Biomarkers and Genomic Medicine (2015) 7, 57e63

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ORIGINAL ARTICLE

Concomitant exposure to cigarette smokeand coal dust induces lung oxidative stressand decreases serum MUC5AC levels in malerats

Nia Kania a,*, Bambang Setiawan b, Edi Widjajanto c,Nurdiana Nurdiana d, M. Aris Widodo d,H.M.S. Chandra Kusuma e

a Research Center for Toxicology, Cancer and Regenerative Medicine, Department of Pathology, UlinGeneral Hospital, Medical Faculty, Lambung Mangkurat University, Banjarmasin, South Kalimantan,Indonesiab Research Center for Toxicology, Cancer and Regenerative Medicine, Department of MedicalChemistry & Biochemistry, Medical Faculty, Lambung Mangkurat University, Banjarmasin, SouthKalimantan, Indonesiac Department of Clinical Pathology, Saiful Anwar General Hospital, Faculty of Medicine,University of Brawijaya, Malang, East Java, Indonesiad Laboratory of Pharmacology, Faculty of Medicine, University of Brawijaya, Malang, East Java,Indonesiae Department of Paediatric, Saiful Anwar General Hospital, Faculty of Medicine,University of Brawijaya, Malang, East Java, Indonesia

Received 5 April 2014; received in revised form 17 June 2014; accepted 23 October 2014Available online 23 December 2014

KEYWORDSEGFR signaling;inflammation;oxidative stress;particulate matter

* Corresponding author. Research CLambung Mangkurat University, Jl. A.

E-mail address: kaniazairin@yahoo

http://dx.doi.org/10.1016/j.bgm.2012214-0247/Copyright ª 2015, Taiwan

Abstract This study aimed to investigate whether concomitant exposure to cigarette smokeand coal dust could activate the epidermal growth factor receptor (EGFR) for MUC5AC expres-sion. Thirty-two male Wistar rats were divided into the following groups (n Z 8 each): controlgroup (C); exposed to cigarette smoke plus coal dust at doses of 6.25 mg/m3 (CS þ CD1);12.5 mg/m3 (CS þ CD2); and 25 mg/m3 (CS þ CD3). The duration of exposure was 21 days, 1hour/day. Lung malondialdehyde level was analyzed colorimetrically. Serum EGF and MUC5ACexpression were measured by ELISA. Expression of lung EGFR and MUC5AC were measured by aconfocal laser scanning microscopy. The level of lung malondialdehyde was higher significantlyin all doses of exposure compared with control group (p < 0.05). The level of serum EGF wassignificantly increased in CS þ CD2 group compared with control group or CS þ CD1 group. The

enter for Toxicology, Cancer and Regenerative Medicine, Ulin General Hospital, Medical Faculty,Yani Km 2 No. 43, Banjarmasin, South Kalimantan, Indonesia..com (N. Kania).

4.10.001Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.

58 N. Kania et al.

expression of EGFR was not significantly different among all the treatment groups (p > 0.05).Serum MUC5AC levels were significantly lower in the two highest doses of coal dust comparedwith the control group. In conclusion, subchronic combined exposure to cigarette smoke andcoal dust induces lung oxidative stress and inflammation and decreases serum MUC5AC level.Copyright ª 2015, Taiwan Genomic Medicine and Biomarker Society. Published by ElsevierTaiwan LLC. All rights reserved.

Introduction

Cigarette smoke contains toxic and/or carcinogenic gasesand chemicals. Chemical composition of cigarette smokedepends on: (1) the type of cigarette; (2) cigarette design,such as the presence/absence of a filter; and (3) individualsmoking patterns. A burning cigarette produces approxi-mately 500 mg (92%) of gas and the remaining 8% is solidparticulates. The gas phase consists mostly of CO2, O2, andN2. Cigarette smoke also contains approximately 1015e1017

oxidants/free radicals and 4700 chemical compounds.Cigarette smoking-derived toxic compounds, including ar-omatic hydrocarbons, have been shown to be lungcarcinogens.1

Lung cancer is a smoking-related disease and cause ofdeath with an increasing incidence.1 Numerous studies havedemonstrated the role of cigarette smoke in an increasedincidence of cancer in asbestos-exposed individuals.2e4

Cigarette-smoke exposure prior to naphthalene exposurecan interfere with repair of bronchial epithelial cells inwhich squamous cells settle in the terminal bronchioles.5

This indicates that combined exposure of cigarette smokeand other toxicants will induce carcinogenicity. To date,there is a lack of studies that analyze the mechanism ofpathophysiology due to exposure to cigarette smoke andcoal dust.

Smoking causes lung inflammation due to the influx ofmacrophages, neutrophils, dendritic cells, and CD8 T-lym-phocytes as a source of inflammatory mediators. In addi-tion, smoking also leads to oxidative stress.6 Inflammationand oxidative stress underlie metaplasia, which are theearliest process prior to the occurrence of lung cancer.Epidermal growth factor receptor (EGFR) expression in thebronchial epithelium increases as a result of the activationof neutrophils and EGFR tyrosine kinase by its ligands.Subsequently, hypersecretion of mucus and goblet cellmetaplasia will result. In vitro and in vivo studies showedthat proinflammatory cytokines would up regulate theexpression of EGFR (transforming growth factor-a or EGF)to induce metaplasia. Activation of EGFR may occurthrough (ligand-dependent mechanism) autophosphor-ylation or (ligand-independent mechanism) transactivation.Subsequently, EGFR activation induces the mucin geneMUC5AC and expression of MUC5AC proteins as well asgoblet cell metaplasia.7 A smoking habit in coal minersexposes them to combined toxicants of cigarette smoke andcoal dust. Individuals often find it difficult to quit smokingso that limiting the number of cigarettes smoked/day isexpected to reduce the emerging effects. Therefore, thepurpose of the present study was to determine whether

concomitant exposure to cigarette smoke and coal dustcould induce oxidative stress and activate the EGFR forMUC5AC expression.

Material and methods

Animals

Thirty-two male Wistar albino rats, 16 weeks of age,weighing 175e200 g, were used for this study. They weredivided into the following groups (n Z 8 rats each): controlgroup (C); exposed to cigarette smoke plus coal dust atdoses of 6.25 mg/m3 (CS þ CD1); exposed to cigarettesmoke plus coal dust at doses of 12.5 mg/m3 (CS þ CD2);and exposed to cigarette smoke plus coal dust at doses of25 mg/m3 (CS þ CD3). The duration of exposure was 21days. Animals were housed in a clean wire cage and main-tained under standard laboratory conditions with temper-ature of 25 � 2�C and 12-hour dark/light cycle. Standarddiet and water were provided ad libitum. Animals wereacclimatized to laboratory conditions for 2 weeks prior tothe experiment. Animal care and experimental procedureswere approved by the institutional ethics committee ofFaculty of Medicine, Brawijaya University, Malang,Indonesia.

Coal dust preparation

Coal dust preparation was performed as described in ourprevious studies.7e10 Two kilograms of sub-bituminous grosscoals obtained from coal mining area in South Kalimantan,Indonesia, were pulverized by Ball Mill, Ring Mill, andRaymond Mill in the Carsurin Coal Laboratory of Banjar-masin. Coal dust particles were then filtered by MeshMicroSieve (BioDesign, New York, NY, USA) to produceparticles with the diameter < 10 mm (PM10) that have beenwell characterized previously.7,10

Cigarette smoke exposure

Smoking exposure was done by smoking pump equipmentthat was designed and available in Pharmacology Labo-ratorium, Medical Faculty, Brawijaya University of Malang.The rats in the control group were exposed to fresh airunder similar conditions. Rats placed into whole-bodyexposure chambers (26 cm � 12 cm � 12 cm3) made fromfiberglass and were exposed to cigarette smoke for 7 min/cigarette, once a day in the morning prior to coal dustexposure, for 21 days. During exposure, the temperature

Cigarette smoke and coal dust 59

was maintained at 22e25�C, and relative humidity wasapproximately 50%.

Coal dust exposure

The concentration of coal dust and procude for exposurewere determined according previous studies.7e11 Theexposure chamber was designed and available in the Lab-oratory of Pharmacology, Faculty of Medicine, BrawijayaUniversity. The procedure work of the chamber is to supplyan ambient resuspended PM10 coal dust that can be inhaledby rats. The chamber was 0.5 m3 and flowed by a 1.5e2 L/minute airstream that adopted the upper ground coal mineenvironmental airstream. To make a more comfortablechamber, oxygen was also provided.7e10

Tissue sampling

After 21 days of exposure, the animals were subjected toeuthanasia by ether inhalation and exsanguinated by car-diac puncture. The lungs were collected, weighed, andwashed with physiological saline. The right lung was his-tologically processed with hematoxylineeosin staining andconfocal microscopy (EGFR and MUC5AC). The left lung washomogenized to measure the malondialdehyde (MDA) levelcolorimetrically. Serum EGF and MUC5AC were measured byenzyme-linked immunosorbent assay technique. All sam-ples were labeled and stored at �80�C until analysis.

MDA analysis

The lung MDA levels were assayed by a previous method.7

Lungs were perfused using ice-cold PBS to obtain cleanand free of blood condition. Then, lungs were homogenizedin KCl buffer (pH 7.6). The homogenate was mixed with 2.5volumes of 10% (w/v) trichloroacetic acid to precipitate theprotein. The precipitate was then centrifuged and the su-pernatant was reacted with 0.67% tetrabutylammonium in aboiling water-bath for 25 minutes. After cooling, theabsorbance of the colored product was read at 532 nm usingthe spectrophotometer. The values obtained werecompared with a series of MDA tetrabutylammonium salt(SigmaeAldrich, St Louis, MO, USA) as standard solutions.

Analysis of EGF and MUC5AC

The serum EGF and MUC5AC ELISA kit were purchased fromUSCNK, Life Science, Inc (Wuhan, Hubei, China). Theanalysis was done according to detail procedures in the kit.

Labeling immunofluorescence staining of EGFR andMUC5AC

Double-labeling immunofluorescence staining of EGFR andMUC5AC was done according to previous studies.12

Statistical analysis

Data are presented as mean � standard deviation and thedifferences between groups were analyzed using one-wayanalysis of variance (ANOVA) with SPSS 15.0 statistical

package for Windows (SPSS, Chicago, IL, USA). Only prob-ability values of p < 0.05 were considered statisticallysignificant and later subjected to Tukey’s posthoc test.

Results

Characteristics of cigarette smoke

The present study used Trubus Alami brand clove cigarettes,which were produced in Tulungagung. These cigarettescontained 2.90 mg of tar and 44.30 mg of nicotine. Carbonmonoxide level of cigarette smoke was 102.33 ppm.13

Lung morphology

Lung morphology after exposure to cigarette smoke andcoal dust is shown in Fig. 1. Subsequent to 21 days ofexposure to cigarette smoke and coal dust, there weremorphological changes with features of intense and massiveinflammation, excess mucus that covered the lumen, andfibrotic process. It was seen that the widened lumen wassurrounded by inflammatory cells, the alveoli were moreintensely inflamed with widening of alveolar lumen, andthe alveolar epithelial bridge was lost, resulting in localemphysema (Fig. 1Be1D; Fig. 1FeH). Fibrogenesis occurredoutside the lumen so that the lumen was surrounded byincreasingly dominant fibrous connective tissues in accor-dance with exposure dosing (Fig. 1BeD).

Levels of MDA

Levels of MDA for different coal dust exposures are pre-sented in Table 1. The level of lung MDA was highersignificantly in all doses of exposure compared with thecontrol group (p < 0.05). MDA level was significantlyelevated in CS þ CD2 or CS þ CD3 compared with CS þ CD1

group (p < 0.05). The level was also increased significantlyin CS þ CD3 over that of the CS þ CD2 group (p < 0.05).

EGF level

Mean levels of serum EGF for different groups is presentedin Table 1. The level of serum EGF was significantlyincreased in the CS þ CD2 group compared with the controlgroup and with CS þ CD1 group (p < 0.05).

EGFR expression

Confocal laser scanning microscopic analysis of EGFRexpression for 21-day exposure to cigarette smoke and coaldust is presented in Table 1 and Fig. 2. The expression ofEGFR was not significant differences among all the treat-ment groups (p > 0.05).

MUC5AC expression

Lung MUC5AC expression levels for all groups can be seen inTable 1 and Fig. 2. The expression of lung MUC5AC was notsignificantly different between groups (p > 0.05). Out ofthe 6.25 mg/m3, 12.5 mg/m3, and 25 mg/m3 doses of coal

Figure 1 Lung histology of rats exposed to cigarette smoke and coal dust for 21 days. (A) In control (nonexposure) we found thecylindrical epithelial coating the intact lumen of bronchoalveolar. (B) After the first dose of exposure, there was no intact bron-choalveolar lumen due to disruption of cuboid or cylindrical cells. (C) After the second dose, there was widening of the bron-choalveolar lumen, almost all cylindrical epithelial transforming into squamous epithelial cells. The fibrotic process was found atthe edge of the bronchoalveolar lumen. (D) After the highest dose of exposure, the bronchoalveolar lumen decline, layered bysquamosa epithelial cells, surrounding by fibrous connective tissue, and mucus in the lumen. (E) In lung parenchyma, the thin wallof adjacent alveolus, we found a minimal connective tissue and no inflammatory cells. (F) In the lung parenchyma of first doseexposure, there was fibrous connective tissue that urgent the alveolus lumen. The wall of alveolus was thicker compared with thecontrol rats. Besides, the massive inflammatory cells was appeared surrounding the alveolus. The mucoid mass was found in thelumen of the alveolus. (G) In parenchyma of second dose, there was a fibrotic process accompanied by massive inflammatory cells,urgent the alveolus lumen become small. In addition, mucoid mass filled the lumen. (H) After the highest dose of exposure, wefound the compaction of parenchyma, localized, minimal alveolus (hematoxylin and eosin staining, magnification 100�).

60 N. Kania et al.

dust exposure, only the two highest doses significantlydecreased the serum MUC5AC expression compared withthe control group (p < 0.05), as seen in Table 1.

Discussion

A burning cigarette produces approximately 500 mg (92%) ofgas and the remaining 8% is solid particulates. Additionally,cigarette smoke contains acetaldehyde, hydroquinone,formaldehyde, benzo(a)pyrene, cresol, nicotine, catechol,

Table 1 Levels of lung and serum biomarkers after 21-day’s ex

Level

0 mg/m3 6.25 m

Lung Malondialdehyde (ng/mL) 0.0211 � 0.0098 0.111Serum EGF (pg/mL) 103.66 � 7.75 108.1Lung EGFR (AU) 826.36 � 57.25 1003.7Lung MUC5AC (AU) 582.06 � 361.65 1035.7Serum MUC5AC (pg/ml) 0.80 � 0.28 0.6

Values are presented as mean � standard deviations: ap < 0.05 comexposed to cigarette smoke plus coal dust at doses of 6.25 mg/m3; cpcoal dust at doses of 12.5 mg/m3.AU Z arbitrary unit; EGF Z epidermal growth factor; EGRF Z epide

acrolein, coumarin, anthracene, nitrogen oxides, and heavymetals.14 In the present study, cigarette smoke contained2.90 mg of tar and 44.30 mg of nicotine. Contents of tar andnicotine used in this study were higher than those (13 mgand 1.4 mg of tar and nicotine, respectively) used in theprior study.15 Tar contains high concentrations of stableradical, which continually accumulate in the smoker’slung.16

Subsequent to 21 days of concomitant exposure tocigarette smoke and coal dust, there were bronchoalveolarmorphological changes with features of intense and massive

posure to cigarette smoke and coal dust.

Doses of coal dust exposure

g/m3 12.5 mg/m3 25 mg/m3

7 � 0.0087a 0.1592 � 0.0032ab 0.2052 � 0.0068abc

4 � 7.41 129.63 � 20.94ab 125.05 � 22.345 � 479.53 716.94 � 175.08 632.43 � 109.624 � 334.44 907.91 � 431.98 649.84 � 427.645 � 0.10 0.39 � 0.11a 0.40 � 0.11a

pared with control group, bp < 0.05 compared with the group< 0.05 compared with the group exposed to cigarette smoke plus

rmal growth factor receptor.

Figure 2 Epidermal growth factor receptor (A1eD1) and MUC5AC expression (A2eD2) as caused by 21 days of exposure tocigarette smoke and coal dust at different doses. (A1, A2) Control; (B1, B2) 21-day exposure to cigarette smoke plus coal dust atdose of 6.25 mg/m3; (C1; C2) 21-day exposure to cigarette smoke plus coal dust at dose of 12.5 mg/m3; (D1; D2) 21-day exposure tocigarette smoke plus coal dust at dose of 25 mg/m3 [rhodamine (epidermal growth factor receptor) and fluorescein isothiocyanate(MUC5AC) staining at 400� magnification on a confocal laser scanning microscope].

Cigarette smoke and coal dust 61

inflammation, mucous secretion that filling the lumen, andfibrotic process. It was apparent that lumen widening wassurrounded by inflammatory cells. Increasingly intenseinflammation occurred in alveolar areas with a widenedalveolar lumen, and broken interalveolar septa wereobserved, resulting in local emphysema. Fibrogenesisoccurred outside the lumen so that the lumen was sur-rounded by increasingly dominant fibrous connective tis-sues in accordance with exposure dosing. Previous studiesconducted cigarette smoke exposure for 1 month, 2months, and 4 months. Goblet cell hyperplasia, mucussecretion in airway epithelium, and damaged interalveolarsepta were observed at 1 month of exposure. Meanwhile,alveolar interstitium filled with collagen fibers wasobserved at 4 months of exposure.16 Our study observedmorphological features similar to those observed at 1month of the above study. Accelerated pulmonary fibro-genesis seen in our study was caused by the interaction ofcigarette smoke and coal dust and the high contents ofactive components in cigarette smoke.

Lung inflammation is defined as small clusters of inflam-matory cells in the alveoli consisting of alveolar macro-phages and lymphocytes. Inflammation has specificpurposes. Acute inflammation protects organs from damage,but chronic inflammation is associated with disease pro-gression.17 In the present study, exposure to cigarette smokeand coal dust (12.5 mg/m3) caused a significant increase inEGF expression compared with control. We hypothesizedthat the interaction of cigarette smoke and coal dust at adose of 12.5 mg/m3 led to an increase in the release of theextracellular domain of pro-EGF into mature EGF mediated

bymetalloproteinases of the ADAM family, particularly ADAM10. Numerous studies have shown an increased activity ofmatrix metalloproteinases (MMPs) as a result of exposure tocigarette smoke.17 Cigarette smoke activates MMPs torelease EGF, which subsequently induces EGFR phosphory-lation to activate mitogen-activated protein kinases. In thepresent study, EGFR expression was not significantlydifferent in different treatment groups so that an increase inEGF was not followed by upregulation of EGFR.18

The present study showed that exposure to cigarettesmoke and coal dust increased pulmonary oxidative stress(p < 0.01). This finding indicates that components ofcigarette smoke and coal dust interact to induce oxidativestress. Cigarette smoke contains 1017 oxidant molecules perpuff of both mainstream and sidestream smoke.19 Gasphase and particulate phase of cigarette smoke containnitric oxide, superoxide radicals, and organic peroxyl rad-icals.19,20 Gas-phase radicals are highly reactive and have ashort half-life. Particulate-phase radicals are relativelystable and consist of hydroquinoneesemiquinoneequinonecomplex. These complexes represent an active redox sys-tem capable of reducing molecular oxygen to form super-oxide radicals. In addition, cigarette smoke also containslong-lived metals, such as nickel and cadmium.19

Free radicals of cigarette smoke will interact with an-tioxidants in the epithelial lining fluid of the airwayepithelium and cellular membrane directly to inducedamage.21 Previous studies found an increase in oxidativedamage to lung tissue that included lipid peroxidation(MDA).6 Inhalation of coal dust will form reactive oxygencompounds via direct and indirect mechanisms. Direct

62 N. Kania et al.

mechanism involves bioactive components of coal dust andindirect mechanism involves the oxidative burst duringactivation of macrophages and polymorphonuclear leuko-cytes.22,23 With the direct mechanism, oxidative capacityof coal dust bioactive component is caused primarily bytransition-metal contents, including Fe, Cr, Mn, Co, Ni, Cu,Zn, and silica. Some of these metals are capable of cata-lyzing the Fenton reaction to produce reactive oxygencompounds.23 Comparison of oxidative stress in healthyindividuals with that of coal miners showed a highly sig-nificant difference in plasma MDA levels.24 With indirectmechanism, superoxide radicals form during phagocytosisof inhaled particles, which will dismutase to form hydrogenperoxide. In the presence of transition metal ions, such asFe or Cu ions, hydrogen peroxide is converted to hydroxylradicals (Fenton or HabereWeiss reaction). The formationof excess reactive oxygen compounds will exceed antioxi-dant capacity, resulting in pulmonary oxidative stress.Furthermore, production of proteolytic and elastase en-zymes, in conjunction with reactive oxygen compounds,will denature proteins and damage carbohydrates and lipidperoxidation and, consequently, lead to changes, such asfibrosis.24 The present study found that fibrosis may beassociated with oxidative stress.

Compared with nonsmokers, the risk of lung cancerincidence in smokers is 22-fold higher in men and 12-foldhigher in women.15 It has been known that the remodelingof the upper and lower respiratory tracts into squamousmetaplasia, resulting in the development of diseases, wasassociated with exposure to cigarette smoke.25 There is nostudy of both the development and incidence of lung can-cer caused by combining exposure to cigarette smoke andcoal dust. Various ingredients of cigarette smoke bringabout an increase in MUC5AC. Exposure to acrolein wouldincrease MUC5AC mRNA expression.26 The benzo(a)pyrenecaused up regulation of MUC5AC.27 Previous studies foundincreased expression of MUC5AC as a result of exposure tocigarette smoke.28 MUC5AC hypersecretion is consistentwith activation of EGFR-AP-1/nuclear factor (NF)-kF andTLR4-AP-1/NF-kB signaling pathways.29 In our study, expo-sure to 25 mg/m3 of cigarette smoke and coal dust led to asignificant decrease in MUC5AC expression compared withcontrol. Our finding indicated that the active componentsof cigarette smoke or coal dust may inhibit the signaling forserum MUC5AC production. Previous studies showed thatlipopolysaccharide exposure is able to induce goblet cellmetaplasia, and that increased MUC5AC expression couldbe inhibited by administration of an MMP inhibitor.30 Inaddition, compound act as inhibitor for NF-kB also potentialto inhibits MUC5AC expression.31 Thus, we hypothesizedthat combined cigarette smoke and coal dust in the presentstudy may produce effects analogous to those of an MMP orNF-kB inhibitors.

Conclusion

Our data suggest that subchronic combined exposure tocigarette smoke and coal dust induces lung oxidative stressand inflammation and also decreases the serum MUC5AClevel.

Conflicts of interest

The authors declare that there are no conflicts of interest.

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