a stellate cell marker of inflamma-tion responsible for induction of fibrosis in the pancreas; TGF-1, a marker of infiltration by TGF--positive immune cells; and collagen-1, a marker of increased extracellular matrix and fibrosis. The expression of -SMA, TGF-1, and collagen-1 was detectable in pancreatic tissues of untreated controls (Figure 3A–C). -SMA expression was observed in the cytoplasm of smooth muscle cells in pancreatic blood vessels and ducts, whereas TGF-1 was mainly expressed in the cytoplasm of endothelial cells in pancreatic vascula-ture. Collagen-1 was distributed in the sheath surrounding pancreatic leaflets. However, as compared to controls, nei-ther the localization nor the expression of -SMA, TGF-1, or collagen-1 was altered significantly at the 3rd, 6th, or 9th week of treatment (not shown), or at the 12th week of treat-ment (Figure 3D–F).

Markers of oxidative stressTo ascertain whether pancreatic tissues of rats chronically exposed to cigarette smoke are more susceptible to oxidative stress, three markers were measured: SOD, an enzyme that inactivates O

2−; GSH-PX, an enzyme that inactivates H

2O

2

and lipid peroxides; and MDA, an established marker of lipid peroxidation. No statistically significant differences in SOD activity or MDA content were observed between the treat-ment groups at 3, 6, 9, or 12 weeks of exposure or between any of these groups and the control group (Figure 2A and C). By contrast, GSH-PX activity in pancreatic tissues of treated rats was found to decrease with the weeks of exposure. At the 12th week, activities were significantly lower than those for the control group (16.3 vs. 21.8 nmol/mg protein), with a statistically significant difference in the mean values also observed (p = 0.002; Figure 2B).

Discussion

The present study utilized the essential features of a rat model of cigarette smoke-induced pancreatitis (Malfertheiner & Schutte, 2006; Wittel et al., 2006a, 2008) to ascertain whether chronic exposure to cigarette smoke promotes oxidative

Figure 1. Histological and pathological features of pancreatic tissues of untreated rats and rats treated with cigarette smoke. (A) Untreated control (H&E stain, ×100); (B) appearance of ductal hyperplasia after 12 weeks of treatment (×100); (C) infiltration of plasma cells and lymphocytes after 12 weeks of treatment (×400).

A

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stress in pancreatic tissue concurrently with inflammation. Findings support the hypothesis that chronic exposure to cigarette smoke is associated with the imposition of oxida-tive stress in the pancreas. A statistically significant decrease in pancreatic GSH-PX activity was observed at 12 weeks of exposure. Although decreases in GSH-PX activity have been observed in the blood (Solak et al., 2005; Li et al., 2007; Zalata ffet al., 2007), urine (Li et al., 2007), and saliva (Kanehira et al., 2006) of subjects exposed to cigarette smoke as opposed to non-exposed subjects, this report is the first to show a decrease in pancreatic GSH-PX activity in response to envi-ronmental cigarette smoke. The suppression of pancreatic GSH-PX activity indicates that the capacity of the tissue to inactivate H

2O

2 and lipid peroxides has been reduced.

Although no measurements of pancreatic ROS were con-ducted in the present study, cigarette smoke has been estab-lished to release free radicals and peroxides to the lung at high concentrations. Furthermore, smoking is clearly associated with the activation of neutrophils and macrophages in the lung (Thorley & Tetley, 2007). These activated cells possess the potential to release ROS to organs such as the pancreas. It is also likely that pancreata of the treated animals were

exposed to inflammatory mediators capable of generating oxidative stress. Statistically significant increases in circulat-ing IL-6 concentrations were observed in these animals, con-sistent with previous observations (Orosz et al., 2007) linking oxidative stress in response to cigarette smoking with the generation of this inflammatory mediator by vascular tissue.

Findings of the present report are also consistent with the induction of inflammation in the pancreas concur-rent with the suppression of GSH-PX activity in this tissue. Manifestations of pancreatic inflammation after 12 weeks of exposure to cigarette smoke included the infiltration of inflammatory plasma cells and lymphocytes into pancreatic tissue of 50% of the experimental animals. The observation that serum IL-6 concentrations were increased after such treatment supports the induction of a generalized inflam-matory response in these animals.

We observed an insignificant trend for pancreatic SOD levels to be increased following cigarette smoke exposure. This contrasts with the significant decrease in GSH-PX lev-els detected. While both of these enzymes are important free radical scavengers, SOD expression is induced by free radicals, while GSH-PX expression (to our knowledge) is not.

Figure 2. Pancreatic SOD and GSH-PX activities, pancreatic MDA contents, and serum IL-6 concentrations for untreated rats and rats exposed to cigarette smoke for 3, 6, 9, and 12 weeks. (A) Pancreatic SOD activity; (B) pancreatic GSH-PX activity; (C) pancreatic MDA content; (D) serum IL-6 con-centration. Findings are presented as mean ± standard deviation. *Significantly different for the control group as compared to the experimental group at 12 weeks (p < 0.013).

30.03 6 9 12Control

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Smoking and oxidative stress in the pancreas 1011

The significant decrease in GSH-PX levels can be attributed to free radical-associated scavenging depletion.

Other markers of oxidative stress in the pancreas were not altered at 12 weeks of treatment with environmental ciga-rette smoke. Pancreatic SOD activity was found to increase modestly, but the increases were not statistically significant. It is conceivable, however, that the expression of this pan-creatic enzyme was marginally increased as a compensatory response to oxidative stress imposed by cigarette smoke, as has been observed in other tissues (Sidel et al., 2007).

The generation of ROS is strongly associated with the pro-gression of inflammation and fibrosis. However, in the present study pancreatic markers of inflammation with fibrosis were unaltered at 12 weeks of treatment with cigarette smoke. No significant changes in expression or localization of -SMA, TGF-1, or collagen-1 were observed. It is possible that 12 weeks of treatment with cigarette smoke were insufficient to fully activate pancreatic stellate cells, to produce detect-able changes in pancreatic -SMA, TGF-1, and collagen-1 at the protein level, and to generate irreversible pancreatic

fibrosis with scarring. In contrast, Wittel et al. (2006a, 2008) observed an increase in pancreatic procollagen-1 gene expression coincident with pancreatic fibrosis and scarring, infiltration of inflammatory cells, and expression of inflam-matory mediators including TGF-1 after rats were treated for 12 weeks with cigarette smoke. It may be relevant that male rats were used in the present study, whereas the stud-ies of Wittel et al. involved female rats. Furthermore, there were a number of methodological differences that may underlie the disparate findings. The most obvious of these is the manner of smoke delivery. Wittel et al. utilized a smoke exposure system to generate the smoke environment. This constituted a smoke generator, a mixer, an animal exposure box, a disposal box, and an air sampling box. There were also differences in exposure time and dosage. In Wittel et al.’s studies, rats were exposed to cigarette smoke for 70 minutes (one 2-second puff every minute) over a period of 12 weeks. The volume of smoke generated was 35 ml for each 2-second period and the dose ranged from 100 to 160 mg/m3.

In summary, rats exposed to cigarette smoke for 12 weeks display evidence for the induction of pancreatic inflamma-tion concurrently with a suppression of pancreatic GSH-PX activity. These findings are consistent with a role for oxida-tive stress in the development of pancreatitis associated with the chronic inhalation of cigarette smoke.

Acknowledgments

Declaration of interest: The authors report no conflicts of interest.

ReferencesAycicek A, Erel I, Kocyigit A. (2005). Decreased total antioxidant capacity and

increased oxidative stress in passive smoker infants and their mothers. Pediatr Int 47:635–639.

Chavez J, Cano C, Souki A, Bermudez V, Medina M, Ciszek A, Amell A, Vargas ME, Reyna N, Toledo A, Cano R, Suarez G, Contreras F, Israili ZH, Hernandez-Hernandez R, Valasco M. (2007). Effect of cigarette smoking on the oxidant/antioxidant balance in healthy subjects. Am J Ther 14:189–193.

Formela LJ, Galloway SW, Kingsnorth AN. (1995). Inflammatory mediators in acute pancreatitis. Br J Surg 82:6–13.

Gomez-Cambronero LG, Sabater L, Pereda J, Cassinello N, Camps B, Vina J, Sastre J. (2002). Role of cytokines and oxidative stress in the pathophysiology of acute pancreatitis: Therapeutical implications. Curr Drug Targets Inflamm Allergy 1:393–403.

Hart AR, Kennedy H, Harvey I. (2008). Pancreatic cancer: A review of the evidence on causation. Clin Gastroenterol Hepatol 6:275–282.

Hayashi I, Morishita Y, Imai K, Nakamura M, Nakachi K, Hayashi T. (2007). High-throughput spectrophotometric assay of reactive oxygen species in serum. Mutat Res 631:55–61.

Kanehira T, Shibata K, Kashiwazaki H, Inoue N, Morita M. (2006). Comparison of antioxidant enzymes in saliva of elderly smokers and non-smokers. Gerodontolgy 23:38–42.

Li N, Jia X, Chen CY, Blumberg JB, Song Y, Zhang W, Zhang X, Ma G, Chen J. (2007). Almond consumption reduces oxidative DNA damage and lipid peroxidation in male smokers. J Nutr 137:2717–2722.

Lindkvist B, Appelros S, Manjer J, Berglund G, Borgstrom A. (2008). A prospective cohort study of smoking in acute pancreatitis. Pancreatology 8:63–70.

Ma A, Qi S, Chen H. (2008). Antioxidant therapy for prevention of inflammation, ischemic reperfusion injuries and allograft rejection. Cardiovasc Hematol Agents Med Chem 6:20–43.

Malfertheiner P, Schutte K. (2006). Smoking—a trigger for chronic inflammation and cancer development in the pancreas. Am J Gastroenterol 101:160–162.

Figure 3. Expression of -SMA, TGF-1, and collagen-1 in pancre-atic tissues of untreated rats and rats exposed to cigarette smoke. (A–C) Expression of -SMA, TGF-1, and collagen-1, respectively, in pancreatic tissues of untreated animals (×400). (D–F) Expression of -SMA, TGF-1, and collagen-1, respectively, in pancreatic tissues of animals exposed to cigarette smoke for 12 weeks (×400).

A B

C D

E F

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oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

ifor

nia

Irvi

ne o

n 10

/26/

14Fo

r pe

rson

al u

se o

nly.

1012 J.-Y. Hao and G. Li

McKay CJ, Glen P, McMillan DC. (2008). Chronic inflammation and pancreatic cancer. Best Pract Res Clin Gastroenterol 22:65–73.

Orosz A, Csiszar A, Labinskyy N, Smith K, Kaminski PM, Ferdinandy P, Wolin MS, Rivera A, Ungvari Z. (2007). Cigarette smoke-induced proinflammatory alterations in the endothelial phenotype: Role of NAD(P)H oxidase activation. Am J Physiol Heart Circ Physiol 282:H130–H139.

Pang B, Wang C, Weng X, Tang X, Zhang H, Niu S, Mao Y, Xin P, Huang X, Zhang H, Zhu J. (2003). Beta carotene protects against chronic bronchitis induced by cigarette smoking. Chin Med J (Engl) 116:514–516.

Schulz HU, Niederau C, Klonowski-Stumpe H, Halangk W, Luthen R, Lippert H. (1999). Oxidative stress in acute pancreatitis. Hepatogastroenterology 46:2736–2750.

Sidel EH, Casselman R, Smith GN. (2007). Effect of cigarette smoke on placental antioxidant enzyme expression. Am J Physiol Regul Integr Comp Physiol 293:R754–R758.

Solak ZA, Kabaroglu C, Cok G, Parildar Z, Bayindir U, Ozmen D, Bayindir O. (2005). Effect of different levels of cigarette smoking on lipid peroxidation, glutathione enzymes and paraoxonase I activity in healthy people. Clin Exp Med 5:99–105.

Talamini G, Bassi C, Falconi M, Sartori N, Vaona B, Bovo P, Benini L, Cavallini G, Pederzoli P, Vantini I. (2007). Smoking cessation at the clinical onset of chronic pancreatitis and risk of pancreatic calcifications. Pancreas 35:320–326.

Thorley AJ, Tetley TD. (2007). Pulmonary epithelium, cigarette smoke, and chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2:409–428.

Wittel UA, Pandey KK, Andrianifahanana M, Johansson SL, Cullen DM, Akhter MP, Brand RE, Prokopczyk B, Batra SK.2006a. Chronic pancreatic inflammation induced by environmental tobacco smoke inhalation in rats. Am J Gastroenterol 101:148–159.

Wittel UA, Singh AP, Henley BJ, Andrianifahanana M, Akhter MP, Cullen DM, Batra SK.2006b. Cigarette-induced differential expression of the genes involved in exocrine function of the rat pancreas. Pancreas 33:364–370.

Wittel UA, Hopt UT, Batra SK. (2008). Cigarette smoke-induced pancreatic damage—experimental data. Langenbecks Arch Surg 393:581–588.

Zalata A, Yahia S, El-Bakary A, El-Sheikha HM. (2007). Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress. Mutat Res 629:140–147.

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