* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674;
Department of Biochemistry and The National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
Received January 5, 2000; accepted February 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: benzo[a]pyrene; MCF-7 cells; estrogen; -napthoflavone; metabolite.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The common phenolic metabolites of B[a]P include the 3-OH, 7-OH, and 9-OH species, which result from hydroxylase activity shown to be mediated through the CYP1A, 2C, and 3A families (Gautier et al., 1996; Monteith et al., 1987
; Shou et al., 1994
). Further metabolism to the (-)-B[a]P-7R-trans-7,8-dihydrodiol (7,8-OH) and the B[a]P-trans-9,10-dihydrodiol (9,10-OH) occurs in the presence of microsomal epoxide hydrolase, which may lead to the formation of the ultimate carcinogen (±)B[a]P-r7,t-8-dihydrodiol-t-9,10-epoxide (BPDE) (Kim et al., 1998
; Slaga et al., 1979
).
The steric resemblance of the PAHs including B[a]P to steroid molecules led to the postulation, as far back as the 1950s, that they would have the ability to act on the same site as steroid hormones (Yang et al., 1961). PAHs have been evaluated for hormonelike activity in both in vivo and in vitro assays (Clemons et al., 1998
; Morreal et al., 1982
, 1979
; Tran et al., 1996
), and demonstrated both estrogenic and antiestrogenic activities. These demonstrated activities might be the consequence of the promiscuous binding capacity of the estrogen receptor, due to the structure of its binding cavity (Brzozowski et al., 1997
).
Gene transactivation assays have been proposed as part of a tiered testing strategy by the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) for the identification of potential endocrine-active chemicals. These in vitro systems are proposed for use in screening for chemicals and mixtures that interact with the estrogen/androgen hormone receptor systems. Some investigators have raised the issue that compounds that require metabolic activation, or that act through nonreceptor-mediated pathways, could potentially be missed in such a system if the target cells lacked the necessary cellular machinery (Gierthy et al., 1996; Loukovaara et al., 1995
; Mousavi and Adlercreutz, 1992
).
Therefore, as part of a larger study investigating the feasibility of incorporating exogenous metabolic capacity into such assays, we investigated the metabolic competence of a model ER- transactivation system utilizing B[a]P as one of our test compounds. This assay utilizes the MCF-7 human breast cancer cell line transiently transfected with a chimeric ER-
expression plasmid and a responsive luciferase reporter. The utility of this system for the analysis of ER-mediated endocrine activity has previously been demonstrated (Zacharewski et al., 1994
), with B[a]P having recently been shown to exhibit significant activity in this assay system (Clemons et al., 1998
). B[a]P is known to bind to the Ah receptor and induce the activity of cytochrome P450 metabolic enzymes (Harris et al., 1988
; Kamps and Safe, 1987
; Merchant et al., 1992
). These properties of B[a]P made it an ideal compound for evaluating the metabolic capabilities of the MCF-7 cell culture system and for exploring the potential interaction of cellular metabolism and ER-mediated events.
In this study, we characterized the metabolite profile of B[a]P in the MCF-7 cell culture system and found the cells to be competent to metabolize B[a]P to a series of mono- and diphenolic products, as has previously been shown in primary cultures of mammary epithelial cells (Bartley and Stampfer, 1985; Moore et al., 1986
; Tischler et al., 1991
). Furthermore, we assessed the estrogenic activity of B[a]P and its metabolites in the ER-
reporter gene assay. The P450 inhibitor and Ah receptor antagonist
-NF (Bauer et al., 1995
; Merchant et al., 1993
; Nesnow et al., 1982
; Rämet et al., 1995
) was also used to further elucidate the relationship between metabolism and the estrogenic activity of B[a]P and its metabolites.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
MCF-7 cells (obtained from Dr. L. Murphy, University of Manitoba, Winnipeg, Manitoba) were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 15 mM HEPES augmented with 50 µg/ml gentamicin, penicillin/streptomycin (100 IU/ml/100 µg/ml), and amphotericin B (2.5 µg/ml). Cells were maintained at 3% CO2 and 95% humidity.
Transfection.
Cells were plated in triplicate in 96-well plates at a density of 68 x 103 cells/well in 5% FBS-DCC (Hyclone, Logan, UT). After attachment and growth for 6 h, the cells were transfected using LipofectinTM (Life Technologies). To each well was added 50 ng of the ß-galactosidase (ß-gal) expression vector pCH110 (Pharmacia, Piscataway, NJ), 150 ng of 17m5-G-Luc, the Gal4-regulated luciferase reporter vector, and 5 ng of Gal4-HEG0, an ER expression vector (both provided by P. Chambon, INSERM, France). The plasmids were transfected in serum-free, antibiotic-free DMEM supplemented with 2 mM L-glutamine. Cells were allowed to incubate overnight at 37°C in a humidified atmosphere of 3% CO2/air. Sixteen to eighteen hours following transfection, the plates were blotted dry on sterile paper towels; the cells were then treated in triplicate with E2, B[a]P and its metabolites, -NF, or DMSO (Sigma) in 5% FBS-DCC. The ER agonist E2 was used over a range of concentrations from 1012 to 108 M, with DMSO as solvent. The pure antiestrogen ICI 182,780 (Wakeling et al., 1991
) was used in all assays in combination with E2 to verify that the reporter gene activity was strictly ER mediated. Following treatment, wells were washed with PBS, and 50 µl lysis buffer (Promega, Madison, WI) was added to each well. Thawing the plates after freezing at 70°C facilitated cell lysis. Aliquots from each well were divided into two 96-well plates for luciferase and ß-gal activity determination. The reference plasmid pCH110 was cotransfected as an internal control to correct for variations in transfection efficiency. The values presented are units of luciferase activity normalized to the ß-gal activity from individual wells. Experiments were evaluated with fold induction being the endpoint of interest. Treatment regimens that resulted in a reduced ß-gal activity relative to that of transfected cultures exposed to E2 under unaltered media conditions were considered cytotoxic and were not used for further analysis. Note that carrier solvent (DMSO) concentrations did not exceed 0.3% in these assays, nor was the osmolality or pH of the E2 or B[a]P dosing solutions changed by ± 6 mOsMol or
0.06 units, respectively, relative to vehicle-treated controls.
Luciferase activity assay.
10 µl of lysate was combined with 100 µl of luciferase assay reagent (Promega), and luminescence was determined immediately using a Packard Topcount NXTTM luminescence counter (Packard Instrument Company, Meriden, CT).
ß-galactosidase activity assay.
The ß-gal activity was measured using a chemiluminescent kit (Tropix Inc, Bedford, MA). ß-gal activity was initiated with 70 µl of galactosidase reaction buffer added to 10 µl of the cell lysate, followed by a 30-min room temperature incubation. After the reaction was stopped by addition of 100 µl of the Accelerator II stop buffer, the chemiluminescence was measured in the Packard Topcount NXTTM luminescence counter.
HPLC analysis of [14C]B[a]P metabolites.
Following 24 h of exposure, 20% v/v acetonitrile was added to the treated cultures. The mixture was allowed to incubate for 5 min at room temperature and medium and lysate were aliquoted into 15-ml tubes. Cell debris was removed by centrifugation at 600 x g for 5 min; the supernatant was used for metabolite analysis. Samples were thawed to room temperature and a portion of each was transferred to an autosampler vial for analysis. An aliquot (400 µl) of each sample was injected using a Waters model 717 Plus autosampler (Waters Corporation, Milford, MA) into a reverse-phase high-pressure liquid chromatographic (HPLC) system. The pumping system was a Hitachi model L-6200A (Hitachi, Ltd., Tokyo, Japan). The separation of sample components was achieved using a 15 cm x 4.6 mm i.d. analytical column packed with 5 µm Zorbax SB-C8 (MAC-MOD Analytical, Inc., Chadds Ford, PA). The mobile phase system consisted of A: 1% acetic acid in water, and B: 1% acetic acid in acetonitrile. The mobile phase gradient program consisted of a 2-min isocratic initial hold at 60/40 A/B, then a 20-min linear gradient down to 10/90 A/B, with a hold at 10/90 A/B for 5 min. An ultraviolet (UV) detector (Kratos SpectroFlow 773, Applied Biosystems, Foster City, CA) set at 254 nm was used to monitor the effluent prior to collection of 15-s fractions using an Isco FOXY 200 fraction collector (Isco, Inc., Lincoln, NE). A 5-ml aliquot of liquid scintillant (Ultima Flo M, Packard, Meriden, CT) was added to each fraction. The fractions were counted for radioactivity using a Beckman LS 6000 IC liquid scintillation counter (Beckman Instruments, Inc., Fullerton, CA) in the 14C mode. Radiochromatograms for each injection were reconstructed from the counted fractions. Metabolite assignments were determined by comparison to authentic standards purchased from the National Cancer Institute Carcinogen Repository (Kansas City, MO).
Data analysis.
Data points are expressed as the mean ± SD of triplicate measurements. The values presented are units of luciferase activity normalized to the ß-gal activity from individual wells. Data were evaluated using the fold induction of the normalized luciferase activity in the B[a]P/E2-treated relative to the DMSO-treated cultures. Unless otherwise stated, experiments were performed at least three times. Data were analyzed for statistical differences between control and treated groups using two-tailed t-test and Dunnett's test relative to the control group for post-hoc comparisons.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The hydroxylated PAHs 3,9-dihydroxy-benz[a]anthracene and 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene were weakly estrogenic in rat bioassays in terms of both in vitro binding to the ER and increased uterine wet weight (Morreal et al., 1979). The 1-OH, 2-OH, 5-OH, 6-OH, 11-OH, and 12-OH, but not the 4-OH metabolites of B[a]P, exhibited competition for binding to the ER in the cytosol of rat uterus (Ebright et al., 1986
). Previous studies have also shown that B[a]P may have a weak mitogenic activity in primary and tumor-derived human mammary epithelial cell cultures (Katdare et al., 1998
; Tannheimer et al., 1997
, 1998
). Recent studies utilizing a chimeric ER-
receptor reporter system demonstrated significant B[a]P activity relative to E2 (Clemons et al., 1998
).
B[a]P was also able to stimulate proliferation as well as alter E2 metabolism of human mammary terminal duct cell cultures (Telang et al., 1997). In addition to the weak estrogenic activity, it has been suggested that PAHs possess antihormonal activity. This has been shown by the ability of benz[a]anthracene-3,9-diols to inhibit estrus in rats (Morreal et al., 1982
), the ability of B[a]P to decrease nuclear ER localization in breast cancer cells (Chaloupka et al., 1992
), and the inhibition of E2- induced reporter activity in a yeast-based ER transactivation system by several PAHs (Tran et al., 1996
). Furthermore, Arcaro et al., 1999, found that B[a]P is not estrogenic in the MCF-7 focus assay. However, they observed that B[a]P could displace E2 in a whole-cell binding, but not in an in vitro competitive binding assay lacking metabolic capability, suggesting an antiestrogenic role for B[a]P metabolites in that system. The agonist activity displayed in our assay relative to the apparent antiestrogenicity in the MCF-7 focus study may be a consequence of our use of a chimeric Gal4 receptor-reporter system, which lacks a functional AF-1 domain (Zacharewski et al., 1994
).
-NF is a well-characterized inhibitor of B[a]P metabolism in in vitro systems (Bauer et al., 1995
; Crespi et al., 1985
; Nesnow et al., 1982
; Rämet et al., 1995
).
-NF has been shown to inhibit B[a]P-induced increases in intracellular Ca2+ levels associated with mitogenic activity in mammary epithelial cells (Tannheimer et al., 1997
) as well as the p53 expression induced by B[a]P, which correlated with DNA damage in breast adenocarcinoma cell lines (Rämet et al., 1995
).
The ability of -NF, the Ah receptor antagonist (Merchant et al., 1993
; Wilhelmsson et al., 1994
; ) and P450 inhibitor, to significantly inhibit the response of B[a]P in our assay was the initial indicator of the possibility that B[a]P metabolites might be responsible for the observed activity. In concert with the observation that the combined activity of E2 and B[a]P could be completely inhibited by ICI but not
-NF demonstrated that the observed responses were mechanistically a consequence of ER-
activation. Together, these data indicated a potential contribution of P450 activity in the estrogenicity displayed by B[a]P, and led to the investigation as to whether the observed activity was produced by hydroxylated B[a]P metabolites.
-NF did not decrease the E2 response itself, with most cases showing a tendency towards increased activity. The inability of
-NF to significantly inhibit the combined E2 and B[a]P response was somewhat surprising and could be a consequence of interactions between the ER- and Ah receptor-signaling pathways. However, further work is required to elucidate the reasons for this observation.
The fact that ICI, but not -NF, was able to block the responses of the hydroxylated B[a]P metabolites is indicative of their being the active estrogenic species in our experiments. Furthermore, the 3-OH and 9-OH metabolites of B[a]P appear to demonstrate significant displacement of E2 from the ER-
in in vitro binding experiments, as well as producing comparable reporter activity (Fertuck et al., manuscript in preparation). The minimal activity produced by
-NF itself in the assay is in keeping with the weak binding of other flavones to the ER (Kuiper et al., 1998
).
Our present hypothesis is that the estrogenlike activity exhibited by B[a]P is predominantly produced by its hydroxylated metabolites. -NF, by binding the Ah receptor, may inhibit the ability of B[a]P to induce its own metabolism, resulting in reduced activity in our assay. The ability of
-NF to reduce the 9-OH-B[a]P-induced luciferase activity may be a consequence of the 9-OH metabolite undergoing further metabolism to more estrogenic species or, alternatively, incurring some degree of displacement by
-NF from the ER.
The 7,8-OH metabolite did not demonstrate significant estrogenic activity, but had a marked effect on cell viability both by visual observation and using a ß-galactosidase activity measure, a technique used previously in a similar system (Maness et al., 1998). The cellular rescue effected by
-NF is in keeping with its ability to inhibit formation of (±)B[a]P-r7,t-8-dihydrodiol-t-9,10-epoxide (BPDE), the ultimate B[a]P metabolite and carcinogen, via the inhibition of the monooxygenase enzyme (Vähäkangas et al., 1979
). This phenomenon has previously been observed in an engineered cell line stably expressing the P4501A1 gene (States et al., 1993
). Reduction in the sensitivity to B[a]P-mediated cytotoxicity also occurred when HepG2 cells were cultured in the presence of
-NF (Babich et al., 1988
).
The fact that metabolites of a ubiquitous environmental contaminant like B[a]P can potentially exhibit estrogenlike activity adds to the plethora of reported effects induced by this well-characterized DNA-reactive/genotoxic carcinogen. This calls for further analysis of the potential interactions of differing pathways, such as the role that Ah receptor activation might play in cumulative estrogen tissue responses, and its potential involvement in disease etiology.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arcaro, K. F., O'Keefe, P. W., Yang, Y., Clayton, W., and Gierthy, J. F. (1999). Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells. Toxicology 133, 115127.[ISI][Medline]
Babich, H., Sardana, M. K., and Borenfreund, E. (1988). Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumor cell line, HepG2. Cell. Biol. Toxicol. 4, 295309.[ISI][Medline]
Baird, W. M., and Ralston, S. L. (1997). Carcinogenic Polycyclic Aromatic Hydrocarbons. In Comprehensive Toxicology, Carcinogens and Anticarcinogens. (G. T. Bowden and S. M. Fischer, Eds.), Vol. 12, pp. 171200. Elsevier-North Holland, Amsterdam.
Bartley, J. C., and Stampfer, M. R. (1985). Factors influencing benzo(a)pyrene metabolism in human mammary epithelial cells in culture. Carcinogenesis 6, 10171022.[Abstract]
Bauer, E., Guo, Z., Ueng, Y-F., Bell, L. C., Zeldin, D., and Guengerich, F. P. (1995). Oxidation of benzo(a)pyrene by recombinant human cytochrome P450 enzymes. Chem. Res. Toxicol. 8, 136142.[ISI][Medline]
Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engström, O., Öhman, L., Greene, G. L., Gustafsson. J-A., and Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389, 753758.[ISI][Medline]
Chaloupka, K., Krishnan, V., and Safe, S. (1992). Polynuclear aromatic hydrocarbon carcinogens as antiestrogens in MCF-7 human breast cancer cells: role of the Ah receptor. Carcinogenesis 13, 22332239.[Abstract]
Clemons, J. H., Allan, L. M., Marvin, C. H., Wu, Z., McCarry, B. E., Bryant, D. W., and Zacharewski, T. R. (1998). Evidence of estrogen- and TCDD-like activities in crude and fractionated extracts of PM10 air particulate material using in vitro gene expression assays. Environ Sci. Technol. 32, 18531860.[ISI]
Crespi, C. L., Altman, J. D., and Marletta, M. A. (1985). Xenobiotic metabolism and mutation in a human lymphoblastoid cell line. Chem. Biol. Interact. 53, 257271.[ISI][Medline]
Ebright, R. H., Wong, J. R., and Chen, L. B. (1986). Binding of z-hydroxybenzo[a]pyrene to estrogen receptors in rat cytosol. Cancer Res. 46, 23492351.[Abstract]
Fertuck, K., Matthews, J., and Zacharewski, T. Phenolic benzo(a)pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression of benzo(a)pyrene, but do not elicit uterotropic effects in vivo. (Manuscript in preparation).
Gautier, J. C., Lecoeur, S., Cosme, J., Perret, A., Urban, P., Beaune, P., and Pompon, D. (1996). Contribution of human cytochrome P450 to benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast. Pharmacogenetics 6, 489499.[ISI][Medline]
Gierthy, J. F., Spink, B. C., Figge, H. L., Pentecost, B. T., and Spink, D. C. (1996). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17 beta-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells. J. Cell Biochem. 60, 17384.[ISI][Medline]
Harris, M., Kamps, C., and Safe, S. (1988). Role of the 4-5s binding protein in the induction of aryl hydrocarbon hydroxylase in the rat. Carcinogenesis 9, 14751479.[Abstract]
Kamps, C., and Safe, S. (1987). Binding of polynuclear aromatic hydrocarbons to the rat 4S cytosolic binding protein: structure-activity relationships. Cancer Lett. 34, 129137.[ISI][Medline]
Katdare, M., Osborne, M. P., and Telang, N. T. (1998). Inhibition of aberrant proliferation and induction of apoptosis in pre-neoplastic human mammary epithelial cells by natural phytochemicals. Oncol. Rep. 5, 311315.[ISI][Medline]
Kim, J. H., Stansbury, K. H., Walker, N. J., Trush, M. A., Strickland, P. T., and Sutter, T. R. (1998). Metabolism of benzo(a)pyrene and benzo(a)pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis 19, 18471853.[Abstract]
Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B., and Gustaffson, J-A. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139, 42524263.
Loukovaara, M., Carson, M., and Adlercreutz, H. (1995). Regulation of sex-hormone-binding globulin production by endogenous estrogens in vitro. Biochem. Biophys. Res. Commun. 206, 895901.[ISI][Medline]
Maness, S. C., McDonnell, D. P., and Gaido, K. W. (1998). Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells. Toxicol. Appl. Pharmacol. 151, 135142.[ISI][Medline]
Merchant, M., Krishnan, V., and Safe, S. (1993). Mechanism of action of -napthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells. Toxicol. Appl. Pharmacol. 120, 179185.[ISI][Medline]
Merchant, M., Wang, X., Kamps, C., Rosengren, R., Morrison, V., and Safe, S. (1992). Mechanism of benzo(a)pyrene-induced Cyp1a-1 gene expression in mouse Hepa 1c1c7 cells: role of the nuclear 6 s and 4 s proteins. Arch. Biochem. Biophys. 292, 250257.[ISI][Medline]
Monteith, D. K., Novotny, A., Michalopoulos, G., and Strom, S. C. (1987). Metabolism of benzo(a)pyrene in primary cultures of human hepatocytes: dose-response over a four-log range. Carcinogenesis 8, 983988.[Abstract]
Moore, C. J., Tricomi, W. A., and Gould, M. N. (1986). Interspecies comparison of polycyclic aromatic hydrocarbon metabolism in human and rat mammary epithelial cells. Cancer Res. 46, 49464952.[Abstract]
Morreal, C. E., Schneider, S. L., Sinha, D. K., and Bronstein, R. E. (1979). Estrogenic properties of 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene in rats. J. Natl. Cancer Inst. 62, 15851588.[ISI][Medline]
Morreal, C. E., Sinha, D. K., Schneider, S. L., Bronstein, R. E., and Dawidzik, J. (1982). Antiestrogenic properties of substituted benz[a]anthracene-3,9-diols. J. Med. Chem. 25, 323326.[ISI][Medline]
Mousavi, Y., and Adlercreutz, H. (1992). Enterolactone and estradiol inhibit each other's proliferative effect on MCF-7 breast cancer cells in culture. J. Steroid Biochem. Mol. Biol. 41, 6159.[ISI][Medline]
Nesnow, S., Easterling, R., Bergman, H., and Roth, R. (1982). Inhibition of benzo(a)pyrene monooxygenase by -napthoflavone may be partially mediated by the metabolite 9-hydroxy-
-napthoflavone. Toxicol. Lett. 14, 713.[ISI][Medline]
Rämet, M., Castrén, K., Järvinen, K., Pekkala, K., Turpeenniemi-Hujanan, T., Soini, Y., Pääkkö, P., and Vähäkangas, K. (1995). p53 protein expression is correlated with benzo(a)pyrene-DNA adducts in carcinoma cell lines. Carcinogenesis 16, 21172124.[Abstract]
Shou, M., Korzekwa, K. R., Crespi, C. L., Gonzalez, F. J., and Gelboin, H. V. (1994). The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzo(a)pyrene and benzo(a)pyrene trans-7,8-dihydrodiol. Mol. Carcinog. 10, 159168.[ISI][Medline]
Slaga, T. J., Bracken, W. J., Gleason, G., Levin, W., Yagi, H., Jerina, D. M., and Conney, A. H. (1979). Marked differences in the skin tumor-initiating activities of the optical enantiomers of the diastereomeric benzo(a)pyrene 7,8-diol-9,10-epoxides. Cancer Res. 39, 6771.[ISI][Medline]
States, J. C., Quan, T., Hines, R. N., Novak, R. F., and Runge-Morris, M. (1993). Expression of human cytochrome P450 1A1 in DNA repair deficient and proficient human fibroblasts stably transformed with an inducible expression vector. Carcinogenesis 14, 16431649.[Abstract]
Tannheimer, S. L., Barton, S. L., Ethier, S. P., and Burchiel, S. W. (1997). Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells. Carcinogenesis 18, 11771182.[Abstract]
Tannheimer, S. L., Ethier, S. P., Caldwell, K. K., and Burchiel, S. W. (1998). Benzo(a)pyrene- and TCDD-induced alterations in tyrosine phosphorylation and insulin-like growth factor signaling pathways in the MCF-10A human mammary epithelial cell line. Carcinogenesis 19, 12911297.[Abstract]
Telang, N. T., Katdare, M., Bradlow, H. L., and Osborne, M. P. (1997). Estradiol metabolism: an endocrine biomarker for modulation of human mammary carcinogenesis. Environ. Health Perspect. 105(Suppl 3), 559564.
Tischler, A. N., Levine, G. A., and Bartley, J. C. (1991). Metabolism of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol in human mammary epithelial cells: feedback inhibition by 7-hydroxybenzo(a)pyrene. Carcinogenesis 12, 15391543.[Abstract]
Tran, D. Q., Ide, C. F., McLachlan, J. A., and Arnold, S. F. (1996). The anti-estrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human estrogen receptor. Biochem. Biophys. Res. Commun. 229, 101108.[Medline]
Vähäkangas, K., Nevasaari, K., Pelkonen, O., and Kärki, N. T. (1979). Effects of various in vitro inhibitors of benzo(a)pyrene metabolism in isolated rat lung perfusions. Acta Pharmacol. Toxicol. 45, 18.[Medline]
Wakeling, A. E., Dukes, M., and Bowler, J. (1991). A potent specific pure antiestrogen with clinical potential. Cancer Res. 51, 38673873.[Abstract]
Wilhelmsson, A., Whitelaw, M. L., Gustaffsson, J-A., and Poellinger, L. (1994). Agonist and antagonist effects of -napthoflavone on dioxin receptor function. Role of the basic region helix-loop-helix dioxin receptor partner factor Arnt. J. Biol. Chem. 269, 1902819033.
Yang, N. C., Castro, A. J., Lewis, M., and Wong, T-W. (1961). Polynuclear aromatic hydrocarbons, steroids and carcinogenesis. Science 134, 386387.[ISI]
Zacharewski, T., Bondy, K., McDonell, P., and Wu, Z. F. (1994). Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17ß-estradiol-induced-pS2 expression. Cancer Res. 54, 27072713.[Abstract]