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The Role of the Ah Receptor and p38 in Benzo[a]pyrene-7,8-dihydrodiol and Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced Apoptosis

2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês

10.1074/jbc.m300780200

1083-351X

Shujuan Chen, Nghia Nguyen, Kumiko Tamura, Michael Karin, Robert H. Tukey,

Toxic Organic Pollutants Impact

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(±) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(±) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death. Benzo[a]pyrene (B[a]P) 1The abbreviations used are: B[a]P, benzo[a]pyrene; PAH, polycyclic aromatic hydrocarbons; BPDE-2, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide; MAP, mitogen-activated protein; ERK, extracellular signal-related kinase; MEF, mouse embryo fibroblasts; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PARP-1, poly(ADP-ribose)polymerase 1; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. is a representative polycyclic aromatic hydrocarbon (PAH) that is generated as a result of combustion and is found in significant concentrations in tobacco smoke (1Pfeifer G.P. Denissenko M.F. Olivier M. Tretyakova N. Hecht S.S. Hainaut P. Oncogene. 2002; 21: 7435-7451Crossref PubMed Scopus (871) Google Scholar). As a ubiquitous environmental contaminant B[a]P is formed as a byproduct of industrialization with traces identified in air- borne particles (2Bostrom C.E. Gerde P. Hanberg A. Jernstrom B. Johansson C. Kyrklund T. Rannug A. Tornqvist M. Victorin K. Westerholm R. Environ. Health Perspect. 2002; 110: 451-488Crossref PubMed Google Scholar), water supplies, as well as food and dietary sources (3Phillips D.H. Mutat. Res. 1999; 443: 139-147Crossref PubMed Scopus (762) Google Scholar). Experiments in animals convincingly demonstrate that B[a]P exposure leads to the generation of tumors (4Kouri R.E. Wood A.W. Levin W. Rude T.H. Yagi H. Mah H.D. Jerina D.M. Conney A.H. J. Natl. Cancer Inst. 1980; 64: 617-623PubMed Google Scholar, 5Hecht S.S. Environ. Mol. Mutagen. 2002; 39: 119-126Crossref PubMed Scopus (176) Google Scholar, 6Rubin H. Carcinogenesis. 2001; 22: 1903-1930Crossref PubMed Scopus (243) Google Scholar) and has been implicated as a human carcinogen. Benzo[a]pyrene is a procarcinogen requiring metabolism and metabolic activation by cytochrome P450 (CYP)-dependent oxidations and epoxide hydrolysis to form the ultimate carcinogens (7Conney A.H. Cancer Res. 1982; 42: 4875-4917PubMed Google Scholar). Tumorigenicity studies have demonstrated that the product generally accepted as the ultimate carcinogen of B[a]P metabolism is the anti or trans isomer of B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE-2) (8Thakker D.R. Yagi H. Lu A. Levin W. Conney A.H. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3381-3385Crossref PubMed Scopus (273) Google Scholar, 9Kapitulnik J. Wislocki P.G. Levin W. Yagi H. Jerina D.M. Conney A.H. Cancer Res. 1978; 38: 354-358PubMed Google Scholar), which can bind to DNA (10Grover P.L. Sims P. Biochem. Pharmacol. 1973; 22: 661-666Crossref PubMed Scopus (53) Google Scholar, 11Sims P. Grover P.L. Swaisland A. Pal K. Hewer A. Nature. 1974; 252: 326-328Crossref PubMed Scopus (1135) Google Scholar, 12Conney A.H. Chang R.L. Jerina D.M. Wei S.J. Drug Metab. Rev. 1994; 26: 125-163Crossref PubMed Scopus (133) Google Scholar) and serve as an initiator of carcinogenesis (1Pfeifer G.P. Denissenko M.F. Olivier M. Tretyakova N. Hecht S.S. Hainaut P. Oncogene. 2002; 21: 7435-7451Crossref PubMed Scopus (871) Google Scholar, 13Levin W. Wood A.W. Chang R.L. Slaga T.J. Yagi H. Jerina D.M. Conney A.H. Cancer Res. 1977; 37: 2721-2725PubMed Google Scholar). The formation of BPDE-2 results from cytochrome P450-dependent oxidation of B[a]P to B[a]P-7,8 oxide, followed by hydration by epoxide hydrolase to B[a]P-7,8-dihydrodiol, which then serves as a substrate for a second CYP-dependent oxidation reaction generating BPDE-2. In tissue culture experiments, B[a]P-7,8-dihydrodiol and BPDE-2 have been shown to initiate programmed cell death or apoptosis (14Jyonouchi H. Sun S. Iijima K. Wang M. Hecht S.S. Carcinogenesis. 1999; 20: 139-145Crossref PubMed Scopus (39) Google Scholar). Thus, the onset of apoptosis by procarcinogens like B[a]P may be viewed in its simplest form to proceed in two independent steps. The first step requires cell-specific metabolism by oxidative and hydrolytic enzymes generating the ultimate carcinogen, and the second step requires the involvement of cellular events that define cell death. Whereas several P450s are capable of metabolizing B[a]P, the oxidation reaction that most efficiently generates B[a]P-dependent mutagenesis is catalyzed by CYP1A1 (15McManus M.E. Burgess W.M. Veronese M.E. Felton J.S. Knize M.G. Snyderwine E.G. Quattrochi L.C. Tukey R.H. Prog. Clin. Biol. Res. 1990; 340: 139-148Google Scholar, 16Shimada T. Gillam E.M.J. Sandhu P. Guo Z. Tukey R.H. Guengerich F.P. Carcinogenesis. 1994; 15: 2523-2530Crossref PubMed Scopus (73) Google Scholar, 17Shimada T. Martin M.V. Pruess-Schwartz D. Marnett L.J. Guengerich F.P. Cancer Res. 1989; 49: 6304-6312PubMed Google Scholar) and CYP1B1 (18Shimada T. Gillam E.M. Oda Y. Tsumura F. Sutter T.R. Guengerich F.P. Inoue K. Chem. Res. Toxicol. 1999; 12: 623-629Crossref PubMed Scopus (156) Google Scholar, 19Shimada T. Hayes C.L. Yamazaki H. Amin S. Hecht S.S. Guengerich F.P. Sutter T.R. Cancer Res. 1996; 56: 2979-2984PubMed Google Scholar). Expressed CYP1A1 and CYP1B1 display similar catalytic activities in converting B[a]P-7,8-dihydrodiol to mutagenic metabolites (20Shimada T. Oda Y. Gillam E.M. Guengerich F.P. Inoue K. Drug Metab. Dispos. 2001; 29: 1176-1182PubMed Google Scholar). Induction of Cyp1a1 and Cyp1b1 in wild-type and Ah receptor-deficient mice by polycyclic aromatic hydrocarbons and polychlorinated biphenyls confirmed that expression of Cyp1a1 and Cyp1b1 is dependent upon the Ah receptor (21Shimada T. Inoue K. Suzuki Y. Kawai T. Azuma E. Nakajima T. Shindo M. Kurose K. Sugie A. Yamagishi Y. Fujii-Kuriyama Y. Hashimoto M. Carcinogenesis. 2002; 23: 1199-1207Crossref PubMed Scopus (191) Google Scholar). Induced by PAHs following activation of the Ah receptor, CYP1 proteins catalyze the formation of B[a]P-oxide as well as the activation of B[a]P-7,8-dihydrodiol to the mutagenic BPDE-2 metabolite. Benzo[a]pyrene is an inducer of the CYP1A1 gene (22Postlind H. Vu T.P. Tukey R.H. Quattrochi L.C. Toxicol. Appl. Pharmacol. 1993; 118: 255-262Crossref PubMed Scopus (138) Google Scholar) and has been shown to stimulate apoptosis in mouse hepa1c1c7 hepatoma cells (23Lei W. Yu R. Mandlekar S. Kong A.N. Cancer Res. 1998; 58: 2102-2106PubMed Google Scholar) and Daudi human B cells (24Salas V.M. Burchiel S.W. Toxicol. Appl. Pharmacol. 1998; 151: 367-376Crossref PubMed Scopus (78) Google Scholar). Thus, since apoptosis by B[a]P is dependent upon metabolism, induction of CYP1 proteins is a requirement. Following induction of CYP1 genes and the oxidative metabolism of B[a]P, B[a]P-7,8-dihydrodiol serves as a substrate for further metabolism by CYP1 proteins to BPDE-2. Because B[a]P is a ligand for the Ah receptor and has been shown to induce CYP1A1 (22Postlind H. Vu T.P. Tukey R.H. Quattrochi L.C. Toxicol. Appl. Pharmacol. 1993; 118: 255-262Crossref PubMed Scopus (138) Google Scholar), it is generally thought that induction of CYP1 genes by B[a]P is the central cellular mechanism leading to the accumulation of CYP1 proteins. Interestingly, there is little information on the potential contribution of the Ah receptor and CYP1 induction in response to B[a]P-7,8-dihydrodiol. For example, in Daudi Human B cells, B[a]P-7,8-dihydrodiol is capable of initiating programmed cell death. Since the formation of B[a]P-7,8-dihydrodiol has limited direct mutagenic potential (25Wislocki P.G. Wood A.W. Chang R.L. Levin W. Yagi H. Hernandez O. Dansette P.M. Jerina D.M. Conney A.H. Cancer Res. 1976; 36: 3350-3357PubMed Google Scholar), the result observed in Daudi Human B cells may indicate that oxidative metabolism of B[a]P-7,8-dihydrodiol to BPDE-2 is required for apoptosis. If B[a]P-7,8-dihydrodiol is a suitable ligand for the Ah receptor, induction of CYP1 genes could underlie the onset of cell death. Apoptotic stimuli by DNA damaging agents leads to mitochondrial disruption and the release of death-promoting factors such as cytochrome c. Evidence suggests that apoptosis and mitochondrial damage is controlled in part by the Bcl-2 family of proteins, some of which inhibit (i.e. Bcl-2 and Bcl-xL) and some of which promote (i.e. Bax and Bak) cytochrome c release. Once released, cytochrome c initiates a self-amplifying cascade of proteolysis among cytosolic caspases, which terminates in cell death (26Roth W. Reed J.C. Nat. Med. 2002; 8: 216-218Crossref PubMed Scopus (64) Google Scholar). The early events involved in nuclear stress initiated by DNA damage leads to the promotion of DNA repair and the activation of poly(ADP-ribose) polymerase-1 (PARP-1) (27Soldani C. Scovassi A.I. Apoptosis. 2002; 7: 321-328Crossref PubMed Scopus (617) Google Scholar), which transfers ADP-ribose to other nuclear proteins involved in DNA repair and transcription (28D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). PARP-1 activation is also felt to be a mediator of cell death. While the actual mechanism of PARP-1 induced cell death is unknown, it has been speculated that consumption of nicotinamide adenine dinucleotide (NAD+) used in PARP-1-initiated ADP-ribosylation leads to depletion of NAD+ and the eventual disruption of mitochondrial function, an event that stimulates cytochrome c release and caspase activation (29Chiarugi A. Moskowitz M.A. Science. 2002; 297: 200-201Crossref PubMed Scopus (134) Google Scholar). Results have indicated that PARP-1, a substrate for caspases, is targeted for cleavage and inactivated during apoptosis, possibly disrupting poly(ADP-ribosyl)ation and eventually allowing for the promotion of nuclear disintegration by endonucleases. In Daudi human B cells, treatment with B[a]P and B[a]P-7,8-dihydrodiol resulted in DNA fragmentation and the cleavage of PARP-1 (24Salas V.M. Burchiel S.W. Toxicol. Appl. Pharmacol. 1998; 151: 367-376Crossref PubMed Scopus (78) Google Scholar), indicating that DNA damage may be the leading initiator of apoptosis. Central to the events leading to apoptosis are the role of cellular signaling pathways in controlling programmed cell death, in particular the phosphorylation cascades that regulate MAP kinases (30Cross T.G. Scheel-Toellner D. Henriquez N.V. Deacon E. Salmon M. Lord J.M. Exp. Cell Res. 2000; 256: 34-41Crossref PubMed Scopus (624) Google Scholar). The MAP kinases include extracellular signal-related kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 kinase. Carbon black particles containing B[a]P have been shown to stimulate the release of tumor necrosis factor α resulting in regulation of MAP kinase activity, a proposed mechanism to induce apoptosis in RAW 264.7 macrophage cells (31Chin B.Y. Choi M.E. Burdick M.D. Strieter R.M. Risby T.H. Choi A.M. Am. J. Physiol. 1998; 275: L942-L949Crossref PubMed Google Scholar). Exposure of 293T and HeLa cells with B[a]P was shown to up-regulate α-PAK-exchange factor in a manner concordant with activation of JNK1, a finding that was tied to B[a]P induction of caspase-mediated apoptosis (32Yoshii S. Tanaka M. Otsuki Y. Fujiyama T. Kataoka H. Arai H. Hanai H. Sugimura H. Mol. Cell. Biol. 2001; 21: 6796-6807Crossref PubMed Scopus (39) Google Scholar). It has also been concluded that oxidative stress induced cell death is regulated in part by p38 and caspase 8 activation, with singlet oxygen-activating p38 upstream of caspase 8-dependent cleavage of Bid (33Zhuang S. Demirs J.T. Kochevar I.E. J. Biol. Chem. 2000; 275: 25939-25948Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). A role for p38 activation in the apoptotic pathway has also been implicated with agents that lead to DNA damage (34Sanchez-Prieto R. Rojas J.M. Taya Y. Gutkind J.S. Cancer Res. 2000; 60: 2464-2472PubMed Google Scholar), a finding that may suggest an important role for p38 in B[a]P-induced cell death since BPDE-2 is genotoxic. In this study, we examined the relationship between the Ah receptor and signal transduction pathways in B[a]P-7,8-dihydrodiol- and BPDE-2-induced apoptosis. An approach is outlined that uses human HepG2 cells, mouse hepa1c1c7 and Arnt-defective BPRc1 cells (35Israel D.I. Whitlock Jr., P. J. Biol. Chem. 1984; 259: 5400-5402Abstract Full Text PDF PubMed Google Scholar) to examine the role of the Ah receptor in both MAP kinase activation and cell death initiated by B[a]P-7,8-dihydrodiol and BPDE-2. Apoptosis is shown to be linked to p38 activation using mouse embryo fibroblasts (MEF) obtained from p38-deficient mice, indicating that p38 plays an important role in BPDE-2 initiated cell death. Chemicals and Reagents—trans-7,8-dihydroxy-7,8-dihydrobenzo-[a]pyrene (B[a]P-7,8-dihydrodiol), Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±) (BPDE-2) were purchased from NCI, Chemical Carcinogens Repositories (National Institutes of Health, Bethesda, MD). All chemicals were dissolved in Me2SO or tetrahydrofuran. The final concentration of Me2SO or tetrahydrofuran in cell cultures was 0.1%. Mitogen-activated protein kinase inhibitors, PD 98059, SB 203580, and U0126 were purchased from Calbiochem (San Diego, CA) and dissolved in Me2SO to appropriate concentrations. All other chemicals were obtained through standard suppliers. The mono- or polyclonal primary antibodies, anti-human/mouse phosphorylated p38, phosphorylated ERK1/2, p38, ERK, and anti-human Bid were purchased from Cell Signaling (Beverly, MA). Anti-human Bcl-xL was from Signal Transduction (San Jose, CA), anti-human/mouse cytochrome c and PARP were from BD PharMingen (San Diego, CA), anti-human/mouse Bak was from Upstate (Waltham, MA). Rabbit anti-human CYP1A1 (36Soucek P. Martin M.V. Ueng Y. Guengerich F.P. Biochemistry. 1995; 34: 16013-16021Crossref PubMed Scopus (29) Google Scholar) was a generous gift from Dr. Fred Guengerich, Vanderbilt University. The horseradish peroxidase-conjugated secondary antibodies were from Sigma. Cell Culture—The human hepatoma cell line, HepG2, was obtained from the American Type Culture Collection. TV101L cells were developed in this laboratory from HepG2 cells and stably express a CYP1A1-luciferase reporter gene (22Postlind H. Vu T.P. Tukey R.H. Quattrochi L.C. Toxicol. Appl. Pharmacol. 1993; 118: 255-262Crossref PubMed Scopus (138) Google Scholar). TV101L cells were grown under the same condition as HepG2 cells but with the addition of 0.8 mg/ml G418. Wild-type mouse hepa1c1c7 and Arnt-defective BPRc1 cells (35Israel D.I. Whitlock Jr., P. J. Biol. Chem. 1984; 259: 5400-5402Abstract Full Text PDF PubMed Google Scholar) were a generous gift from Dr. James Whitlock, Stanford University. Wild-type MEF and those deficient in p38 have been described previously (37Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Transient transfection of BPRc1 cells with a full-length human Arnt cDNA (pArnt/CMV4) was conducted as previously described (38Yueh M.F. Huang Y.H. Chen S. Nguyen N. Tukey R.H. J. Biol. Chem. 2003; PubMed Google Scholar). All cell lines in this study were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and supplemented with penicillin/streptomycin (10,000 units/ml). Cells were incubated in a humidified incubator under 5% CO2 at 37 °C. All experiments included solvent-treated control cultures. Apoptosis Assay—Detection of apoptotic oligonucleosomal DNA fragmentation was performed as described (39Wani M.A. Zhu Q.Z. El Mahdy M. Wani A.A. Carcinogenesis. 1999; 20: 765-772Crossref PubMed Scopus (54) Google Scholar). Approximately 1 × 106 HepG2 cells were exposed to different concentrations of B[a]P-7,8-dihydrodiol for 24 h and the cells collected by trypsinization and pelleted at 1,000 × g for 5 min. Cell pellets were resuspended in 55 μl of lysis buffer (20 mm EDTA, 10 mm Tris-HCl, pH 8.0, 0.8% SDS) and treated with 20 μl RNase A (10 mg/ml) at 37 °C for 1 h, followed by the addition of 25 μl of proteinase K (20 mg/ml) and then incubated at 55 °C overnight. Lysates were extracted with an equal volume of phenol/chloroform/isoamyl alchohol (25:24:1) and DNA precipitated with 2 volumes of ice-cold absolute ethanol. The DNA was collected in a microcentrifuge and resuspended in 20 μl of 10 mm Tris-HCl, pH 8.0, 10 mm EDTA. DNA samples were subjected to electrophoresis in 1.5% agarose gels. Confirmation of apoptosis was quantified by measurement of externalized phosphatidylserine residues as detected using annexin V-FITC (BD PharMingen). After exposure to appropriate concentrations of chemicals, cells were collected and washed with ice-cold phosphate-buffered saline and then suspended in 500 μl of annexin V binding buffer. A 100-μl aliquot was taken, 5 μl of annexin V-FITC was added, and the mixture incubated for 15 min at room temperature in the dark. After the addition of 400 μl of binding buffer, the cells were acquired on a FACS Calibur flow cytometer and analyzed using CELLQuest software. The results are shown as a histogram with annexin V-positive cells calculated as apoptotic cells. Cell Viability Assay (MTT Assay)—Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay (40Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (47998) Google Scholar). After treatment with various concentrations of appropriate chemicals, the culture medium was replaced with serum-free medium containing 0.5 mg/ml MTT, and cultures were incubated for an additional 3 h. The blue MTT formazan was dissolved in 1 ml of isopropyl alcohol with 0.04% HCl, and the absorbance values were determined at a 570-nm test wavelength and a 630-nm reference wavelength using a DU 640B spectrophotometer (Beckman Coulter). The results are displayed as percent of viable cells compared with the vehicle control. Western Blot Analysis—All Western blots were performed using Nu-PAGE Bis-Tris gel electrophoresis as outlined by the supplier (Invitrogen). For total cellular protein, cells were lysed in buffer containing 25 mm Hepes, pH 7.5, 0.3 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton X-100, 20 mm β-glycerophosphate, 0.5 mm DTT, 1 mm sodium orthovanadate, 0.1 μm okadaic acid, and 1 mm phenylmethylsulfonyl fluoride. Protein concentrations of the cell lysates were determined by Bio-Rad analysis according to the manufacturer's instruction. A 30-μg aliquot was boiled for 5 min in loading buffer and resolved on a 10% Bis-Tris gel under denaturing conditions and the proteins transferred to polyvinylidene difluoride membrane using a semidry transfer system (Norvex). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline for1hat room temperature, followed by incubation with primary antibodies in Tris-buffered saline overnight at 4 °C. Membranes were washed and exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Each membrane was again washed, and the conjugated horseradish peroxidase was detected using the ECL Plus Western blotting detection system (Amersham Biosciences) and scanned with a Molecular Dynamics Storm 840 scanner. For Western blot analysis of microsomal CYP1A1, cells were scraped from the plates and suspended in 0.25 m sucrose (1:5, v/v). Cell suspensions were homogenized 20 times in a Kontes Potter-Elvehjem tissue grinder and the suspensions centrifuged at 5,000 × g in a Sorvall RT 6000B-refrigerated centrifuge. The supernatant was collected and centrifuged at 150,000 × g for 1 h in a Beckman TL100 tabletop ultracentrifuge. The microsomal pellet was suspended in 500 μl of 50 mm Tris-HCl, pH 7.4, 10 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride. A 10-μg aliquot was processed by Western blot analysis as outlined above. Detection of CYP1A1 was performed using a rabbit anti-human CYP1A1 antibody. Cytochrome c Release Analysis—The conditions used for cytochrome c release have been outlined (41Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4530) Google Scholar). Cells were collected and incubated in 1 ml of isotonic buffer containing 250 mm sucrose, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 0.1 mm phenylmethylsulfonyl fluoride with protease inhibitors. After 15 min on ice, the cells were homogenized and then centrifuged at 1,000 × g for 10 min at 4 °C in a microcentrifuge. The supernatant was centrifuged at 10,000 × g for 15 min in a microcentrifuge, followed by a final centrifugation at 100,000 × g for 1 h in a Beckman TL100 tabletop centrifuge. The resulting supernatant (S-100) was stored at -80 °C and used for Western blot analysis. RT-PCR for CYP1A1 Gene Transcripts—Total RNA was extracted from cells using acidic phenol/quanidinium isothiocyanate solution (TRIzol, Invitrogen). 3 μg of total RNA was denatured together with oligo(dT) primer at 70 °C for 10 min. The synthesis of cDNA has been outlined (42Strassburg C.P. Manns M.P. Tukey R.H. J. Biol. Chem. 1998; 273: 8719-8726Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). For amplification of CYP1A1, two primers were generated. The forward and reverse primers were obtained from DNA sequence (accession number AF253322) of the CYP1 locus (43Corchero J. Pimprale S. Kimura S. Gonzalez F.J. Pharmacogenetics. 2001; 11: 1-6Crossref PubMed Scopus (91) Google Scholar). The forward primer was 5′-GGTTGTGGTCTAGCGCCGG-3′ (bases 7661–7680) and the reverse primer was 5′-CCTCCCAGCGGGCAATGGTC-3′ (bases 5822–5842). In a reaction volume of 96 μl containing 3 mm MgCl2, 50 mm KCl, 20 mm Tris-HCl, pH 8.4, and 0.2 mm of each dNTP, 2 mm of each primer, and 5 units of VENT (exo-) DNA polymerase, cycling was carried out at 94 °C (60 s), 59 °C (60 s), and 72 °C (60 s) for 35 cycles. The protocol was preceded by an incubation of 5 min at 95 °C and followed by an extended elongation time of 7 min at 72 °C. Luciferase Activity Assay—Luciferase assays were carried out as previously described (44Chen Y.-H. Tukey R.H. J. Biol. Chem. 1996; 271: 26261-26266Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). TV101L cells were treated and lysed on plates in a buffer containing 1% Triton, 25 mm Tricine, pH 7.8, 15 mm MgSO4, 4 mm EDTA, and 1 mm DTT. Cell lysates were centrifuged at 14,000 × g in a microcentrifuge for 10 min at 4 °C, and supernatants were used for luciferase and protein assays. A cell extract aliquot of 10 μl was mixed with 300 μl of reaction mixture, which contained 15 mm potassium phosphate buffer, pH 7.8, 15 mm MgSO4, 2 mm ATP, 4 mm EDTA, 25 mm Tricine, and 1 mm DTT. Reactions were started by adding 100 μl of luciferin (0.3 mg/ml) and light output measured for 10 s at 24 °C using a Monolight 2001 luminometer (Analytical Luminescence Laboratory). The results were normalized by protein concentrations and expressed as fold induction of vehicle control. B[a]P-7,8-dihydrodiol and BPDE-2 Initiate Apoptosis in HepG2 Cells—DNA fragmentation and mitochondrial release of cytochrome c (41Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4530) Google Scholar) are well characterized biochemical markers of apoptosis. The treatment of HepG2 cells with B[a]P-7,8-dihydrodiol induced apoptosis in a dose-dependent fashion as demonstrated by DNA fragmentation (Fig. 1A), as well as cytochrome c release (Fig. 1B). Other key regulatory proteins involved in controlling cytochrome c release are the Bcl-2 family of proteins, some of which promote cell survival such as Bcl-2, or induce cell death, such as Bax and Bid. Early markers of apoptosis are characterized by the activation of caspase 8 by death receptors and the resulting cleavage and activation of the pro-apoptotic protein Bid (45Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3832) Google Scholar). B[a]P-7,8-dihydrodiol treatment leads to Bid cleavage as demonstrated in Fig. 1C. Concordant with Bid cleavage are increases in the pro-apoptotic Bak protein (Fig. 1C), which has been shown to accelerate cytochrome c release (45Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3832) Google Scholar). In addition, B[a]P-7,8-dihydrodiol stimulates a reduction in anti-apoptotic proteins such as Bcl-xL (Fig. 1C), which serves to resist mitochondrial release of cytochrome c. Thus, changes in the levels of expression of the Bcl-2 family of proteins by B[a]P-7,8-dihydrodiol is concordant with the observed accumulation of cytosolic cytochrome c. Apoptosis is executed through the activation of caspases by cytochrome c (46Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3106) Google Scholar). To examine if the increases in DNA fragmentation and mitochondrial cytochrome c release are related to cytochrome c-dependent caspase activation, we assessed the degradation of specific protein products known to be caspase targets. Poly(ADP-ribosyl)ation by PARP-1 is activated in response to DNA damage, inducing transcriptional activation of genes involved in targeted cell death (29Chiarugi A. Moskowitz M.A. Science. 2002; 297: 200-201Crossref PubMed Scopus (134) Google Scholar). PARP-1 activation also rapidly depletes NAD+ pools, which is felt to have a detrimental impact on metabolic pathways such as glycolysis and mitochondrial respiration resulting in mitochondrial disruption and cytochrome c release. Thus, PARP-1 degradation is the result of caspase activation and is a good marker for caspase-dependent apoptosis. The treatment of HepG2 cells with increasing concentrations of B[a]P-7,8-dihydrodiol leads to cleavage of PARP-1 (Fig. 1D), concordant in a dose-dependent fashion with changes observed in the Bcl-2 family of proteins and cytochrome c release. B[a]P-7,8-dihydrodiol is metabolized to the ultimate carcinogen, BPDE-2. Treatment of HepG2 cells with BPDE-2 at concentrations comparable to B[a]P-7,8-dihydrodiol initiated cell death as demonstrated with annexin V-