Department of Biochemistry and Molecular Biology, and National Food Safety and Toxicology Center, Michigan State University, Lansing, Michigan 48824
Received August 16, 2000; accepted October 16, 2000
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ABSTRACT |
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Key Words: benzo[a]pyrene; polycyclic aromatic hydrocarbon; metabolism; estrogen receptor; receptor isoform; endocrine disrupter.
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INTRODUCTION |
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Compounds of diverse structure have the ability to bind to the estrogen receptor isoforms ER and ERß, though they often share some general structural features with E2 and other endogenous estrogens. Recent crystallization of the ligand binding domain (LBD) of both ER
and ERß in the presence of agonist or antagonist has revealed the nature of the binding pocket, which is both lined with hydrophobic amino acids and contains specific residues for hydrogen bonding with the hydroxyl groups of the ligand (Brzozowski et al., 1997
; Pike et al., 1999
). Interestingly, some 4- or 5-ring carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are isosteric to sex steroids (see Fig. 1
) and are able to induce tumor formation in estrogen-responsive tissues such as the mammary gland, ovary, and uterus (Grover et al., 1980
; Huggins et al., 1967
; Yang et al., 1961
). Furthermore, B[a]P has been reported to adversely affect ovarian follicle growth, ovulation, and corpora lutea formation (Mattison et al., 1989
; Swartz and Mattison, 1985
) and to produce a weak estrus-like response in the rodent uterus (Cook and Dodds, 1933
).
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The majority of B[a]P studies to date have examined the mutagenicity and carcinogenicity of B[a]P metabolites. B[a]P is known to be biotransformed to polar metabolites by a subset of the cytochrome P450 metabolizing enzymes. It is predominantly metabolized by the CYP1A1 isozyme, but 1A2, 2A1, 3A4, and 1B1 can also contribute, with at least four other isozymes playing minor roles (Hall et al., 1989; Kim et al., 1998
; Larsen et al., 1998
; Parke and Ioannides, 1990
; Shou et al., 1994
). The specific metabolites formed in many in vitro and in vivo systems have been identified, and although there are quantitative differences in the amount of each metabolite formed in cells from different sources, the identities of the metabolites remain essentially constant regardless of the species or tissue type: 1-, 3-, 7-, and 9-OH-B[a]P, 4,5-, 7,8-, and 9,10-diOH-B[a]P, and 1,6-, 3,6-, and 6,12-B[a]P-dione (Estabrook et al., 1980
; Gelboin, 1980
; Pelkonen and Nebert, 1982
).
B[a]P has recently been shown to induce in vitro gene expression mediated though the D, E, and F domains of human ER (Gal4-hER
def) (Clemons et al., 1998
), whereas it reportedly acts in an antiestrogenic manner in other in vitro systems (Arcaro et al., 1999
; Tran et al., 1996
). In this study, the possible in vitro and in vivo estrogenic activity of B[a]P and its major metabolites was further examined.
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MATERIALS AND METHODS |
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Construction of plasmids.
The plasmid pGEX-hERdef was constructed by PCR amplification of the human ER
(Dr. P. Chambon, IGBMC, Illkirch, France) DEF domains (amino acids 264595) as previously described (Matthews and Zacharewski, 1999). The pGal4-mERßdef was constructed by PCR amplification of amino acids 159485 of the mERß (kindly provided by Dr. V. Giguère, Montreal, Quebec, Canada), using forward primer (5`-aaaagaattcctcgagcctgcgacttcgcaagtgttacgaa-3`) and reverse primer (5`-aaaagcggccgcagatcttcactgtgaatggaggttctggga-3`). The fragment was digested with XhoI and ClaI and ligated into the similarly digested eukaryotic expression vector containing the DNA binding domain of the yeast transcription factor Gal4, pG4MpolyII. PCR amplification was performed essentially as previously described (Gillesby and Zacharewski, 1999
) using Vent DNA polymerase in a reaction mixture containing Thermopol buffer, 200 µM dNTPs, 1 mM MgSO4, 500 nM primer, and 1.25 IU of polymerase. The mixture was heated to 94°C for 5 min, followed by 35 rounds of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 45 s. The sequence of each construct was confirmed by restriction enzyme digest and ABI/Prism automated sequencing (Perkin Elmer Applied Biosystems, Foster City, CA).
Competitive ligand binding assay.
The method used for the competitive binding assays has recently been described in detail (Matthews and Zacharewski, 2000), but is outlined briefly as follows. Experiments were performed using either a bacterially expressed fusion protein consisting of glutathione-S-transferase and the D, E, and F domains of human ER
(GST-hER
def, > 85% purity) (Matthews and Zacharewski, 2000
) or full-length human ERß (hERß, > 80% purity; Panvera, Madison, WI). The receptor was first diluted in TEGD buffer (10 mM Tris pH 7.6, 1.5 mM EDTA, 1 mM DTT, and 10% [v/v] glycerol) containing 1 mg/ml BSA as a carrier protein. An aliquot (240 µl) was incubated at 4°C for 2 h with 5 µl of 2.5 nM [3H]E2 and 5 µl of unlabeled competitor (10 pM to 1 µM final concentration of E2, or 60 nM to 20 µM final concentration of B[a]P or metabolite). The [3H]E2 and all competitor compounds were dissolved in DMSO, with the DMSO concentration in the final mixture not exceeding 4%. The fusion protein preparation was diluted in order to ensure 10,000 dpm of total binding in each tube.
Each concentration was tested in quadruplicate and at least three independent experiments were performed. Results are expressed as percent specific binding of [3H]E2 versus log of competitor concentration. Analysis was performed using nonlinear regression with the single-site competitive binding option of GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA). Reported IC50 values denote the calculated concentration of test compound required to displace 50% of the [3H]E2 from the receptor protein.
Cell culture.
MCF-7 human breast cancer cells were kindly provided by Dr. L. Murphy (University of Manitoba, Winnipeg, Manitoba, Canada). Cells were maintained in DMEM supplemented with 10% FBS and with 20 mM HEPES, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 50 µg/ml gentamicin. Cells were grown at 37°C in a 4% CO2 humidified environment.
Transfection and reporter gene assay.
Transfections were performed essentially as previously described (Clemons et al., 1998). MCF-7 cells were plated in 6-well culture dishes at approximately 50% confluency in 2 ml of media supplemented with 6% dextran-coated charcoal-stripped FBS and allowed to attach for 6 h. Cells were then transiently transfected with three plasmids, using the calcium phosphate coprecipitation method (Sambrook et al., 1989
): 1.5 µg 17m5-G-Luc (provided by Dr. P. Chambon), 0.2 µg Gal4-hER
def (Gal4 linked to D, E, and F domains of hER
; also known as Gal4-HEG0) or Gal4-mERßdef (Gal4 linked to D, E, and F domains of mouse ERß) , and 0.2 µg pCMV-lacZ, a ß-galactosidase expression vector used for normalizing transfection efficiency across wells (Amersham Pharmacia). After 18 h, cells were rinsed twice with sterile phosphate-buffered saline and fresh media was added. Cells were then treated by adding 2 µl of test compound dissolved in DMSO to 2 ml of media, so that the total concentration of DMSO did not exceed 0.1% unless otherwise noted. The cells were harvested after 24 h of treatment, and luciferase and ß-galactosidase activity was measured using standard protocols (Brasier et al., 1989
; Sambrook et al., 1989
).
Each treatment was performed in duplicate, and two aliquots were assayed from each well, so that means and standard deviations were calculated using four measurements. Independent experiments were performed at least three times, and results are expressed as fold induction, representing luciferase activity of treated cells divided by that of solvent-treated cells, normalized for ß-galactosidase activity. GraphPad Prism 3.0 was used for graphical analyses, including EC50 values, which denote the concentration of test compound required to cause 50% of the maximal response induced by E2.
Uterotrophic assay.
C57BL/6 and DBA/2 mice ovariectomized by the vendor on postnatal day 20 were obtained from Charles River Laboratories (Raleigh, NC). The mice were kept in cages containing cellulose fiber chips (Aspen Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY) in a 23°C HEPA-filtered environment with 3040% humidity and a 12-h light/dark cycle. Animals were allowed free access to deionized water and Harlen Teklad 22/5 Rodent Diet 8640 (Madison, WI). Mice were acclimatized for 4 days prior to dosing. On the fourth day, animals were weighed, and doses of EE and B[a]P were prepared in sesame oil (Loriva, Ronkonkoma, NY) based on the average weight of the animals. Beginning the following day, each mouse was gavaged with a 0.1-cc dose of 0.1 mg/kg/day EE, or 0.1, 1, or 10 mg/kg/day B[a]P for 3 consecutive days. At 24 h following the third treatment, animals were sacrificed by cervical dislocation, and uteri were excised, separated from attached connective tissue, blotted, weighed, and stored in RNAlater storage solution (Ambion Inc., Austin, TX) at 80°C until further use. Studies with 3- and 9-OH-B[a]P were performed as described above for B[a]P, except that solutions were injected subcutaneously with a 25-gauge needle at a dose level of 1, 5, 10, or 20 mg/kg/day.
RT-PCR of lactoferrin mRNA.
Uteri were thawed in the storage solution, transferred to a tube containing 0.5 ml Trizol (Life Technologies), minced with sterile scissors, and homogenized using a Polytron tissue homogenizer (Kinematica, Lucerne, Switzerland) for three pulses of 15 s each at 85% output. Total RNA extraction was performed by the Trizol method according to the manufacturer's instructions.
Samples were reverse transcribed and then PCR amplified using specific primers for lactoferrin or for ß-actin in separate tubes. The primers used were as follows, with restriction enzyme sites (to allow subcloning and sequencing to verify the identity of the amplified product) underlined and indicated in parentheses: LF forward 5`-aaaagaattcggatccgggacacttcgtccatacctgaa-3` (EcoRI, BamHI); LF reverse 5`-aaaactcgaggcggccgcgctatcacatcctgctgctttttat-3` (XhoI, NotI); A forward 5`-aaaaggatccaagcttctgaagtaccattgaacatggca-3` (BamHI, HindIII); A reverse 5`-aaaactcgaggcggccgctgtcacgcacgatttccctctcag-3` (XhoI, NotI).
The lactoferrin primer pair was designed to amplify a 632 bp product from nt 436 to 1067 of the cDNA, and the actin primer pair was designed to amplify a 436 bp product from nt 121 to 556. A 1 µg aliquot of RNA was incubated with 20 pmol reverse primer for 12 min at 70°C, cooled quickly on ice, then mixed with First Strand Buffer, 200 IU SuperScript II reverse transcriptase, 10 nmol each dNTP, 200 nmol DTT, and 20 IU RNase inhibitor, in a 20 µl final reaction volume. The mixture was then incubated for 50 min at 42°C followed by 15 min at 70°C using a RoboCycler Gradient 96 PCR machine (Stratagene, La Jolla, CA).
Lactoferrin and actin forward primers were end labeled with [32P]dATP to facilitate quantitation of PCR products. A mixture of PNK buffer, 75 IU PNK, 50 nmol of primer, and 5 µl [
32P]dATP in DEPC-treated water was incubated at 37°C for 30 min, an additional 50 IU of PNK was added, and the mixture was incubated another 30 min. Final reaction volume was 300 µl. Unincorporated nucleotides were removed using a MicroSpin G-25 spin column (Amersham/Pharmacia) according to the manufacturer's directions.
A 2-µl aliquot (10%) of the cDNA product was then combined with PCR Buffer, 2.5 IU Taq DNA polymerase, 75 nmol MgCl2, 10 nmol each dNTP, 20 pmol reverse primer, 5 pmol labeled forward primer, and 15 pmol unlabeled forward primer. The 50 µl reaction mixture was subjected to the following cycling temperature program: 3 min at 94°C; 24 cycles of 30 sec at 94°C, 30 sec at 67°C, 60 sec at 72°C; 10 min at 72°C. Samples were run on a 5% acrylamide gel, which was dried and exposed to a Molecular Dynamics (Sunnyvale, CA) storage phosphor screen for 16 h. The signals were quantitated using a Molecular Dynamics Storm 820 scanner and ImageQuaNT v. 4.2a software. Lactoferrin levels were normalized using ß-actin mRNA levels, which were assumed to be unaffected by treatment.
Statistical analyses.
Increases in uterine weight above that of vehicle-treated controls were determined using an analysis of covariance with body weight at time of sacrifice as a covariate, followed by the Fischer's multiple pairwise comparison procedure of Systat v. 5.04 (Systat, Inc., Evanston, IL). A log transformation was performed if group variances (body weight or uterine weight) had p < 0.01 using Bartlett's test for homogeneity.
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RESULTS |
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Uterotrophic Assay and Expression of Uterine Lactoferrin mRNA
Mice were dosed with B[a]P by oral gavage to simulate dietary exposure. Although EE (0.1 mg/kg) caused a statistically significant increase in uterine wet weight (6-fold; p < 0.001) in both the DBA/2 and C57BL/6 strains, B[a]P was not found to cause an increase in uterine wet weight or uterine lactoferrin mRNA expression at any of the concentrations studied (Table 2 and Fig. 5
). Similarly, when 3- and 9-OH-B[a]P were administered to mice by subcutaneous injection, in order to bypass first-pass metabolic effects, EE caused a 6-fold and 10-fold increase in uterine weight in DBA/2 and C57BL/6 mice, respectively (p < 0.05), whereas no response was seen in 3- and 9-OH-B[a]P-treated mice, as summarized in Table 3
. This lack of response was also reflected in the absence of changes in uterine lactoferrin mRNA in either mouse strain, as shown in Figure 5
. EE, B[a]P, and B[a]P metabolites were not found to adversely affect body weight during the dosing periods (data not shown), and body weight was not found to be a significant covariate in any of the experiments at the p < 0.02 level of significance.
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DISCUSSION |
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B[a]P itself was not able to bind to either receptor isoform, but induced hER- and mERß-mediated luciferase reporter expression in MCF-7 cells. The ability of monohydroxylated B[a]P metabolites to bind and induce gene expression mediated through both ER isoforms suggests that these metabolites are responsible for the increase in reporter gene expression observed in MCF-7 cells treated with B[a]P. This is consistent with previous studies reporting that B[a]P is not able to compete appreciably with [3H]E2 for binding to recombinant full-length hER
(Tran et al., 1996
), but is able to compete for ER binding in an MCF-7 whole-cell binding assay (Arcaro et al., 1999
). Similar results would be expected using hERß rather than mERß, as these two receptors share a high degree of similarity (89% amino acid identity in the DEF region).
Though it is known that the P450 content of cultured cells may differ greatly from that of the cells of an intact organism (Dipple and Bigger, 1983), MCF-7 cells are known to express several P450 isozymes, including 1A1, 1A2, and 1B1 (Li et al., 1998
; Spink et al., 1998
), and to exhibit aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase activity (Harris et al., 1989
). The data presented here suggest that B[a]P-exposed MCF-7 cells are biotransforming the compound to estrogenic metabolites. It has recently been shown that MCF-7 cells leave much of the B[a]P unmetabolized, but also produce several hydroxylated metabolites including 3- and 9-OH-B[a]P, and that Gal4-hER
defmediated induction of reporter gene expression caused by these two metabolites is abrogated by treatment with
-naphthoflavone, a P450 inhibitor (Charles et al., 2000
). Dihydrodiol and dione metabolites were ineffective at either binding or inducing ER-mediated gene expression. The finding that monohydroxylated B[a]P metabolites act as weak ER agonists is consistent with studies showing that high-affinity ER ligands such as E2 and the hydroxylated PAH 3,9-dihydroxy-benz[a]anthracene tend to possess a hydroxyl group at each end of the molecule (Schneider et al., 1976
), while the removal of either hydroxyl group from E2 results in detectable but greatly reduced estrogenic activity (Chernayaev et al., 1975
; Odum et al., 1997
).
The effects of B[a]P were also examined in vivo to evaluate its ER-mediated effects in the context of an intact organism, which includes serum binding proteins and a full complement of oxidative and conjugative enzymes of metabolism. Increased uterine weight, which is considered to be a hallmark of E2 activity, and increased lactoferrin mRNA expression are both accepted, sensitive markers of estrogen action (Korach and McLachlan, 1995; Odum et al., 1997
; Teng et al., 1989
). Orally administered B[a]P did not cause a detectable increase in uterine weight or lactoferrin mRNA levels in ovariectomized immature C57BL/6 (PAH-responsive) or DBA/2 (PAH-nonresponsive) mouse strains at the doses used. The use of differentially responsive mouse strains is reflective of the heterogeneity of metabolic pathways in the human population (Camus et al., 1984
), where responsiveness refers to ability of ligandaryl hydrocarbon receptor (AhR) complexes to induce the expression of specific P450 isozymes. The resulting increased P450 activity induces metabolism of a variety of PAHs that are ligands for the AhR, including B[a]P (Bigelow and Nebert, 1982
; Piskorska-Pliszczynska et al., 1986
). As a result, these compounds are able to induce their own metabolism, particularly in animals possessing highly inducible CYP1A1, such as the C57BL/6 strain (Harper et al., 1991
; Niwa et al., 1975
).
B[a]P is known to be distributed rapidly within the body (Moir et al., 1998), and its metabolites can readily penetrate cell membranes (Brown and Chee, 1992
; Pelkonen and Nebert, 1982
). Although a large proportion of administered B[a]P remains unmetabolized (Moore and Gould, 1984
), 3- and 9-OH-B[a]P are known to be major metabolites in C57BL/6 and DBA/2 mouse strains, whether B[a]P is given as a single dose or is able to cause induction of P450 enzymes by treatment over a period of several days (Legraverend et al., 1984
; Wang et al., 1976
). The lack of uterotrophic or lactoferrin mRNA induction effect observed in B[a]P-treated mice in the present study therefore suggests one or more of the following:
The lack of response following sc injection of up to 20 mg/kg of 3- or 9-OH-B[a]P is evidence against the first possibility and suggests that the metabolites are excreted quickly or are not acting as estrogens on the specific uterine end points examined.
These results show that ER binding and ER-mediated gene expression studies of B[a]P did not accurately predict the lack of observed in vivo estrogenicity in the C57BL/6 or DBA/2 mouse uterus. Moreover, though the present study provided little evidence of nonadditive interactions between B[a]P metabolites and E2 in MCF-7 cells, other researchers have reported contrasting findings. For example, it has been reported that B[a]P can antagonize the estrogenic activities of E2 in yeast expressing hER (Tran et al., 1996
) and cause a decrease in E2-induced MCF-7 cell proliferation (Arcaro et al., 1999
) and nuclear ER levels (Chaloupka et al., 1992
). It has been hypothesized that many of the antiestrogenic effects observed with PAHs are mediated through the AhR. B[a]P and many other PAHs exhibit high binding affinity for the AhR (Bigelow and Nebert, 1982
; Piskorska-Pliszczynska et al., 1986
), a ligand-dependent transcription factor that modulates the expression of specific genes by binding to the xenobiotic response element (XRE, also known as DRE). Binding of a PAH or other ligand to the AhR could produce an antiestrogenic response by increasing the rate at which E2 is transformed to less active metabolites, or by allowing the AhR-ligand complex to alter the transcription rate of E2-inducible genes by binding to XRE sequences in the promoters of these genes (Duan et al., 1999
; Gillesby and Zacharewski, 1998
; Zacharewski and Safe, 1998
).
Importantly, the published reports demonstrating an antiestrogenic effect of B[a]P do not account for interactions of B[a]P metabolites with ERß, which has been shown to be absent or nearly absent in MCF-7 cells (Kuiper et al., 1997). hER
and hERß are fairly well conserved in the regions of the LBD forming the ligand binding pocket, and although some studies have reported that many compounds display a similar affinity for each isoform, others have demonstrated significant differences with certain ligands (Barkhem et al., 1998
; Kuiper et al., 1997
; Pike et al., 1999
). In addition, ERß has been found to have a slightly lower affinity for the ERE, as well as for E2, compared with ER
, although their abilities to transactivate reporter genes when bound to E2 are very similar (MacGregor and Jordan, 1998
). The in vitro preference of monohydroxylated B[a]P metabolites for ERß over ER
in the present study, as well as the distinct behavior of the two isoforms in the cotreatment experiments, is therefore of particular interest, as the extent to which these two isoforms have overlapping or distinct roles is currently an area of intense research. Interestingly, in this study individual metabolites showed distinct behaviors that emphasize the importance of hydroxyl group placement, as illustrated, for example, by the greater EC50 value for 3-OH-B[a]P relative to its IC50 in the ERß test systems, whereas the reverse was true for 7- and 9-OH-B[a]P. The lack of correlation between relative binding affinity and the level of reporter gene induction may be due, in part, to ligand-induced allosteric changes that affect ER complex:DNA interactions (Christman et al., 1995
). The ligand may also induce conformational changes that affect interactions with coactivators (Paige et al., 1999
), which could influence the induction of gene expression. For example, genistein exhibited a 30-fold greater affinity for hERß relative to hER
, but induced comparable levels of hER
- and hERß-mediated reporter gene expression (Kuiper et al., 1997
, 1998
). This may partially explain the lack of correlation between relative binding affinity and gene expression observed for some B[a]P metabolites.
Moreover, the heightened combined ability of B[a]P (through its metabolites) and E2 to induce reporter gene expression in the ER compared to the ERß test system was surprising. B[a]P affects a number of cellular processes, and therefore the apparent synergistic effects may be due to cross talk between mechanisms that converge at ER
but not ERß. Although both receptors have a similar structure, hERß shares only 58% amino acid sequence identity with hER
in the ligand binding domain (Domain E) (Pace et al., 1997
). This difference may partially account for the enhanced activity with ER
but not ERß.
No evidence of estrogenic effects of B[a]P was found in the mouse uterus, suggesting that in vitro studies of estrogenic activity should be interpreted with caution. Furthermore, these findings suggest that as further information is gained regarding the biological properties of ERß, appropriate in vivo studies should be designed that examine effects in tissues such as the ovaries and bone, which, unlike the uterus, express high levels of ERß. Future studies will be designed to clarify these issues, with the aim of developing in vitro assays that accurately predict in vivo estrogenic responses.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J.-A., and Nilsson, S. (1998). Differential response of estrogen receptor alpha and estrogen receptor beta to partial estrogen agonists/antagonists. Mol. Pharmacol. 54, 105112.
Bigelow, S. W., and Nebert, D. W. (1982). The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor. Toxicol. Lett. 10, 109118.[ISI][Medline]
Brasier, A. R., Tate, J. E., and Habener, J. F. (1989). Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7, 11161122.[ISI][Medline]
Brown, S., and Chee, C. T. Y. (1992). Benzopyrene metabolism and transport by cells in culture. Biochem. Soc. Trans. 20, 279S.[Medline]
Brzozowski, A. M., Pike, A. C. W., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, O., Ohman, 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]
Camus, A.-M., Aitio, A., Sabadie, N., Wahrendorf, J., and Bartsch, H. (1984). Metabolism and urinary excretion of mutagenic metabolites of benzo[a]pyrene in C57 and DBA mice strains. Carcinogenesis 5, 3539.[Abstract]
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]
Charles, G. D., Bartels, M. J., Zacharewski, T. R., Gollapudi, B. B., Freshour, N. L., and Carney, E. W. (2000). Activity of benzo[a]pyrene and its hydroxylated metabolites in an ER reporter gene assay. Toxicol. Sci. 55, 320326.
Chernayaev, G. A., Barkova, T. I., Egorova, V. V., Sorokina, I. B., Ananchenko, S. N., Mataradze, G. D., Sokolova, N. A., Rozen, V. B. (1975). A series of optical structural and isomeric analogs of estradiol: A comparative study of the biological activity and affinity to cytosol receptor of rabbit uterus. J. Steroid. Biochem. 6, 14831488.[ISI][Medline]
Christman, J. K., Nehls, S., Polin, L., and Brooks, S. C. (1995). Relationship between estrogen structure and conformational changes in estrogen receptor/DNA complexes. J. Steroid. Biochem. Mol. Biol. 54, 201210.[ISI][Medline]
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]
Cook, J. W., and Dodds, E. C. (1933). Sex hormones and cancer-producing compounds. Nature 131, 205206.
Dipple, A., and Bigger, C. A. H. (1983). Metabolic properties of in vitro systems. Ann. N. Y. Acad. Sci. 407, 2633.[ISI][Medline]
Duan, R., Porter, W., Samudio, I., Vyhlidal, C., Kladde, M., and Safe, S. (1999). Transcriptional activation of c-fos protooncogene by 17ß-estradiol: Mechanism of aryl hydrocarbon receptor-mediated inhibition. Mol. Endocrinol. 13, 15111521.
Ebright, R. H., Wong, J. R., and Chen, L. B. (1986). Binding of 2-hydroxybenzo[a]pyrene to estrogen receptors in rat cytosol. Cancer Res. 46, 23492351.[Abstract]
Estabrook, R. W., Saeki, Y., Chacos, N., Capdevila, J., and Prough, R. A. (1980). Polycyclic hydrocarbon metabolism: a plethora of phenomena. Adv. Enzyme Regul. 19, 317.[Medline]
Gelboin, H. V. (1980). Benzopyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes. Physiol. Rev. 60, 11071166.
Giguère, V., Tremblay, A., and Tremblay, G. B. (1998). Estrogen receptor beta: re-evaluation of estrogen and antiestrogen signaling. Steroids 63, 335339.[ISI][Medline]
Gillesby, B. E., and Zacharewski, T. R. (1998). Exoestrogens: Mechanisms of action and strategies for identification and assessment. Environ. Toxicol. Chem. 17, 314.[ISI]
Gillesby, B. E., and Zacharewski, T. R. (1999). pS2 (TFF1) mRNA levels in human breast tumor samples: Correlation with clinical and histological prognostic markers. Breast Cancer Res. Treat. 56, 253265.[ISI][Medline]
Grimmer, G., Brune, H., Dettbarn, G., Heinrich, U., Jacob, J., Mohtashamipur, E., Norpoth, K., Pott, F., and Wenzel-Hartung, R. (1988). Urinary and faecal excretion of chrysene and chrysene metabolites by rats after oral, intraperitoneal, intratracheal or intrapulmonary application. Arch. Toxicol. 62, 401405.[ISI][Medline]
Grover, P. L., MacNicoll, A. D., Sims, P., Easty, G. C., and Neville, A. M. (1980). Polycyclic hydrocarbon activation and metabolism in epithelial cell aggregates prepared from human mammary tissue. Int. J. Cancer 26, 467475.[ISI][Medline]
Hall, M., Forrester, L. M., Parker, D. K., Grover, P. L., and Wolf, C. R. (1989). Relative contribution of various forms of cytochrome P450 to the metabolism of benzo[a]pyrene by human liver microsomes. Carcinogenesis 10, 18151821.[Abstract]
Harper, P. A., Golas, C. L., and Okey, A. B. (1991). Ah receptor in mice genetically "nonresponsive" for cytochrome P4501A1 induction: cytosolic Ah receptor, transformation to the nuclear binding state, and induction of aryl hydrocarbon hydroxylase by halogenated and nonhalogenated aromatic hydrocarbons in embryonic tissues and cells. Mol. Pharmacol. 40, 818826.[Abstract]
Harris, M., Piskorska-Pliszczynska, J., Zacharewski, T., Romkes, M., and Safe, S. (1989). Structure-dependent induction of aryl hydrocarbon hydroxylase in human breast cancer cell lines and characterization of the Ah receptor. Cancer Res. 49, 45314535.[Abstract]
Hattemer-Frey, H. A., and Travis, C. C. (1991). Benzo[a]pyrene: environmental partitioning and human exposure. Toxicol. Ind. Health 7, 141157.[ISI][Medline]
Huggins, C. B., Pataki, J., and Harvey, R. G. (1967). Geometry of carcinogenic polycyclic aromatic hydrocarbons. Proc. Natl. Acad. Sci. U.S.A. 58, 22532260.[ISI][Medline]
Jacob, J., and Grimmer, G. (1996). Metabolism and excretion of polycyclic aromatic hydrocarbons in rat and in human. Cent. Eur. J. Public Health 4, 3339.
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 [published erratum appears in Carcinogenesis (1999) 20(3), 515]. Carcinogenesis 19, 18471853.[Abstract]
Korach, K. S. (1994). Insights from the study of animals lacking functional estrogen receptor. Science 266, 15241527.[ISI][Medline]
Korach, K. S., and McLachlan, J. A. (1995). Techniques for detection of estrogenicity. Environ. Health Perspect. 103, 58.[ISI]
Kuiper, G. G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J.-A. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors and ß. Endocrinology 138, 863870.
Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B., and Gustafsson, J.-A. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 42524263.
Larsen, M. C., Angus, W. G., Brake, P. B., Eltom, S. E., Sukow, K. A., and Jefcoate, C. R. (1998). Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: Role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism. Cancer Res. 58, 23662374.[Abstract]
Legraverend, C., Guenthner, T. M., and Nebert, D. W. (1984). Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity. Teratology 29, 3547.[ISI][Medline]
Li, W., Harper, P. A., Tang, B. K., and Okey, A. B. (1998). Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene. Biochem. Pharmacol. 56, 599612.[ISI][Medline]
MacGregor, J. I., and Jordan, V. C. (1998). Basic guide to the mechanisms of antiestrogen action. Pharmacol. Rev. 50, 151196.
Matthews, J., and Zacharewski, T. (2000). Differential binding affinities of PCBs, HO-PCBs and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss) and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay. Toxicol. Sci. 53, 326339.
Mattison, D. R., Singh, H., Takizawa, K., Thomford, P. J. (1989). Ovarian toxicity of benzo(a)pyrene and metabolites in mice. Reprod. Toxicol. 3, 115126.[ISI][Medline]
Moir, D., Viau, A., Chu, I., Withey, J., and McMullen, E. (1998). Pharmacokinetics of benzo[a]pyrene in the rat. J. Toxicol. Environ. Health 53, 507530.[ISI]
Moore, C. J., and Gould, M. N. (1984). Differences in mediated mutagenesis and polycyclic aromatic hydrocarbon metabolism in mammary cells from pregnant and virgin rats. Carcinogenesis 5, 103108.[Abstract]
Niwa, A., Kumaki, K., Nebert, D. W., and Poland, A. P. (1975). Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse. Distinction between the "responsive" homozygote and heterozygote at the Ah locus. Arch. Biochem. Biophys. 166, 559564.[ISI][Medline]
Odum, J., Lefevre, P. A., Tittensor, S., Paton, D., Routledge, E. J., Beresford, N. A., Sumpter, J. P., Ashby, J. (1997). The rodent uterotrophic assay: Critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay. Regul. Toxicol. Pharmacol. 25, 176188.[ISI][Medline]
Pace, P., Taylor, J., Suntharalingam, S., Coombes, R. C., and Ali, S. (1997). Human estrogen receptor beta binds DNA in a manner similar to and dimerizes with estrogen receptor alpha. J. Biol. Chem. 272, 2583225838.
Paige, L. A., Christensen, D. J., Gron, H., Norris, J. D., Gottlin, E. B., Padilla, K. M., Chang, C.-Y., Ballas, L. M., Hamilton, P. T., McDonnell, D. P., Fowlkes, D. M. (1999). Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc. Natl. Acad. Sci. U.S.A. 96, 39994004.
Parke, D. V., and Ioannides, C. (1990). The role of metabolism studies in the safety evaluation of new chemicals. Acta Pharm. Jugosl. 40, 363382.[ISI]
Pelkonen, O., and Nebert, D. W. (1982). Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol. Rev. 34, 189222.[ISI][Medline]
Pike, A. C., Brzozowski, A. M., Hubbard, R. E., Bonn, T., Thorsell, A.-G., Engstrom, O., Ljunggren, J., Gustafsson, J.-A., Carlquist, M. (1999). Structure of the ligand-binding domain of oestrogen receptor ß in the presence of a partial agonist and a full antagonist. Embo J. 18, 46084618.
Piskorska-Pliszczynska, J., Keys, B., Safe, S., and Newman, M. S. (1986). The cytosolic receptor binding affinities and AHH induction potencies of 29 polynuclear aromatic hydrocarbons. Toxicol. Lett. 34, 6774.[ISI][Medline]
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.). Cold Spring Harbor Laboratory Press, Plainview, NY.
Schneider, S. L., Alks, V., Morreal, C. E., Sinha, D. K., and Dao, T. L. (1976). Estrogenic properties of 3,9-dihydroxybenz[a]anthracene, a potential metabolite of benz[a]anthracene. J. Natl. Cancer Inst. 57, 13511354.[ISI][Medline]
Selkirk, J. K., MacLeod, M. C., Mansfield, B. K., Nikbakht, P. A., and Dearstone, K. C. (1983). Species heterogeneity in the metabolic processing of benzo[a]pyrene. Basic Life Sci. 24, 283294.[Medline]
Shou, M., Korzekwa, K. R., Crespi, C. L., Gonzalez, F. J., and Gelboin, H. V. (1994). Metabolism of benzo[a]pyrene by seven cDNA expressed human cytochromes P450. Polycycl. Arom. Comp. 7, 19.
Spink, D. C., Spink, B. C., Cao, J. Q., DePasquale, J. A., Pentecost, B. T., Fasco, M. J., Li, Y., and Sutter, T. R. (1998). Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 19, 291298.[Abstract]
Swartz, W. J., and Mattison, D. R. (1985). Benzo(a)pyrene inhibits ovulation in C57BL/6N mice. Anat. Rec. 212, 268276.[ISI][Medline]
Teng, C. T., Pentecost, B. T., Chen, Y. H., Newbold, R. R., Eddy, E. M., and McLachlan, J. A. (1989). Lactotransferrin gene expression in the mouse uterus and mammary gland. Endocrinology 124, 992999.[ISI][Medline]
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, 102108.[ISI][Medline]
van Schooten, F. J., Moonen, E. J. C., van der Wal, L., Levels, P., and Kleinjans, J. C. S. (1997). Determination of polycyclic aromatic hydrocarbons (PAH) and their metabolites in blood, feces, and urine of rats orally exposed to PAH contaminated soils. Arch. Environ. Contam. Toxicol. 33, 317322.[ISI][Medline]
Wang, I. Y., Rasmussen, R. E., Petrakis, N. L., and Wang, A.-C. (1976). Enzyme induction and the difference in the metabolite patterns of benzo[a]pyrene produced by various strains of mice. In Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis (R. I. Freudenthal and P. W. Jones, Eds.), Vol. 1, pp. 7789. Raven Press, New York.
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., and Safe, S. (1998). Antiestrogenic activity of TCDD and related compounds. In Modulation of Endocrine Systems by Xenobiotics (K. Korach, Ed.). pp. 431448. Marcel Dekker, Inc., New York.