The cooked food derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine is a potent oestrogen: a mechanistic basis for its tissue-specific carcinogenicity

Sandra N. Lauber, Simak Ali1 and Nigel J. Gooderham2

Molecular Toxicology, Biomedical Sciences and 1 Cancer Medicine, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK

2 To whom correspondence should be addressed at: Molecular Toxicology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, London SW7 2AZ, UK. Tel: +44 20 7594 3188; Fax: +44 20 7594 3050; Email: n.gooderham{at}ic.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces tumours of the breast, colon and prostate in rats. Here we show that in addition to its well-established genotoxicity, which can be detected at concentrations >10–6 M, PhIP is also oestrogenic. In COS-1 cells transiently transfected with an oestrogen-responsive reporter gene, PhIP (10–10–10–6 M) mediated transcription through oestrogen receptor (ER) {alpha}, but not ER-ß, and inhibition by the pure ER antagonist ICI 182 780 demonstrated a requirement for a functional ER. In contrast, the structurally related food-derived carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) failed to induce reporter gene transcription. Additionally, we show that in a hormonally responsive breast cancer cell line (MCF-7 cells), PhIP induced transcriptional activation using endogenously expressed ER. Examination of the genotoxic potential of PhIP using a model mammalian cell mutation assay (hprt locus) demonstrated that the genetic toxicology of PhIP was readily detectable, but separate, in terms of effective concentration, from its oestrogenic activity. To determine whether the oestrogenicity of PhIP could mediate oestrogen-dependent responses such as cell growth, we examined the growth of hormonally responsive cells (MCF-7 cells). We show that PhIP can stimulate cell proliferation and, again, this was dependent upon a functional ER. Using ligand blotting, we further show that PhIP can stimulate the expression of progesterone receptor (PR-A and PR-B) and c-MYC and activate the MAPK signal transduction pathway. These responses were similar to that produced by oestradiol, in terms of temporal aspects, potency and a requirement for a functional ER. Each of these dose-dependent mitogenic responses occurred at concentrations of PhIP (~10–9–10–11M) that are likely to be equivalent to systemic human exposure via consumption of cooked meat. Thus PhIP can induce cellular responses that encompass altered gene expression and mitogenesis. We suggest that the combination of genetic toxicology and oestrogen-like promotion of genomic and cellular events provide a mechanism for the tissue-specific tumorigenicity of this compound.

Abbreviations: CAT, chloramphenicol acetyltransferase; DCC-FCS, dextran-coated charcoal-stripped fetal calf serum; DMEM, Dulbecco-Vogt's modified Eagle's medium; E2, 17-ß-oestradiol; ELISA, enzyme-linked immunosorbent assay; EMS, ethyl methane sulphonate; ER, oestrogen receptor; ERE, oestrogen-responsive element; ERK, extracellular regulated kinase; FBS, fetal bovine serum; FCS, fetal calf serum; ß-gal, ß-galactosidase; HA, heterocyclic amines; MAPK, mitogen-activated protein kinase; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; MEM, minimal essential medium; pen/strep, 100 ß-gal IU/ml penicillin/100 µg/ml streptomycin; PBS, phosphate-buffered saline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PR, progesterone receptor; 6-TG, 6-thioguanine


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiology has identified diet as a major factor in the aetiology of cancer (1) and particularly the consumption of meat (2). The cooking of meat generates a family of genotoxic compounds known as the heterocyclic amines (HA) (3). One of the most abundantly formed cooked meat HA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), is readily activated to its mutagenic N-hydroxy derivative by human CYP1 enzymes (4). PhIP is carcinogenic in animal models, where it induces colon, prostate and breast cancers (5,6). In particular, PhIP has been shown to preferentially induce breast over colon cancer in female rats. The finding that PhIP specifically induces mammary tumours in female rats is interesting when taken together with the recent demonstration that in human mammary epithelial cells N-hydroxylated PhIP can act as a substrate for oestrogen sulfotransferase, resulting in a highly activated metabolite that covalently binds to DNA (7). These observations suggest the possibility that PhIP shows sufficient similarity to oestrogen to be recognized by other proteins that bind oestrogen.

Oestrogens elicit important cellular responses in many target tissues, including the reproductive organs, bone and the cardiovascular system, and are involved in some brain functions. Oestrogens have also been implicated in the development and/or regulation of malignancies associated with the breast (8), ovaries (9), endometrium (10), prostate (11) and colon (12). In the case of breast cancer there is evidence linking prolonged exposure to oestrogens with increased risk of breast cancer. Risk factors include early menarche, late first full-term pregnancy, late menopause, the use of certain oral contraceptives and oestrogen replacement therapy (8).

The principal effects of oestrogen are mediated by oestrogen receptor (ER)-{alpha} and ER-ß, members of the nuclear receptor superfamily of transcription factors, which regulate the expression of target genes upon binding to cognate ligands. As demonstrated by gene knockout studies, ER-{alpha} is required for reproductive development in females, including normal mammary gland development (13), whereas ER-ß knockout mice are apparently normal for mammary gland development, although recent evidence suggests that terminal mammary gland differentiation may be affected in these mice (14). Approximately two-thirds of breast tumours are ER-{alpha}-positive, while ER-ß-positivity correlates with response to endocrine therapies, comprising competitive oestrogen antagonists such as tamoxifen (15), as well as aromatase inhibitors that prevent oestrogen synthesis, such as anastrazole (8,16). A significant role for ER-ß in breast cancer is less clear, although it is present in most primary breast tumours. As well as being important in breast cancer progression, a role for ER-{alpha} in breast cancer initiation has been suggested by the finding that ER-{alpha} overexpression in benign breast epithelium correlates with increased breast cancer risk (17). In addition to the link between prolonged lifetime exposure to oestrogens and an increased risk of breast cancer, it has been proposed that oestrogenic chemicals in foods, as well as industrial chemicals, may contribute to breast cancer risk (18). Here we show that PhIP can stimulate ER-{alpha}-induced activation of oestrogen-regulated genes and induce a proliferative response in oestrogen-dependent MCF-7 breast cancer cells. We therefore hypothesize that in addition to its mutagenic effects, PhIP may act as an oestrogen to influence the development of oestrogen-dependent cancer.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
COS-1 green monkey kidney fibroblast cells and MCF-7 and T47D human breast cancer cells were obtained from the European Collection of Cell Cultures (Porton Down, UK), while V79 h1A2 cells were a kind gift from Dr J.Doehmer (Institut für Toxikologie und Umwelthygeine, Technische Universität, München, Germany). Dulbecco-Vogt's modified Eagle's medium (DMEM), minimal essential medium (MEM), phenol red-free DMEM and MEM, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin were obtained from Invitrogen (Paisley, UK). PhIP and 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) were purchased from Toronto Research Chemicals (Toronto, Canada) and ICI 182,780 from AstraZeneca (Macclesfield, UK). Vectors were kindly provided by Professor P.Chambon (Universite Louis Pasteur, Strasbourg, France). Chloramphenicol acetyltransferase (CAT) and ß-galactosidase (ß-gal) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boehringer-Mannheim (UK). Antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The Dual-luciferase Reporter Assay System was purchased from Promega (Southampton, UK). All other chemicals and reagents were obtained from Sigma (Poole, UK).

Cell culture
COS-1 cells (European Collection of Cell Cultures) were propagated in DMEM supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and 100 IU/ml penicillin/100 µg/ml streptomycin (pen/strep). MCF-7 cells (European Collection of Cell Cultures) were maintained in MEM containing 10% FCS, 2 mM L-glutamine, 1% non-essential amino acids and pen/strep. T47D cells were maintained in RPMI 1640 supplemented with 5% FCS, 2 mM L-glutamine and pen/strep. MELN cells (a gift from Dr P.Balaguer, INSERM, Montpellier, France) were routinely maintained in DMEM supplemented with 5% FBS and pen/strep. For experiments, cells were transferred to phenol red-free medium in which the FCS was stripped with dextran-coated charcoal (DCC-FCS), to remove all oestrogenic influences. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2.

Transcriptional activation of the ER-dependent reporter gene 17-M-ERE-G-CAT
COS-1 cells seeded in phenol red-free DMEM supplemented with 5% DCC-FCS were transfected by calcium phosphate co-precipitation with 0.4 µg of an oestrogen-responsive reporter construct (17-M-ERE-G-CAT), 0.1 µg of either a wild-type human ER-{alpha} construct (HEG0) or ER-ß construct (hERß), along with 0.05 µg of ß-gal expression vector (pSGßlacZ). Vectors were kindly provided by Prof. P.Chambon (Strasbourg, France) and have been described elsewhere (19). Bluescript vector M13+ DNA (BSM; Stratagene) was used as carrier DNA to make a total of 2 µg DNA/well. Ligands dissolved in ethanol, were added 30 min after the addition of the precipitates. In some experiments, the cells were pretreated for 30 min with the pure anti-oestrogen ICI 182,780 (10–7 M) before addition of the ligands. 17-ß-Oestradiol (E2) at a concentration of 10–7 M was used as the positive control. The precipitates were removed 16 h post-transfection by washing in medium and fresh ligands were added. Cells were harvested after a further 24 h and assayed for CAT protein using a CAT ELISA kit (Boehringer-Mannheim). Transfection efficiency was normalized using the internal ß-gal control. To examine whether PhIP could utilize an endogenous ER, MCF-7 cells (which constitutively express ER-{alpha}) were transfected with 17-M-ERE-G-CAT alone and treated with PhIP or E2.

Transcriptional activation of the integrated ERE–luciferase reporter gene in MELN cells
MELN cells were used for studies of transcriptional activation of integrated ERE reporter genes via constitutively expressed ER-{alpha}. MELN cells are derived from MCF-7 cells (which constitutively express ER-{alpha}) stably transfected with the ERE-ßGlob-Luc-SVNeo plasmid (20). Cells were plated at a density of 50 000/well in 24-well plates in phenol-red free DMEM supplemented with 5% dextran-coated charcoal-stripped fetal calf serum (DCC-FCS) and pen/strep, 24 h prior to treatment. The following day the seeding medium was removed and replaced with medium containing ligands. Ligands were prepared by serial dilution in phenol red-free DMEM supplemented with 5% DCC-FCS and pen/strep, to give a final concentration of 10–7–10–12 M ligand. The volume of medium placed in each well was 500 µl. Cells were treated with ligands for 24 h, then luciferase activity was determined using Promega's Dual-Luciferase® Reporter Assay System.

Cell proliferation assay
Cells (MCF-7) were seeded into 24-well plates at 1 x 105 cells/well in phenol red-free MEM supplemented with 5% DCC-FCS. After 24 h the medium was changed and the cells were treated with different concentrations of PhIP and E2 in the presence or absence of ICI 182,780. After 2 days the ligands were washed off and replaced with fresh medium. The cells were allowed to grow for a further 4 days, after which the relative cell number was estimated using the resazurin reduction assay (21).

Mammalian cell hprt locus mutagenicity assay
The mammalian cell hprt locus mutagenicity assay was performed following the method of Yadollahi-Farsani et al. (22). The cells used in the assay were V79 h1A2 cells, which are V79 Chinese hamster cells genetically engineered for stable expression of human cytochrome P450 1A2 (23).

Cell survival was measured by clonogenicity assay, assessing the colony-forming ability of treated cells under non-selective conditions. Colonies comprising >50 cells (clones of ~1.5 mm diameter) were scored and recorded as colony-forming survivors.

Resistance to the lethal effects of 6-thioguanine (6-TG) (5 µg/ml) was used to select for hprt mutant clones. Colonies comprising >50 cells (clones of ~1.5 mm diameter) were scored and recorded as hprt mutants.

SDS–PAGE and ligand blotting
For determination of progesterone receptor and extracellular regulated kinase (ERK), cells were scraped from flasks in the presence of 1 ml of extraction buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, pH 7.4, 1 mM sodium fluoride and 50 µg/ml leupeptin) and incubated at 4°C for 15 min. For determination of c-MYC, cells were harvested by lysis in NP40 buffer (10 mM Tris–HCl, pH 7.4, 10 mM sodium chloride, 3 mM magnesium chloride, 0.5% NP40) to isolate the nuclei. Lysates were cleared by centrifugation at 14 000 g for 15 min at 4°C. The supernatants were added to equal volumes of sample buffer (2% SDS, 60 mM Tris, pH 6.8, 10% glycerol, 100 mM dithiothreitol, 0.01% bromophenol blue) and stored at –20°C until required. SDS–PAGE was undertaken using 10–12.5% polyacrylamide gels according to the method of Laemmli (24).

Electrophoresed proteins were transferred to nitrocellulose membranes by electroblot transfer in ice-cold transfer buffer (48 mM Tris base, 39 mM glycine, 20% v/v methanol) at 450 mA for 1 h 15 min. Membranes were incubated in blocking buffer [phosphate-buffered saline (PBS) containing 0.01% Tween 20 and 5% non-fat dry milk] for 1 h at room temperature, then washed in PBS and incubated with primary antibody overnight at 4°C with constant agitation. The following primary antisera were used, rabbit polyclonal anti-human progesterone receptor (PR) (1:1000 dilution), rabbit polyclonal anti-human c-MYC (1:500 dilution), rabbit polyclonal anti-human ERK 1&2 (1:1000 dilution), mouse monoclonal anti-human ppERK 1&2 (1:1000 dilution), mouse monoclonal anti-human ß-actin (1:10 000 dilution). Probed membranes were washed three times for 10 min in PBS containing 0.01% Tween 20 to remove non-specifically bound antibody before being incubated with a peroxidase-conjugated secondary antibody for 1 h (horseradish peroxidase-conjugated goat polyclonal anti-mouse or anti-rabbit antibody) (1:1000–2500 dilution). Proteins were visualized using the Pierce SuperSignal chemi-illuminescence detection system, following the manufacturer's protocol and Hyperfilm (Kodak, UK).

Statistical analysis
One way ANOVA and the Kruskal–Wallis test were used for statistical analysis.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Transcriptional activation of ER-dependent reporter gene 17-M-ERE-G-CAT
Binding of ligand to the ER may not in itself be sufficient to elicit oestrogenic responses; there needs to be transformation and translocation of the ER, followed by binding of co-activators and subsequent transcription. Receptor–ligand binding assays cannot account for such steps and, for this reason, a reporter gene assay was chosen to assess oestrogenic activity. Agonist activity was assessed by ability to induce expression of the reporter gene 17-M-ERE-G-CAT, in which expression of the bacterial CAT gene is placed under transcriptional control of elements of the rabbit ß-globin promoter and an oestrogen-responsive element (ERE).

In cells transfected with ER-{alpha}, PhIP induced transcriptional activation of the CAT reporter gene at concentrations ranging from 10–6 to 10–10 M in a dose-dependent manner (Figure 1A). In the presence of the pure oestrogen antagonist ICI 182,780, reporter gene transcription by PhIP, like E2, was inhibited (Figure 1B). In transfections using only the empty expression vector pSG5 (no ER), neither E2 nor PhIP could activate the target gene (Figure 2A). Together these results demonstrate that gene activation of this ERE-containing model reporter gene by PhIP requires a functional ER. To determine whether PhIP could work via an endogenous ER, the breast cancer cell line MCF-7 (constitutive ER-{alpha} expression) was used; both PhIP (10–7 M) and E2 (10–7 M) were able to stimulate expression of the reporter gene (Figure 2B), demonstrating that constitutively expressed ER-{alpha} could also be used.



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Fig. 1. Transcriptional activation of the ER-dependent reporter gene 17-M-ERE-G-CAT with ER-{alpha} and PhIP in COS-1 cells. (A) In the presence of ER-{alpha}. COS-1 cells were transfected with a wild-type human ER-{alpha} expression vector (HEG0) and an oestrogen-responsive reporter gene (17-M-ERE-G-CAT) in oestrogen-deficient medium and treated with E2 (10–7 M) or PhIP (10–6–10–11 M). Values are means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with ethanol control). (B) Inhibition of transcriptional activation with the anti-oestrogen ICI 182,780. Results are shown as means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with cells treated with ligand alone).

 


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Fig. 2. Transcriptional activation of the ER-dependent reporter gene 17-M-ERE-G-CAT in COS-1 and MCF-7 cells. (A) In the presence of pSG5 or pSG5 HEG0 (ER-{alpha}). COS-1 cells were transfected with an oestrogen-responsive reporter gene (17-M-ERE-G-CAT) and either wild-type human ER-{alpha} expression vector (HEG0) or the empty expression vector pSG5, in oestrogen-deficient medium. They were then treated with E2 (10–7 M) or PhIP (10–7 M). Values are means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with ethanol control). (B) In MCF-7 cells. MCF-7 cells were transfected with an oestrogen-responsive reporter gene (17-M-ERE-G-CAT) and cultured in oestrogen-deficient medium. They were then treated with E2 (10–7 M) or PhIP (10–7 M). Values are means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with ethanol control).

 
The ability of PhIP to act through the second receptor subtype, ER-ß, was investigated using COS-1 cells and transfection with the hERß construct. In this system, PhIP had no activity at any of the concentrations tested, yet E2 produced a good transcriptional response (Figure 3A). Furthermore, the E2-induced response at 10–8 M in these cells was not altered in the presence of a molar excess of PhIP, demonstrating that PhIP was neither an ER-ß agonist nor antagonist.



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Fig. 3. Transcriptional activation of the ER-dependent reporter gene 17-M-ERE-G-CAT with ER-ß and PhIP and ER-{alpha} and MeIQx in COS-1 cells. (A) Inability of PhIP to induce reporter gene transcription in COS-1 cells transfected with ER-ß. Values are means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with ethanol control). (B) MeIQx does not stimulate transcription of the oestrogen-responsive reporter gene 17-M-ERE-G-CAT in COS-1 cells transfected with ER-{alpha}. Values are means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with ethanol control).

 
The oestrogenicity of the related cooked food-derived carcinogenic heterocyclic amine MeIQx was also examined. Unlike PhIP, MeIQx did not significantly stimulate transcription of the reporter gene over the ethanol control in ER-{alpha}-transfected cells (Figure 3B).

Transcriptional activation of ER-dependent reporter gene ERE-ßGlob-Luc in MELN cells
To confirm the oestrogenic activity of PhIP in a non-transfected cell system, we used MELN cells, which incorporate stable expression of the luciferase gene under control of the EREglobin promoter sequence and constitutively express native ER-{alpha}. The cells were highly responsive to E2 (Figure 4), producing maximal activity at ≥10–10 M. They were also very responsive to PhIP, but required concentrations that were three orders of magnitude higher (Figure 4). Nevertheless, PhIP produced substantial transactivation at concentrations >10–8 M.



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Fig. 4. Transcriptional activation of the ER-dependent reporter gene ERE-gGlob-Luc in MELN cells. MELN cells were plated in oestrogen-deficient medium (5% DCSS). They were then treated with E2 or PhIP for 24 h, then luciferase activity was determined. Values are means ± SD of three separate transfections, each performed in triplicate (*P < 0.01 significantly different compared with ethanol control).

 
Mammalian cell cytotoxicity and mutagenicity at the hprt locus
An established mammalian cell mutation assay using the hamster fibroblast cell line V79 h1A2 (22) was employed to determine whether the concentrations of PhIP that elicit oestrogenic activity could also induce a measurable mutagenic response. V79 cell lines have been extensively used in mutagenicity and cytotoxicity studies since they have a very stable karyotype and possess a functional X-linked hprt locus as a target for mutation. The V79h1A2 cell line has the added advantage of expressing human CYP1A2, the enzyme required to oxidize PhIP to genotoxic derivatives.

Incubation of V79 h1A2 cells with PhIP (10–7 M) for 24 h did not have a significant effect on cell survival (96 ± 16% of ethanol control) (Figure 5A). However, increasing the dose to 10–4 M greatly reduced survival (20.7 ± 10% of control). Positive control cells treated with ethyl methane sulphonate (EMS) (200 µg/ml) also showed reduced survival (55 ± 16% of ethanol control). (Figure 5A).



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Fig. 5. Effect of PhIP on survival and mutation frequency at the hprt locus in V79h1A2 cells. (A) Cell survival. Cells treated with ethanol or EMS (200 µg/ml) served as negative and positive controls, respectively. Cells were plated immediately after treatment (24 h) in non-selective medium (no 6-TG) and colonies scored after 7 days. Values are means ± SD (n = 3). (B) Mutation frequency. Cells treated with ethanol or EMS (200 µg/ml) served as negative and positive controls, respectively. Cells were plated immediately after treatment (24 h) in non-selective medium for 7 days, then transferred to medium containing 6-TG (5 µg/ml) and surviving colonies were scored after a further 14 days. Values (corrected for survival) are means ± SD (n = 3).

 
Treatment of the V79h1A2 cells with the direct acting mutagen EMS (200 µg/ml) induced a substantial increase in mutation frequency (360 ± 88 mutant colonies per 106 survivors). In contrast, there was no significant increase in mutation frequency over the ethanol control in cells treated with 10–7 M PhIP (Figure 5B), a concentration that induced a good oestrogenic response. At higher concentrations of PhIP (10–5–10–4 M) a much more pronounced increase in mutation frequency was noted (24 ± 9 and 173 ± 46 mutant colonies per 106 surviving cells, respectively).

Effect on MCF-7 cell proliferation
We next examined the effect of PhIP on cell proliferation using a modified E-SCREEN assay (25). MCF-7 breast cancer cells are growth inhibited in a medium containing dextran-coated charcoal-stripped human serum and will only proliferate when exposed to added E2 or oestrogenic compounds (25). We measured proliferation of viable cells using the resazurin reduction assay (21) and found that like E2, PhIP provoked a concentration-dependent increase in cell proliferation (Figure 6) that was attenuated by ICI 182,780 (10–7 M).



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Fig. 6. Effect of PhIP on growth of MCF-7 cells. MCF-7 cells were cultured in phenol red-free medium containing 5% DCC-FCS as described in Materials and methods. After treatment with ligand, the relative cell number was estimated using the resazurin reduction assay (21). The open triangles show the effect of including ICI 182,780 (10–7 M) with PhIP (10–9 M). Each point represents the mean ± SD of four separate experiments, each performed in triplicate. (**P < 0.01, *P < 0.05 significantly different compared with ethanol control; +P < 0.01 significantly different compared with ligand alone).

 
Effect of PhIP on E2-mediated protein expression
To assess the ability of PhIP to promote ER-mediated transcription of an endogenous gene, its effects on the induction of PR, an E2-regulated protein, were evaluated by western blotting with protein extracts obtained from T47D breast cancer cells, a cell line that is known to induce PR upon stimulation with E2 (26). A significant dose-dependent enhancement of both PR-A and PR-B expression was observed after treatment with both E2 and PhIP that was antagonized by ICI 182,780 (Figure 7A). PhIP-induced PR induction was rapid, detectable after 1 h of treatment, and persisted for at least 48 h (Figure 7B). Again, this effect was reversed by concomitant treatment with ICI 182,780.



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Fig. 7. Immunoblot showing the dose-dependent effect of PhIP on progesterone receptor (PR) expression in T47D cells. (A) PR-A and PR-B protein levels were evaluated in protein extracts obtained from T47D cells treated with E2 or PhIP ± ICI 182,780 (10–7 M) for 24 h. PR-A and PR-B resolved as bands of 82 and 110 kDa, respectively. (B) Temporal effect of PhIP (10–7 M) on PR-A expression in T47D cells.

 
MCF-7 cells are known to respond to E2 with increased c-MYC expression (27). Figure 8 shows the effects of E2 (10–8 M) on c-MYC protein induction and demonstrates that PhIP (10–8 M) increases c-MYC levels in a similar time-frame and with similar intensity to E2. The proteins resolved as two bands at ~62 and 66 kDa (c-MYC2 and c-MYC1, respectively) (2830).



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Fig. 8. Immunoblot showing the temporal effect of E2 (10–8 M) and PhIP (10–8 M) on c-MYC expression in MCF-7 cells.

 
Effect of PhIP on signal transduction
E2 can stimulate the mitogen-activated protein kinase (MAPK) signaling pathway in MCF-7 cells (31). As shown in Figure 9, treatment of MCF-7 cells with PhIP for 24 h, like E2, resulted in increased levels of activated ERK (ppERK 1&2), whereas levels of total ERK remained unaffected. This effect was partially inhibited by concurrent treatment with the antioestrogen ICI 182,780 (Figure 9).



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Fig. 9. Immunoblot showing the effect of E2 and PhIP ± ICI 182,780 (10–7 M) treatment for 24 h on MAPK phosphorylation status in MCF-7 cells. Phosphorylated ERK-1 and ERK-2 resolved as bands of 42 and 44 kDa, respectively. Levels of total ERK-1 and ERK-2 were detected on the same blot to control for loading variations.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
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 Discussion
 References
 
The ER is unique among the steroid receptors in its ability to bind a diverse range of non-steroidal compounds such as insecticides, phthalate esters and other xenobiotics (32,33). Reports have suggested an association between these environmental oestrogens and a variety of disease states, including cancer, reproductive deformities and impaired fertility.

Both E2 and PhIP (10–6–10–10 M) activated ER-{alpha}-dependent gene transcription in COS-1 cells transiently transfected with an oestrogen-responsive reporter gene (17 M-ERE-G-CAT) and an ER-{alpha} expression vector. Co-administration of the complete anti-oestrogen ICI 182,780 inhibited this activity, strongly indicating that transactivation of the reporter gene was mediated via the ER. Confirmation of this response in the human breast cancer cell line MCF-7 demonstrated that PhIP, like E2, was capable of using the endogenous ER to effect transactivation and experiments using a non-transfected cell system (MELN cells) also confirmed PhIP-mediated transactivation, although the sensitivity of this assay was much lower compared with the COS-1 cell assay. These data suggest that PhIP may be a new candidate non-steroidal environmental oestrogen.

The oestrogenic potency of PhIP is better than many environmental oestrogens studied to date in similar reporter gene systems. For example, the dietary antioxidant butylated hydroxyanisole causes stimulatory effects on transcriptional activity of ER in vitro only at concentrations of ≥10–5 M (34), whereas the lowest observable effect measured with PhIP was between 10–8 and 10–10 M. Even if the oestrogenic effects of PhIP alone are not sufficient to elicit a response in vivo, they may add to the total oestrogenic burden upon the body. Several studies have suggested that environmental oestrogens may act cumulatively and that it is appropriate to look at the activity of combinations of xenoestrogens as well as measuring the effects of individual compounds (35,36).

While the genotoxicity of high dose PhIP (>10–6 M) has been well characterized (reviewed in 3,37,38), few studies have examined the biological effects exerted by the compound at concentrations likely to result from the consumption of cooked meat. More importantly, little is known about the possible chronic effects of PhIP on humans if ingested at low concentrations over a lifetime. To our knowledge, accurate quantitation of plasma PhIP after consumption of a cooked meat meal has not been reported, but cooked meat has been shown to contain up to 50 ng PhIP/g meat (37,39,40), thus intakes of between 1 and 50 µg PhIP/day are feasible. PhIP consumed in meat is extensively absorbed (41), but if first pass hepatic metabolism is high, it is possible that the majority of plasma PhIP would be in the form of metabolites. Certainly, the majority of urinary PhIP-derived material is in the form of metabolites and in humans only 1–5% of the ingested dose appears as unchanged amine (41). Without accurate estimates of plasma PhIP it is very difficult to estimate circulating levels of the compound, yet given the levels of consumption (µg) and of excreted unchanged compound in the urine, plasma levels of 10–9–10–11 M are conceivable, with the possibility of localized levels being higher. Indeed, the report by DeBruin et al. (42) that PhIP is present in human breast milk supports this proposal.

Another cooked meat-derived heterocyclic amine, MeIQx, shares structural similarities with PhIP, namely an exocyclic amino group and an imidazole ring fused to an aromatic moiety (Figure 10), yet it was unable to stimulate reporter gene transcription, indicating that it is devoid of oestrogenic activity. It is perhaps significant that compared with PhIP, MeIQx has a quite different carcinogenic profile in rats, inducing tumours in the liver and Zymbal gland (43), whereas PhIP induces tumours at hormonally dependent sites, the breast, prostate and colon (5,6). Coincidently, in a recent epidemiological study Sinha et al. (40) examined the association between dietary heterocyclic amine intake and the risk of developing breast cancer in women participating in the Iowa Women's Health Study. An elevated risk of breast cancer was observed with increased PhIP consumption but there was no evidence for an association with MeIQx consumption.



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Fig. 10. Chemical structures of MeIQx and PhIP.

 
Unlike E2, PhIP showed no transcriptional activity with ER-ß at any of the concentrations tested. Furthermore, the activity of E2 at 10–8 M was not attenuated in the presence of a molar excess of PhIP, suggesting that PhIP is neither an ER-ß agonist nor antagonist. Although the DNA-binding domains of ER-{alpha} and ER-ß show a high degree of homology, the ligand-binding domains show only 59% homology (44), which may account for the selective ability of PhIP to act via the former but not the latter. Although both ER-{alpha} and ER-ß function as ligand-modulated transcription factors, their tissue distributions are different, with both forms being expressed in most E2-responsive human tissues but with ER-{alpha} the dominant form in the breast and ER-ß the dominant form in the gastrointestinal tract (45,46). Also the activity of oestrogens is not always the same when acting on the two subtypes (46). Thus, it is likely that at least some of the biological roles of ER-{alpha} and ER-ß are different. Indeed, it has been proposed that the two subtypes sometimes mediate opposing physiological effects. For example, Gustafsson (44) suggests that while ER-{alpha}-mediated activity is thought mainly to be involved in promoting cell proliferation, ER-ß-mediated activity appears to inhibit this process. Similarly, studies of the mammary glands of female ER-ß knockout mice have revealed overexpression of the proliferative antigen Ki67 and severe cystic breast disease as the mice age (47). The higher incidence of colorectal cancer in females, the high frequency of this tumour in patients with breast cancer and the presence of oestrogen receptors in colorectal mucosa together suggest an involvement of E2 in either colon carcinogenesis or tumour progression. Several recent epidemiological studies have indicated that oestrogens given to post-menopausal women protect against cancer of the colon and that this protection might possibly be afforded by the expression of ER-ß in the colon (48). Further evidence for the protective role of ER-ß includes reports that there is a selective loss of this receptor in colorectal cancer and prostate cancer (49,50). The mechanism of the putative protective role of ER-ß against cell proliferation and carcinogenesis has yet to be elucidated. The exclusivity of PhIP for ER-{alpha}, as noted in the present study, is therefore particularly interesting. The selectivity for one ER subtype as displayed by PhIP is not uncommon, for example the phytoestrogen genistein has been shown to preferentially bind with 30-fold greater affinity to ER-ß than ER-{alpha} (51).

In the present study the ability of PhIP to induce mutation in V79h1A2 mammalian cells was examined using an hprt mutation assay. These cells (which express human CYP1A2) have the ability to activate PhIP and a clear mutagenic effect was noted at concentrations >10–6 M, but not below this. This may simply be a reflection of the comparative sensitivity of this genotoxicity assay, but given this caveat it is clear from the data that the oestrogenic effects of PhIP can occur at concentrations that are several orders of magnitude lower than those required for demonstration of its genotoxic effects. Since the Km of PhIP for human CYP1A2 is ~10–5 M (4,52,53) and the Kd for ER-{alpha} appears to be in the nM range, it might be expected that the ER-{alpha} protein would more efficiently compete with CYP1A2 for limited concentrations of PhIP. It could be argued that the lipophilicity of PhIP would favour a membrane localization and presumably increase accessibility to CYP enzymes, yet the lipophilicity of PhIP is remarkably similar to that of oestradiol and thus identical arguments could be applied to the cellular distribution of oestradiol.

The effect of PhIP on the biology of the whole cell was examined using the E-SCREEN assay (25), which is based on the ability of breast adenocarcinoma MCF-7 cells to proliferate in response to oestrogens or oestrogen-like compounds. This ER-{alpha}-positive cell line is growth inhibited in medium containing dextran-coated charcoal-stripped human serum and will only proliferate when exposed to E2 or oestrogenic compounds such as endosulfane, dieldrin and toxaphene (25). Under such conditions of growth inhibition PhIP stimulated the growth of these cells almost as well as E2 and attenuation of growth by ICI 182,780 strongly implies a role for the ER, further supporting the proposal that PhIP is acting as an oestrogen at the cellular level.

We therefore examined the ability of PhIP to induce biochemical changes that may be associated with its E2-like mediated proliferation effects. Consistent with its activation of the ER, PhIP up-regulated PR expression and this induction was inhibited by the co-administration of an anti-oestrogen (ICI 182,780). The up-regulation of PR in breast cancer cells is considered to be mainly due to transcriptional events and is regarded as a marker of oestrogen responsiveness (26,5456). There is compelling evidence that, like E2, progesterone and synthetic progestins may play an important role in breast cell proliferation and cancer (57). Indeed, progesterone may be more important than oestrogen as a driver of proliferation (5861). Thus, by up-regulating PR PhIP may sensitize breast cancer cells to the proliferative effects of progestins.

Stimulation of the expression of proto-oncogenes such as c-MYC has been suggested as one of the mechanisms accounting for the mitogenic action of E2 (62). Amplification of c-MYC has been repeatedly observed in various tumours and cell lines; indeed, 50–100% of breast cancer cases have increased levels of c-MYC proteins (6365). The c-MYC proto-oncogene encodes a nuclear protein essential for DNA replication and is one of the key genes required for a cell to progress through the cell cycle (66,67). This function is thought to be elicited mainly via activation of transcription of target genes that are regulators of the cell cycle, such as the cyclins (68). In the normal cell c-MYC expression is tightly regulated in response to growth signals. Non-proliferating, quiescent cells express low or undetectable levels of c-MYC protein, but this expression is rapidly induced following mitotic stimulation. PhIP was found to increase c-MYC protein levels in MCF-7 cells with a time course and intensity similar to E2-mediated induction. E2 induction of c-MYC is thought to occur directly, via an ERE half-site Sp1 element in the c-MYC gene promoter, and the finding that PhIP induces c-MYC protein within 1 h suggests that it too works by this direct mechanism. The finding that PhIP can stimulate the expression of this proto-oncogene may partially explain the proliferative action of PhIP in MCF-7 breast cancer cells.

Several recent reports have demonstrated that E2 stimulates the MAPK signal transduction pathway in a number of model systems, including CaCo-2 human colon carcinoma cells, rat osteoblasts and MCF-7 cells (31,69,70). Cross-talk between MAPK and the ER exists at several levels. Firstly, E2 may activate the MAPK pathway through non-genomic effects via membrane-associated ERs and activation of RAS (71), RAF-1 (31) and protein kinase C (72). Secondly, MAPK can directly phosphorylate Ser118 of the ER and enhance its transcriptional activity (73). Thirdly, E2 may stimulate the expression of growth factors, which in turn activate MAPK. The consequence of this is phosphorylation at tyrosine and threonine sites on the activation loop of ERK-1 and ERK-2 via RAS and RAF signaling. The ERKs then stimulate downstream events involved in gene regulation by phosphorylating and activating key transcription factors for genes involved in cell proliferation in the nucleus. For example, ERK-2 phosphorylates the steroid co-activator Src-1, which then binds to the cyclic AMP response element-binding protein to enhance ER-mediated gene activation.

The ability of PhIP to activate the MAPK pathway suggests that, like E2, it can influence cell proliferation not only by stimulating ER-mediated gene expression (e.g. by increasing c-MYC protein levels), but also possibly through non-genomic effects. Since the anti-oestrogen ICI 182,780 only partially inhibited PhIP-mediated ERK phosphorylation, it would appear that PhIP is not acting solely through the ER.

In conclusion, PhIP possesses oestrogenic activity at low concentrations and this activity is mediated by the ER-{alpha} subtype, supporting the idea that exposure to PhIP, even at low doses, could result in oestrogenic effects. We suggest that the well-established and unequivocable genetic toxicology of PhIP coupled with its oestrogenic activity could drive clonal expansion and promote growth of the initiated phenotype. Together these characteristics may explain the site-specific carcinogenicity of PhIP and are intriguing factors when considering the causality of food-derived cancer in humans.


    Acknowledgments
 
We gratefully acknowledge the help provided by J.Pike in setting up the transfection assays. These studies were supported by funds provided by the Department of Health and Food Standards Agency, UK.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 14, 2004; revised July 15, 2004; accepted August 10, 2004.