help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0096
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
148/10/5112    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J. Y.
Right arrow Articles by Kim, J.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J. Y.
Right arrow Articles by Kim, J.-M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*BENZO(A)PYRENE
Endocrinology Vol. 148, No. 10 5112-5122
Copyright © 2007 by The Endocrine Society

Benzo[a]pyrene Induces Apoptosis in RL95-2 Human Endometrial Cancer Cells by Cytochrome P450 1A1 Activation

Ji Young Kim, Jin-Yong Chung, Ji-Eun Park, Seung Gee Lee, Yoon-Jae Kim, Moon-Seok Cha, Myung Seok Han, Hye-Jeong Lee, Young Hyun Yoo and Jong-Min Kim

Departments of Anatomy and Cell Biology (J.Y.K., J.-Y.C., J.-E.P., S.G.L., Y.-J.K., Y.H.Y., J.-M.K.), Obstetrics and Gynecology (M.-S.C., M.S.H.), and Pharmacology (H.-J.L.), College of Medicine, Dong-A University, Busan 602-714, Korea

Address all correspondence and requests for reprints to: Jong-Min Kim, Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Dongdaeshin-dong 3-1, Seo-gu, Busan 602-714, Korea. E-mail: jmkim7{at}dau.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Benzo[a]pyrene (B[a]P) has been shown to be an inducer of apoptosis in some cell types. To date, due to the lack of an appropriate model system, studies of the cellular and biochemical mechanism(s) by which B[a]P induces apoptosis have been focused on Hepa1c1c7 cells. Moreover, the precise relationship between the bioactivation of B[a]P by CYP1A1 or CYP1B1 and the occurrence of cytotoxicity-mediated apoptosis requires further elucidation. In the present study, we showed that B[a]P-induced apoptosis in RL95-2 cells is accompanied by the activation of caspases. In addition, the mitochondrial changes, including the decrease of mitochondrial potential and the release of mitochondrial cytochrome c and second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein binding protein with low PI (Smac/DIABLO) into the cytosol, support the suggestion that the mitochondrial pathway is robustly associated with B[a]P-evoked apoptosis. This study showed the involvement of the nuclear translocation of mitochondrial apoptosis-inducing factor in B[a]P-induced apoptosis of RL95-2 cells. Exposure to B[a]P up-regulates aryl hydrocarbon receptor, heat-shock protein 90, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and epoxide hydrolase significantly, which might be prerequisites for the conversion of B[a]P to B[a]P-7,8-dihydroxy-9,10-epoxide. Although both CYP1A1 and CYP1B1 proteins were up-regulated significantly by B[a]P, only CYP1A1 exhibited activity. Thus, CYP1A1 is believed to be a central oxidative enzyme that is ultimately required for formation of B[a]P-7,8-dihydroxy-9,10-epoxide from B[a]P in RL95-2 cells. Altogether, our data showed that RL95-2 cells are susceptible to apoptosis by exposure to B[a]P and that B[a]P-evoked apoptosis is mediated predominantly by the activation of CYP1A1. Here we suggest that RL95-2 cells are an excellent model for the investigation of xenobiotic mechanisms associated with CYP1A1 as well as CYP1B1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BENZO[a]PYRENE (B[a]P), a polyaromatic hydrocarbon compound, is a major toxicant in diesel exhaust, charcoal-broiled food, industrial waste byproducts, and cigarette smoke (1, 2). B[a]P is believed to act as a potent genotoxin and was among the first pure compounds recognized to exhibit carcinogenic activity in mice (3). To date, the mutagenic and carcinogenic properties of B[a]P in vitro have been demonstrated in many cell types (4, 5). B[a]P is also known to have a xenoestrogenic action (6, 7) and is currently considered to be an endocrine disruptor (8). In addition to these effects, B[a]P has been shown to induce apoptosis in Hepa1c1c7 hepatoma cells (9), Daudi human B cells (10), human ectocervical cells (11), A20.1 murine B cells (12), primary human macrophages (13), and ovarian follicle cells (14, 15). Among these, only Hepa1c1c7 cells have been used to study the mechanism of B[a]P-induced apoptosis (16, 17). Therefore, these findings need to be confirmed in other cell types and require supporting data, especially regarding the bioactivation of B[a]P by the enzymes associated with B[a]P metabolism.

B[a]P, in cells, binds to and activates a ligand-dependent transcription factor termed aryl-hydrocarbon receptor (AhR) (18). Ligand-bound AhR translocates to the nucleus and forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT). Ligand-activated AhR-ARNT complexes can interact with specific promoter elements (termed xenobiotic response elements), which induce the transcription of a group of genes involved in the metabolism of B[a]P, including cytochromes P450 CYP1A1, CYP1A2, and CYP1B1 (18, 19, 20, 21). Several P450 enzymes are associated with key steps in the oxidation of B[a]P, namely 7,8-epoxidation of B[a]P and 9,10-epoxidation of B[a]P-7,8-diol. CYP1A1 has been demonstrated to be the most active in these oxidations in mammals (22, 23, 24). CYP1A1 can activate B[a]P to B[a]P-7,8-oxide, which, through hydration by epoxide hydrolase, is metabolized to (+/–)-B[a]P-trans-7,8-dihydrodiol (B[a]P-7,8-DHD), which may then serve as a substrate for a second CYP-dependent oxidation reaction, generating the ultimate carcinogenic metabolite B[a]P-7,8-dihydroxy-9,10-epoxide (BPDE). In the nucleus, BPDE may bind covalently to DNA, which may result in misreplication, mutagenesis, and apoptosis (25).

The uterine endometrium has been identified as a potential target tissue of B[a]P action (26) as well as retaining the enzymes required for B[a]P bioactivation (27). Moreover, epidemiological investigation has shown that cigarette smoking reduces the relative risk of endometrial cancer, compared with nonsmokers (28). In this context, we chose the RL95-2 uterine endometrial cells as a model system for studying B[a]P-induced cytotoxicity and cell death and hypothesized that the exposure of B[a]P would cause apoptosis in this cell type via CYP1A1 or CYP1B1 activation. Here we report for the first time that the human endometrial RL95-2 cells are susceptible to apoptosis by exposure to B[a]P and that the B[a]P-induced apoptosis might be provoked ultimately by BPDE that is metabolized from B[a]P via CYP1A1 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
B[a]P (purity ~98%), {alpha}-naphthoflavone, 7,12-dimethylbenz[a]anthracene, dimethyl sulfoxide (DMSO), Hoechst 33258, glutaraldehyde, phenylmethyl-sulfonyl fluoride, paraformaldehyde, propidium iodide (PI), RNase A, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carboncyanine iodide (JC-1), 3–[4,5-dimethylthiazol-2-yl]2.5-diphenyltetrazolium bromide; thiazolyl blue (MTT), pyrene, 2,3',4,5'-tetramethoxystilbene (TMS), antiactin, CYP1A1, and CYP1B1 isozymes were purchased from Sigma (St. Louis, MO). BPDE was obtained from the Midwest Research Institute in the National Cancer Institute Repository (Kansas City, MO). Anti-AhR, apoptosis-inducing factor (AIF), cytochrome c, heat-shock protein (Hsp90), and CYP1A1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cleaved caspase-8, caspase-9, caspase-3, and caspase-7 came from Cell-Signaling Technology (Beverly, MA). Anti-poly(ADP-ribose) polymerase (PARP) and voltage-dependent anion channel (VDAC) were purchased from Calbiochem (San Diego, CA). Anti-X-linked inhibitor of apoptosis protein (XIAP) was from BD Pharmingen (San Diego, CA). Anti-second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein binding protein with low PI (Smac/DIABLO) was purchased from ProScience Inc. (Poway, CA). Anti-CYP1B1 was obtained from Gentest (San Jose, CA) and as a gift from Dr. Young-Jin Chun (Chung-Ang University, Seoul, Korea). Anti-epoxide hydrolase (EH) was purchased from Oxford Biochemicals Inc. (Oxford, MI). Anti-BPDE was obtained from Trevigen (Gaithersburg, MD). Antirabbit and -mouse Ig conjugated with horseradish peroxidase were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Cell culture
RL95-2 cells (human endometrial adenocarcinoma cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM nutrient mixture F-12 HAM (Sigma) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1.2 g/liter sodium bicarbonate supplemented with 10 µg/ml penicillin-streptomycin (Invitrogen). The cells were incubated in a humidified incubator at 37 C with 5% CO2 and were exposed to B[a]P when the confluency reached 20%.

MTT cell viability assay
Cells were seeded in 12-well plates at a density of 5 x 105 cells/well. After treatment at an appropriate time (24, 48, or 96 h), the culture medium was removed and replaced with a medium containing 0.5 mg MTT dissolved in PBS (pH 7.2). After 4 h, the formed crystals were dissolved with 200 µl DMSO. The intensity of the color in each well was measured at a wavelength of 490 nm using a microplate reader (BIOTEK EL-312e; Bio-Tek Instruments, Winsooki, VT).

Flow cytometric cell death assay
The cells were harvested, fixed with 95% ethanol for 24 h, incubated with 0.05 mg/ml PI and 1 µg/ml RNase A at 37 C for 30 min, and analyzed by flow cytometry, using an Epics XL and analysis software (EXPO32; Beckman Coulter, Fullerton, CA). The cells belonging to the sub-G1 population were considered to be apoptotic cells; the percentage of each phase of the cell cycle was determined.

Annexin V cell death assay
The cells were stained using the Annexin V-fluorescein isothiocyanate apoptosis detection kit (BD Biosciences, San Jose, CA) according to the manufacturer’s protocol. Stained cells were analyzed by flow cytometry.

Hoechst 33258 staining
The cells were stained in Hoechst 33258 (4 µg/ml) for 30 min at 37 C, fixed for 10 min in 4% paraformaldehyde, and then observed under an Axiophot microscope (Zeiss, Jena, Germany).

Transmission electron microscopy (TEM)
For TEM, cells were seeded in 10-cm Falcon dishes (1 x 107 cells/dish) for 24 h before adding 10 µM B[a]P for an additional 96 h. Total (floater + attached; ~5 x 107) cells were harvested and collected by centrifugation and washed with Na/K phosphate buffer [0.1 M (pH 7.3)]. Primary fixation was done with 1% glutaraldehyde in 0.15 M HEPES (pH 7.3) at 37 C for 5 min. After primary fixation the cells were washed repeatedly with phosphate buffer and subjected to secondary fixation with 1% OsO4 plus 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.3) for 2 h on ice, embedded in Epon 812, sectioned (60 nm) tangentially, and examined at 100 kV in an electron microscope (Hitachi H-600, Tokyo, Japan).

Western blot analysis
Whole-cell lysates were prepared by incubating cell pellets in lysis buffer [30 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, 25 mM NaF, 10 mM Na4P2O7] for 30 min on ice. After the insoluble fractions were removed by centrifugation at 14,000 rpm at 4 C for 30 min, the supernatants were collected and protein concentration was determined with a BCA protein assay kit (Pierce Biotechnology, Woburn, MA). The same amounts of proteins (~30 µg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were incubated for 1 h at room temperature (RT) with a primary antibody in Tris-buffered saline containing 0.05% Tween 20 (pH 7.4) in the presence of 5% nonfat dry milk. After the membranes were washed in Tris-buffered saline containing 0.05% Tween 20, secondary antibody reactions were performed with an appropriate source of antibody conjugated with horseradish peroxidase. The signals were detected with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) in the LAS-3000 detector (Fujifilm, Tokyo, Japan). Immunoblotting for ß-actin was performed in every experiment as an internal control.

Immunocytochemistry
Harvested cells were attached on the slide glass by cytospin centrifugation. The cells were fixed with 4% paraformaldehyde, washed with PBS, and incubated with 0.2% Triton X-100. Then the cells were incubated with the appropriate primary antibody in 1% BSA at RT. For secondary antibody reaction, the cells were incubated with an appropriate fluorescence-conjugated secondary antibody at RT. For counterstaining of the nucleus, if required, cells were incubated with PI (50 µg/ml) at RT. Finally, cells were mounted and observed under a confocal microscope (LSM510; Carl Zeiss).

Measurement of mitochondrial membrane potential (MMP)
The cells (5 x 105) were incubated with 1 µM JC-1 dye at 37 C for 15 min, washed and resuspended with PBS, and then the fluorescence [red (585/590 nm); green (510/527 nm)] measured by flow cytometer.

Preparation of mitochondrial fractions
The cells (5 x 107) were washed in Tris-based, Mg2+/Ca2+-deficient (TD) buffer [135 mM NaCl, 5 mM KCl, 25 mM Tris-Cl (pH 7.6)] and allowed to swell for 15 min in ice-cold hypotonic Ca2+ reticulocyte standard (CaRSB) buffer [10 mM NaCl, 1.5 mM CaCl2, 10 mM Tris-Cl (pH 7.5), protease inhibitors]. Cells were Dounce homogenized with 30 strokes, and mitochondria stabilization buffer [210 mM mannitol, 70 mM sucrose, 5 mM EDTA, 5 mM Tris (pH 7.6)] was added. After removing nuclear contaminants (690 x g for 15 min), the supernatant was centrifuged at 20,800 x g for 15 min. Finally, the pellet (mitochondria) was directly diluted with lysis buffer [5 M NaCl, 1 M Tris-Cl (pH 7.6), 5% Triton X-100, protease inhibitors], and the mitochondria (pellet) and supernatant (cytosol) were applied for protein analysis.

Caspase-3, -8, and -9 activity assays
A fluorometric assay kit (CLONTECH, Palo Alto, CA), which contains fluorogenic substrates specific for different caspases (3, 8, 9) immobilized in the wells, was used to evaluate enzyme activity. Ten micrograms of the extracted proteins in homogenization buffer (50 mM Tris HCl, 150 mM NaCl, 10% glycerin, and 1% Triton X-100) were added to the wells. The plate was incubated in the fluorescence plate reader at 37 C for 3 h, and fluorescence was read every 10 min. The activity was determined by fluorometric detection (excitation, 380 nm; emission, 460 nm) and the negative control (blank, without sample) was subtracted from all the samples. Results at 2 h were selected, as the manufacturer suggested. Baseline values of negative controls and samples with specific inhibitors did not increase during the 2-h interval.

Isolation of microsomes
The cells (6 x 106) were resuspended in isotonic homogenization buffer (10 mM HEPES, 1 mM EDTA, 2 mM dithiothreitol), spun down at 17,900 x g for 5 min, and swelled in isotonic homogenization buffer for 5 min at 4 C. After 30 strokes in a Dounce homogenizer, the lysates were centrifuged for 30 min at 7,800 x g, and the supernatants were again centrifuged for 60 min at 17,900 x g. The microsomal fraction was obtained from the pellet.

Ethoxyresorufin O-deethylase (EROD) assay
The enzymatic activity of CYP1 was measured by the EROD assay. Isolated microsomal proteins (15 µg) were mixed with the assay buffer [100 mM HEPES, 5 mM MgCl2, 5 µM ethoxyresorufin substrate (pH 7.8)], and the reaction was initiated by the addition of 0.25 mM of nicotinamide adenine dinucleotide phosphate (NADPH). Resorufin formation was calculated by comparing the fluorescence with that of a resorufin standard dilution series. The conversion of ethoxyresorufin to resorufin was measured with a FluoroCount (Packard Bioscience, Waltham, MA) at 530 nm excitation and 585 nm emission.

CYP1A1 and CYP1B1 enzyme activity assays
Cellular proteins were isolated with lysis buffer consisting of 30 mM NaCl, 50 mM Tris-HCl (pH 7.6), 5% Triton X-100, and 100 mM phenylmethyl-sulfonyl fluoride. The enzymatic activities of CYP1A1 and CYP1B1 were measured by P450-Glo assay kits (Promega, Madison, WI) as per the manufacturer’s instruction manual. Briefly, isolated proteins (30 µg) were mixed with the 4 x cytochrome P450/KPO4/substrate reaction mixture (CYP1A1: 0.5 pM CYP1A1 isozyme, 400 mM KPO4,120 µM luciferin-chloroethyl ether; CYP1B1: 1 pM CYP1B1 isozyme, 400 mM KPO4, 80 µM luciferin-conjugated equine estrogen), and 2x NADPH regeneration mixture (2.6 mM NADP+, 6.6 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, 6.6 mM MgCl2). The sample and the 4x cytochrome P450/KPO4/substrate reaction mixture were added to a 96-well plate. After preincubating the plate at 37 C for 10 min, the 2x NADPH regeneration mixture was added to each reaction. The plate was incubated at 37 C for 30 min, and the reconstituted luciferin detection reagent was added. Again the plate at RT was incubated for 20 min and the luminescence recorded using a luminometer (type 392; Amersham Bioscience, Piscataway, NJ).

Statistics
Data were expressed as the mean ± SD of three or four separate experiments. Where appropriate, data were subjected ANOVA followed by Duncan’s post hoc test. Means were considered significantly different at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Benzo[a]pyrene can induce cell death in endometrial carcinoma RL95-2 cells
To investigate whether B[a]P does have a cytotoxic effect on endometrial carcinoma RL95-2 cells, the cells were exposed to various concentrations of B[a]P (0.001–100 µM) for up to 96 h. The dose-response experiments showed that 10 and 100 µM B[a]P evoked a significant level of cell death (>40%) 96 h after treatment (Fig. 1Go, A and B-a) and that 10 µM B[a]P is considered to be optimal for the time-course experiments in this study (Fig. 1BGo-b).


Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 1. Effects of B[a]P on cell viability and apoptosis in RL95-2 endometrial cancer cells. A, Cell viability shown in cells treated with various concentrations of B[a]P (0.001–100 µM) for 24, 48, and 96 h and determined by MTT assay. B, Cell death after treatment with increasing concentrations (0.001–100 µM) of B[a]P for 96 h (a) or at different time points with 10 µM B[a]P treatment (b). Cell death was assessed by flow cytometry. The percentage of cells with a sub-G1 DNA content was taken as a measure of cell death. At least three independent experiments were performed, and data shown are the mean ± SD. *, P < 0.001, compared with 0 µM or 0 h control; #, P < 0.05, compared with 0 h control. C, Identification of apoptosis in RL95-2 endometrial cancer cells induced by exposure to B[a]P. Cells were treated with 10 µM B[a]P for 96 h. Con, Control. a and b, Cell death quantified by sub-G1 analysis of the cell cycle; c and d, cell death identified by the annexin V cell death assay. i, viable (live); ii, necrotic; iii, early apoptotic; iv, and late apoptotic regions; e and f, cellular morphology observed with a phase-contrast microscope; arrows denote membrane blebbing; original magnification, x800; g and h, nuclear morphology stained by Hoechst 33258; arrows point to fragmented nuclei; original magnification, x800; i and j, ultrastructural nuclear morphology observed with a TEM; arrows indicate condensed nuclear chromatin; original magnification, x8000. FI, Fluorescent intensity.

 
B[a]P-induced cell death exhibits apoptotic characteristics
B[a]P-induced RL95-2 cell death was characterized by flow cytometry, annexin V staining, phase-contrast microscopy, Hoechst 33258 staining, and electron microscopy (Fig. 1CGo). The flow cytograms showed that B[a]P-treated cells exhibit a much greater sub-G1 population (Fig. 1CGo-b; 47%) than that of the control (Fig. 1CGo-a; ~2%). Annexin V staining verified that B[a]P-treated cells die by apoptosis (Fig. 1CGo-d). Cellular membrane blebbing and nuclear fragmentation, typical morphological characteristics of apoptosis, were evidently seen in B[a]P-treated cells by phase-contrast microscopy and Hoechst 33258 staining, respectively (Fig. 1Go, C-f and C-h), which were rare in the control (Fig. 1Go, C-e and C-g). Finally, the ultrastructural morphology showed chromatin crescents as well as disruptions of cellular organelles in B[a]P-treated cells (Fig. 1CGo-j), but the intact nuclear and cytoplasmic integrity was seen in control cells (Fig. 1CGo-i).

Mitochondrial alteration is associated with B[a]P-induced apoptosis
Apoptosis is often accelerated by a decrease in MMP (29) and the releases of mitochondrial proteins such as cytochrome c (30), Smac/DIABLO (31, 32), and AIF (33) into the cytosol. In the present study, the exposure of B[a]P resulted in the depolarization of MMP (Fig. 2AGo), the disruption of mitochondrial morphology (Fig. 2BGo), and the release of mitochondrial cytochrome c and Smac/DIABLO proteins into the cytosol (Fig. 2Go, C and D; see also supplemental Fig. S1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Furthermore, the release and/or translocation of mitochondrial AIF into the cytosol and/or nucleus after exposure to B[a]P was seen immunocytochemically (Fig. 2DGo-f; see also supplemental Fig. S1A-f). This indicates that B[a]P-evoked apoptosis might involve a caspase-independent pathway.


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 2. Mitochondrial alterations and caspase-dependent apoptosis in RL95-2 endometrial cancer cells after exposure to B[a]P. A, Change of MMP ({Delta}{psi}m). The RL95-2 cells were treated with B[a]P (10 µM) for up to 96 h, stained with JC-1, and the MMP was analyzed by flow cytometry. #, P < 0.01, compared with 0 h control; *, P < 0.001, compared with 0 h control. B, Representative electron micrographs of RL95-2 cells. The cells were treated with B[a]P (10 µM) for 96 h, fixed, and TEM images of the selected fields for mitochondria were acquired. Arrows denote mitochondria. a, Control (Con); b, B[a]P treated. Original magnification, x20,000. C, Western blot analysis of mitochondrial cytochrome c and Smac/DIABLO release into the cytosol after exposure to B[a]P. The RL95-2 cells were exposed to B[a]P (10 µM) for the indicated lengths of time up to 96 h. Equal amounts of protein isolated from the mitochondria (10 µg) were separated by SDS-PAGE and immunoblotted using a cytochrome c or Smac/DIABLO antibody. Voltage-dependent anion channel (VDAC) is indicated as an internal loading control for mitochondrial proteins. D, Immunocytochemical localizations of cytochrome c, Smac/DIABLO, and AIF. The RL95-2 cells were treated with B[a]P (10 µM) for 96 h. The cells were then cytospun, fixed, and immunostained with a cytochrome c, Smac/DIABLO, or AIF antibody. Original magnification, x800. E, Caspase-dependent apoptosis after exposure to B[a]P (10 µM), as shown by Western blots of caspase-8, -9, -3, -7, and PARP cleavages. The RL95-2 cells were exposed to B[a]P (10 µM) for the indicated periods of time up to 96 h. Equal amounts of protein (30 µg) were separated by SDS-PAGE and immunoblotted using a cytochrome c or Smac/DIABLO antibody. Actin was used as an internal (loading) control. F, Localizations of active caspase-8, -9, and -3 in cells after exposure to B[a]P for 96 h. Control, Con. Microphotographs were taken using confocal microscopy. Original magnification, x800.

 
B[a]P-induced apoptosis is accompanied by caspase activations
To clarify whether B[a]P-induced RL95-2 cell apoptosis occurs in a caspase-dependent manner, the activation of initiator (-8 and -9) and effector (-3 and -7) caspases was analyzed by Western blotting and immunocytochemistry using active form-specific antibodies. As shown in Fig. 2EGo, the exposure of B[a]P provoked the activation of caspase-8, -9, -3, and -7 as well as the cleavage of PARP. In addition, immunocytochemical localizations of these proteins confirmed that activated caspase-9, -8, -3, and -7 are markedly present in B[a]P-treated cells (Fig. 2FGo; see also supplemental Fig. S1B). Caspase-8 and caspase-9 activity assays further showed that both caspases are activated by exposure to B[a]P (Fig. 3AGo). Next, we verified the role of caspase activation in B[a]P-induced apoptosis using a pancaspase inhibitor (zVAD-fmk) or caspase-3 inhibitor [N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aldehyde (DEVD-CHO)]. The pretreatment of zVAD-fmk or DEVD-CHO significantly suppressed B[a]P-induced apoptosis (Fig. 3BGo), caspase-3 activity (Fig. 3CGo), and cleavages of caspase-3 and PARP (Fig. 3DGo). Although both inhibitors abolished the B[a]P-provoked caspase-3 acitivity (Fig. 3CGo), they did not completely suppress the B[a]P-induced apoptosis (Fig. 3BGo), suggesting that the caspase-independent pathway might be also operating in the cells.


Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 3. Suppression of B[a]P-induced apoptosis in RL95-2 endometrial cancer cells by caspase inhibitors. The RL95-2 cells were exposed to B[a]P (10 µM) for 48 or 96 h in the absence or presence of caspase inhibitors (10 µM each zVAD-fmk or 10 µM DEVD-CHO). A, Caspase-8 and -9 activities measured spectrofluorometrically using a caspase-8 or caspase-9-specific substrate (z-IETD-AFC or Ac-LEHD-AFC). In each reaction, caspase-8 or caspase-9 inhibitor (Ac-IETD-CHO or Ac-LEHD-CMK) was added to confirm the specific activity. B, Cell death assessed by flow cytometry. The percentage of cells with a sub-G1 DNA content was taken as a measure of cell death. At least three independent experiments were performed and data shown are the mean ± SD. *, P < 0.001, compared with the 96-h B[a]P-treated group. C, Caspase-3 activity measured spectrofluorometrically using a caspase-3-specific substrate (N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide), as described in Materials and Methods. At least three independent experiments were performed and data shown are the mean ± SD. *, P < 0.001, compared with the 96-h B[a]P-treated group. D, Western blot analyses for caspase-3 and PARP cleavages. Equal amounts of protein (30 µg) were separated by SDS-PAGE and immunoblotted using a cleaved form-specific antibody for caspase-3 or a PARP antibody. Actin expression was examined as a loading control.

 
Up-regulation of AhR and CYP1A1 activation is a prerequisite for B[a]P-induced apoptosis
Ligation of AhR by B[a]P or other polyaromatic hydrocarbons and subsequent formation of the complex with Hsp90 and ARNT are known to promote the expression of CYP1A1 or CYP1B1 (23, 24, 25, 26). To explore the mechanism(s) by which B[a]P induces apoptosis, the changes of AhR, Hsp90, CYP1A1, and CYP1B1 proteins in total cell lysates after exposure to B[a]P were investigated. In this study, we found that RL95-2 cells do not retain sufficient endogenous levels of AhR, Hsp90, CYP1A1, and CYP1B1 proteins (Figs. 4Go and 5Go). However, the exposure to B[a]P caused prompt and significant increases in AhR, Hsp90 (Fig. 4Go, A and B; see also supplemental Fig. S2), CYP1A1, and CYP1B1 (Fig. 5Go, A and B; see also supplemental Fig. S3). In addition, we showed that the expression of EH increases after exposure to B[a]P (Fig. 5Go, A and B).


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 4. Changes in AhR and Hsp90 protein expression in RL95-2 endometrial cancer cells after exposure to B[a]P. A, Western blot analyses of AhR and Hsp90 proteins after treatment with B[a]P (10 µM) for the indicated lengths of time up to 96 h. Equal amounts of protein (30 µg) were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Actin expression was examined as a loading control. B, Immunolocalization of AhR and Hsp90. Cells were treated for 48 h with 10 µM B[a]P, cytospun, fixed, and immunostained with antibodies as indicated. The images were taken by a confocal microscope. a, Control: AhR; b, B[a]P treated: AhR; c, control: Hsp90; d, B[a]P treated: Hsp90. Original magnification, x800.

 

Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 5. Relationship between the expression and activity of B[a]P-metabolizing enzymes and cell death in RL95-2 endometrial cancer cells after exposure to B[a]P. A, Changes in CYP1A1, CYP1B1, and EH protein expression detected by Western blotting. The RL95-2 cells were exposed to B[a]P (10 µM) for the indicated lengths of time up to 96 h. Equal amounts (30 µg) of proteins from the total cell lysates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Actin is indicated as a loading control for the total cell lysates. B, Immunolocalization of CYP1A1, CYP1B1, and EH. Cells were treated for 48 h with 10 µM B[a]P, cytospun, fixed, and immunostained with antibodies as indicated. The images were taken by a confocal microscope. a, Control (Con): CYP1A1; b, B[a]P treated: CYP1A1; c, control: CYP1B1; d, B[a]P treated, CYP1B1; e, Control: EH; f, B[a]P treated: EH. Original magnification, x800. C, Alteration of CYP1A1 and CYP1B1 proteins and activities in isolated microsomes. The RL95-2 cells were exposed to B[a]P (10 µM) for the indicated periods of time up to 96 h. a, Western blot analyses of microsomal CYP1A1 and CYP1B1. Equal amounts (15 µg) of proteins from the microsomal lysates were separated by SDS-PAGE and immunoblotted using the CYP1A1 or CYP1B1 antibodies. b, Change of CYP1 activity monitored by EROD assay, as described in Materials and Methods. At least three independent experiments were performed and data shown are the mean ± SD. In D and E, cells were treated with either B[a]P (10 µM) or vehicle (0.1% DMSO) for 48 h in the absence or presence of {alpha}-NF (25 µM), TMS (1 µM), and pyrene (25 µM). 7,12-Dimethylbenz[a]anthracene (DMBA; 1 µM) was treated as a positive induction control for CYP1B1 acitivity. D, Changes in CYP1A1 and CYP1B1 activity, measured by fluorogenic-specific enzyme activity assays, as described in Materials and Methods. The data are expressed as mean ± SD of three independent experiments performed in duplicate. *, Significantly different from B[a]P treated alone (P < 0.001). E, Cell death determined by sub-G1 analysis of flow cytometry (A). The data are expressed as mean ± SD of three independent experiments. *, Significantly different from B[a]P treated alone (P < 0.001).

 
We isolated the microsomes from cells to examine the activities of the enzymes CYP1A1 and CYP1B1. Western blot analysis showed that the isolated microsomes contain abundant amounts of proteins CYP1A1 and CYP1B1, but CYP1A1 appears to be expressed more promptly as well as abundantly after exposure to B[a]P (Fig. 5CGo-a). The EROD assay is the standard measurement of polyaromatic hydrocarbon-activating capacity, to which all 3 CYP1 isozymes (CYP1A1, CYP1A2, and CYP1B1) contribute. The EROD assay initially showed that exposure to B[a]P increases CYP1 activity significantly and that the activity was abolished by the presence of {alpha}-naphthoflavone ({alpha}-NF) (Fig. 5CGo-b). However, the CYP1A1- or CYP1B1-specific enzyme activity assay revealed that, despite up-regulation of the CYP1B1 proteins, CYP1B1 activity was not induced by exposure to B[a]P and that CYP1A1 activity was suppressed only by {alpha}-NF and not by TMS or pyrene, a CYP1B1-specific inhibitor (Fig. 5DGo). Finally, we found that TMS or pyrene is incapable of suppressing B[a]P-induced apoptosis (Fig. 5EGo), suggesting that B[a]P could be converted to the cytotoxic metabolites (i.e. BPDE) in RL95-2 cells mainly by activation of CYP1A1.

B[a]P-induced apoptosis in RL95-2 cells is evoked by BPDE molecules
To confirm whether B[a]P is ultimately converted to BPDE at the cellular level, we demonstrated that BPDE molecules are visualized by an immunocytochemical method using a BPDE-specific monoclonal antibody. As shown in Fig. 6AGo, B[a]P-treated cells exhibited the same intense immunoreactivity for BPDE (Fig. 6AGo-b) as seen in BPDE-treated cells (Fig. 6AGo-c), whereas control cells did not show the immunoreactivity (Fig. 6AGo-a). Importantly, however, BPDE immunoreactivity was seen faintly in B[a]P-treated cells in the presence of {alpha}-NF (Fig. 6AGo-d) but was seen clearly in TMS-primed or pyrene-primed cells (Fig. 6Go, A-e and 6A-f). This indicates that B[a]P is metabolized to BPDE by the activation of CYP1A1. In addition, BPDE-induced apoptosis occurred significantly in a shorter period time (at 12 or 24 h) than that induced by B[a]P (Fig. 6BGo), which again confirms that B[a]P-induced apoptosis in RL95-2 cells is evoked by BPDE.


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 6. Cellular detection of BPDE molecules in RL95-2 cells after exposure to B[a]P and induction of apoptosis by BPDE. A, The RL95-2 cells were treated with B[a]P (10 µM) for 96 h in the absence or presence of {alpha}-NF (25 µM), TMS (1 µM), and pyrene (25 µM). BPDE (1 µM) was treated for 96 h as a positive control. For immunocytochemical detection of BPDE molecules, harvested cells were cytospun, fixed, and immunostained with a BPDE-specific monoclonal antibody. The images were taken by a confocal microscope. a, Control (Con); b, B[a]P treated; c, BPDE treated; d, {alpha}-NF plus B[a]P treated; e, TMS plus B[a]P treated; f, pyrene plus B[a]P treated: EH. Original magnification, x1600. B, The RL95-2 cells were treated with BPDE (1 µM) for 12 and 24 h. Cell death was assessed by flow cytometry. The percentage of cells with a sub-G1 DNA content was taken as a measure of cell death. Three independent experiments were performed and data shown are the mean ± SD. *, P < 0.001, compared with 0 h control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
B[a]P is primarily considered to be involved in mutagenesis and carcinogenesis (7, 8). However, it has been demonstrated that B[a]P can induce apoptosis in Hepa1c1c7 cells (9) and Daudi human B cells (10). To date, the studies on cellular and biochemical mechanism(s) by which B[a]P induces apoptosis have been focused on mouse hepatoma Hepa1c1c7 cells (16, 17). The precise relationship between the bioactivation of B[a]P by CYP1A1 or CYP1B1 and the occurrence of cytotoxicity-mediated apoptosis is in need of further elucidation. Moreover, the exploration of another cellular model for studying B[a]P-induced apoptosis is required for the determining the apoptogenic function or role of B[a]P. In this study, we demonstrated that RL95-2 endometrial adenocarcinoma cells are susceptible to apoptosis induced by exposure to B[a]P and that B[a]P-evoked apoptosis in RL95-2 cells is predominantly mediated by the activation of CYP1A1, not CYP1B1.

Cytotoxic chemicals often induce apoptotic cell death (34). Two apoptotic pathways have been proposed. One is involved with cell death receptors (TNF-R or Fas) in cellular membrane, in which binding of ligand to the receptor activates initiator caspase-8 and, in turn, activates downstream effector caspases (caspase-3 and caspase-7) (35). The other is associated with mitochondrial alterations such as a decrease in mitochondrial membrane potential and release of cytochrome c from the mitochondrial membrane, followed by activation of effector caspases via activation of caspase-9 (36). Our data showed that B[a]P-induced apoptosis in RL95-2 cells is accompanied by the activation of caspase-8 as well as caspase-9, followed by activation of caspase-3 and caspase-7. In addition, mitochondrial changes, including the decrease of membrane potential, the release of cytochrome c and Smac/DIABLO into the cytosol, and the disruption of ultrastructural morphology support the suggestion that the mitochondrial (intrinsic) pathway is robustly involved with B[a]P-induced apoptosis. However, the suppression of caspase-3 activity by caspase inhibitors was not associated with the total repression of cell death induced by B[a]P, indicating that B[a]P-induced apoptosis is not entirely caspase dependent. The AIF has been suggested to engage in caspase-independent apoptosis (37). This study also showed the possibility of the nuclear translocation of mitochondrial AIF after exposure to B[a]P, which suggests that AIF might be associated with apoptosis that was not suppressed by caspase inhibitors.

The most important objective for studying B[a]P-induced apoptosis in the RL95-2 cellular system is to discover the possible B[a]P signaling and action mechanisms that are involved in provoking the cytotoxicity that causes apoptosis. In general, B[a]P is thought to bind AhR (21). AhR is a ligand-activated transcription factor that controls the expression of a group of genes whose functions are linked to the metabolism of dietary drugs and potentially hazardous agents (38). AhR exists as cytoplasmic aggregates bound to two molecules of the 90 kDa heat-shock protein Hsp90 (39). Upon ligand binding, AhR dissociates from Hsp90 and the ligand-receptor complex translocates to the nucleus. Then the activated AhR dimerizes with ARNT and binds to a class of promoter DNA sequences, called xenobiotic-response elements, of target genes (i.e. CYP1A1 and CYP1B1) to activate their transcription (21, 40).

In this study, we found that the exposure of RL95-2 cells to B[a]P up-regulates the protein levels of AhR, Hsp90, CYP1A1, CYP1B1, and EH significantly, but most of these proteins were at near-basal levels in the control cells. Compared with other cell types tested (data not shown), RL95-2 cells have a strong ability to express these proteins in response to B[a]P. It is suggested that the increase and accumulation of AhR and Hsp90 in response to exposure to B[a]P treatment might contribute coordinately to the expression of CYP1A1 and CYP1B1. Although EH activity was not determined in this study, based on the up-regulation of EH after exposure to B[a]P, it appears to involve the conversion process of B[a]P-7,8-oxide to B[a]P-7,8-DHD, an important intermediate molecule of B[a]P metabolism (23). It has been recognized that, whereas CYP1A1 is dominantly expressed in hepatic cells (41), CYP1B1 is found mainly in extrahepatic tissues, such as the adrenal, breast, kidney, and lung (24, 42). Interestingly, although RL95-2 cells originated from extrahepatic tissue, CYP1A1 was far more active than CYP1B1 after exposure to B[a]P, at least in terms of the involvement of B[a]P metabolism in cytotoxicity-mediated apoptosis. Both CYP1A1 and CYP1B1 are known to be associated with the activations of B[a]P to B[a]P-7,8-oxide as well as B[a]P-7,8-DHD to BPDE (43). However, our results demonstrated that the expression of CYP1B1 is up-regulated significantly by the exposure to B[a]P, but the activity remained at the basal level. This suggests that the status of increased CYP1B1 expression does not represent an increase in its activity. Thus, CYP1A1 is believed to be a central oxidative enzyme that is ultimately required for BPDE formation from B[a]P in RL95-2 cells.

Using an immunohistochemical detection method for the BPDE molecules (44, 45), the evidence for conversion of B[a]P to BPDE was visualized by immunofluorescence cytochemistry combined with highly sensitive confocal microscopy. In our cellular model, BPDE immunoreactivity in B[a]P-treated cells was verified by comparing the immunoreactivity of BPDE-treated cells (positive control) as well as the disappearance of immunoreactivity in the presence of {alpha}-NF. Finally, BPDE molecules provoked RL95-2 cell apoptosis remarkably in a shorter period time than that induced by B[a]P. This again indicates that B[a]P-induced apoptosis in RL95-2 cells is evoked by BPDE.

Altogether, our data show that the exposure of RL95-2 endometrial cancer cells to B[a]P results in both caspase-dependent and caspase-independent apoptosis, and that CYP1A1 plays a key role in B[a]P-provoked cytotoxicity and cell death in this cellular system. We believe that RL95-2 cells are an excellent cellular model for the investigation of xenobiotic mechanisms associated with CYP1A1 as well as CYP1B1. Further research on B[a]P-induced apoptotic mechanisms in RL95-2 cells might be helpful for the development of therapeutic strategies for CYP1A1-related cancers.


   Footnotes
 
This work was supported by grants from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (02-PJ1-PG3-20708-0014) and Korea Research Foundation Grant 2004-005-E00006.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: Ac-DEVD-CHO, N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aldehyde; AhR, aryl hydrocarbon receptor; AIF, apoptosis-inducing factor; ARNT, AhR nuclear translocator; B[a]P, benzo[a]pyrene; B[a]P-7,8-DHD, (+/–)-B[a]P-trans-7,8-dihydrodiol; BPDE, B[a]P-7,8-dihydroxy-9,10-epoxide; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; EH, epoxide hydrolase; EROD, ethoxyresorufin O-deethylase; Hsp90, heat-shock protein 90; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carboncyanine iodide; MMP, mitochondrial membrane potential; MTT, thiazolyl blue; NADPH, nicotinamide adenine dinucleotide phosphate; {alpha}-NF, {alpha}-naphthoflavone; PAH, polycyclic aromatic hydrocarbon; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; RT, room temperature; Smac/DIABLO, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein binding protein with low PI; TEM, transmission electron microscopy; TMS, 2,3',4,5'-tetramethoxystilbene.

Received January 22, 2007.

Accepted for publication July 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hattemer-Frey HA, Travis CC 1991 Benzo[a]pyrene: environmental partitioning and human exposure. Toxicol Ind Health 7:141–157[Medline]
  2. Knize MG, Salmon CP, Pais P, Felton JS 1999 Food heating and the formation of heterocyclic aromatic amine and polycyclic aromatic hydrocarbon mutagens/carcinogens. Adv Exp Med Biol 459:179–193[Medline]
  3. Cook JW, Hewett CL, Hieger I 1933 The isolation of a cancer-producing hydrocarbon from coal tar. J Chem Soc 395–405
  4. Caruso JA, Reiners Jr JJ, Emond J, Shultz T, Tainsky MA, Alaoui-Jamali M, Batist G 2001 Genetic alteration of chromosome 8 is a common feature of human mammary epithelial cell lines transformed in vitro with benzo[a]pyrene. Mutat Res 473:85–99[Medline]
  5. Walle T, Walle UK, Sedmera D, Klausner M 2006 Benzo[a]pyrene-induced oral carcinogenesis and chemoprevention: studies in bioengineered human tissue. Drug Metab Dispos 34:346–350[Abstract/Free Full Text]
  6. Dodge JA, Glasebrook AL, Magee DE, Phillips DL, Sato M, Short LL, Bryant HU 1996 Environmental estrogens: effects on cholesterol lowering and bone in the ovariectomized rat. J Steroid Biochem Mol Biol 59:155–161[CrossRef][Medline]
  7. Santodonato J 1997 Review of the estrogenic and antiestrogenic activity of polycyclic aromatic hydrocarbons: relationship to carcinogenicity. Chemosphere 34:835–848[Medline]
  8. Hoyer PB 2001 Reproductive toxicology: current and future directions. Biochem Pharmacol 62:1557–1564[CrossRef][Medline]
  9. Lei W, Yu R, Mandlekar S, Kong AN 1998 Induction of apoptosis and activation of interleukin 1ß-converting enzyme/Ced-3 protease (caspase-3) and c-Jun NH2-terminal kinase 1 by benzo(a)pyrene. Cancer Res 58:2102–2106[Abstract/Free Full Text]
  10. Salas VM, Burchiel SW 1998 Apoptosis in Daudi human B cells in response to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol. Toxicol Appl Pharmacol 151:367–376[CrossRef][Medline]
  11. Rorke EA, Sizemore N, Mukhtar H, Couch LH, Howard PC 1998 Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells. Int J Oncol 13:557–563[Medline]
  12. Burchiel SW, Davis DA, Ray SD, Barton SL 1993 DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma. Fundam Appl Toxicol 21:120–124[CrossRef][Medline]
  13. van Grevenynghe J, Sparfel L, Le Vee M, Gilot D, Drenou B, Fauchet R, Fardel O 2004 Cytochrome P450-dependent toxicity of environmental polycyclic aromatic hydrocarbons towards human macrophages. Biochem Biophys Res Commun 317:708–716[CrossRef][Medline]
  14. Matikainen T, Perez GI, Jurisicova A, Pru JK, Schlezinger JJ, Ryu HY, Laine J, Sakai T, Korsmeyer SJ, Casper RF, Sherr DH, Tilly JL 2001 Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals. Nat Genet 28:355–360[CrossRef][Medline]
  15. Matikainen TM, Moriyama T, Morita Y, Perez GI, Korsmeyer SJ, Sherr DH, Tilly JL 2002 Ligand activation of the aromatic hydrocarbon receptor transcription factor drives Bax-dependent apoptosis in developing fetal ovarian germ cells. Endocrinology 143:615–620[Abstract/Free Full Text]
  16. Solhaug A, Refsnes M, Lag M, Schwarze PE, Husoy T, Holme JA 2004 Polycyclic aromatic hydrocarbons induce both apoptotic and anti-apoptotic signals in Hepa1c1c7 cells. Carcinogenesis 25:809–819[Abstract/Free Full Text]
  17. Ko CB, Kim SJ, Park C, Kim BR, Shin CH., Choi S, Chung SY, Noh JH, Jeun JH, Kim NS, Park R 2004 Benzo(a)pyrene-induced apoptotic death of mouse hepatoma Hepa1c1c7 cells via activation of intrinsic caspase cascade and mitochondrial dysfunction. Toxicology 199:35–46[CrossRef][Medline]
  18. Hankinson O 1995 The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 35:307–340[CrossRef][Medline]
  19. Denison MS, Whitlock Jr JP 1995 Xenobiotic-inducible transcription of cytochrome P450 genes. J Biol Chem 270:18175–18178[Free Full Text]
  20. Swanson HI 2002 DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation. Chem Biol Interact 141:63–76[CrossRef][Medline]
  21. Nebert DW, Puga A, Vasiliou V 1993 Role of the Ah receptor and the dioxin-inducible [Ah] gene battery in toxicity, cancer, and signal transduction. Ann NY Acad Sci 685:624–640[Abstract]
  22. Conney AH 1982 Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. Cancer Res 42:4875–4917[Free Full Text]
  23. Gelboin HV 1980 Benzo {alpha}-pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes. Physiol Rev 60:1107–1166[Free Full Text]
  24. Shimada T, Hayes CL, Yamazaki H, Amin S, Hecht SS, Guengerich FP, Sutter TR 1996 Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res 56:2979–2984[Abstract/Free Full Text]
  25. Kaina B 2003 DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling. Biochem Pharmacol 66:1547–1554[CrossRef][Medline]
  26. Khorram O, Garthwaite M, Golos T 2002 Uterine and ovarian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA expression in benign and malignant gynaecological conditions. Mol Hum Reprod 8:75–80[Abstract/Free Full Text]
  27. Igarashi T, Osuga U, Tsutsumi O, Momoeda M, Ando K, Matsumi H, Takai Y, Okagaki R, Hiroi H, Fujiwara O, Yano T, Taketani Y 1999 Expression of Ah receptor and dioxin-related genes in human uterine endometrium in women with or without endometriosis. Endocr J 46:765–772[Medline]
  28. Lesko SM, Rosenberg L, Kaufman DW, Helmrich SP, Miller DR, Strom B, Schottenfeld D, Rosenshein NB, Knapp RC, Lewis J 1985 Cigarette smoking and the risk of endometrial cancer. N Engl J Med 313:593–596[Abstract]
  29. Richter C, Schweizer M, Cossarizza A, Franceschi C 1996 Control of apoptosis by the cellular ATP level. FEBS Lett 378:107–110[CrossRef][Medline]
  30. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD 1997 The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132–1136[Abstract/Free Full Text]
  31. Du C, Fang M, Li Y, Li L, Wang X 2000 Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102:33–42[CrossRef][Medline]
  32. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL 2000 Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102:43–53[CrossRef][Medline]
  33. Susin SA, Zamazami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M, Kroemer G 1996 Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 184:1331–1341[Abstract/Free Full Text]
  34. Raffray M, Cohen GM 1997 Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death? Pharmacol Ther 75:153–177[CrossRef][Medline]
  35. Ashkenazi A, Dixit VM 1998 Death receptors: signaling and modulation. Science 281:1305–1308[Abstract/Free Full Text]
  36. Green DR, Reed JC 1998 Mitochondria and apoptosis. Science 281:1309–1312[Abstract/Free Full Text]
  37. Lorenzo HK, Susin SA, Penninger J, Kroemer G 1999 Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ 6:516–524[CrossRef][Medline]
  38. Burbach KM, Poland A, Bradfield CA 1992 Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc Natl Acad Sci USA 89:8185–8189[Abstract/Free Full Text]
  39. Carver LA, Bradfield CA 1997 Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo. J Biol Chem 272:11452–11456[Abstract/Free Full Text]
  40. Whitlock Jr JP 1999 Induction of cytochrome P4501A1. Annu Rev Pharmacol Toxicol 39:103–125[CrossRef][Medline]
  41. Fujii-Kuriyama Y, Imataka H, Sogawa K, Yasumoto K, Kikuchi Y 1992 Regulation of CYP1A1 expression. FASEB J 6:706–710[Abstract]
  42. Sutter TR, Tang YM, Hayes CL, Wo YY, Jabs EW, Li X, Yin H, Cody CW, Greenlee WF 1994 Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2. J Biol Chem 269:13092–13099[Abstract/Free Full Text]
  43. Jiang H, Shen YM, Quinn AM, Penning TM 2005 Competing roles of cytochrome P450 1A1/1B1 and aldo-keto reductase 1A1 in the metabolic activation of (+/–)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene in human bronchoalveolar cell extracts. Chem Res Toxicol 18:365–374[CrossRef][Medline]
  44. Zenzes MT, Bielecki R, Reed TE 1999 Detection of benzo(a)pyrene diol epoxide-DNA adducts in sperm of men exposed to cigarette smoke. Fertil Steril 72:330–335[CrossRef][Medline]
  45. Zenzes MT, Puy LA, Bielecki R, Reed TE 1999 Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa. Mol Hum Reprod 5:125–131[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
148/10/5112    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J. Y.
Right arrow Articles by Kim, J.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J. Y.
Right arrow Articles by Kim, J.-M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*BENZO(A)PYRENE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals