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Exposure to Asphalt Fumes Activates Activator Protein-1 through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Mouse Epidermal Cells

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m309023200

ISSN

1083-351X

Autores

Cuiling Ma, Jin Wang, Jia Luo,

Tópico(s)

Insect Resistance and Genetics

Resumo

Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3β. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis. Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3β. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis. Millions of tons of asphalt are produced every year and extensively used in the paving and roofing industries (1Institute Asphalt United States asphalt usage report. Asphalt Institute, College Park, MD1989Google Scholar). It has been estimated that ∼2 million workers are exposed to asphalt fumes (1Institute Asphalt United States asphalt usage report. Asphalt Institute, College Park, MD1989Google Scholar). It was reported that road-paving workers can be exposed to 0.1–2 mg/m3 of bitumen fumes, which can include 10–200 ng/m3 benzo(a)pyrene (2Burstyn I. Kromhout H. Kauppinen T. Heikkila P. Boffetta P. Ann. Occup. Hyg. 2000; 44: 43-56Crossref PubMed Scopus (132) Google Scholar). The worker exposure routes are mainly through inhalation and skin contamination. Prolonged, extensive exposure to asphalt fumes has been reported to be associated with several adverse health effects (3Lutes C.C. Thomas R.J. Burnette R. Evaluation of emission from paving asphalts. Final report to US EPA. Acurex Enviromental Corp., Research Triangle Park, NC1994Google Scholar). A major health concern from exposure to asphalt fumes is the potential exposure to carcinogens. Epidemiological studies indicate that there is an increased risk for lung, stomach, nonmelanoma skin cancer, and leukemia in the roofer population (4Brandt H. Lafontaine M. Kriech A.J. de Groot P. Bonnet P. Binet S. Wissel H. Morele Y. Nunge H. Castegnaro M. Ann. Occup. Hyg. 2000; 44: 31-41Crossref PubMed Scopus (35) Google Scholar). Experimental studies using animal and in vitro models demonstrate that laboratory-generated condensates from roofing asphalt fumes are genotoxic and produce skin tumors in mice (5Machado M.L. Beatty P.W. Fetzer J.C. Glickman A.H. McGinnis E.L. Fundam. Appl. Toxicol. 1993; 21: 492-499Crossref PubMed Scopus (3) Google Scholar, 6Sivak A. Niemeier R. Lynch D. Beltis K. Simon S. Salomon R. Latta R. Belinky B. Menzies K. Lunsford A. Cooper C. Ross A. Bruner R. Cancer Lett. 1997; 117: 113-123Crossref PubMed Scopus (69) Google Scholar). Exposure to several other asphalt-based paints causes the formation of DNA adducts in the skin and lung of mice as well as in human skin fibroblast (7Binet S. Pfohl-Leszkowicz A. Brandt H. Lafontaine M. Castegnaro M. Sci. Total Environ. 2002; 300: 37-49Crossref PubMed Scopus (78) Google Scholar). However, the information regarding the potential carcinogenic effect of asphalt fumes is very limited.Chemical carcinogenesis is a complex process that can be divided experimentally into three stages: initiation, promotion, and progression. Initiation is associated with irreversible, carcinogen-mediated DNA mutation. In contrast, promotion is a reversible process in which there are increases in the rate of cell replication and/or alterations in gene expression. Progression represents the final genetic changes associated with the conversion of benign tumors into fully malignant cells. JB6 P+ mouse epidermal cell line (Cl 41), originally derived from primary mouse epidermal cells, offers an excellent model to investigate the molecular events that are associated with tumor promotion. These cells undergo a response analogous to second stage tumor promotion in mouse skin when treated with various tumor promoters. For example, exposure of JB6 P+ cells to 12-O-tetradecanoylphorbol 13-acetate or epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; AP-1, activator protein 1; PI3K, phosphatidylinositol-3 kinase; MAPK, mitogen-activated protein kinase; GSK-3β, glycogen synthase kinase-3β; JNK, c-Jun NH2-terminal kinase; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; PAH, polycyclic aromatic hydrocarbon; PTEN, tensin homologue deleted on chromosome-10; PtdIns, phosphatidyl inositol; PtdInsP2, phosphatidylinositol biphosphate; PtdInsP3, phosphatidylinositol triphosphate; PH, Pleckstrin homology; d-JNKI, cell-permeable peptide inhibitor of JNK, dextrorotatory form.1The abbreviations used are: EGF, epidermal growth factor; AP-1, activator protein 1; PI3K, phosphatidylinositol-3 kinase; MAPK, mitogen-activated protein kinase; GSK-3β, glycogen synthase kinase-3β; JNK, c-Jun NH2-terminal kinase; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; PAH, polycyclic aromatic hydrocarbon; PTEN, tensin homologue deleted on chromosome-10; PtdIns, phosphatidyl inositol; PtdInsP2, phosphatidylinositol biphosphate; PtdInsP3, phosphatidylinositol triphosphate; PH, Pleckstrin homology; d-JNKI, cell-permeable peptide inhibitor of JNK, dextrorotatory form. induces phenotype of anchorage-independent growth and tumorigenicity in vivo (8Colburn N.H. 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Natl. Acad. Sci. U. S. A. 2001; 98: 7510-7515Crossref PubMed Scopus (97) Google Scholar).AP-1 is a basic leucine zipper transcription factor that is composed of homodimer or heterodimer proteins of the Jun, Fos, or ATF families (14Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2282) Google Scholar). AP-1 regulates the expression of a diverse array of genes, including those involved in cell growth, proliferation, and transformation (14Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2282) Google Scholar, 15Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3249) Google Scholar, 16Young M.R. Yang H.S. Colburn N.H. Trends Mol. Med. 2003; 9: 36-41Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It has been demonstrated that the activation of AP-1 is essential for the promotion of skin tumorigenesis (17Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar, 18Huang C. Ma W.Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (204) Google Scholar, 19Young M.R. Li J.J. Rincon M. Flavell R.A. Sathyanarayana B.K. Hunziker R. Colburn N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9827-9832Crossref PubMed Scopus (367) Google Scholar, 20Hsu T.C. Young M.R. Cmarik J. Colburn N.H. Free Radic. Biol. Med. 2000; 28: 1338-1348Crossref PubMed Scopus (249) Google Scholar, 21Dhar A. Young M.R. Colburn N.H. Mol. Cell Biochem. 2002; 234–235: 185-193Crossref PubMed Scopus (171) Google Scholar). The activation of AP-1 is regulated by multiple signaling pathways, including phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways. A recent study indicates that AP-1 activity is also regulated by glycogen synthase kinase-3β (GSK-3β) signaling (22Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1304) Google Scholar). These signal pathways have been shown to play a critical role in promotion of skin tumorigenesis (18Huang C. Ma W.Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (204) Google Scholar, 23Huang C. Ma W.Y. Dong Z. Mol. Cell. Biol. 1996; 16: 6427-6435Crossref PubMed Scopus (152) Google Scholar, 24Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (186) Google Scholar, 25Cohen C. Zavala-Pompa A. Sequeira J.H. Shoji M. Sexton D.G. Cotsonis G. Cerimele F. Govindarajan B. Macaron N. Arbiser J.L. Clin. Cancer Res. 2002; 8: 3728-3733PubMed Google Scholar, 26Leis H. Segrelles C. Ruiz S. Santos M. Paramio J.M. Mol. Carcinog. 2002; 35: 180-185Crossref PubMed Scopus (59) Google Scholar, 27Lin S. Li J.J. Fujii M. Hou D.X. Int. J. Oncol. 2003; 22: 829-833PubMed Google Scholar). The current study was designed to evaluate the effect of exposure to asphalt fumes on AP-1 activity and associated intracellular signaling. Asphalt fumes were generated from a dynamic asphalt-fume generation system that simulates road-paving conditions (28Wang J. Lewis D.M. Castranova V. Frazer D.G. Goldsmith T. Tomblyn S. Simpson J. Stone S. Afshari A. Siegel P.D. Anal. Chem. 2001; 73: 3691-3700Crossref PubMed Scopus (34) Google Scholar). We demonstrate here that exposure to asphalt fumes induces AP-1 transactivation in JB6 P+ cells as well as in skin and primary keratinocytes derived from transgenic mice expressing AP-1 reporter gene; it selectively activates PI3K/Akt and c-Jun NH2-terminal kinase (JNK) pathways. Furthermore, exposure to asphalt fumes promotes anchorage-independent growth in soft agar. These results provide an important insight into the potential role of exposure to asphalt fumes in tumor promotion.MATERIALS AND METHODSMaterials—Reagent grade dichloromethane (CH2Cl2, 99.9+%), hexane, and perdeuterated anthracene were purchased from Aldrich. The PAH mix reference material was purchased from Supelo (Bellefonte, PA). The test asphalt was the type used by the paving industry (Hot Performance Grade Asphalt PG 64-22). The glass-fiber filters (20.3 × 25.4 cm) were purchased from Gelman Sciences (Ann Arbor, Michigan). Solid phase extraction cartridges of EnvirElut PAH (500 ng/2.8 ml) were purchased from Varian (Harbor City, CA). Poly(tetrafluoroethylene tubes (30 ml) and glass tubes (10 ml) were purchased from Fisher Scientific. Target vials (1.5 mm with 200-μl inserts) were obtained from Alltech Associates, Inc. (Deerfield, IL). Extraction of asphalt fumes from collection media was performed by ultrasonic extraction (FS-220, Ultrasonicator 320W; Fisher Scientific). Extracts were reduced under a nitrogen stream using a TurboVap LV evaporator (Zymark). High purity helium was purchased from Butler Gas Products Co. (Mckees Rocks, PA), and used as gas chromatography/mass spectrometry (Hewlett Packard, Wilmington, DE) carrier gas. The GC column was HP-5 MS, 95% dimethylpolysiloxane, nonpolar, 30-m length, 0.53 mm inner diameter (Hewlett Packard). All antibodies except anti-actin and anti-p110 subunit of PI3K antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Anti-actin and anti-p110 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Preparation of Asphalt Fume Samples—Generation of asphalt fumes was conducted in the National Institute for Occupational Safety and Health inhalation facility. A dynamic asphalt fume generation system was employed to provide test asphalt fumes (28Wang J. Lewis D.M. Castranova V. Frazer D.G. Goldsmith T. Tomblyn S. Simpson J. Stone S. Afshari A. Siegel P.D. Anal. Chem. 2001; 73: 3691-3700Crossref PubMed Scopus (34) Google Scholar). A computer control system has been designed to improve the system's performance and to simplify its operation. The test asphalt is representative of the type used by the paving industry throughout the midwestern United States. The asphalt fumes were generated at 150 °C, and collected on glass-fiber filters. The fumes sample preparation involved desorption, filtration, and preconcentration. In the experiment, the asphalt fumes collected on filter were transferred to a poly(tetrafluoroethylene) tube, and dichloromethane/hexane (50/50) was added. Ultrasonic extraction was performed using an FS-220 Ultrasonicator (320W). After desorption of asphalt fumes from the collection medium, the extract was filtered. Preconcentration was performed under nitrogen using TurboVap LV Evaporator. Sample extracts were reconstituted with dichloromethane.Culture and Treatment of Mouse Epidermal Cell Lines—JB6 P+ mouse epidermal cell line (Cl 41) were grown in Eagle's minimal essential medium containing 10% fetal bovine serum (FBS), 2 mm l-glutamine, and 25 μg/ml gentamicin at 37 °C with 5% CO2. The stable transfectants of Cl 41 cells expressing AP-1-luciferase reporter (Cl 41 AP-1) and dominant-negative PI3K regulatory p85 subunit (JB6DNp85) have been described previously (24Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (186) Google Scholar). Cells were exposed to asphalt fumes (10–320 μg/ml) for specified periods. For blocking intracellular kinase activity, cells were treated with either PI3K inhibitor (10 μm LY294002 or 100 nm wortmannin; Sigma), GSK-3β inhibitor (10 μm 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione; Calbiochem), or JNK inhibitor (1 μm d-JNKI; Alexis Biochemicals, San Diego, CA) 30 min before exposure to asphalt fumes.AP-1-luciferase Reporter Transgenic Mice and Keratinocyte Culture—2X TRE-luciferase reporter transgenic mice were originally established by Rincon and Flavell (29Rincon M. Flavell R.A. EMBO J. 1994; 13: 4370-4381Crossref PubMed Scopus (246) Google Scholar). A C57BL/6 male mouse carrying the 2X TRE-luciferase transgene was crossed with DBA/2 female mice (SASCO, Omaha, NE) (18Huang C. Ma W.Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (204) Google Scholar). The F1 offspring were screened by testing both the basal and 12-O-tetradecanoylphorbol 13-acetate-induced levels of luciferase activity for the presence of the AP-1-luciferase reporter gene. Mice were housed under temperature-, humidity-, and light-controlled conditiona. Food and water were available ad libitum.Primary mouse epidermal keratinocytes were isolated from the epidermis of newborn AP-1 transgenic mice by a method described previously (30Hakkinen L. Koivisto L. Larjava H. Methods Cell Sci. 2001; 23: 189-196Crossref PubMed Scopus (58) Google Scholar, 31Hodivala-Dilke K. Methods Mol. Biol. 2002; 188: 139-144PubMed Google Scholar). Briefly, the trunk skin of newborn mice was floated dermis side down on 0.25% trypsin/EDTA in Dulbecco's phosphate-buffered saline solution overnight at 4 °C, and the epidermal layer was separated. The epidermis was minced in keratinocyte essential medium (Sigma), and then the cell suspension was filtered through a 70-μm cell strainer to remove the stratum corneum. Keratinocytes were collected by centrifugation (500 × g, 10 min) and seeded into fibronectin/collagen-coated dishes. Cells were maintained in a keratinocyte growth medium (Sigma) for 4 h with 5% CO2 at 37 °C. Unattached cells were removed by a gentle wash with warm keratinocyte growth medium. Adhesive keratinocytes were cultured in this medium at 37 °C in a humidified atmosphere of 5% CO2.Measurement of AP-1 Activity—AP-1 transactivation in JB6 P+ epidermal cells was determined by assaying the activity of the luciferase reporter (24Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (186) Google Scholar). Briefly, cells were cultured in 96-well plates and grown in a medium containing 10% FBS. The plates were incubated at 37 °C in a humidified atmosphere of 5% CO2. For assaying AP-1 activity, subconfluent cultures were maintained in a medium containing 0.1% FBS for 24 h and subsequently treated with asphalt fumes with or without protein kinase inhibitors for specified durations. After treatment, cellular protein was extracted with a 1× lysis buffer supplied in the luciferase assay kit (Promega), and luciferase activity was measured with a Monolight luminometer (Analytical Luminescence Laboratory, Sparks, MD). AP-1 activity (luciferase activity) was calculated and expressed relative to the untreated cultures. AP-1 activity was also measured in purified primary keratinocytes and the skin of transgenic mice expressing AP-1 luciferase reporter gene. Primary epidermal keratinocytes were exposed to asphalt fumes (10 μg) for 6 h, and AP-1 activity was determined. For assay of AP-1 activity in mouse skin, a small piece of tail skin (2 mm2) was removed either before or after asphalt painting, and cellular protein was extracted with a 2× lysis buffer, then luciferase activity was assayed as above.MTT Assay—The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was employed to determine the number of viable cells in culture (Roche Molecular Biochemicals) (32Sun Y. Lin H. Zhu Y. Ma C. Ye J. Luo J. J. Cell. Physiol. 2002; 192: 225-233Crossref PubMed Scopus (18) Google Scholar). The assay is based on the cleavage of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. Briefly, the cells were plated into 96-well microtiter plates and exposed to either asphalt fumes (10–320 μg/ml) or Me2SO (control) for 48 h. After exposure to asphalt fumes, 10 μl of MTT labeling reagent were added to each well, and the plates were incubated at 37 °C for 4 h. The cultures were then solubilized, and spectrophotometric absorbance of the samples was detected by a microtiter plate reader. The wavelength to measure absorbance of formazan product is 570 nm, with a reference wavelength of 750 nm.PI3K Activity—Cells were washed with ice-cold phosphate-buffered saline, scraped from the plates, and centrifuged at 4000 cpm for 5 min. The cell pellet was incubated for 30 min on ice in lysis buffer (150 mm NaCl, 100 mm Tris-HCl, pH 8.0, 1% Triton X-100, 5 mm EDTA, and 10 mm NaF) supplemented with 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium vanadate, 2 mm leupeptin, and 2 mm aprotinin, and centrifuged at 15,000 × g for 15 min. PI3K was immunoprecipitated using a method described previously (33Luo J. Miller M.W. J. Neurosci. 1999; 19: 10014-10025Crossref PubMed Google Scholar). Briefly, 400 μg of total protein was incubated with 20 μl of protein A/G plus agarose for 1 h at 4 °C, followed by spinning at 3000 cpm for 3 min. The supernatant was then incubated with 10 μl of antibody directed against p110 subunit of PI3K (Santa Cruz Biotechnology) overnight at 4 °C. Protein A/G-agarose beads (30 μl) were added for an additional 1 h. The beads were then pelleted and washed sequentially, five times, with 20 mm Tris, pH 7.5, 100 mm NaCl, and 1 mm EDTA) and once with 20 mm HEPES. PI3K activity assay was performed using phosphatidylinositol as substrate in a final volume of 50 μl containing 20 mm HEPES, pH 7.5, 10 mm MgCl, 2 μCi of [γ32P]ATP, 60 μm ATP, and 0.2 mg/ml sonicated phosphatidylinositol. Reaction was carried out for 15 min at room temperature and extracted by the addition of 80 μl of 1 m HCl and 160 μl of chloroform/methanol (1:1). After centrifugation at 3000 cpm for 2 min, the organic phase (bottom layer) was collected and dried in with a vacuum drier (SpeedVac, Thermo Savant, Holbrook, NY). The samples were dissolved in 10 μl of chloroform and separated on a thin layer chromatography plate. Incorporation of 32P into phosphorylated lipids was detected by autoradiography for 1–2 days.Immunoblotting—The immunoblotting procedure for detecting phosphorylation and expression of signal proteins was performed as described previously (33Luo J. Miller M.W. J. Neurosci. 1999; 19: 10014-10025Crossref PubMed Google Scholar). Briefly, cells were washed with phosphate-buffered saline and lysed with radioimmunoprecipitation assay buffer for 10 min, solubilized cells were centrifuged, the supernatant was collected, and the protein concentration was determined. Aliquots of the protein (40 μg) were loaded onto the lanes of an SDS 10.0% polyacrylamide gel. The proteins were separated by electrophoresis, and the separated proteins were transferred to nitrocellulose membranes. The membranes were blocked with either 5% nonfat dry milk or 5% bovine serum albumin (for detection of phosphorylation) in 0.010 m phosphate-buffered saline, pH 7.4, and 0.05% Tween 20 at room temperature for 1 h to block nonspecific immunoreactivity. Subsequently, the membranes were incubated with primary antibodies directed against signal proteins for 1.5 h at room temperature. After two quick washes in phosphate-buffered saline and 0.05% Tween 20, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) diluted at 1:2000 in phosphate-buffered saline and 0.05% Tween 20 for 1 h. The immune complexes were detected by the enhanced chemiluminescence method (Amersham Biosciences). In some cases, the blots were stripped and re-probed with an anti-actin antibody (Santa Cruz Biotechnology).Anchorage-independent Growth—The cell growth matrix consists of two layers of basal medium Eagle agar in 6-well culture trays. The base layer (2 ml) contained 10% FBS and 0.5% basal medium Eagle agar. The top layer (0.5 ml) contained 10% FBS, 0.33% basal medium Eagle agar, and JB6 P+ or JB6DNp85 cell suspension (0.5 × 104). EGF (30 ng/ml) and asphalt fumes (10 ng/ml) were applied in both the top and bottom layers. The cultures were maintained at 37 °C with an atmosphere of 5% CO2 for 14 days, and the number of induced cell colonies was counted under a microscope. Colonies, which contain 8 or more cells, were counted in four 0.5-cm2 areas randomly chosen with respect to distance from the center of the well, and the count was multiplied by the appropriate factor to give the colony number/well.Statistical Analysis—Differences among treatment groups were tested using an analysis of variance. Differences in which p was less than 0.05 were considered statistically significant. In cases where significant differences were detected, specific post hoc comparisons between treatment groups were examined with Student-Newman-Keuls tests.RESULTSDetermination of Asphalt Fume Concentration—The concentration of asphalt fumes was determined by gas chromatography/mass spectrometry with positive electron ionization. The instrument was calibrated with the use of a mixture of 16 reference PAHs, and perdeuterium anthracene was used as internal standard. The total ion chromatograms were acquired with a 3-min solvent delay. Separation was performed on a HP-5 MSD capillary column (30-m length, 0.53 mm inner diameter) with a temperature program from 50–310 °C at an increasing rate of 5 °C/min. Calibration curves were developed with five-point measurements. The recovery of the asphalt fumes were evaluated by adding stable isotope perdeuterated anthracene in the samples and determined by gas chromatography/mass spectrometry. The analytical results were recorded as profiles of total ion chromatogram over specific ranges of mass-to-charge ratios. A typical total ion chromatogram acquired is displayed in Fig. 1A. The major components were observed over a range of molecular size of m/z 51–365 (Fig. 1, B and C), and eluted from capillary column at retention times of 13–52 min. The relative recovery of the internal standard can account for losses of the analytes during sample preparation and detection. The total asphalt fume concentration was determined and diluted to 41.41 mg/ml with Me2SO for cell exposure. In some cases, asphalt fumes were dissolved in ethanol for comparison.Exposure to Asphalt Fumes Activates AP-1—MTT assay was performed to determine whether asphalt fumes, at a pharmacologically relevant concentration range, affect cell viability. As shown in Fig. 2, at the concentration less than 40 μg/ml, asphalt fumes did not significantly affect the viability of JB6 P+ cells; however, at high concentrations (>40 μg/ml), asphalt fumes caused cell death regardless of serum concentration in the culture medium. In all the subsequent studies, therefore, a non-toxic concentration of asphalt fumes (20 μg/ml) was used. Exposure to asphalt fumes significantly stimulated AP-1 activity in JB6 P+ cells, and maximal activation occurred at 12 to 24 h after treatment with asphalt fumes (Fig. 3A). Furthermore, exposure to asphalt fumes dramatically increased AP-1 activity in the skin and purified keratinocytes of transgenic mice expressing AP-1 reporter gene (Fig. 3B).Fig. 2Effect of asphalt fumes on the viability of JB6 P+ cells. Cells were grown in either serum-free medium or medium containing 10% serum and exposed to asphalt fumes (AF; 0–320 μg/ml) dissolved in Me2SO for 48 h. The controls were exposed to Me2SO. The viability of cells was determined by MTT assay as described under “Materials and Methods.” Each data point is the mean of five replicates ± S.E. (bars). *, p < 0.05, statistically significant difference between control and asphalt fume-treated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Effects of asphalt fumes on AP-1 activity. A, AP-1 activity in JB6 P+ cells. Cells were exposed to asphalt fumes (AF, 0 or 20 μg/ml) for 6–48 h. AP-1 activity was determined as described under “Materials and Methods.” Each data point is the mean of four replicates ± S.E. (bars). B, AP-1 activity in transgenic mice expressing AP-1 reporter gene. Skin of transgenic mice was painted with asphalt fumes for 24 h. Equal areas of skin (2 mm2) were removed before and after asphalt fume painting and assayed for AP-1 activity. Purified keratinocytes from transgenic mice were cultured as described under “Materials and Methods” and exposed to asphalt fumes (10 μg/ml, 6 h). AP-1 activity in keratinocytes was determined as described above. Each data point is the mean of four independent trials ± S.E. (bars). *, p < 0.05, statistically significant difference between control and asphalt fume-trea

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