Inability to confirm estrogenicity of the heterocyclic amine PhIP in two in vitro assays

Toxicology in Vitro 24 (2010) 1757–1763

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Inability to confirm estrogenicity of the heterocyclic amine PhIP in two in vitro assays Richard M. Evans a, Sinikka Rahte a,b, Andreas Kortenkamp a,* a b

The School of Pharmacy, University of London, Centre for Toxicology The School of Pharmacy, University of London, Centre for Pharmacognosy and Phytotherapy

a r t i c l e

i n f o

Article history: Received 7 September 2009 Accepted 18 December 2009 Available online 29 December 2009 Keywords: 2-Amino-1-methyl-6-phenylimidazo-[4,5b]pyridine PhIP Endocrine disruption In vitro cell-based assay Food estrogens

a b s t r a c t 2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is a heterocyclic amine which is found in food after cooking and which is a known mutagen. Reports from several laboratories have proposed that PhIP has estrogenic activity, which would classify PhIP as a xenoestrogen with human exposure via food. We tested PhIP in two cell-based assays for estrogenicity, both based on human cell lines but utilising different outcome measures: ERLUX (reporter-gene activation) and ESCREEN (cell proliferation). PhIP was inactive in both assays at concentrations spanning the picomolar to micromolar range. To eliminate supplier differences as an explanation for the disparity between these results and positive findings in the literature, we purchased PhIP from three suppliers and found no detectable estrogenic activity in any batch. 1H NMR spectroscopy confirmed the chemical identity of the tested stock solutions. Correct assay performance was confirmed by including positive and vehicle controls on every assay plate, and by demonstrating the expected responses to a panel of known estrogens (estradiol, bisphenol A, genistein). Our results differ from those in the literature and, whilst the exact reason for this is unknown, we discuss possible explanations of the disparity. Our results provide no in vitro evidence for the classification of PhIP as an estrogen. Ó 2010 Published by Elsevier Ltd.

1. Introduction PhIP (2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine, CAS 105650-23-5, see Fig. 4A) belongs to the group of heterocyclic amines (HCAs), which includes several mutagens/carcinogens of potential importance to human health. PhIP is one of the most prevalent HCAs in food and was initially identified as being of concern due its mutagenic/carcinogenic potential (Felton et al., 1986b). However, PhIP has more recently been identified as estrogenic (Lauber et al., 2004) with a potential role in breast cancer (Lauber and Gooderham, 2007), raising the possibility that PhIP could be classified as a xenoestrogen and thus be of potential concern as an endocrine disruptor. The potential for human health risk from HCAs in food was identified over 30 years ago, see reviews by (Sugimura et al., 2004) and (Turesky, 2007). Humans are exposed to HCAs through the diet, particularly through consumption of meat and fish which have been cooked in such a way as to produce HCAs in the food. PhIP is typically present in cooked food at levels of around 35 ng/g,

* Corresponding author. Address: Centre for Toxicology, The School of Pharmacy, University of London, 29-39 Brunswick Square, London, WC1N 1AX, UK. Tel.: +44 (0) 20 7753 5908; fax: +44 (0) 20 7753 5811. E-mail address: [email protected] (A. Kortenkamp). 0887-2333/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.tiv.2009.12.017

although higher levels may be encountered in particular conditions. For example, concentrations are increased by increasing cooking temperature and are higher in cooked meat than in cooked fish (Skog, 2002). The estimated total daily intake of HCAs, of which PhIP is the most abundant, ranges from 0 to 15 lg per person (Skog, 2002) and the daily dietary intake of PhIP alone has been estimated to range from 280 to 460 ng per person (Byrne et al., 1998). PhIP has been identified as one of the main mutagens in cooked beef (1.5  9 cm patties fried at 250 or 300 °C, 6 min per side). Despite having a low mutagenic potency, as defined by ability to cause reversions in the Ames/Salmonella mutagenicity test, PhIP was considered to have the potential to make a significant contribution to carcinogenicity because of its abundance (Felton et al., 1986a). Other mutagens identified in the same study included the HCAs 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and trimethylimidazopyridine (TMIP). Human dietary exposure to genotoxins in cooked foods is not limited to HCAs and includes acrylamide, nitrosamines and polyaromatic hydrocarbons (PAHs) (Jagerstad and Skog, 2005). HCAs, including PhIP, can be detected in the urine of healthy volunteers eating a normal diet, but not in parenterally fed hospital inpatients; indicating that HCAs are not endogenously produced and that a normal diet can produce continuous exposure to HCAs

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(Ushiyama et al., 1991). In milk samples from healthy women, PhIP was detectable in most samples and was present at concentrations up to 59 pg/mL (DeBruin et al., 2001). Humans exposed to a single dose of PhIP in a meal showed different metabolic profiles and different total urine excretion amounts, indicating that human exposure may vary depending on digestion, diet and genetics (Felton et al., 2002). The identification of PhIP as an estrogen was first made by the Gooderham group, who have reported estrogenicity in a number of in vitro assays, using a variety of different cell types and endpoints: including COS-1 cells transiently transfected with the estrogen receptor a isoform (ERa) and a chloramphenicol acetyl transferase (CAT) reporter-gene; MELN cells stably transfected with a luciferase reporter-gene under the control of an estrogen-response element (ERE); and MCF7 cell proliferation assays (Gooderham et al., 2002; Lauber et al., 2004; Gooderham et al., 2007). These results were particularly important since PhIP was reported to be potent, with activity in the low nanomolar to high picomolar range, similarly to estradiol (Lauber et al., 2004). Interestingly, the dose–response characteristics were not always simple sigmoid relationships, but could be decidedly non-monotonic (Gooderham et al., 2007); and PhIP was also reported to be exquisitely selective for the ERa isoform compared to ERb (Gooderham et al., 2002; Lauber et al., 2004). Of significant importance to human health is that the association of carcinogenicity/mutagenicity and estrogenicity was suggested to provide mechanistic support for a role for PhIP in breast cancer (Lauber and Gooderham, 2007). Confirmation of the findings of the Gooderham group was provided by Bennion et al. who used computational docking and NMR to show that PhIP interacts with ERa, and confirmed the potential estrogenicity of PhIP by using proliferation of, and luciferase induction in, MCF7 cells (Bennion et al., 2005). As part of a larger crystallographic analysis of the estrogen receptor, a third group also showed that PhIP was active in MCF7 cells transiently transfected with a luciferase reporter-gene under the control of the ERE (Nettles et al., 2008). Initially, we examined the estrogenicity of PhIP because we intended to include it in a larger panel of endocrine disrupting chemicals (EDCs) to examine the effects of mixtures composed of compounds from different chemical groupings; if PhIP is estrogenic then the group of HCAs should be considered for inclusion. We also wished to compare the responses obtained for PhIP with full dose– response curves for estradiol (the endogenous ligand for the estrogen receptor), bisphenol A (a widely used plasticiser and a xenoestrogen) and genistein (a phytoestrogen), in order to be able to assess the relevance of any detected response in the context of the effects of the endogenous ligand and known EDCs within the same assays and under the same experimental conditions. We tested PhIP in two established cell-based assays for estrogenicity, both based on human breast cancer cell lines but differing in the outcome measure. The ERLUX assay uses T 47D cells stably transfected with a luciferase reporter-gene coupled with an estrogenresponse element (ERE); and the ESCREEN assay uses the proliferative or mitogenic response of an estrogen-sensitive MCF7 cell line (MCF7-BUS). Both assays have the advantages of being based on human backgrounds, and have the complexity of cell-based systems, for example including signalling or enzyme systems that are not present in isolated receptor binding studies or in yeastbased assays.

2. Methods PhIP was purchased from three commercial suppliers: Apollo Scientific Ltd. (Cat. # OR1700T, purity >90%, www.apolloscientific.co.uk), MP Biomedicals (Cat. # 154190, purity >98%,

www.mpbio.com) and Toronto Research Chemicals Inc. (Cat. # A617000, purity 98%, www.trc-canada.com). Cell culture reagents were purchased from Invitrogen (Paisley, UK). Estradiol and bisphenol A were purchased from Sigma (Poole, UK) and genistein from Alfa Aesar (Heysham, UK). 2.1. T47D-Kbluc cell line for estrogenicity, reporter-gene endpoint (ERLUX) T47D-Kbluc cells were obtained from the ATCC and the protocol established by the depositing authors was followed (Wilson et al., 2004). Cells were routinely grown in RPMI (10% FCS). For seven days prior to experiments, cells were maintained in low estrogen conditions by the use of pre-assay media (RPMI, 10% charcoal-dextran stripped FCS, no antibiotics). For experiments, cells were seeded in white polystyrene 96 well plates at a density of 10,000 cells/well and allowed to attach for 24 h before removal of media, and application of test chemicals. Test chemicals were dissolved in ethanol to give stock solutions of millimolar concentrations. Test and control solutions were obtained by dilution of ethanolic stocks in dosing media (phenol red-free RPMI, 5% charcoal-dextran stripped FCS, no antibiotics), and in all cases the final concentration of ethanol was 0.5%. The positive control was 1 nM estradiol. Positive and vehicle controls were run as eight replicate wells per plate, and compounds were tested in a dilution series comprising eight concentrations, each concentration tested in triplicate. As recommended by Wilson et al. two additional controls were also included on each plate: 1) vehicle control plus an antiestrogen, ICI 182,780 (1 lM), and 2) positive control plus ICI 182,780 (1 lM). These additional controls were used to monitor the background level of estrogenicity, and full experiments were excluded if vehicle showed high levels of estrogenicity or if the positive could not be suppressed by the antiestrogen (data not shown). A crude measure of toxicity was provided by comparing values for treatments that were not positive, with the value of the vehicle control. Toxicity would be expected to decrease these small (but non-zero) values towards zero, and this was not observed for any of the tested chemicals (data not shown). 24 h after application of test and control solutions, a volume of Steady-Glo assay reagent (Promega) equal to the volume of culture media was added and plates were incubated for 10 min, with shaking, to allow for cell lysis. Plates were then loaded into a plate reader (FLUOstar Optima, BMG Labtech) and incubated for a further 10 min in the dark, followed by measurement of luminescence. To reduce variation, the temperature of the plate reader chamber was maintained at 27 °C throughout. 2.2. MCF7 cell line assay for estrogenicity, mitogenic endpoint (ESCREEN) MCF7-BUS cells were a kind gift (A. Soto, Boston) and the established ESCREEN method was followed (Soto et al., 1995) using the adapted 96 well format (Silva et al., 2007). Cells were cultured in DMEM (5% FCS). For experiments, cells were seeded in clear polystyrene 96 well plates at a density of 2500 cells/well and allowed to attach for 24 h before washing with rinse media (phenol redfree DMEM, no supplements). Estrogen deprivation (by use of charcoal-dextran stripped serum and removal of phenol red) was not used prior to seeding because this results in an almost complete lack of attachment of cells. Test chemicals were dissolved in ethanol to give stock solutions of millimolar concentrations. Test and control solutions were diluted prior to application in dosing media (phenol red-free DMEM, 10% charcoal-dextran stripped FCS). The final concentration of ethanol was 0.5% in test and control wells. The positive control was 25 nM estradiol and the final concentration of ethanol in all wells was 0.5%. The raw value of vehicle con-

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2.3. Data handling and analysis Raw results from either assay were normalised by subtraction of the mean value of on-plate vehicle controls and then division by the mean value of on-plate positive controls. Experiments were performed on different days meaning that results for different dilution series and different batches of the test chemicals were obtained on different days. Use of normalisation meant that data from different experiments could be reliably combined and compared, as discussed previously (Rajapakse et al., 2004). Data was expressed relative to the positive control, rather than as fold change from the vehicle response, for two reasons. Firstly, to anchor the response data to a meaningful response, namely the maximal response of the cognate ligand (estradiol), and secondly, to avoid spurious changes on the response axis due to the very low background estrogenicity which was achieved in vehicle controls in both assay systems. When the vehicle response is numerically small, chance variations that are not experimentally significant can nonetheless significantly affect the apparent fold change of positive test chemicals. Dose–response relationships were produced by fitting either a linear (PhIP) or sigmoid (E2, bisphenol A, genistein; 4-parameter logistic equation) model (Prism 4, GraphPad software Inc.) to the normalised data. Due to the lack of any evidence of a positive effect of PhIP, a detailed statistical comparison of PhIP to the results of testing estradiol, bisphenol A or genistein was not appropriate or required. A clear separation between vehicle and positive controls is a prerequisite for any assay, and for both assays employed here the separation was sufficiently large (Figs. 1 and 2) that statistical criteria were not required other than the quality criteria (described above for each assay) for rejection of plates if background estrogenicity was unacceptably high. For active chemicals, we calculated EC50, defined as the concentration producing an effect of 50% of the positive control (estradiol, 1 nM (ERLUX) or 25 nM (ESCREEN)), and relative potency (RP), defined as the ratio of EC50 Test chemical to EC50 Estradiol. 2.4. NMR Proton nuclear magnetic resonance (1H NMR) spectroscopy was used for the purposes of verifying the chemical identity of PhIP stock solutions prepared from PhIP purchased from different suppliers. 1H NMR spectra (average of 128 scans) were taken in deuterated DMSO using a Bruker Avance instrument (400 MHz). Spectra were calibrated to solvent residuals (DMSO = 2.5 ppm).

Normalised ELUX response

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M Fig. 1. ERLUX results for PhIP and three known estrogens. Figure shows ERLUX results from replicate experiments for PhIP (n = 4) and a panel of three estrogens: estradiol (E2, n = 4), genistein (GEN, n = 6) and bisphenol A (BPA, n = 8). Within each experiment, tested concentrations were run in triplicate. Data are shown as fits to an appropriate linear (PhIP) or non-linear (estradiol, genistein, bisphenol A) regression model with 95% confidence intervals (CI, dashed lines). For PhIP, unaveraged data points are also shown for each tested concentration (gray symbols, shape indicates supplier: square (ICN), diamond (Apollo), circle (TRC); each test used a different but overlapping dilution series, hence the data points do not align at eight discrete concentrations). Normalised values for controls (‘‘CON”) are indicated next to the y-axis (mean ± 95%CI, data shown separately for pooled data from plates testing estradiol (square), genistein (up triangle), bisphenol A (down triangle), PhIP (circle)), the positive control was response to 1 nM estradiol (set to 1.0) and the vehicle control was the response to 0.5% ethanol (set to 0.0).

Normalised ESCRE EEN response

trols was monitored for any indication of increasing background estrogenicity, raw values were typically 0.06–0.08 optical density units (ODU) and experiments were rejected if the vehicle value (averaged per plate) exceeded 0.1 ODU. Inclusion of an antiestrogen control to demonstrate that background estrogenicity was low was not considered necessary because the background absorption of vehicle wells was similar to that of wells containing media only. At all stages, media removal from cells was carried out gently and in a controlled fashion by use of an electronic multichannel pipette set to the lowest speed possible. Controls were run as eight replicate wells per plate and compounds were tested in a dilution series comprising eight concentrations, each concentration tested in two replicate wells per plate. The plate layout was designed to reduce variation due to evaporation and spreading of test chemicals, and had been previously optimised in the laboratory (Silva et al., 2007). After application of test solutions, plates were incubated for 120 h before fixation with 10% trichloroacetic acid and sulforhodamine B (SRB) staining to measure protein and allow the indirect quantification of cell number.

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M Fig. 2. ESCREEN results for PhIP and three known estrogens. Figure shows ESCREEN results from replicate experiments for testing of PhIP (n = 4) and a panel of three estrogens: estradiol (E2, n = 8), genistein (GEN, n = 7) and bisphenol A (BPA, n = 9). Within each experiment tested concentrations were run in duplicate. Data are shown as fits to an appropriate linear (PhIP) or non-linear (estradiol, genistein, bisphenol A) regression model with 95% confidence intervals (CI, dashed lines). The reason for the estradiol regression fit exceeding 1.0 is probably a slight diminution in positivity when the maximal concentration of estradiol (around 1 nM) is exceeded, for example in the positive control (25 nM). For PhIP, unaveraged data points are also shown for each tested concentration (gray symbols, shape indicates supplier: square (ICN), diamond (Apollo), circle (TRC); each test used a different but overlapping dilution series, hence the data points do not align at eight discrete concentrations). Normalised values for controls (‘‘CON”) are indicated next to the yaxis (mean ± 95%CI, data shown separately for pooled data from plates testing E2 (square), genistein (up triangle), bisphenol A (down triangle), PhIP (circle)), the positive control was response to 25 nM estradiol (set to 1.0) and the vehicle control was the response to 0.5% ethanol (set to 0.0).

3. Results 3.1. ERLUX Consideration of potential estrogenicity using the ERLUX reporter-gene assay, revealed no detectable estrogenicity for PhIP at any

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of the tested concentrations (0.1 pM–5 lM). Negative results for PhIP were obtained in four independent experiments, using batches of PhIP from three different suppliers. Data from replicate experiments are shown in Fig. 1. Fig. 1 also presents data from comparable experiments in which the ERLUX assay, under identical experimental conditions as used for the examination of PhIP, successfully detected the estrogenicity of three well known estrogens: estradiol (EC50: 5  1013 M; RP: 1 (by definition), bisphenol A (EC50: 5  107 M; RP: 1,000,096) and genistein (EC50: 3  108 M; RP: 63,096). It is interesting to note that both bisphenol A and genistein produced supramaximal effects, i.e. maximal effects that were greater than the maximal effect of estradiol; however this is a known feature of the ERLUX assay (Wilson et al., 2004), and is also observed in similar reporter-gene assays (van der Woude et al., 2005; Legler et al., 1999). Inclusion of the antiestrogen ICI 182,780 (1 lM) completely abolished the observed response to estradiol, bisphenol A and genistein (data not shown).

3.2. ESCREEN Examination of the potential estrogenicity of PhIP in the ESCREEN assay revealed no indication of estrogenicity at any of the tested concentrations (0.5 pM–10 lM). Negative results were obtained in four independent experiments, using batches of PhIP from three different suppliers. Replicate experimental results are shown in Fig. 2 and, for comparison, Fig. 2 also includes replicate results from experiments in which three known estrogens were tested: estradiol (EC50: 6  1012 M; RP: 1(by definition), bisphenol A (EC50: 2  107 M; RP: 25,119), and genistein (EC50: 4  108 M; RP: 6,310). The order of potency was the same in both the ERLUX and ESCREEN assays: E2 > genistein > bisphenol A. We also examined whether PhIP might potentiate the effects of estrogens rather than being directly estrogenic itself because the presence of estrogens in experimental media is a known issue for estrogenicity assays. However in the ESCREEN assay described herein, this possibility was already substantially reduced by the use of charcoal-dextran stripped serum and the exclusion of phenol red from the dosing media. Nonetheless we examined the ESCREEN response to PhIP in the presence of increasing concentrations of estradiol. Fig. 3 shows that concentrations of PhIP up to 5 lM had no detectable effect on the observed estrogenicity of estradiol.

Normalised ESCREEN N effect

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PhIP concentration (Log, M) Fig. 3. Effect of estradiol on ESCREEN response to PhIP. Figure shows ESCREEN results when increasing concentrations of PhIP, indicated along the x-axis, were tested in the presence of estradiol at concentrations of 0 (ethanol, open circles), 0.1, 0.6 and 3.6 pM (filled circles). The size of the filled circles is proportional to the estradiol concentration. Data are single values from one experimental plate.

3.3. NMR 1

H NMR spectroscopy was applied to samples of PhIP stock solutions prepared from PhIP purchased from two different suppliers (Apollo Scientific Ltd. and Toronto Research Chemicals Inc.). 1H NMR analysis confirmed that the chemical identity of the substances tested as PhIP was indeed PhIP, see Fig. 4, and showed that the spectra of PhIP batches from the two different suppliers were consistent with one another (data not shown).

4. Discussion In summary, we did not obtain any in vitro experimental support for the identification for PhIP as an estrogen in either the ERLUX (reporter-gene endpoint) or ESCREEN (mitogenic endpoint) assays. Both assays performed reliably and correctly identified three established estrogens. The design of both assays included positive and vehicle controls on each plate and thus provided confidence that each assay was performing as expected on each experimental occasion, and allowed results from different experiments to be reliably compared. 1H NMR analysis confirmed the chemical identity of the tested compounds to be PhIP. Our inability to find estrogenic activity of PhIP contrasts with reports in the literature, and the reason for this disparity has not been identified. We now discuss this disparity, and possible explanations in detail. We eliminated one possible explanation for the disparity between our experimental results and the publications from other groups, namely that different background levels of estrogen could influence the ability to observe agonist behaviour. If the assay system contains estrogens, test agents that are not in fact estrogenic but that positively modulate the effect of estrogens, by whatever mechanism, may produce an increase in the assay endpoint and may then be inaccurately labelled as estrogenic. However, we found that PhIP did not affect the ESCREEN response to estradiol when estradiol was included at concentrations up to that evoking an approximately 50% effect when applied alone. Supplier differences were also eliminated as an explanation for the disparity by testing multiple batches from different suppliers, including the supplier (Toronto Research Chemicals) used in published studies in which positive results were reported (Lauber et al., 2004; Bennion et al., 2005). Because our results differ from several published studies, it is important to examine possible reasons for this disparity, especially the possibility that our results may be false negative findings. We consider it unlikely that our results constitute a false negative since we employed two assays, each of which used a different outcome measure (ERLUX: reporter-gene expression, luminescence; ESCREEN: cell proliferation, cell number) and a different exposure time (ERLUX: 1 day, ESCREEN: 5 days); and in both assays the estrogenicity of several known estrogens was detected correctly. We also addressed the possibility of batch differences between suppliers, and the potential effect of varying background levels of estrogens in the assays. In the following paragraphs we consider other possible reasons for the disparity between our results and published results, including 1) the characteristics of the MCF7 sub-line used, 2) subtle experimental differences, and 3) any requirement for metabolic activation. MCF7 sub-lines. One possible explanation for the differences seen in cell proliferation assays could be the exact sub-line of MCF7 cells that were used. The ESCREEN experiments described herein deliberately employed the MCF7-BUS sub-line because MCF7 sub-lines are known to vary in their responsiveness to estradiol and have been shown to differ genomically from each other (Villalobos et al., 1995; Jones et al., 2000). A comparison of four MCF7 sub-lines, BUS, ATCC, BB and BB104, showed substantial

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A

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Fig. 4. 1H NMR results confirming chemical identity of PhIP. 1H NMR spectrum of a stock solution of PhIP (Apollo Scientific Ltd.) that was also tested in the in vitro estrogenicity assays. The 1H NMR spectrum is consistent with the chemical structure of PhIP, shown in A, and with spectra published in the literature and provided by the manufacturer (Toronto Research Chemicals). The complete 1H NMR spectrum is shown in B. Residual solvent signals for DMSO and water are visible (d = 2.5 and 3.3 respectively).

differences in estrogen-related growth between the lines and led to the recommendation that MCF7-BUS sub-line should be used for ESCREEN assays, or for similar assays in which the proliferative response to estrogens is examined (Villalobos et al., 1995). Similarly Jones et al. compared three different MCF7 sub-lines, BUS, SOP and UCL, and found that the estradiol concentration required to evoke a half-maximal response was approximately 10 pM for all three sublines, but the maximal proliferative effect could vary up to 10-fold between sub-lines: BUS > SOP > UCL (Jones et al., 2000). MCF7 sublines with greater proliferative responses, such as MCF7-BUS, might be expected to produce better signal–noise ratios and facilitate the confident identification of positive responses when used in assays such as the ESCREEN. Subtle experimental differences: We cannot eliminate subtle experimental differences as the explanation for the disparity between these results and published results because we did not replicate the assays and experimental conditions of previously published studies. The aim of the experiments published herein was similar to that of published studies, namely the robust identification of any estrogenic activity of PhIP in vitro, but we did not attempt to exactly reproduce earlier studies, choosing instead to select suitable assays from those available. Consequently we employed a cell proliferation assay using the sensitive MCF7-BUS sub-line and not other less sensitive sub-lines, see preceeding paragraph, and we employed a stably transfected reporter-gene assay which has been publicly deposited (Wilson et al., 2004) instead of transiently transfected systems which can produce less stable responses than permanently transfected lines. Transiently and stably-transfected systems each have advantages depending on the application, it could be considered that transiently transfected systems are ideal for exploration of mechanisms, for example when

the ability to ‘plug-in’ different receptor subtypes and response elements is invaluable (Koohi et al., 2007). However for standardised assays to detect a defined activity (e.g. estrogenicity) stablytransfected systems allow the variability associated with transfection to be removed, and also allow for the selection post-transfection of the system with the greatest sensitivity for the endpoint being studied. Duration of exposure to test chemicals is an experimental factor that might alter the ability of an assay to detect a given activity, however our ERLUX protocol used the same duration of exposure (24 h) as published studies using similar assays. Our ESCREEN protocol included a five day duration of exposure, and some of the published studies stated that they used a similar duration but replenished the applied solutions after two days (Gooderham et al., 2007; Lauber et al., 2004), or used a shorter duration (two or three days) but without replenishment (Bennion et al., 2005). We choose not to use additional media changes because of the risk that this manipulation can increase variability, for example due to cell detachment during media removal or addition, and because of the need to use a standardised protocol that was also suitable for the control and exemplar EDCs that were tested in parallel. Overall, duration of exposure cannot explain our negative ERLUX results, compared to published studies; and, whilst duration of exposure did differ somewhat between our ESCREEN protocol and the protocols used in published studies with positive results, we would not expect the differences to explain the complete lack of positive effect in our studies. Metabolic activation: It has been shown that the mutagenicity of PhIP is dependent on prior metabolism (Buonarati and Felton, 1990), particularly via CYP1A2 (Zhao et al., 1994). However metabolism has not been identified as a prerequisite for estrogenicity of

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PhIP; indeed it has been noted that PhIP is able to produce cancers in mice lacking CYP1A2, and suggested that, in such an instance, PhIP is acting as an estrogen and without the requirement of metabolism, at least not via CYP1A2 (Lauber and Gooderham, 2007). However, even if metabolic activation were required for PhIP to show estrogenicity it seems unlikely that the required metabolism would be absent in both T47D-Kbluc and MCF7-BUS cells, but not in the cell lines used in published studies. Furthermore, a dependency on metabolic activation might be expected to produce variability in assay outcome, but might not be expected to abolish activity in some cases whilst preserving it in others. Similarly, although we did not specifically assess the cytotoxicity of PhIP, a level of cytotoxicity that exactly masked the putative estrogenicity seems unlikely and our data for co-application of PhIP and estradiol (Fig. 3) would have revealed any cytotoxicity to MCF7-BUS cells. Our data show that PhIP concentrations up to the micromolar range did not negatively affect the proliferative response to estradiol, consistent with a lack of cytotoxicity of PhIP on these cells. There are several literature reports of interest in the context of the negative results found in our studies. Firstly, our negative results are consistent with an in vivo study which also failed to detect any estrogenicity of PhIP. Intraperitoneal administration of PhIP at 50 mg/kg/day for three days to ovariectomised 6 week old rats resulted in no estrogenic response as determined by a lack of increase in uterine weight, stromal thickness, epithelial cell height or 5-bromo-20 -deoxyuridine (BrdU) positive cell counts (Kawamori et al., 2001). The protocol employed was essentially equivalent to that of a rat uterotrophic assay, in which estrogenicity is measured as an increase in weight of estrogen-sensitive tissues, namely the uterus and which has been considered to be the ‘gold standard’ for identification of estrogens (Clode, 2006). Secondly, a recent in vitro study found that, in MCF7 cells of an undefined sub-line, PhIP did not up-regulate estrogen-responsive proteins (estrogen receptor a, c-myc, p53 and ERK) or affect cell viability (as measured by Ki67 immunostaining) (Immonen et al., 2009). A small increase in cell proliferation (increase of 19% over vehicle control) was seen with low nanomolar concentrations of PhIP, however this effect was very dependent on culture conditions and showed atypical dose–response characteristics. The cell proliferation assay may have suffered from exhibiting only a small response to estradiol (a maximum increase of only 34% over control) which is likely due to the use of an insensitive MCF7 sub-line. The decision to use an individual well instead of a plate as the unit of independent observation may also have affected the ease with which statistical significance was demonstrated. In the reports of both Kawamori et al. and Immonen et al. PhIP was compared to estradiol, the cognate estrogen receptor ligand, but not to other candidate EDCs, so it could be proposed that their assays may have lacked sensitivity to identify weakly estrogenic chemicals. A strength of the current studies is the comparison of PhIP to two EDCs, bisphenol A and genistein, showing that the assays used were sensitive enough to detect the effects of weakly estrogenic substances as well as the cognate ligand. In conclusion, human exposure to heterocyclic amines, including PhIP, remains of concern because of their mutagenic potential. However our in vitro data provide no support for considering PhIP or, as a consequence, the wider group of heterocyclic amines, to be estrogenic. We consider that further in vitro testing using different, less well established systems is unlikely to be illuminating, and poses the very real risk that spurious positive results will be detected using endpoints whose meaning is uncertain. Interpretation of the meaning of poorly established endpoints is very much more problematic than the interpretation of the well characterized, experimentally and theoretically rigorous endpoints of an assay such as the ESCREEN. The OECD is currently developing an

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