Induction of Fibroblast Growth Factor-9 and Interleukin-1 Gene Expression by Motorcycle Exhaust Particulate Extracts and Benzo(a)pyrene in H
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《毒物学科学杂志》
Institute of Toxicology and Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
Division of Environmental Health and Occupational Medicine, National Health Research Institute, Kaohsiung, Taiwan, ROC
ABSTRACT
Motorcycle exhaust particulates (MEP) contain carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene. This study has determined the ability of MEP to alter the expression of select genes from drug metabolism, cytokine, oncogene, tumor suppressor, and estrogen signaling families of human lung adenocarcinoma CL5 cells. cDNA microarray analyses and confirmation studies were performed using CL5 cells treated with 100 μg/ml MEP extract for 6 h. The results showed that MEP increased the mRNA levels of metabolic enzymes CYP1A1 and CYP1B1, proinflammatory cytokines interleukin (IL)-1, IL-6, and IL-11, fibroblast growth factor (FGF)-6 and FGF-9, vascular endothelial growth factor (VEGF)-D, oncogene fra-1, and tumor suppressor p21. In contrast, MEP decreased tumor suppressor Rb mRNA in CL5 lung epithelial cells. Treatment with 10 μM benzo(a)pyrene for 6 h altered gene expression profiles, in a manner similar to those by MEP. Induction of IL-1, IL-6, IL-11, and FGF-9 mRNA by MEP and benzo(a)pyrene was concentration and time dependent. Cotreatment with 2 mM N-acetylcysteine blocked the MEP- and benzo(a)pyrene-mediated induction. Treatment with MEP or benzo(a)pyrene increased IL-6 and IL-11 releases to CL5 cell medium. Incubation of human lung fibroblast WI-38 with MEP- or benzo(a)pyrene-induced CL5 conditioned medium for 4 days stimulated cell growth of the fibroblasts. Inhalation exposure of rats to 1:10 diluted motorcycle exhaust 2 h daily for 4 weeks increased CYP1A1, FGF-9, and IL-1 mRNA in lung. This present study shows that MEP and benzo(a)pyrene can induce metabolic enzyme, inflammatory cytokine, and growth factor gene expression in CL5 cells and stimulate lung epithelium-fibroblast interaction.
Key Words: motorcycle exhaust particulate; benzo(a)pyrene; fibrobalst growth factor; interleukin; lung epithelial cell.
INTRODUCTION
The emissions of motorcycle exhaust (ME) are a major source of air pollution in areas where motorcycles are a popular means of transportation. The 2- and 4-stroke motorcycle engines have smaller capacity and poorer combustion efficiency than diesel and gasoline engines. ME also contains higher levels of carcinogen benzene than exhaust from gasoline and diesel engines (Jemma et al., 1995). The motorcycle commuters, therefore, are exposed to higher levels of benzene, as compared to car commuters (Chan et al., 1993). Carcinogenic polycyclic aromatic hydrocarbons (PAH) such as benzo(a)pyrene, benz(a)anthacene, and benzo (g,h,i)perylene have been detected in the organic solvent extracts of ME particulates (MEP) from 2-stroke engine (Ueng et al., 2000). The 2-stroke engine is distinctively different from other vehicle engines in that the former requires mixing motor oil with fuel prior to combustion. Studies of exhaust from this type of engine are of environmental and toxicological significance because, in addition to motorcycles, the 2-stroke engines are also widely used in a variety of applications including outboard boat motors, snowmobiles, lawn mowers, and trimmers.
ME and MEP have many toxicological properties. For example, ME inhalation exposure increased lipid peroxidation and decreased cytochrome P450 (CYP) 2B1 protein in rat lung (Ueng et al., 2004). Treatment of human lung cancer cells with organic extracts of MEP increased oxidative stress and DNA damage and decreased gap junctional intercellular communication (Kuo et al., 1998). Furthermore, MEP extract was found to enhance vasoconstriction in organ culture of rat aortas and induce inflammation and hyperresponsiveness in mouse airways (Lee et al., 2004; Tzeng et al., 2003).
Airway epithelium is a physical barrier to inhaled particulates and toxicants and a critical cell type in the pathogenesis of lung disease and cancer. The lung epithelial cells are responsive to the stimulatory effects of diesel exhaust particulates (DEP), which induce production of cytokines and mediators in human bronchial epithelial cells (Kawasaki et al., 2001; Steerenberg et al., 1998). Induction of these inflammatory mediators is believed to be one of the etiological factors for asthma and chronic obstructive pulmonary disease (Mills et al., 1999). Information regarding the effect of MEP on cytokine production by lung epithelium remains to be explored. Airway epithelium is a target site of cancers such as lung adenocarcinoma, which has a high female-to-male ratio and a large proportion of nonsmokers. Gene and environment interactions could possibly contribute to the high female susceptibility to lung adenocarcinoma. CYP enzymes are involved in activation of pulmonary PAH carcinogens and mammary carcinogen 17-estradiol. MEP induced CYP1A1 and CYP1B1 expression in CL5 female lung epithelial adenocarcinoma cell line (Wang et al., 2002). Oncogene, tumor suppressor, and estrogen signaling genes may play significant roles in female lung carcinogenesis. MEP interactions with these genes still need to be elucidated.
The major objective of our studies was to investigate the interaction of MEP with genes important in the development of lung disease and cancer in human lung epithelial cells. In this regard, CL5 cells were treated with MEP extracts, and cDNA microarrays analyses were conducted using arrays consisting of 255 genes selected from the metabolic enzyme, cytokine, oncogene, tumor suppressor, and estrogen signaling families. Gene alteration and bioactivity studies were extended to include the prototypic chemical carcinogen benzo(a)pyrene, for mechanistic and comparison purposes.
MATERIALS AND METHODS
Preparations of MEP extract and washed MEP.
Organic solvent extracts of MEP were prepared as described previously (Ueng et al., 2000). A 1992 Yamaha Cabin motorcycle with a 2-stroke 50-cc engine and a variable carburetor was used. An aerosol monitor model 8520 DUSKTRAK (TSI, Inc., Shoreview, MN) was used to determine the original concentrations of ME particles. The mean values of particles concentrations were: PM1, 118 mg/m3; PM2.5, 216 mg/m3; and PM10, 228 mg/m3. MEP were collected on 0.5-μm quartz filters. Soxhlet extraction was carried out in dark for 24 h using dichloromethane:hexane (1:1). The MEP extract was rotor-evaporated to dryness and stored in the dark at –20°C until analysis. The washed MEP were prepared essentially as described by Yin et al. (2004). MEP were suspended in acetone by sonication of the quartz filters. After solvent removal, MEP were washed consecutively using sterile phosphate-buffered solution, pH 7.4, dichloromethane, and acetone:methanol (1:1) mixture. The washed MEP were air dried, weighed, and stored in the dark at –20°C until analysis.
Gas chromatography/mass spectrometry (GC/MS) analysis of MEP extract.
The MEP extract was analyzed for PAH using a Hewlett-Packard 6890 gas chromatograph and a 5973 mass spectrometer equipped with a 7673 autosampler and an HP-5MS capillary column (60 m x 0.25 mm ID, 0.25 μm film thickness). The GC/MS operation conditions were as described previously (Ueng et al., 2000). Helium was used as the GC carrier gas. Following sample injection, the GC column was held at 80°C for 0.1 min, temperature programmed to 190°C at 10°C/min, 260°C at 3.5°C/min, and 300°C at 1.4°C/min. The MS was operated under 280°C detector temperature at 70 eV in the scan and selected ion models for qualitative and quantitative analyses, respectively. The GC column was calibrated with a standard U.S. Environmental Protection Agency 610 Polynuclear Aromatic Hydrocarbons Mixture consisting of 16 priority PAH pollutants (Supelco, Inc., Bellefonte, PA). Each PAH of MEP extract was quantified by means of an external calibration curve built from standard PAH solutions of 2, 5, 10, and 50 μg/ml. If the target PAH analyte exceeded the linear range of the calibration standards, the MEP extract sample was further diluted and reanalyzed.
Cells and treatments.
The human lung epithelium cell line CL5 was derived from a lung adenocarcinoma tumor specimen of a 40-year-old women patient at the Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. The cell line has been single-cell cloned and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.0 g/l sodium bicarbonate at 37°C in a humidified atmosphere of 5% CO2. Human bronchial epithelial BEAS-2B cells immortalized with SV40 (American Type Culture Collection, Manassas, VA) were gifts from Dr. Pinpin Lin, Institute of Toxicology, Chung Shan Medical University, Taichung, Taiwan. BEAS-2B cells were maintained in serum-free LHC-9 medium with glutamine (BioSource International Inc., Rockville, MD). WI-38 human normal lung fibroblast (American Type Culture Collection) was obtained from Food Industry Research and Development Institute, Hsinchu, Taiwan. WI-38 cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 1.5 g/l sodium bicarbonate.
Lung cells were used when the monolayer reached near confluence. The cell density was about 10 x 106 cells/dish in 10-cm culture dishes for treatment. MEP extract or test compound was dissolved in dimethyl sulfoxide (DMSO) and added to the medium so that DMSO concentration in the medium was less than 0.1%. Control cells were treated with 0.1% DMSO in medium. Cell viability was determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Carmichael et al., 1987). Peroxide formation was determined using the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) (Lebel et al., 1992).
cDNA probe preparation and cDNA microarray analysis.
Gene expression profile was analyzed using nonradioactive GEArray pathway-specific expression arrays (SuperArray Inc., Bethesda, MD). The five arrays used were the human drug metabolism and common cytokine gene arrays, consisting of 96 genes each, and the human cancer/oncogene, cancer/tumor suppressor, and estrogen signaling pathway arrays, each consisting of 23 genes. There were cDNA fragments of 255 individual genes on these nylon-membrane arrays, and their gene tables are available (www.superarray.com). Total RNA was isolated from CL5 cells as described previously (Wang et al., 2001) and converted to biotinylated cDNA probes by reverse transcription with a dNTP mix containing biotin-dUTP. Biotinylated cDNA probes were hybridized to gene-specific cDNA fragments spotted on the membranes following manufacture's protocol. The GEArray membrane was then blocked with GEAblocking solution and incubated with alkaline phosphatase conjugated streptavidin. The relative gene expression levels were detected by chemiluminescence signal using the alkaline phosphatase substrate, CDP-Star, and X-ray film. The relative abundance of a particular transcript was estimated by comparing its signal intensity to the signals derived from internal hybridization controls -actin and GAPDH. Image analysis and spot quantitation were carried out using GenePix 3.0 program (Axon Instruments, Union City, CA).
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Five μg total RNA were reverse transcribed using 1X RT, 2.2 mM MgCl2, 2.0 mM dNTP, 0.2 U/ml RNAsin, 0.5 mM random hexamer primers, and 0.3 U/μl MMLV reverse transcriptase in 25 μl reactions using a 2-step cycle: 70°C, 5 min and 37°C, 2 h. Reverse transcription reagents were purchased from Promega Corp., Madison, WI. The resulting cDNA was used in subsequent real-time RT-PCR reactions with fluorescence detection using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Reaction was carried out in microAmp 96-well reaction plates, SYBR Green PCR Master Mix 2X, DNA polymerase, dNTPs with dUTP, forward and reverse primers (0.15 μM each) (Invitrogen Corp., Carlsbad, CA), and 200 ng cDNA in a final volume of 25 μl. Amplification parameters were: denaturation at 94°C 10 min, followed by 45 cycles of 95°C, 15 s; 60°C, 60 s. All primers and probes were designed using PrimerExpress software (Table 1). Samples were analyzed in triplicate, and -actin was used as an endogenous control.
Quantitation of mRNA transcription was performed using a relative quantitation method with standard curves constructed from five log RNA concentrations of a specific gene or -actin and their respective CT values. The input amount of a specific gene was calculated from its standard curve and normalized to the input amount of -actin calculated from its standard curve. The relative difference between treatment and control groups was calculated from the ratio of the amount of specific gene to that of -actin in each group.
RT-PCR analysis.
Two μg total RNA were isolated from CL5, BEAS-2B cells, or rat lung. cDNA synthesis and PCR were conducted as described previously (Wang et al., 2001). PCR primers for CYPs, cytokines, and internal controls were synthesized (Gibco/BRL, Life Technologies, Inc., Gaithersburg, MD) according to the published sequences (Table 2). All reactions were conducted with -actin or cyclophilin primers as internal controls. PCR products were separated on 2% agarose gels and stained with ethidium bromide. Intensity of PCR product was quantitated using an IS-1000 Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA) and normalized against the intensity of internal control -actin or cyclophilin. Relative intensity of target gene PCR product from treated cell culture or rats was calculated by dividing its intensity by the corresponding intensity from control cells or rats.
Preparation of cell lysate.
CL5 cells were harvested by scraping and washed in a phosphate buffered saline solution. The following procedures were carried out at 4°C. Cell suspension was centrifuged at 1,500 rpm for 3 min. The cell pellet was washed and sonicated in 0.1 M potassium phosphate buffer, pH 7.4. Cell homogenate was centrifuged at 9,000 x g for 20 min, and the resulting supernatant, cell lysate, was stored at –80°C prior to analyses of cytokines concentrations.
Enzyme-linked immunosorbent assay (ELISA).
CL5 cells in maintenance medium were plated at 1 x 105 cells per well in 6-well tissue culture plates. After seeding overnight, maintenance medium was changed to experimental medium consisting of RPMI 1640 medium supplemented with 1% charcoal-treated fetal calf serum. After 12 h incubation, the medium was removed, and 1 ml fresh experimental medium containing 100 μg/ml MEP extract or 10 μM benzo(a)pyrene was added to each well. To control cultures, 0.1% DMSO in fresh medium was added. Twenty-four h after treatment, the cell-conditioned medium was collected by centrifugation and stored at –20°C until ELISA. IL-1, IL-6, and IL-11 levels of conditioned media were determined by ELISA using human Quantikine human kits (R&D Systems Inc., Minneapolis, MN). Concentrations of these cytokines in cell lysate were similarly determined.
Bioactivity assay.
CL5 cells in maintenance medium were plated in 6-cm tissue culture dishes at 5 x 105 cells per dish. After seeding overnight, maintenance medium was changed to experimental medium consisting of RPMI 1640 medium supplemented with 1% charcoal-treated fetal calf serum. After 12 h incubation, the medium was removed and 5 ml fresh experimental medium containing 100 μg/ml MEP extract or 10 μM benzo(a)pyrene was added to each dish. To control cultures, 0.1% DMSO in fresh medium was added. Twenty-four h after treatment, the CL5 cell-conditioned medium was collected by centrifugation and stored at –20°C until use. WI-38 cells in maintenance medium were seeded into 24-well culture plates at 1 x 104 cells per well. After seeding overnight, fibroblasts were serum-deprived for 16 h with serum-free MEM with supplements. Following serum-deprivation, the serum-free medium was replaced with experimental medium, prepared by 1:1 dilution of CL5 cell-conditioned medium with serum-free MEM with supplements. The experimental medium was replaced at 48 h after incubation. Cell growth of WI-38 fibroblast was determined at 96 h. Cells were fixed and stained with sulforhodamine B as described by Skehan et al. (1990). The bound dye was solubilized, and its absorbance was read at 490 nm using an ELISA reader.
Experimental animals and ME inhalation exposure.
Seven-week-old female Wistar rats were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. Before experiments began, the animals were allowed 1 week of acclimation at the animal quarter. In ME inhalation studies, the animals were exposed to 1:10 diluted ME using a head-nose-only inhalation chamber (Technical and Scientific Equipment GMBH, Bad Hamburg, Germany). The animals were exposed to ME from 9 to 10 A.M. and 4 to 5 P.M. daily, Monday through Friday, for 4 weeks. Control rats were exposed to clean air only. The animal maintenance and the inhalation chamber and exposure conditions were described previously (Ueng et al., 2004). The exposure chamber atmospheres were measured using an aerosol monitor model 8520 DUSKTRAK with a cutoff at 10 μm and a combustion analyzer model CA-6200 (TSI, Inc.). The control and ME exposure atmospheres components and their mean concentrations were: particles, 0.5 and 21.5 mg/m3; carbon monoxide, 0.2 and 5.8 ppm; carbon dioxide, 0 and 0.3%; nitric oxide, 0 and 4.5 ppm; nitric dioxide, 0 and 0 ppm; and oxygen, 20.5 and 20.3%, respectively. Animals were killed within 24 h after the last exposure. The Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine approved all animal care and experimental procedures.
Statistical analysis.
The statistical significance of difference between control and treatment groups was evaluated by the Student's t-test of paired data. A p-value <0.05 was considered statistically significant.
RESULTS
Qualitative GC/MS analysis of MEP extract identified the presence of 14 PAH in the chemical mixture (Table 3). Carcinogenic benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-c,d)pyrene, and benzo(g,h,i)perylene were identified in the MEP extract. The results of quantitative GC/MS analysis showed that benzo(a)pyrene was one of the more abundant carcinogenic PAH in MEP extract. The amounts of these carcinogenic PAH were smaller than those of noncarcinogenic PAH such as naphthalene and phenanthrene. For comparison purposes, benzo(a)pyrene was chosen as the component compound of MEP extract in the following studies.
A preliminary study was first conducted to determine the optimal conditions for MEP treatment. In this study, CL5 cells were treated with increasing concentrations of MEP extract for various time periods. RT-PCR analysis was performed to determine the effect of MEP on CYP1A1, the marker of exposure and effect. MTT assay was then carried out to determine the effect on cytotoxicity. It was found that treatment with 100 μg/ml MEP extract for 6 h produced a near maximal inductive effect on CYP1A1 and minimal cytotoxic effects (data not shown). These treatment conditions were used in the subsequent cDNA microarray studies.
In the drug metabolism array study, a visual comparison of the membranes of controls and MEP-treated CL5 cells demonstrated that MEP-treated cells showed increases of CYP1A1, CYP3A7, and UGT2B gene expressions (Fig. 1). Image analysis of transcript intensity indicated that MEP increased CYP1A1, CYP3A7, UGT2B, and UGT1A1 mRNA by 9-, 3-, 4-, and 2-fold, respectively (Table 4). In the cytokine array study, MEP elevated expression of fibroblast growth factor (FGF)-6, FGF-9, IL-1, and IL-22 genes (Fig. 2). Image analysis further showed that MEP increased FGF-6, FGF-9, and vascular endothelial growth factor (VEGF)-D mRNA by 4-, 2-, and 9-fold, respectively; IL-1 and IL-22 by 5- and 6-fold; and TNFSF10 by 5-fold. The results of oncogene, tumor suppressor, and estrogen signaling pathway arrays studies indicated that MEP extract produced 4-, 6-, and 4-fold increases of oncogenes fra-1, c-src, and SHC and resulted in 2-fold increases of tumor suppressor p21 and estrogen response gene COX7RP (Table 4). In contrast, MEP decreased tumor suppressors p53 and Rb expression by about 60%. The data from these five arrays studies collectively showed that MEP up-regulated the expression of 15 genes and down-regulated 2 genes. No remarkable alterations on the expressions of the other 238 genes were detected on the arrays.
To evaluate the validity of gene alterations identified in the arrays, confirmation studies were carried out using CL5 cells treated with 100 μg/ml MEP extract for 6 h. Additional cells were treated with 10 μM benzo(a)pyrene, a constituent of MEP, for 6 h. Under this treatment condition maximal effects on CYP1A1 mRNA and minimal effects on cytotoxicity could be induced (data not shown). Total RNA was prepared from the control and treated cells, and real-time RT-PCR analysis was performed. This study showed that MEP produced an 11-fold increase of CYP1A1; 3-, 5-, 6-, and 3-fold increases of IL-1, FGF-6, FGF-9, and VEGF-D; and 2-fold increases of fra-1 and p21 mRNA, respectively (Table 5). Furthermore, MEP resulted in a 47% decrease of Rb expression. These gene alterations were in agreement with those identified in the arrays. The real-time RT-PCR data indicated that MEP did not produce marked effects on CYP3A7, IL-22, TNFSF10, SHC, p53, or COX7RP mRNA, in variation from the array data. In these confirmation studies, the effects of MEP and benzo(a)pyrene on five related genes were determined. MEP induced the expression of CYP1B1, IL-6, and IL-11 by 4-, 2-, and 3-fold, without affecting IL-15 and VEGF-C. Treatment of CL5 cells with benzo(a)pyrene increased the expression of metabolic enzymes CYP1A1 and CYP1B1; proinflammatory cytokines IL-1, IL-6, IL-11, and IL-15; growth factors FGF-6, FGF-9, and VEGF-D; oncogene fra-1; and tumor suppressor p21. Benzo(a)pyrene decreased tumor suppressor Rb expression, without affecting CYP3A7, IL-22, TNFSF10, VEGF-C, SHC, or COX7RP (Table 5). The degrees of alterations of these 12 genes induced by benzo(a)pyrene were in general similar to those induced by MEP, except that the PAH produced a 2-fold increase of IL-15 mRNA level which was not altered by MEP.
Concentration-response and time-course studies were carried out to better characterize the inductive effects of MEP and benzo(a)pyrene on FGF-9, IL-1, IL-6, and IL-11 mRNA. In concentration-response studies, CL5 cells were treated with increasing concentrations of MEP extract or benzo(a)pyrene for 6 h. The results of RT-PCR analysis showed that 1, 10, 100, and 200 μg/ml MEP extract produced concentration-dependent increases of FGF-9 mRNA (Fig. 3, top). Near-maximal increases were observed at 100 and 200 μg/ml MEP. Treatment with 0.1, 1, 10, and 50 μM benzo(a)pyrene increased FGF-9 expression in a concentration-dependent manner, showing a maximal increase at 50 μM (Fig. 3, bottom). Similar to the induction kinetics observed with FGF-9, MEP and benzo(a)pyrene also produced concentration-dependent increases of IL-1, IL-6, and IL-11 mRNA with maximal increases at 100 or 200 μg/ml MEP and 50 μM benzo(a)pyrene, respectively (Table 6). In time-course studies, CL5 cells were treated with 100 μg/ml MEP extract or 10 μM benzo(a)pyrene for 3 to 48 h. The results of RT-PCR analysis showed that MEP produced time-dependent increases of FGF-9 mRNA at 3 and 6 h following treatment (Fig. 4, top). These increases declined time-dependently at 12, 24, and 48 h. In parallel to MEP, benzo(a)pyrene resulted in maximal increases of FGF-9 at 3 and 6 h with gradual decline at the subsequent time points (Fig. 4, bottom). Similar time courses of inductive effects were also observed with IL-1, IL-6, and IL-11 (Table 7). As with these inflammatory cytokines, IL-15 mRNA was likewise induced concentration- and time-dependently by benzo(a)pyrene (data not shown).
Because CL5 is a tumor-derived cell line, it was important to examine the effects of MEP and benzo(a)pyrene on gene expression using a noncancerous human lung epithelial cell line. In this study, human bronchial epithelial BEAS-2B cells were treated with MEP extract or benzo(a)pyrene for 6 h, total RNA was isolated, and RT-PCR analysis was conducted. It was noted that treatment with 1, 10, and 100 μg/kg MEP produced concentration-dependent increases of CYP1A1 and CYP1B1 mRNA (Fig. 5, left). It was also found that treatment with 0.1, 1, and 10 μM benzo(a)pyrene increased CYP1A1 and CYP1B1 concentration-dependently (Fig. 5, right). Thus the inductive effects of MEP and benzo(a)pyrene in BEAS-2B cells were quite similar to the respective effects found in CL-5 cells. However, MEP extract and benzo(a)pyrene had no marked effects on the proinflammatory cytokines IL-, IL-6, IL-11, and IL-15; growth factors FGF-6, FGF-9, and VEGF-D; oncogene fra-1; and tumor suppressors p21 and Rb mRNA levels in the BEAS-2B cells (data not shown).
Studies were performed to investigate the effects of MEP and benzo(a)pyrene on peroxide production and to explore its possible mechanistic role in cytokine induction of CL5 cells. In these studies, CL5 cells were treated with 2 mM N-acetylcysteine, 100 μg/ml MEP extract, and 10 μM benzo(a)pyrene, respectively or in combination, for 6 h, peroxide formation was determined by DCFH oxidation method (Lebel et al., 1992), and cytokine mRNA levels were analyzed by RT-PCR procedures. The antioxidant N-acetylcysteine resulted in a 52% decrease of peroxide production, and the MEP extract produced a 32% increase, as compared to the controls (Fig. 6). Peroxide formation in cells cotreated with MEP extract and N-acetylcysteine was similar to the formation observed in control cells. Benzo(a)pyrene increased peroxide production by 27%. Cotreatment with benzo(a)pyrene and the antioxidant reduced the benzo(a)pyrene-mediated increase of peroxides. The results of RT-PCR analysis showed that N-acetylcysteine had no marked effects on IL-1, IL-6, FGF-9, and VEGF-D mRNA of CL5 cells (Fig. 7). Treatment with MEP extract elevated mRNA levels of these cytokines as expected. Cotreatment with MEP and N-acetylcysteine reduced the MEP-elevated mRNA levels of IL-1, IL-6, FGF-9, and VEGF-D to their respective levels as in controls. Similarly, cotreatment with benzo(a)pyrene and the antioxidant reduced the benzo(a)pyrene-elevated cytokines mRNA levels. These results clearly indicated that increase of peroxide formation was involved in cytokine induction by MEP and benzo(a)pyrene.
To further investigate the induction properties, the abilities of MEP and benzo(a)pyrene in increasing protein productions of IL-1, IL-6, and IL-11 were examined. CL5 cells were treated with 100 μg/ml MEP extract or 10 μM benzo(a)pyrene for 24 h, and cytokine productions in culture medium and cell lysate were determined by ELISA. It was found that MEP produced no effect on IL-1 and 57% and 51% increases of IL-6 and IL-11 production, respectively, in culture medium (Table 8). The effects of benzo(a)pyrene on these cytokine productions were qualitatively similar to the effects of MEP. Production of IL-6 was higher than that of IL-1 and IL-11 in control and treated-cell culture media. In cell lysate, MEP resulted in a 28% increase, no effect, and a 41% increase of IL-1, IL-6, and IL-11 concentrations, respectively. Benzo(a)pyrene produced 32%, 99%, and 38% increases of the respective cytokine concentrations. IL-1 concentration in cell lysate was greater than the corresponding concentrations of IL-6 and IL-11. These ELISA data indicated a general pattern that MEP and benzo(a)pyrene induced intracellular concentrations of IL-1, IL-6, and IL-11 and increased releases of IL-6 and IL-11 to cell medium.
The subsequent bioactivity study was conducted to investigate the effects of increased cytokine and growth factor production on the cell growth of WI-38 human lung fibroblast. Conditioned medium was collected from CL5 cells treated with 100 μg/ml MEP or 10 μM benzo(a)pyrene for 24 h. Exposure of WI-38 cells to this MEP-induced conditioned medium for 4 days produced a 36% increase of cell growth of the fibroblasts (Fig. 8). Exposure to benzo(a)pyrene-induced conditioned medium resulted in a 51% increase of WI-38 cell growth. Treatment of WI-38 cells with 50 ng/ml recombinant human FGF-9, a positive control, for 4 days increased cell growth by 57%. These bioactivity data showed that MEP and benzo(a)pyrene enhanced the ability of CL5 epithelial cells to stimulate the growth of lung fibroblast.
The following studies were done to determine the abilities of washed MEP and benzo(e)pyrene, an isomer of benzo(a)pyrene, to induce gene expression in CL5 cells. CYP1A1, FGF-9, and IL-1 were selected to represent drug metabolism, growth factor, and inflammatory cytokine families, respectively. CL5 cells were treated with 100 μg/ml washed MEP or 10 μM benzo(e)pyrene for 6 h. Additional cells were treated with 100 μg/ml MEP or 10 μM benzo(a)pyrene for 6 h, for comparison purposes. Total RNA was isolated, and RT-PCR analysis was carried out. The results showed that washed MEP and benzo(e)pyrene had no marked effects on CYP1A1, FGF-9, or IL-1 mRNA levels, unlike the inductive effects of MEP and benzo(a)pyrene (Fig. 9). In concentration-response and time-course studies, treatment with 1, 10, and 100 μg/ml washed MEP or 0.1, 1, 10, and 50 μM benzo(e)pyrene for 6 h and treatment with 100 μg/ml washed MEP or 10 μM benzo(e)pyrene for 3, 6, and 12 h did not increase the metabolic enzyme and cytokines mRNA. These treatment conditions did not show cytotoxicity based on MTT assay (data not shown).
The above gene expression studies were conducted mostly using female lung adenocarcinoma CL5 cells treated with MEP extract in vitro; therefore it was of interest to investigate the ability of the environmental mixture ME to induce CYP1A1, FGF-9, and IL-1 mRNA in vivo. In this regard, female rats were exposed to 1:10 diluted ME by inhalation for 1 h each in the morning and afternoon daily, Monday through Friday, for 4 weeks, aiming to provide more environmentally realistic conditions. The ME inhalation exposure had no effects on body weight or lung, liver, and kidney relative tissue weights (Table 9). RT-PCR analysis of lung RNA showed that ME inhalation exposure produced 3-fold increases of CYP1A1 and FGF-9 and a 2-fold increase of IL-1 mRNA levels, respectively (Fig. 10). The results of this rat inhalation study and those of cell culture studies showed that the metabolism, growth factor, and proinflammatory cytokine genes expression in lung cells were upregulated by ME in vivo and MEP extract in vitro.
DISCUSSION
Findings from this present study show that MEP extract and benzo(a)pyrene can alter the expression of an array of genes belonging to the metabolic enzyme, proinflammtory cytokine, growth factor, oncogene, and tumor suppressor families in human lung epithelial cells. To the best of our knowledge, this report is the first to show environment and gene interactions of IL-1, IL-11, FGF-9, and VEGF-D. IL-1 is an intracellular messenger in epithelial and endothelial cells where the autocrine plays a regulatory role in cell differentiation. IL-11 is a member of IL-6-type cytokines, which are involved in acute-phase response to injury and activation of target genes associated with cell differentiation, proliferation, and survival (Heinrich et al., 2003). FGF-9 belongs to the FGF superfamily, which regulates cell differentiation, proliferation, and migration during embryonic development and controls tissue repair and response to injury in adult organism (Ornitz and Itoh, 2001). FGF-9 may also play an oncogenic role in human lung cancer (Matsumoto-Yoshitomi et al., 1997). The VEGF family is known to promote endothelial cell proliferation and vascular permeability. VEGF-D induces both tumor angiogenesis and lymphangiogenesis and increases lymphatic spread of tumors (Stacker et al., 2001). These bioactivities indicate that MEP and benzo(a)pyrene elevate the expression of genes whose products are important regulators of defense mechanisms, cell growth, and tumor progression during the multiple development stages of lung disease and cancer.
This study also demonstrates that gene alteration properties of MEP mimic the properties of benzo(a)pyrene, a constituent of MEP. Therefore the biological effects of MEP may be attributed, at least in part, to benzo(a)pyrene and related PAH present in MEP. Induction of IL-1, IL-6, FGF-9, and VEGF-D by MEP and benzo(a)pyrene was associated with increased peroxide formation and blocked by the antioxidant N-acetylcysteine in CL5 cells. These data and the CYP1A1 and CYP1B1 induction data together strongly suggest a possible sequent series of events leading to upregulation of proinflammatory cytokines and growth factors. The events involve (1) induction of metabolic enzymes by MEP and benzo(a)pyrene, which increases metabolic activation of protoxicants and formation of reactive oxygen species, and (2) the increased cellular oxidative stress in turn would activate, via signaling pathways, those transcription factors such as activator protein-1 and nuclear factor-kappa B, which could induce the expression of proinflammatory cytokines and growth factors. Additional studies are required to confirm this hypothesis.
MEP and benzo(a)pyrene decreased Rb mRNA in the human lung cells, unlike the increases observed with the other genes. The exact reasons for the decrease are still not clear. The tumor suppressor Rb plays a crucial role in cell cycle control in which phosphorylation of Rb protein is necessary for cell progression though G1 phase (Shackelford et al., 1999). The decrease of Rb expression possibly indicated that exposure to environmental chemicals might contribute to dysregulation of cell cycle controls by altering the expression of Rb and related cell cycle regulators. Benzo(a)pyrene elevated the level of IL-15 mRNA in CL5 cells. IL-15 plays unique roles in both innate and adaptive immune cell homeostasis (Lodolce et al., 2002). The significance of benzo(a)pyrene induction of IL-15 in lung epithelial cells remains to be further elucidated. Dissimilar to their counterparts of CL5 cells, the proinflammatory cytokines and growth factors of BEAS-2B cells were refractory to the stimulatory effects of MEP and benzo(a)pyrene. Concentration-response and time-course studies using additional cancer and noncancer cell lines such as normal human bronchial cells will be required to determine whether this dissimilarity was a reflection of differences in the induction kinetics in these two specific cell lines or an indication of selectivity of gene induction in cancer cells.
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent arylhydrocarbon receptor agonist, is associated with chronic obstructive pulmonary disease and increased risk to lung cancer in humans (Steenland et al., 1999). cDNA microarray analysis of human lung adenocarcinoma A549 cells revealed that 68 out of 2091 genes changed their expression levels at 24 h following treatment of the cells with 0.1, 1, and 10 nM TCDD (Martinez et al., 2002). With these gene changes there was induction of metabolic enzymes CYP1A1 and CYP1B1, cytokines IL-8 and leukemia inhibitory factor, and growth factors FGF-2 and VEGF. In parallel to TCDD, benzo(a)pyrene and MEP induced the same metabolic enzymes and the genes of the same families in human lung adenocarcinoma CL5 cells. These findings suggest the possibility that TCDD, benzo(a)pyrene, and MEP induced similar changes in gene categories of lung adenocarcinomal cell lines. Given that CYP1A1 induction is a hallmark indicating the biochemical effect of arylhydrocarbon receptor agonists, these present findings also suggest that it would be important to investigate the role of arylhydrocarbon receptors in the integrated pathways leading to formation and progression of lung adenocarcinoma.
Exposures to diesel exhaust and DEP have been associated with respiratory and allergic diseases such as asthma. DEP has been reported to promote release of specific cytokines, chemokines, and related mediators, which initiates a cascade resulting in airway inflammation (Pandya et al., 2002). Exposure of BEAS-2B cells to 40 to 300 μg/ml DEP for 24 or 48 h produced increases of IL-6 and chemokine IL-8 production (Steerenberg et al., 1998). Treatment of BEAS-2B cells with 5 and 25 μg/ml DEP for 24 h increased production of granulocyte macrophage-colony stimulating factor and regulated on activation, normal T cells expressed and secreted as well as IL-8 (Kawasaki et al., 2001). Similar to DEP, MEP induced IL-6 expression in CL5 cells. However, the results of cytokine array studies indicated that MEP had no marked effects on IL-8 or granulocyte macrophage-colony stimulating factor in CL5 cells (data not shown). Further studies will be needed to better define the effects of MEP on those cytokines, chemokines, and mediators inducible by DEP in order to properly assess the respiratory health risk of ME exposure, relative to that of DE exposure.
The present study showed that MEP and benzo(a)pyrene increased the releases of IL-6 and IL-11, but not of IL-1, by CL5 cells. IL-1 and its immature form, pro-IL-1, were found to remain mainly in the cytoplasm and carried out their activities intracellularly (Roux-Lombard, 1998). Accordingly, the present ELISA data also demonstrated that MEP and benzo(a)pyrene increased IL-1 production in CL5 cell lysate, but not in culture medium. FGF-9, which was originally called glial-activating factors, was discovered as a secreted factor from human glioma cell line. Expression of FGF-9 in COS cells demonstrated that it was glycosylated and efficiently secreted (Miyamoto et al., 1993). However, the effects of MEP and benzo(a)pyrene on FGF-9 protein expression of CL5 cells still need to be explored.
A major finding in the present study was the stimulation of cell growth in human lung fibroblast by conditioned medium from MEP- or benzo(a)pyrene-induced CL5 epithelial cells. An underlying basis for the stimulatory effect was the expression of receptors for the proinflammatory cytokine and growth factor induced by the environmental chemicals. The lung cell types which could express receptors for IL-1, IL-6, FGF-9, and VEGF-D included macrophage, mast cells, and endothelium, in addition to epithelium and fibroblast (Heinrich et al., 2003; Roux-Lombard, 1998). With such wide distribution of the receptors, it is not unreasonable that MEP and benzo(a)pyrene can stimulate epithelial cells interactions with a variety of cells, including fibroblast, macrophage, and other cell types, that play a role in maintaining the homeostasis of the microenvironment in the lung.
In the present studies, CL5 cells were treated with 100 μg/ml MEP extract or 10 μM benzo(a)pyrene in cell medium for 6 h. The following calculation was done to further assess the significance of the concentrations of the environmental chemicals used. The yield of MEP extract from MEP was 56% (g/g). Consequently, 100 μg/ml MEP extract was equivalent to 179 μg/ml (1.79 x 105 mg/m3 ) MEP in CL5 cell medium. A typical PM10 concentration in ME was 228 mg/ m3. Therefore the MEP concentration in cell medium would be at least 785 times higher than the concentration in ME. Benzo(a)pyrene concentration in MEP extract was 20.2 ng/mg (Table 3). Ten μM benzo(a)pyrene in CL5 cell medium was equivalent to 125 mg/ml MEP extract, which would be 1250-fold higher than the 100 μg/ml MEP extract used to treat CL5 cells. The results of these calculation analysis indicated that the MEP extract and benzo(a)pyrene concentrations for treatment of CL5 cells were not compatible with the environmental levels that humans may be exposed to. Extrapolation of these findings with the treated CL5 cells to adverse health effects associated with human exposure requires further experimental studies and physiological toxicokinetic modeling and considerations.
The present findings with CL5 cells have provided new mechanistic and predictive information regarding the gene regulation properties of MEP. This is supported by the findings of experimental animal study that inhalation exposure to ME under environmentally relevant conditions induced CYP1A1, FGF-9, and IL-1 genes expression in rat lung (Fig. 10). The induction by ME in rat lung also suggests that the MEP-mediated induction of metabolism, growth factor, and inflammatory cytokine genes in human lung adenocarcinoma CL5 cells is not just an artifact of a tumor cell line. The induction in CL5 cells indicates several possible consequences, such as that, upon exposure to MEP, the lung tumor cells might increase production of cytokines including the proangiogenic FGF-9, which would increase the invasiveness of the tumor cells. Induction of the same metabolic enzyme and cytokines in CL5 cells and rat lung by MEP and ME emphasizes that it may be necessary to study their differential toxicological effects on normal and tumor cells.
In summary, MEP and benzo(a)pyrene induce an array of altered gene expression including induction of genes involved in metabolic activation, inflammation, and angiogenesis in lung epithelial cells. The findings on induction of FGF-9, VEGF-D, IL-1, and IL-11 have further elucidated the roles of environment and gene interactions which may be important in the promotion of lung diseases including cancer.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
The authors thank the technical assistance of Ms. T.-C. Tien and Ms. S.-C. Chen. This work was supported by grants DOH91-0543–003B, NHRI92A1-NSCLC19-5, and NHRI93A1-NSCLC19-5 from the Department of Health, ROC. Conflict of interest: none declared.
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Division of Environmental Health and Occupational Medicine, National Health Research Institute, Kaohsiung, Taiwan, ROC
ABSTRACT
Motorcycle exhaust particulates (MEP) contain carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene. This study has determined the ability of MEP to alter the expression of select genes from drug metabolism, cytokine, oncogene, tumor suppressor, and estrogen signaling families of human lung adenocarcinoma CL5 cells. cDNA microarray analyses and confirmation studies were performed using CL5 cells treated with 100 μg/ml MEP extract for 6 h. The results showed that MEP increased the mRNA levels of metabolic enzymes CYP1A1 and CYP1B1, proinflammatory cytokines interleukin (IL)-1, IL-6, and IL-11, fibroblast growth factor (FGF)-6 and FGF-9, vascular endothelial growth factor (VEGF)-D, oncogene fra-1, and tumor suppressor p21. In contrast, MEP decreased tumor suppressor Rb mRNA in CL5 lung epithelial cells. Treatment with 10 μM benzo(a)pyrene for 6 h altered gene expression profiles, in a manner similar to those by MEP. Induction of IL-1, IL-6, IL-11, and FGF-9 mRNA by MEP and benzo(a)pyrene was concentration and time dependent. Cotreatment with 2 mM N-acetylcysteine blocked the MEP- and benzo(a)pyrene-mediated induction. Treatment with MEP or benzo(a)pyrene increased IL-6 and IL-11 releases to CL5 cell medium. Incubation of human lung fibroblast WI-38 with MEP- or benzo(a)pyrene-induced CL5 conditioned medium for 4 days stimulated cell growth of the fibroblasts. Inhalation exposure of rats to 1:10 diluted motorcycle exhaust 2 h daily for 4 weeks increased CYP1A1, FGF-9, and IL-1 mRNA in lung. This present study shows that MEP and benzo(a)pyrene can induce metabolic enzyme, inflammatory cytokine, and growth factor gene expression in CL5 cells and stimulate lung epithelium-fibroblast interaction.
Key Words: motorcycle exhaust particulate; benzo(a)pyrene; fibrobalst growth factor; interleukin; lung epithelial cell.
INTRODUCTION
The emissions of motorcycle exhaust (ME) are a major source of air pollution in areas where motorcycles are a popular means of transportation. The 2- and 4-stroke motorcycle engines have smaller capacity and poorer combustion efficiency than diesel and gasoline engines. ME also contains higher levels of carcinogen benzene than exhaust from gasoline and diesel engines (Jemma et al., 1995). The motorcycle commuters, therefore, are exposed to higher levels of benzene, as compared to car commuters (Chan et al., 1993). Carcinogenic polycyclic aromatic hydrocarbons (PAH) such as benzo(a)pyrene, benz(a)anthacene, and benzo (g,h,i)perylene have been detected in the organic solvent extracts of ME particulates (MEP) from 2-stroke engine (Ueng et al., 2000). The 2-stroke engine is distinctively different from other vehicle engines in that the former requires mixing motor oil with fuel prior to combustion. Studies of exhaust from this type of engine are of environmental and toxicological significance because, in addition to motorcycles, the 2-stroke engines are also widely used in a variety of applications including outboard boat motors, snowmobiles, lawn mowers, and trimmers.
ME and MEP have many toxicological properties. For example, ME inhalation exposure increased lipid peroxidation and decreased cytochrome P450 (CYP) 2B1 protein in rat lung (Ueng et al., 2004). Treatment of human lung cancer cells with organic extracts of MEP increased oxidative stress and DNA damage and decreased gap junctional intercellular communication (Kuo et al., 1998). Furthermore, MEP extract was found to enhance vasoconstriction in organ culture of rat aortas and induce inflammation and hyperresponsiveness in mouse airways (Lee et al., 2004; Tzeng et al., 2003).
Airway epithelium is a physical barrier to inhaled particulates and toxicants and a critical cell type in the pathogenesis of lung disease and cancer. The lung epithelial cells are responsive to the stimulatory effects of diesel exhaust particulates (DEP), which induce production of cytokines and mediators in human bronchial epithelial cells (Kawasaki et al., 2001; Steerenberg et al., 1998). Induction of these inflammatory mediators is believed to be one of the etiological factors for asthma and chronic obstructive pulmonary disease (Mills et al., 1999). Information regarding the effect of MEP on cytokine production by lung epithelium remains to be explored. Airway epithelium is a target site of cancers such as lung adenocarcinoma, which has a high female-to-male ratio and a large proportion of nonsmokers. Gene and environment interactions could possibly contribute to the high female susceptibility to lung adenocarcinoma. CYP enzymes are involved in activation of pulmonary PAH carcinogens and mammary carcinogen 17-estradiol. MEP induced CYP1A1 and CYP1B1 expression in CL5 female lung epithelial adenocarcinoma cell line (Wang et al., 2002). Oncogene, tumor suppressor, and estrogen signaling genes may play significant roles in female lung carcinogenesis. MEP interactions with these genes still need to be elucidated.
The major objective of our studies was to investigate the interaction of MEP with genes important in the development of lung disease and cancer in human lung epithelial cells. In this regard, CL5 cells were treated with MEP extracts, and cDNA microarrays analyses were conducted using arrays consisting of 255 genes selected from the metabolic enzyme, cytokine, oncogene, tumor suppressor, and estrogen signaling families. Gene alteration and bioactivity studies were extended to include the prototypic chemical carcinogen benzo(a)pyrene, for mechanistic and comparison purposes.
MATERIALS AND METHODS
Preparations of MEP extract and washed MEP.
Organic solvent extracts of MEP were prepared as described previously (Ueng et al., 2000). A 1992 Yamaha Cabin motorcycle with a 2-stroke 50-cc engine and a variable carburetor was used. An aerosol monitor model 8520 DUSKTRAK (TSI, Inc., Shoreview, MN) was used to determine the original concentrations of ME particles. The mean values of particles concentrations were: PM1, 118 mg/m3; PM2.5, 216 mg/m3; and PM10, 228 mg/m3. MEP were collected on 0.5-μm quartz filters. Soxhlet extraction was carried out in dark for 24 h using dichloromethane:hexane (1:1). The MEP extract was rotor-evaporated to dryness and stored in the dark at –20°C until analysis. The washed MEP were prepared essentially as described by Yin et al. (2004). MEP were suspended in acetone by sonication of the quartz filters. After solvent removal, MEP were washed consecutively using sterile phosphate-buffered solution, pH 7.4, dichloromethane, and acetone:methanol (1:1) mixture. The washed MEP were air dried, weighed, and stored in the dark at –20°C until analysis.
Gas chromatography/mass spectrometry (GC/MS) analysis of MEP extract.
The MEP extract was analyzed for PAH using a Hewlett-Packard 6890 gas chromatograph and a 5973 mass spectrometer equipped with a 7673 autosampler and an HP-5MS capillary column (60 m x 0.25 mm ID, 0.25 μm film thickness). The GC/MS operation conditions were as described previously (Ueng et al., 2000). Helium was used as the GC carrier gas. Following sample injection, the GC column was held at 80°C for 0.1 min, temperature programmed to 190°C at 10°C/min, 260°C at 3.5°C/min, and 300°C at 1.4°C/min. The MS was operated under 280°C detector temperature at 70 eV in the scan and selected ion models for qualitative and quantitative analyses, respectively. The GC column was calibrated with a standard U.S. Environmental Protection Agency 610 Polynuclear Aromatic Hydrocarbons Mixture consisting of 16 priority PAH pollutants (Supelco, Inc., Bellefonte, PA). Each PAH of MEP extract was quantified by means of an external calibration curve built from standard PAH solutions of 2, 5, 10, and 50 μg/ml. If the target PAH analyte exceeded the linear range of the calibration standards, the MEP extract sample was further diluted and reanalyzed.
Cells and treatments.
The human lung epithelium cell line CL5 was derived from a lung adenocarcinoma tumor specimen of a 40-year-old women patient at the Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. The cell line has been single-cell cloned and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.0 g/l sodium bicarbonate at 37°C in a humidified atmosphere of 5% CO2. Human bronchial epithelial BEAS-2B cells immortalized with SV40 (American Type Culture Collection, Manassas, VA) were gifts from Dr. Pinpin Lin, Institute of Toxicology, Chung Shan Medical University, Taichung, Taiwan. BEAS-2B cells were maintained in serum-free LHC-9 medium with glutamine (BioSource International Inc., Rockville, MD). WI-38 human normal lung fibroblast (American Type Culture Collection) was obtained from Food Industry Research and Development Institute, Hsinchu, Taiwan. WI-38 cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 1.5 g/l sodium bicarbonate.
Lung cells were used when the monolayer reached near confluence. The cell density was about 10 x 106 cells/dish in 10-cm culture dishes for treatment. MEP extract or test compound was dissolved in dimethyl sulfoxide (DMSO) and added to the medium so that DMSO concentration in the medium was less than 0.1%. Control cells were treated with 0.1% DMSO in medium. Cell viability was determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Carmichael et al., 1987). Peroxide formation was determined using the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) (Lebel et al., 1992).
cDNA probe preparation and cDNA microarray analysis.
Gene expression profile was analyzed using nonradioactive GEArray pathway-specific expression arrays (SuperArray Inc., Bethesda, MD). The five arrays used were the human drug metabolism and common cytokine gene arrays, consisting of 96 genes each, and the human cancer/oncogene, cancer/tumor suppressor, and estrogen signaling pathway arrays, each consisting of 23 genes. There were cDNA fragments of 255 individual genes on these nylon-membrane arrays, and their gene tables are available (www.superarray.com). Total RNA was isolated from CL5 cells as described previously (Wang et al., 2001) and converted to biotinylated cDNA probes by reverse transcription with a dNTP mix containing biotin-dUTP. Biotinylated cDNA probes were hybridized to gene-specific cDNA fragments spotted on the membranes following manufacture's protocol. The GEArray membrane was then blocked with GEAblocking solution and incubated with alkaline phosphatase conjugated streptavidin. The relative gene expression levels were detected by chemiluminescence signal using the alkaline phosphatase substrate, CDP-Star, and X-ray film. The relative abundance of a particular transcript was estimated by comparing its signal intensity to the signals derived from internal hybridization controls -actin and GAPDH. Image analysis and spot quantitation were carried out using GenePix 3.0 program (Axon Instruments, Union City, CA).
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Five μg total RNA were reverse transcribed using 1X RT, 2.2 mM MgCl2, 2.0 mM dNTP, 0.2 U/ml RNAsin, 0.5 mM random hexamer primers, and 0.3 U/μl MMLV reverse transcriptase in 25 μl reactions using a 2-step cycle: 70°C, 5 min and 37°C, 2 h. Reverse transcription reagents were purchased from Promega Corp., Madison, WI. The resulting cDNA was used in subsequent real-time RT-PCR reactions with fluorescence detection using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Reaction was carried out in microAmp 96-well reaction plates, SYBR Green PCR Master Mix 2X, DNA polymerase, dNTPs with dUTP, forward and reverse primers (0.15 μM each) (Invitrogen Corp., Carlsbad, CA), and 200 ng cDNA in a final volume of 25 μl. Amplification parameters were: denaturation at 94°C 10 min, followed by 45 cycles of 95°C, 15 s; 60°C, 60 s. All primers and probes were designed using PrimerExpress software (Table 1). Samples were analyzed in triplicate, and -actin was used as an endogenous control.
Quantitation of mRNA transcription was performed using a relative quantitation method with standard curves constructed from five log RNA concentrations of a specific gene or -actin and their respective CT values. The input amount of a specific gene was calculated from its standard curve and normalized to the input amount of -actin calculated from its standard curve. The relative difference between treatment and control groups was calculated from the ratio of the amount of specific gene to that of -actin in each group.
RT-PCR analysis.
Two μg total RNA were isolated from CL5, BEAS-2B cells, or rat lung. cDNA synthesis and PCR were conducted as described previously (Wang et al., 2001). PCR primers for CYPs, cytokines, and internal controls were synthesized (Gibco/BRL, Life Technologies, Inc., Gaithersburg, MD) according to the published sequences (Table 2). All reactions were conducted with -actin or cyclophilin primers as internal controls. PCR products were separated on 2% agarose gels and stained with ethidium bromide. Intensity of PCR product was quantitated using an IS-1000 Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA) and normalized against the intensity of internal control -actin or cyclophilin. Relative intensity of target gene PCR product from treated cell culture or rats was calculated by dividing its intensity by the corresponding intensity from control cells or rats.
Preparation of cell lysate.
CL5 cells were harvested by scraping and washed in a phosphate buffered saline solution. The following procedures were carried out at 4°C. Cell suspension was centrifuged at 1,500 rpm for 3 min. The cell pellet was washed and sonicated in 0.1 M potassium phosphate buffer, pH 7.4. Cell homogenate was centrifuged at 9,000 x g for 20 min, and the resulting supernatant, cell lysate, was stored at –80°C prior to analyses of cytokines concentrations.
Enzyme-linked immunosorbent assay (ELISA).
CL5 cells in maintenance medium were plated at 1 x 105 cells per well in 6-well tissue culture plates. After seeding overnight, maintenance medium was changed to experimental medium consisting of RPMI 1640 medium supplemented with 1% charcoal-treated fetal calf serum. After 12 h incubation, the medium was removed, and 1 ml fresh experimental medium containing 100 μg/ml MEP extract or 10 μM benzo(a)pyrene was added to each well. To control cultures, 0.1% DMSO in fresh medium was added. Twenty-four h after treatment, the cell-conditioned medium was collected by centrifugation and stored at –20°C until ELISA. IL-1, IL-6, and IL-11 levels of conditioned media were determined by ELISA using human Quantikine human kits (R&D Systems Inc., Minneapolis, MN). Concentrations of these cytokines in cell lysate were similarly determined.
Bioactivity assay.
CL5 cells in maintenance medium were plated in 6-cm tissue culture dishes at 5 x 105 cells per dish. After seeding overnight, maintenance medium was changed to experimental medium consisting of RPMI 1640 medium supplemented with 1% charcoal-treated fetal calf serum. After 12 h incubation, the medium was removed and 5 ml fresh experimental medium containing 100 μg/ml MEP extract or 10 μM benzo(a)pyrene was added to each dish. To control cultures, 0.1% DMSO in fresh medium was added. Twenty-four h after treatment, the CL5 cell-conditioned medium was collected by centrifugation and stored at –20°C until use. WI-38 cells in maintenance medium were seeded into 24-well culture plates at 1 x 104 cells per well. After seeding overnight, fibroblasts were serum-deprived for 16 h with serum-free MEM with supplements. Following serum-deprivation, the serum-free medium was replaced with experimental medium, prepared by 1:1 dilution of CL5 cell-conditioned medium with serum-free MEM with supplements. The experimental medium was replaced at 48 h after incubation. Cell growth of WI-38 fibroblast was determined at 96 h. Cells were fixed and stained with sulforhodamine B as described by Skehan et al. (1990). The bound dye was solubilized, and its absorbance was read at 490 nm using an ELISA reader.
Experimental animals and ME inhalation exposure.
Seven-week-old female Wistar rats were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. Before experiments began, the animals were allowed 1 week of acclimation at the animal quarter. In ME inhalation studies, the animals were exposed to 1:10 diluted ME using a head-nose-only inhalation chamber (Technical and Scientific Equipment GMBH, Bad Hamburg, Germany). The animals were exposed to ME from 9 to 10 A.M. and 4 to 5 P.M. daily, Monday through Friday, for 4 weeks. Control rats were exposed to clean air only. The animal maintenance and the inhalation chamber and exposure conditions were described previously (Ueng et al., 2004). The exposure chamber atmospheres were measured using an aerosol monitor model 8520 DUSKTRAK with a cutoff at 10 μm and a combustion analyzer model CA-6200 (TSI, Inc.). The control and ME exposure atmospheres components and their mean concentrations were: particles, 0.5 and 21.5 mg/m3; carbon monoxide, 0.2 and 5.8 ppm; carbon dioxide, 0 and 0.3%; nitric oxide, 0 and 4.5 ppm; nitric dioxide, 0 and 0 ppm; and oxygen, 20.5 and 20.3%, respectively. Animals were killed within 24 h after the last exposure. The Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine approved all animal care and experimental procedures.
Statistical analysis.
The statistical significance of difference between control and treatment groups was evaluated by the Student's t-test of paired data. A p-value <0.05 was considered statistically significant.
RESULTS
Qualitative GC/MS analysis of MEP extract identified the presence of 14 PAH in the chemical mixture (Table 3). Carcinogenic benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-c,d)pyrene, and benzo(g,h,i)perylene were identified in the MEP extract. The results of quantitative GC/MS analysis showed that benzo(a)pyrene was one of the more abundant carcinogenic PAH in MEP extract. The amounts of these carcinogenic PAH were smaller than those of noncarcinogenic PAH such as naphthalene and phenanthrene. For comparison purposes, benzo(a)pyrene was chosen as the component compound of MEP extract in the following studies.
A preliminary study was first conducted to determine the optimal conditions for MEP treatment. In this study, CL5 cells were treated with increasing concentrations of MEP extract for various time periods. RT-PCR analysis was performed to determine the effect of MEP on CYP1A1, the marker of exposure and effect. MTT assay was then carried out to determine the effect on cytotoxicity. It was found that treatment with 100 μg/ml MEP extract for 6 h produced a near maximal inductive effect on CYP1A1 and minimal cytotoxic effects (data not shown). These treatment conditions were used in the subsequent cDNA microarray studies.
In the drug metabolism array study, a visual comparison of the membranes of controls and MEP-treated CL5 cells demonstrated that MEP-treated cells showed increases of CYP1A1, CYP3A7, and UGT2B gene expressions (Fig. 1). Image analysis of transcript intensity indicated that MEP increased CYP1A1, CYP3A7, UGT2B, and UGT1A1 mRNA by 9-, 3-, 4-, and 2-fold, respectively (Table 4). In the cytokine array study, MEP elevated expression of fibroblast growth factor (FGF)-6, FGF-9, IL-1, and IL-22 genes (Fig. 2). Image analysis further showed that MEP increased FGF-6, FGF-9, and vascular endothelial growth factor (VEGF)-D mRNA by 4-, 2-, and 9-fold, respectively; IL-1 and IL-22 by 5- and 6-fold; and TNFSF10 by 5-fold. The results of oncogene, tumor suppressor, and estrogen signaling pathway arrays studies indicated that MEP extract produced 4-, 6-, and 4-fold increases of oncogenes fra-1, c-src, and SHC and resulted in 2-fold increases of tumor suppressor p21 and estrogen response gene COX7RP (Table 4). In contrast, MEP decreased tumor suppressors p53 and Rb expression by about 60%. The data from these five arrays studies collectively showed that MEP up-regulated the expression of 15 genes and down-regulated 2 genes. No remarkable alterations on the expressions of the other 238 genes were detected on the arrays.
To evaluate the validity of gene alterations identified in the arrays, confirmation studies were carried out using CL5 cells treated with 100 μg/ml MEP extract for 6 h. Additional cells were treated with 10 μM benzo(a)pyrene, a constituent of MEP, for 6 h. Under this treatment condition maximal effects on CYP1A1 mRNA and minimal effects on cytotoxicity could be induced (data not shown). Total RNA was prepared from the control and treated cells, and real-time RT-PCR analysis was performed. This study showed that MEP produced an 11-fold increase of CYP1A1; 3-, 5-, 6-, and 3-fold increases of IL-1, FGF-6, FGF-9, and VEGF-D; and 2-fold increases of fra-1 and p21 mRNA, respectively (Table 5). Furthermore, MEP resulted in a 47% decrease of Rb expression. These gene alterations were in agreement with those identified in the arrays. The real-time RT-PCR data indicated that MEP did not produce marked effects on CYP3A7, IL-22, TNFSF10, SHC, p53, or COX7RP mRNA, in variation from the array data. In these confirmation studies, the effects of MEP and benzo(a)pyrene on five related genes were determined. MEP induced the expression of CYP1B1, IL-6, and IL-11 by 4-, 2-, and 3-fold, without affecting IL-15 and VEGF-C. Treatment of CL5 cells with benzo(a)pyrene increased the expression of metabolic enzymes CYP1A1 and CYP1B1; proinflammatory cytokines IL-1, IL-6, IL-11, and IL-15; growth factors FGF-6, FGF-9, and VEGF-D; oncogene fra-1; and tumor suppressor p21. Benzo(a)pyrene decreased tumor suppressor Rb expression, without affecting CYP3A7, IL-22, TNFSF10, VEGF-C, SHC, or COX7RP (Table 5). The degrees of alterations of these 12 genes induced by benzo(a)pyrene were in general similar to those induced by MEP, except that the PAH produced a 2-fold increase of IL-15 mRNA level which was not altered by MEP.
Concentration-response and time-course studies were carried out to better characterize the inductive effects of MEP and benzo(a)pyrene on FGF-9, IL-1, IL-6, and IL-11 mRNA. In concentration-response studies, CL5 cells were treated with increasing concentrations of MEP extract or benzo(a)pyrene for 6 h. The results of RT-PCR analysis showed that 1, 10, 100, and 200 μg/ml MEP extract produced concentration-dependent increases of FGF-9 mRNA (Fig. 3, top). Near-maximal increases were observed at 100 and 200 μg/ml MEP. Treatment with 0.1, 1, 10, and 50 μM benzo(a)pyrene increased FGF-9 expression in a concentration-dependent manner, showing a maximal increase at 50 μM (Fig. 3, bottom). Similar to the induction kinetics observed with FGF-9, MEP and benzo(a)pyrene also produced concentration-dependent increases of IL-1, IL-6, and IL-11 mRNA with maximal increases at 100 or 200 μg/ml MEP and 50 μM benzo(a)pyrene, respectively (Table 6). In time-course studies, CL5 cells were treated with 100 μg/ml MEP extract or 10 μM benzo(a)pyrene for 3 to 48 h. The results of RT-PCR analysis showed that MEP produced time-dependent increases of FGF-9 mRNA at 3 and 6 h following treatment (Fig. 4, top). These increases declined time-dependently at 12, 24, and 48 h. In parallel to MEP, benzo(a)pyrene resulted in maximal increases of FGF-9 at 3 and 6 h with gradual decline at the subsequent time points (Fig. 4, bottom). Similar time courses of inductive effects were also observed with IL-1, IL-6, and IL-11 (Table 7). As with these inflammatory cytokines, IL-15 mRNA was likewise induced concentration- and time-dependently by benzo(a)pyrene (data not shown).
Because CL5 is a tumor-derived cell line, it was important to examine the effects of MEP and benzo(a)pyrene on gene expression using a noncancerous human lung epithelial cell line. In this study, human bronchial epithelial BEAS-2B cells were treated with MEP extract or benzo(a)pyrene for 6 h, total RNA was isolated, and RT-PCR analysis was conducted. It was noted that treatment with 1, 10, and 100 μg/kg MEP produced concentration-dependent increases of CYP1A1 and CYP1B1 mRNA (Fig. 5, left). It was also found that treatment with 0.1, 1, and 10 μM benzo(a)pyrene increased CYP1A1 and CYP1B1 concentration-dependently (Fig. 5, right). Thus the inductive effects of MEP and benzo(a)pyrene in BEAS-2B cells were quite similar to the respective effects found in CL-5 cells. However, MEP extract and benzo(a)pyrene had no marked effects on the proinflammatory cytokines IL-, IL-6, IL-11, and IL-15; growth factors FGF-6, FGF-9, and VEGF-D; oncogene fra-1; and tumor suppressors p21 and Rb mRNA levels in the BEAS-2B cells (data not shown).
Studies were performed to investigate the effects of MEP and benzo(a)pyrene on peroxide production and to explore its possible mechanistic role in cytokine induction of CL5 cells. In these studies, CL5 cells were treated with 2 mM N-acetylcysteine, 100 μg/ml MEP extract, and 10 μM benzo(a)pyrene, respectively or in combination, for 6 h, peroxide formation was determined by DCFH oxidation method (Lebel et al., 1992), and cytokine mRNA levels were analyzed by RT-PCR procedures. The antioxidant N-acetylcysteine resulted in a 52% decrease of peroxide production, and the MEP extract produced a 32% increase, as compared to the controls (Fig. 6). Peroxide formation in cells cotreated with MEP extract and N-acetylcysteine was similar to the formation observed in control cells. Benzo(a)pyrene increased peroxide production by 27%. Cotreatment with benzo(a)pyrene and the antioxidant reduced the benzo(a)pyrene-mediated increase of peroxides. The results of RT-PCR analysis showed that N-acetylcysteine had no marked effects on IL-1, IL-6, FGF-9, and VEGF-D mRNA of CL5 cells (Fig. 7). Treatment with MEP extract elevated mRNA levels of these cytokines as expected. Cotreatment with MEP and N-acetylcysteine reduced the MEP-elevated mRNA levels of IL-1, IL-6, FGF-9, and VEGF-D to their respective levels as in controls. Similarly, cotreatment with benzo(a)pyrene and the antioxidant reduced the benzo(a)pyrene-elevated cytokines mRNA levels. These results clearly indicated that increase of peroxide formation was involved in cytokine induction by MEP and benzo(a)pyrene.
To further investigate the induction properties, the abilities of MEP and benzo(a)pyrene in increasing protein productions of IL-1, IL-6, and IL-11 were examined. CL5 cells were treated with 100 μg/ml MEP extract or 10 μM benzo(a)pyrene for 24 h, and cytokine productions in culture medium and cell lysate were determined by ELISA. It was found that MEP produced no effect on IL-1 and 57% and 51% increases of IL-6 and IL-11 production, respectively, in culture medium (Table 8). The effects of benzo(a)pyrene on these cytokine productions were qualitatively similar to the effects of MEP. Production of IL-6 was higher than that of IL-1 and IL-11 in control and treated-cell culture media. In cell lysate, MEP resulted in a 28% increase, no effect, and a 41% increase of IL-1, IL-6, and IL-11 concentrations, respectively. Benzo(a)pyrene produced 32%, 99%, and 38% increases of the respective cytokine concentrations. IL-1 concentration in cell lysate was greater than the corresponding concentrations of IL-6 and IL-11. These ELISA data indicated a general pattern that MEP and benzo(a)pyrene induced intracellular concentrations of IL-1, IL-6, and IL-11 and increased releases of IL-6 and IL-11 to cell medium.
The subsequent bioactivity study was conducted to investigate the effects of increased cytokine and growth factor production on the cell growth of WI-38 human lung fibroblast. Conditioned medium was collected from CL5 cells treated with 100 μg/ml MEP or 10 μM benzo(a)pyrene for 24 h. Exposure of WI-38 cells to this MEP-induced conditioned medium for 4 days produced a 36% increase of cell growth of the fibroblasts (Fig. 8). Exposure to benzo(a)pyrene-induced conditioned medium resulted in a 51% increase of WI-38 cell growth. Treatment of WI-38 cells with 50 ng/ml recombinant human FGF-9, a positive control, for 4 days increased cell growth by 57%. These bioactivity data showed that MEP and benzo(a)pyrene enhanced the ability of CL5 epithelial cells to stimulate the growth of lung fibroblast.
The following studies were done to determine the abilities of washed MEP and benzo(e)pyrene, an isomer of benzo(a)pyrene, to induce gene expression in CL5 cells. CYP1A1, FGF-9, and IL-1 were selected to represent drug metabolism, growth factor, and inflammatory cytokine families, respectively. CL5 cells were treated with 100 μg/ml washed MEP or 10 μM benzo(e)pyrene for 6 h. Additional cells were treated with 100 μg/ml MEP or 10 μM benzo(a)pyrene for 6 h, for comparison purposes. Total RNA was isolated, and RT-PCR analysis was carried out. The results showed that washed MEP and benzo(e)pyrene had no marked effects on CYP1A1, FGF-9, or IL-1 mRNA levels, unlike the inductive effects of MEP and benzo(a)pyrene (Fig. 9). In concentration-response and time-course studies, treatment with 1, 10, and 100 μg/ml washed MEP or 0.1, 1, 10, and 50 μM benzo(e)pyrene for 6 h and treatment with 100 μg/ml washed MEP or 10 μM benzo(e)pyrene for 3, 6, and 12 h did not increase the metabolic enzyme and cytokines mRNA. These treatment conditions did not show cytotoxicity based on MTT assay (data not shown).
The above gene expression studies were conducted mostly using female lung adenocarcinoma CL5 cells treated with MEP extract in vitro; therefore it was of interest to investigate the ability of the environmental mixture ME to induce CYP1A1, FGF-9, and IL-1 mRNA in vivo. In this regard, female rats were exposed to 1:10 diluted ME by inhalation for 1 h each in the morning and afternoon daily, Monday through Friday, for 4 weeks, aiming to provide more environmentally realistic conditions. The ME inhalation exposure had no effects on body weight or lung, liver, and kidney relative tissue weights (Table 9). RT-PCR analysis of lung RNA showed that ME inhalation exposure produced 3-fold increases of CYP1A1 and FGF-9 and a 2-fold increase of IL-1 mRNA levels, respectively (Fig. 10). The results of this rat inhalation study and those of cell culture studies showed that the metabolism, growth factor, and proinflammatory cytokine genes expression in lung cells were upregulated by ME in vivo and MEP extract in vitro.
DISCUSSION
Findings from this present study show that MEP extract and benzo(a)pyrene can alter the expression of an array of genes belonging to the metabolic enzyme, proinflammtory cytokine, growth factor, oncogene, and tumor suppressor families in human lung epithelial cells. To the best of our knowledge, this report is the first to show environment and gene interactions of IL-1, IL-11, FGF-9, and VEGF-D. IL-1 is an intracellular messenger in epithelial and endothelial cells where the autocrine plays a regulatory role in cell differentiation. IL-11 is a member of IL-6-type cytokines, which are involved in acute-phase response to injury and activation of target genes associated with cell differentiation, proliferation, and survival (Heinrich et al., 2003). FGF-9 belongs to the FGF superfamily, which regulates cell differentiation, proliferation, and migration during embryonic development and controls tissue repair and response to injury in adult organism (Ornitz and Itoh, 2001). FGF-9 may also play an oncogenic role in human lung cancer (Matsumoto-Yoshitomi et al., 1997). The VEGF family is known to promote endothelial cell proliferation and vascular permeability. VEGF-D induces both tumor angiogenesis and lymphangiogenesis and increases lymphatic spread of tumors (Stacker et al., 2001). These bioactivities indicate that MEP and benzo(a)pyrene elevate the expression of genes whose products are important regulators of defense mechanisms, cell growth, and tumor progression during the multiple development stages of lung disease and cancer.
This study also demonstrates that gene alteration properties of MEP mimic the properties of benzo(a)pyrene, a constituent of MEP. Therefore the biological effects of MEP may be attributed, at least in part, to benzo(a)pyrene and related PAH present in MEP. Induction of IL-1, IL-6, FGF-9, and VEGF-D by MEP and benzo(a)pyrene was associated with increased peroxide formation and blocked by the antioxidant N-acetylcysteine in CL5 cells. These data and the CYP1A1 and CYP1B1 induction data together strongly suggest a possible sequent series of events leading to upregulation of proinflammatory cytokines and growth factors. The events involve (1) induction of metabolic enzymes by MEP and benzo(a)pyrene, which increases metabolic activation of protoxicants and formation of reactive oxygen species, and (2) the increased cellular oxidative stress in turn would activate, via signaling pathways, those transcription factors such as activator protein-1 and nuclear factor-kappa B, which could induce the expression of proinflammatory cytokines and growth factors. Additional studies are required to confirm this hypothesis.
MEP and benzo(a)pyrene decreased Rb mRNA in the human lung cells, unlike the increases observed with the other genes. The exact reasons for the decrease are still not clear. The tumor suppressor Rb plays a crucial role in cell cycle control in which phosphorylation of Rb protein is necessary for cell progression though G1 phase (Shackelford et al., 1999). The decrease of Rb expression possibly indicated that exposure to environmental chemicals might contribute to dysregulation of cell cycle controls by altering the expression of Rb and related cell cycle regulators. Benzo(a)pyrene elevated the level of IL-15 mRNA in CL5 cells. IL-15 plays unique roles in both innate and adaptive immune cell homeostasis (Lodolce et al., 2002). The significance of benzo(a)pyrene induction of IL-15 in lung epithelial cells remains to be further elucidated. Dissimilar to their counterparts of CL5 cells, the proinflammatory cytokines and growth factors of BEAS-2B cells were refractory to the stimulatory effects of MEP and benzo(a)pyrene. Concentration-response and time-course studies using additional cancer and noncancer cell lines such as normal human bronchial cells will be required to determine whether this dissimilarity was a reflection of differences in the induction kinetics in these two specific cell lines or an indication of selectivity of gene induction in cancer cells.
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent arylhydrocarbon receptor agonist, is associated with chronic obstructive pulmonary disease and increased risk to lung cancer in humans (Steenland et al., 1999). cDNA microarray analysis of human lung adenocarcinoma A549 cells revealed that 68 out of 2091 genes changed their expression levels at 24 h following treatment of the cells with 0.1, 1, and 10 nM TCDD (Martinez et al., 2002). With these gene changes there was induction of metabolic enzymes CYP1A1 and CYP1B1, cytokines IL-8 and leukemia inhibitory factor, and growth factors FGF-2 and VEGF. In parallel to TCDD, benzo(a)pyrene and MEP induced the same metabolic enzymes and the genes of the same families in human lung adenocarcinoma CL5 cells. These findings suggest the possibility that TCDD, benzo(a)pyrene, and MEP induced similar changes in gene categories of lung adenocarcinomal cell lines. Given that CYP1A1 induction is a hallmark indicating the biochemical effect of arylhydrocarbon receptor agonists, these present findings also suggest that it would be important to investigate the role of arylhydrocarbon receptors in the integrated pathways leading to formation and progression of lung adenocarcinoma.
Exposures to diesel exhaust and DEP have been associated with respiratory and allergic diseases such as asthma. DEP has been reported to promote release of specific cytokines, chemokines, and related mediators, which initiates a cascade resulting in airway inflammation (Pandya et al., 2002). Exposure of BEAS-2B cells to 40 to 300 μg/ml DEP for 24 or 48 h produced increases of IL-6 and chemokine IL-8 production (Steerenberg et al., 1998). Treatment of BEAS-2B cells with 5 and 25 μg/ml DEP for 24 h increased production of granulocyte macrophage-colony stimulating factor and regulated on activation, normal T cells expressed and secreted as well as IL-8 (Kawasaki et al., 2001). Similar to DEP, MEP induced IL-6 expression in CL5 cells. However, the results of cytokine array studies indicated that MEP had no marked effects on IL-8 or granulocyte macrophage-colony stimulating factor in CL5 cells (data not shown). Further studies will be needed to better define the effects of MEP on those cytokines, chemokines, and mediators inducible by DEP in order to properly assess the respiratory health risk of ME exposure, relative to that of DE exposure.
The present study showed that MEP and benzo(a)pyrene increased the releases of IL-6 and IL-11, but not of IL-1, by CL5 cells. IL-1 and its immature form, pro-IL-1, were found to remain mainly in the cytoplasm and carried out their activities intracellularly (Roux-Lombard, 1998). Accordingly, the present ELISA data also demonstrated that MEP and benzo(a)pyrene increased IL-1 production in CL5 cell lysate, but not in culture medium. FGF-9, which was originally called glial-activating factors, was discovered as a secreted factor from human glioma cell line. Expression of FGF-9 in COS cells demonstrated that it was glycosylated and efficiently secreted (Miyamoto et al., 1993). However, the effects of MEP and benzo(a)pyrene on FGF-9 protein expression of CL5 cells still need to be explored.
A major finding in the present study was the stimulation of cell growth in human lung fibroblast by conditioned medium from MEP- or benzo(a)pyrene-induced CL5 epithelial cells. An underlying basis for the stimulatory effect was the expression of receptors for the proinflammatory cytokine and growth factor induced by the environmental chemicals. The lung cell types which could express receptors for IL-1, IL-6, FGF-9, and VEGF-D included macrophage, mast cells, and endothelium, in addition to epithelium and fibroblast (Heinrich et al., 2003; Roux-Lombard, 1998). With such wide distribution of the receptors, it is not unreasonable that MEP and benzo(a)pyrene can stimulate epithelial cells interactions with a variety of cells, including fibroblast, macrophage, and other cell types, that play a role in maintaining the homeostasis of the microenvironment in the lung.
In the present studies, CL5 cells were treated with 100 μg/ml MEP extract or 10 μM benzo(a)pyrene in cell medium for 6 h. The following calculation was done to further assess the significance of the concentrations of the environmental chemicals used. The yield of MEP extract from MEP was 56% (g/g). Consequently, 100 μg/ml MEP extract was equivalent to 179 μg/ml (1.79 x 105 mg/m3 ) MEP in CL5 cell medium. A typical PM10 concentration in ME was 228 mg/ m3. Therefore the MEP concentration in cell medium would be at least 785 times higher than the concentration in ME. Benzo(a)pyrene concentration in MEP extract was 20.2 ng/mg (Table 3). Ten μM benzo(a)pyrene in CL5 cell medium was equivalent to 125 mg/ml MEP extract, which would be 1250-fold higher than the 100 μg/ml MEP extract used to treat CL5 cells. The results of these calculation analysis indicated that the MEP extract and benzo(a)pyrene concentrations for treatment of CL5 cells were not compatible with the environmental levels that humans may be exposed to. Extrapolation of these findings with the treated CL5 cells to adverse health effects associated with human exposure requires further experimental studies and physiological toxicokinetic modeling and considerations.
The present findings with CL5 cells have provided new mechanistic and predictive information regarding the gene regulation properties of MEP. This is supported by the findings of experimental animal study that inhalation exposure to ME under environmentally relevant conditions induced CYP1A1, FGF-9, and IL-1 genes expression in rat lung (Fig. 10). The induction by ME in rat lung also suggests that the MEP-mediated induction of metabolism, growth factor, and inflammatory cytokine genes in human lung adenocarcinoma CL5 cells is not just an artifact of a tumor cell line. The induction in CL5 cells indicates several possible consequences, such as that, upon exposure to MEP, the lung tumor cells might increase production of cytokines including the proangiogenic FGF-9, which would increase the invasiveness of the tumor cells. Induction of the same metabolic enzyme and cytokines in CL5 cells and rat lung by MEP and ME emphasizes that it may be necessary to study their differential toxicological effects on normal and tumor cells.
In summary, MEP and benzo(a)pyrene induce an array of altered gene expression including induction of genes involved in metabolic activation, inflammation, and angiogenesis in lung epithelial cells. The findings on induction of FGF-9, VEGF-D, IL-1, and IL-11 have further elucidated the roles of environment and gene interactions which may be important in the promotion of lung diseases including cancer.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
The authors thank the technical assistance of Ms. T.-C. Tien and Ms. S.-C. Chen. This work was supported by grants DOH91-0543–003B, NHRI92A1-NSCLC19-5, and NHRI93A1-NSCLC19-5 from the Department of Health, ROC. Conflict of interest: none declared.
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