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Effects of estradiol, benzophenone-2 and benzophenone-3 on the expression pattern of the estrogen receptors (ER) alpha and beta, the estrogen receptor-related receptor 1 (ERR1) and the aryl hydrocarbon receptor (AhR) in adult ovariectomized rats
Toxicology 205 (2004) 123–130
Effects of estradiol, benzophenone-2 and benzophenone-3 on the expression pattern of the estrogen receptors (ER) alpha and beta, the estrogen receptor-related receptor 1 (ERR1) and the aryl hydrocarbon receptor (AhR) in adult ovariectomized rats Christiane Schlecht, Holger Klammer, Hubertus Jarry, Wolfgang Wuttke∗ Department of Clinical and Experimental Endocrinology, University of Goettingen, Robert-Koch-Strasse 40, D-37099 Goettingen, Germany Available online 30 July 2004
Abstract The estrogen receptors (ERs) are members of a super family of ligand-activated transcription factors mediating estrogenic responses. A close functional kinship was found for the structurally related estrogen receptor-related receptor1 (ERR1), a constitutively active transcription factor. The aryl hydrocarbon receptor (AhR) mediates the toxic and estrogenic effects of a wide variety of environmental contaminants and industrial pollutants. Both the ERR1 and the AhR are known to modulate the ER’s signalling pathways in multiple ways. Organic chemicals with a certain structural relationship to steroid hormones often induce a tissue- or cell-specific variety of responses distinct from estrogenic responses and this may involve ERR1 and AhR. The UV-screens benzophenone-2 and benzophenone-3 (BP2, BP3), structurally related to known steroid receptor ligands, are used in cosmetics and plastics to improve product stability and durability. Both BP2 and BP3 were shown to exert uterotrophic effects and BP2 was shown to bind to the estrogen receptors. Whether such effects are also exerted in other organs is unknown. Therefore, an approach to a multi-organic risk assessment for these substances was made by measuring the gene-expression of the four mentioned receptors in the pituitary, the uterus and the thyroid after a five-day treatment in comparison to estradiol. Though BP2 seems to exert an estrogen-like effect while BP3 does not, there are regulatory effects on receptor expression for both substances that indicate a kind of endocrine disruption that is not assessed by “classical” estrogenic markers. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: ER␣; ER; AhR; ERR1; 2,2 ,4,4 -Tetrahydroxybenzophenone; 2-Hydroxy 4-methoxybenzophenone; Estradiol; Endocrine disruptor; Gene expression; Rat
1. Introduction Abbreviations: E2 , 17-estradiol; BP2, benzophenone-2; BP3, benzophenone-3; IGF1, insulin-like growth factor; HAH, halogenated aromatic hydrocarbons; C3, complement protein 3; LH, luteinizing hormone; PAH, polycyclic aromatic hydrocarbons ∗ Corresponding author. Tel.: +49 551 396714; fax: +49 551 396518. E-mail address: [email protected] (W. Wuttke).
Estrogens mediate their effects on growth, differentiation and functioning of target tissues through the two known estrogen receptors (ERs) alpha and beta (Green et al., 1986; Kuiper et al., 1996). These receptors belong to a large family of ligand-activated
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transcription factors (Mangelsdorf et al., 1995). Amongst other steroid receptors, this family comprises the estrogen receptor-related receptors (ERR) (Gigu`ere et al., 1988; Eudy et al., 1998). The ERRs display a close structural and functional relationship to the estrogen receptors (Lu et al., 2001). Taken together with their broad expression (Gigu`ere, 2002), they might play a major role in modulating anti/estrogenic responses. Because no endogenous ligand is known, the ERRs are called orphan receptors. The estrogen receptor-related receptor1 (ERR1) is supposed to be a constitutively active transcription factor (Chen et al., 2001). The aryl hydrocarbon receptor (AhR) belongs to the family of basic helix-loop–helix per-arnt-sim transcription factors and shares only a small structural relationship with the steroid receptors. Initially identified by Poland et al. (1979), it became known as the mediator of the toxic and anti/estrogenic effects of dioxin. The AhR is very promiscuous and a wide variety of ligands including halogenated aromatic hydrocarbons (HAHs) like dibenzofurans, biphenyls und dioxins, polycyclic aromatic hydrocarbons (PAHs) from incomplete combustions and possible endogenous ligands like tryptophan derivatives bind to this receptor (Denison and Nagy, 2003). For both receptors, ERR1 and AhR, multiple mechanisms of interfering with the estrogen receptor signalling pathway are known (as reviewed in Gigu`ere, 2002; Safe and McDougal, 2002) and both may be involved in mediating and/or modulating signal transduction and may be in part responsible for tissue- and cell-specific effects of industrial chemicals and environmental pollutants. Cosmetic products may contain chemical UV filters to improve product stability and durability as well as to protect human skin from harmful effects of UV radiation. Another use for UV filters is to prevent plastic products from light-induced damage. From the 12 known derivatives of benzophenone (BP), which are used as UV filters, the test substances benzophenone-2 (BP2; 2,2 ,4,4 tetrahydroxybenzophenone) and benzophenone-3 (BP3; 2-hydroxy-4-methoxybenzophenone) are frequently used in cosmetics and plastics. BP3 has been shown to exert a uterotrophic effect in vivo, to stimulate cell proliferation of MCF-7 breast cancer cells and to increase the secretion of tumour marker pS2 in vitro (Schlumpf et al., 2001). Additionally, it is absorbed through the skin after a 4-h application
of sunscreen products (Hayden et al., 1997; Felix et al., 1998) and via the gastro-intestinal tract (Kadry et al., 1995). A possible bioaccumulation has been postulated after BP3 was found in human milk, a marker for bioaccumulation in human adipose tissue (Pittet and Ferrer, 1997). Despite an estrogenic effect of BP3 itself, it is likely that the chemical is metabolized in vivo to the more potent estrogenic benzophenone-1 as it occurs in vitro after incubation with the liver S9 extract (Takatori et al., 2003). After a comparison of 517 chemical structures for their ability to activate the ER␣ in a yeast-two-hybrid assay, estrogenic structures seem to need an aromatic ring with a hydroxyl group in para position of the rest of the molecule and a hydrophobic moiety in the meta position (Nishihara et al., 2000). With the potency of BP1 with two hydroxyl groups in ortho and para positions on one of the aromatic rings, it is likely that BP2 with four hydroxyl groups displays at least a similar estrogenic potency. Indeed, BP2 has been positively tested in a reporter gene assay for ER␣, as well as in a uterotrophic assay (Yamasaki et al., 2003). Due to a few mutations in its ligand-binding domain, ERR1 is not able to bind 17-estradiol (E2 ; Horard and Vanacker, 2003) but the close structural kinship allows the assumption that estrogen receptor-related chemicals might also be ERR1 ligands (Chen et al., 2001). The AhR on the other hand is very promiscuous and has also to be considered to be a possible target of environmental chemicals, especially as the strongest known ligand dioxin shares an astonishing similarity with benzophenones. The risk of being exposed to benzophenones is considerable. The content of a single UV filter in a cosmetic product, regulated by EU Council Directive 76/768/EEC, may not exceed 10% but a total content of 29.3% has been found (Rastogi, 2002). To rate benzophenones as endocrine disruptors, they have to exert adverse effects like increased rate of cancer, reproductive system abnormalities, immune system deficiencies or excert adverse effects on other metabolic parameters (Melnick, 1999). Though non-receptor-mediated mechanisms by endocrine disruptors are known (reviewed in Cato et al., 2002), the primary sites of action are nuclear receptors. Therefore, our first approach was the evaluation of the expression of nuclear receptor transcripts in various estrogen-responsible organs in ovariectomized adult rats. After a five-day treatment
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with the chemicals BP2 and BP3 in two different dosages with a 17-estradiol and a negative control group, the expression of the four receptors ER␣, ER, ERR1 and AhR mRNA was measured in the pituitary, uterus and thyroid. Additionally, some “classical” markers of estrogenic action have been tested.
2. Materials and methods 2.1. Chemicals 17-Estradiol was obtained from Sigma-Aldrich GmbH (Taufkirchen, Germany); benzophenone-2 (UVINUL D50) was obtained from BASF ChemTrade GmbH (Burgbernheim, Germany) and benzophenone3 (EUSOLEX 4360) was obtained from Merck KG (Frankfurt, Germany). Benzophenone-2, benzophenone-3 and E2 were dissolved in olive oil. The applied dosages were 250 and 1000 mg/kg bodyweight for BP2 and BP3 while 17-estradiol was applied at a concentration of 0.6 mg/kg bodyweight. Pure olive oil served as control. 2.2. Animals Female Sprague–Dawley rats were purchased from Winkelmann (Borchen, Germany). Animals were housed with a light period from 6:00 to 18:00 h, at 23 ◦ C, a relative humidity of 55% with water and food ad libitum for a period of 3 weeks. The animals received soy-free chow (Sniff, Soest, Germany). Two weeks after bilateral ovariectomy, the test solutions were administered per gavage daily at 5:30 h for five days. Three to 4 hours after the last treatment, the animals were decapitated under CO2 anaesthesia. The trunk blood was collected and organs were removed, immediately frozen in liquid nitrogen and stored at −80 ◦ C. 2.3. RNA isolation and RT-PCR Total RNA was isolated using the RNeasy Total RNA Kit (Qiagen, Hilden, Germany) following the manufacturers instructions and DNA was removed by using DNase (Qiagen, Hilden, Germany). Reverse transcription was carried out with M-MLV Reverse Transcriptase RNase H Minus, Point Mutant including Re-
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combinant RNasin Ribonuclease Inhibitor (Promega, Madison WI, USA) and Random Primers (Invitrogen, Karlsruhe, Germany). RT-PCR was performed using the ABI PRISM 7700 Sequence Detector (PE Applied Biosystems, Warrington, UK) and the QPCR core kit (Eurogentec, Seraing, Belgium). The ER␣ PCR was set up according to Ponglikitmongkol et al. (1988), the ER PCR to Kuiper et al. (1996), the AhR PCR to Lovekamp-Swan et al. (2003) and the C3 PCR to Misumi et al. (1990). Primers for ERR1 (accession no. NM007953) were designed using the Primer Express software (PE Applied Biosystems, Warrington, UK). Sequences were exon spanning to avoid amplification of genomic DNA and resulted in a 143 bp amplicon. Sequences are ERR1 forward: 5 -TCC CAG GCT TCT CCT CAC TGT-3 , ERR1 reverse: 5 -TCA TCT AGG ACC AGG TCC TCA GC-3 , ERR1 probe: 5 -FAM CCC AGC GCT CAC TGC CAC TGC TAMRA-3 . 2.4. Data analysis and statistics Data are presented as mean plus S.E.M., n = 11. The relative transcript abundancy shows transcript counts of the control group after 40 cycles calculated on the basis of standard curves. For relative expression graphs, the control group was set 100% and all data were set in correlation to the control. Statistical differences were determined by student’s t-test with P < 0.05 as the level of significance.
3. Results The relative gene expression of the four receptors (Fig. 1) revealed that the ER␣ is the dominant receptor in the pituitary and in the uterus while it is almost not detectable in the thyroid. While expressed at a very low level in the pituitary and the uterus, the ER gene was expressed at a much higher level in the thyroid than ER␣ mRNA. The ERR1 showed a moderate transcript expression in the pituitary and the uterus and was the dominant receptor in the thyroid. The AhR mRNA was only expressed at a low (pituitary and thyroid) to moderate (uterus) level compared to the other receptors. Some of the “classical” markers of estrogenic action have been studied to evaluate the estrogenic potency of BP2 and BP3 at a few estrogenic end points.
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relative transcript abundancy
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400000
Pituitary Thyroid Uterus
300000 200000 100000 75000
50000
25000
0 AhR
ERR1
ER alpha
ER beta
Fig. 1. Relative expression of nuclear receptor mRNA. Total transcript counts after 40 cycles are shown. Mean plus S.E.M., n = 11. Values are only comparable between different receptors to a certain degree since separate standards were used.
The only data shown here are the uterine weight (Fig. 2). There was a significant increase in uterine weight in both treatment groups of BP2 comparable to the effect of estradiol, while in the BP3 treatment groups, no effect was seen. Other parameters were measured by our laboratory but are not shown here. The parameters were animal weight, food intake, vaginal cytology, C3 and IGF1 expression in the uterus and the LH  subunit expression in the pituitary. All data correlated with the effects seen in the uterine weight. Upon treatment with the high doses of BP2 and BP3, the AhR mRNA expression was significantly decreased in the pituitary (Fig. 3). The same decrease, though not significant, was observed in the uterus. A profound
Fig. 2. Uterine wet weight after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
significant decrease of AhR mRNA expression in the thyroid was induced by the high dosage group of BP2. In tendency also, the low dosage of BP2 and E2 reduced AhR mRNA levels while BP3 did not exert an effect on AhR expression. In the pituitary, there was no measurable effect in all treatment groups on the expression of ERR1 mRNA (Fig. 4). The expression of ERR1 mRNA in the thyroid was decreased slightly but significantly by both high dosage treatment groups of BP2 and BP3 as well as by E2 . The opposite effect, a strong increase of the uterine gene expression of ERR1, was seen in the E2 group and in both BP2 groups. BP3 did not alter uterine ERR1 mRNA levels. The strongest effect on the ER␣ gene expression (Fig. 5) in the pituitary was seen in both BP3 treatment groups where the expression was decreased and in the high dosage group, close to the detection limit. There was no clear effect of the test compounds on the expression of ER␣ mRNA in the thyroid. In the uterus, the significant effect was determined in the high dosage group of BP2, with decreased ER␣ gene expression. E2 had a decreasing effect on the ER mRNA level in the pituitary (Fig. 6), while both BP2 and BP3 were without an effect. The gene expression in the thyroid was increased in the high dosage group of BP2 and, in tendency, in the low dosage BP2 and E2 group. The uterine ER gene expression was decreased by E2 , by the high dosage of BP2 and by the low dosage of BP3.
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Fig. 3. AhR mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
Fig. 4. ERR1 mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
Fig. 5. ER␣ mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
4. Discussion With this experiment, we demonstrate that 17estradiol has a different impact on the expression pattern of some nuclear receptors, which are often the first and, therefore, the key targets of endocrine act-
ing chemicals. Additionally, we show for the first time that benzophenones have an impact on the gene expression of the two estrogen receptors as well as on the receptors ERR1 and AhR. The abundancy of the aryl hydrocarbon receptor was highest in the uterus and significantly lower in the
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Fig. 6. ER mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
thyroid and in the pituitary gland. Its function in these organs is yet unknown but a role in development is probable, since the AhR knockout mice show a significantly high mortality of their offspring. The dominant expression of the ERR1 in the thyroid may lead to the conclusion that a mediating or at least a modulating role in signal transduction is probable but this has not been reported yet. Hence, a role in energy metabolism and bone development has been postulated for the ERR1 (Gigu`ere, 2002). The abundancy of both estrogen receptors (correlating with Kuiper et al., 1997) in the tested organs suggests that ER␣ may be more important in the uterus and anterior pituitary where this receptor type was highly expressed whereas almost no ER␣ transcripts were detectable in the thyroid gland. An opposite distribution was seen for the ER transcripts which were high in the thyroid and low in the uterus and pituitary gland. There is now growing evidence that ER␣ activation mediates proliferating signals whereas activation of ER causes differentiation of cells (F¨orster et al., 2002). This may shed some light on the functions of the receptor subtypes: in the uterus and pituitary where constant remodelling during estrous cycle and pregnancy is going on, ER␣ may be required for proliferation whereas in the thyroid gland, a high degree of differentiation is required at all times; hence, ER is the dominant ER subtype. Benzophenone and hydroxylated benzophenones like benzophenone 2 and 3 are known to exert E2 -like effects in the uterus, i.e. to stimulate uterine weight (Schlumpf et al., 2001; Yamasaki et al., 2003). The uterus is proposed by the OECD as the most important screening tool with most reliable end points of estro-
genic actions. Our results indicate that according to the OECD protocol BP2 would be classified as a strong estrogenic-acting chemical. The non-hydroxylated diphenylketone benzophenone was shown to have estrogenic activities in reporter gene assays as well as in the uterotrophic assay (Yamasaki et al., 2002) while O-alkylated benzophenone subtypes like BP12 did not exert any activity. Benzophenone was also shown not to bind to ERs of either subtype, which may suggest that the mother substance benzophenone needs to be hydroxylated in vivo before acquiring estrogenic activity and that alkylation prevents binding to ERs because of the unavailability of hydroxyl groups. Hence, the alkylated benzophenones remain inactive. BP3 was devoid of any uterotrophic effect and would therefore not be classified as an endocrine disruptor following the OECD protocol. The lacking effect of BP3 in the uterotrophic assay is in apparent contrast to data published by Schlumpf et al. (2001). However, these authors observed effects only in immature rats at doses of 1500 mg/kg/day and above, which are much higher than the doses used in the present experiments. This may also explain why BP3 with no hydroxyl group in para position but with an O-alkyl group turned out to be non-estrogenic at the applied concentrations. In the pituitary, where E2 reduced ER gene expression, both doses of BP2 proved to be ineffective in modulating ER transcript levels. This indicates that BP2 did not exert an estrogenic activity in the pituitary on ER gene expression. On the other hand, BP2 and BP3 proved to be active to reduce AhR gene expression in the pituitary gland, an effect not seen in the
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E2 -treated animals. Hence, other than ER␣- or ERmediated mechanisms may also be initiated by BP2 and BP3. However, the downregulating effect of BP3 on ER␣ mRNA in the pituitary gland suggests that it has effects in estrogen-receptive organs which are not yet unravelled. A reduced ER␣ gene expression in the pituitary may indicate less proliferation. BP2 and BP3 at the higher dose significantly inhibited AhR gene expression in the pituitary, an effect not observed in the E2 -treated animals. Hence, BP2 and BP3 most likely acted via non-ER-mediated mechanisms. It was interesting to observe a mild stimulation of ER under E2 and the low BP2 dosage but a profound stimulation under the high BP2 exposure in the thyroid. This exaggerated response of ER gene expression to the BP2 stimulus was accompanied by largely increased prolactin levels (data not shown) and points again to the differentiating effects of ER. The suppression of the AhR transcripts in the thyroid gland was seen in both the E2 - and the BP2-treated animals, though statistical significance was only achieved in animals treated with the high dose of BP2. Therefore, any substance addressing the AhR may become less active in the thyroid gland of estrogenized animals. Whether this is a beneficial or an adverse effect cannot be judged at the present time because functions of the AhR in the thyroid gland are unknown. In several cell lines, activation of the AhR was shown to reduce ER␣-mediated estrogenic activity (reviewed in Safe and McDougal, 2002). If this would take place in the thyroid gland, it would not reflect on ER␣ transcripts because these remained unchanged under all treatment regimes. In the thyroid gland, all three test substances E2 , BP2 and BP3 downregulated ERR1 gene expression moderately. The effects mediated via ERR1 in the thyroid gland are unknown at present. Surprisingly, BP3, which was devoid of any uterotrophic effect, inhibited ER␣ transcript expression and also mildly the ER gene expression in the uterus. Whether these effects are of functional significance for the uterus remains unknown at present and deserves further investigation. Reduced ER in the uterus may be indicative for less differentiation and allowing more activity of ER␣-induced proliferation. No significant effects of any of the three treatment compounds were observed on gene expression of the AhR in the uterus. The large upregulation of ERR1 in the uterus by E2 and BP2 suggests a profound role of this transcription factor
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in the regulatory mechanisms of this organ. This might not be an essential role for the ERR1, since knockout mice are fertile (Luo et al., 2003). The investigated chemical substances affected the expression of the four measured nuclear receptors in different ways, showing the possibility for endocrine disruptors to evolve adverse effects in all organs, tissues and cells which express nuclear receptors. Our data suggest that E2 and BP2 have generally similar effects in the three organs tested while BP3 exerts effects which are not covered by the uterotrophic assay but might prove to be adverse. Effects mediated via AhR and ERR1 in these organs are unknown, so the physiological relevance of our findings remains to be established.
Acknowledgement This work was supported by the European Commission (EURISKED, contract no. EVK1-CT200200128).
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Effects of estradiol, benzophenone-2 and benzophenone-3 on the expression pattern of the estrogen receptors (ER) alpha and beta, the estrogen receptor-related receptor 1 (ERR1) and the aryl hydrocarbon receptor (AhR) in adult ovariectomized rats Christiane Schlecht, Holger Klammer, Hubertus Jarry, Wolfgang Wuttke∗ Department of Clinical and Experimental Endocrinology, University of Goettingen, Robert-Koch-Strasse 40, D-37099 Goettingen, Germany Available online 30 July 2004
Abstract The estrogen receptors (ERs) are members of a super family of ligand-activated transcription factors mediating estrogenic responses. A close functional kinship was found for the structurally related estrogen receptor-related receptor1 (ERR1), a constitutively active transcription factor. The aryl hydrocarbon receptor (AhR) mediates the toxic and estrogenic effects of a wide variety of environmental contaminants and industrial pollutants. Both the ERR1 and the AhR are known to modulate the ER’s signalling pathways in multiple ways. Organic chemicals with a certain structural relationship to steroid hormones often induce a tissue- or cell-specific variety of responses distinct from estrogenic responses and this may involve ERR1 and AhR. The UV-screens benzophenone-2 and benzophenone-3 (BP2, BP3), structurally related to known steroid receptor ligands, are used in cosmetics and plastics to improve product stability and durability. Both BP2 and BP3 were shown to exert uterotrophic effects and BP2 was shown to bind to the estrogen receptors. Whether such effects are also exerted in other organs is unknown. Therefore, an approach to a multi-organic risk assessment for these substances was made by measuring the gene-expression of the four mentioned receptors in the pituitary, the uterus and the thyroid after a five-day treatment in comparison to estradiol. Though BP2 seems to exert an estrogen-like effect while BP3 does not, there are regulatory effects on receptor expression for both substances that indicate a kind of endocrine disruption that is not assessed by “classical” estrogenic markers. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: ER␣; ER; AhR; ERR1; 2,2 ,4,4 -Tetrahydroxybenzophenone; 2-Hydroxy 4-methoxybenzophenone; Estradiol; Endocrine disruptor; Gene expression; Rat
1. Introduction Abbreviations: E2 , 17-estradiol; BP2, benzophenone-2; BP3, benzophenone-3; IGF1, insulin-like growth factor; HAH, halogenated aromatic hydrocarbons; C3, complement protein 3; LH, luteinizing hormone; PAH, polycyclic aromatic hydrocarbons ∗ Corresponding author. Tel.: +49 551 396714; fax: +49 551 396518. E-mail address: [email protected] (W. Wuttke).
Estrogens mediate their effects on growth, differentiation and functioning of target tissues through the two known estrogen receptors (ERs) alpha and beta (Green et al., 1986; Kuiper et al., 1996). These receptors belong to a large family of ligand-activated
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transcription factors (Mangelsdorf et al., 1995). Amongst other steroid receptors, this family comprises the estrogen receptor-related receptors (ERR) (Gigu`ere et al., 1988; Eudy et al., 1998). The ERRs display a close structural and functional relationship to the estrogen receptors (Lu et al., 2001). Taken together with their broad expression (Gigu`ere, 2002), they might play a major role in modulating anti/estrogenic responses. Because no endogenous ligand is known, the ERRs are called orphan receptors. The estrogen receptor-related receptor1 (ERR1) is supposed to be a constitutively active transcription factor (Chen et al., 2001). The aryl hydrocarbon receptor (AhR) belongs to the family of basic helix-loop–helix per-arnt-sim transcription factors and shares only a small structural relationship with the steroid receptors. Initially identified by Poland et al. (1979), it became known as the mediator of the toxic and anti/estrogenic effects of dioxin. The AhR is very promiscuous and a wide variety of ligands including halogenated aromatic hydrocarbons (HAHs) like dibenzofurans, biphenyls und dioxins, polycyclic aromatic hydrocarbons (PAHs) from incomplete combustions and possible endogenous ligands like tryptophan derivatives bind to this receptor (Denison and Nagy, 2003). For both receptors, ERR1 and AhR, multiple mechanisms of interfering with the estrogen receptor signalling pathway are known (as reviewed in Gigu`ere, 2002; Safe and McDougal, 2002) and both may be involved in mediating and/or modulating signal transduction and may be in part responsible for tissue- and cell-specific effects of industrial chemicals and environmental pollutants. Cosmetic products may contain chemical UV filters to improve product stability and durability as well as to protect human skin from harmful effects of UV radiation. Another use for UV filters is to prevent plastic products from light-induced damage. From the 12 known derivatives of benzophenone (BP), which are used as UV filters, the test substances benzophenone-2 (BP2; 2,2 ,4,4 tetrahydroxybenzophenone) and benzophenone-3 (BP3; 2-hydroxy-4-methoxybenzophenone) are frequently used in cosmetics and plastics. BP3 has been shown to exert a uterotrophic effect in vivo, to stimulate cell proliferation of MCF-7 breast cancer cells and to increase the secretion of tumour marker pS2 in vitro (Schlumpf et al., 2001). Additionally, it is absorbed through the skin after a 4-h application
of sunscreen products (Hayden et al., 1997; Felix et al., 1998) and via the gastro-intestinal tract (Kadry et al., 1995). A possible bioaccumulation has been postulated after BP3 was found in human milk, a marker for bioaccumulation in human adipose tissue (Pittet and Ferrer, 1997). Despite an estrogenic effect of BP3 itself, it is likely that the chemical is metabolized in vivo to the more potent estrogenic benzophenone-1 as it occurs in vitro after incubation with the liver S9 extract (Takatori et al., 2003). After a comparison of 517 chemical structures for their ability to activate the ER␣ in a yeast-two-hybrid assay, estrogenic structures seem to need an aromatic ring with a hydroxyl group in para position of the rest of the molecule and a hydrophobic moiety in the meta position (Nishihara et al., 2000). With the potency of BP1 with two hydroxyl groups in ortho and para positions on one of the aromatic rings, it is likely that BP2 with four hydroxyl groups displays at least a similar estrogenic potency. Indeed, BP2 has been positively tested in a reporter gene assay for ER␣, as well as in a uterotrophic assay (Yamasaki et al., 2003). Due to a few mutations in its ligand-binding domain, ERR1 is not able to bind 17-estradiol (E2 ; Horard and Vanacker, 2003) but the close structural kinship allows the assumption that estrogen receptor-related chemicals might also be ERR1 ligands (Chen et al., 2001). The AhR on the other hand is very promiscuous and has also to be considered to be a possible target of environmental chemicals, especially as the strongest known ligand dioxin shares an astonishing similarity with benzophenones. The risk of being exposed to benzophenones is considerable. The content of a single UV filter in a cosmetic product, regulated by EU Council Directive 76/768/EEC, may not exceed 10% but a total content of 29.3% has been found (Rastogi, 2002). To rate benzophenones as endocrine disruptors, they have to exert adverse effects like increased rate of cancer, reproductive system abnormalities, immune system deficiencies or excert adverse effects on other metabolic parameters (Melnick, 1999). Though non-receptor-mediated mechanisms by endocrine disruptors are known (reviewed in Cato et al., 2002), the primary sites of action are nuclear receptors. Therefore, our first approach was the evaluation of the expression of nuclear receptor transcripts in various estrogen-responsible organs in ovariectomized adult rats. After a five-day treatment
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with the chemicals BP2 and BP3 in two different dosages with a 17-estradiol and a negative control group, the expression of the four receptors ER␣, ER, ERR1 and AhR mRNA was measured in the pituitary, uterus and thyroid. Additionally, some “classical” markers of estrogenic action have been tested.
2. Materials and methods 2.1. Chemicals 17-Estradiol was obtained from Sigma-Aldrich GmbH (Taufkirchen, Germany); benzophenone-2 (UVINUL D50) was obtained from BASF ChemTrade GmbH (Burgbernheim, Germany) and benzophenone3 (EUSOLEX 4360) was obtained from Merck KG (Frankfurt, Germany). Benzophenone-2, benzophenone-3 and E2 were dissolved in olive oil. The applied dosages were 250 and 1000 mg/kg bodyweight for BP2 and BP3 while 17-estradiol was applied at a concentration of 0.6 mg/kg bodyweight. Pure olive oil served as control. 2.2. Animals Female Sprague–Dawley rats were purchased from Winkelmann (Borchen, Germany). Animals were housed with a light period from 6:00 to 18:00 h, at 23 ◦ C, a relative humidity of 55% with water and food ad libitum for a period of 3 weeks. The animals received soy-free chow (Sniff, Soest, Germany). Two weeks after bilateral ovariectomy, the test solutions were administered per gavage daily at 5:30 h for five days. Three to 4 hours after the last treatment, the animals were decapitated under CO2 anaesthesia. The trunk blood was collected and organs were removed, immediately frozen in liquid nitrogen and stored at −80 ◦ C. 2.3. RNA isolation and RT-PCR Total RNA was isolated using the RNeasy Total RNA Kit (Qiagen, Hilden, Germany) following the manufacturers instructions and DNA was removed by using DNase (Qiagen, Hilden, Germany). Reverse transcription was carried out with M-MLV Reverse Transcriptase RNase H Minus, Point Mutant including Re-
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combinant RNasin Ribonuclease Inhibitor (Promega, Madison WI, USA) and Random Primers (Invitrogen, Karlsruhe, Germany). RT-PCR was performed using the ABI PRISM 7700 Sequence Detector (PE Applied Biosystems, Warrington, UK) and the QPCR core kit (Eurogentec, Seraing, Belgium). The ER␣ PCR was set up according to Ponglikitmongkol et al. (1988), the ER PCR to Kuiper et al. (1996), the AhR PCR to Lovekamp-Swan et al. (2003) and the C3 PCR to Misumi et al. (1990). Primers for ERR1 (accession no. NM007953) were designed using the Primer Express software (PE Applied Biosystems, Warrington, UK). Sequences were exon spanning to avoid amplification of genomic DNA and resulted in a 143 bp amplicon. Sequences are ERR1 forward: 5 -TCC CAG GCT TCT CCT CAC TGT-3 , ERR1 reverse: 5 -TCA TCT AGG ACC AGG TCC TCA GC-3 , ERR1 probe: 5 -FAM CCC AGC GCT CAC TGC CAC TGC TAMRA-3 . 2.4. Data analysis and statistics Data are presented as mean plus S.E.M., n = 11. The relative transcript abundancy shows transcript counts of the control group after 40 cycles calculated on the basis of standard curves. For relative expression graphs, the control group was set 100% and all data were set in correlation to the control. Statistical differences were determined by student’s t-test with P < 0.05 as the level of significance.
3. Results The relative gene expression of the four receptors (Fig. 1) revealed that the ER␣ is the dominant receptor in the pituitary and in the uterus while it is almost not detectable in the thyroid. While expressed at a very low level in the pituitary and the uterus, the ER gene was expressed at a much higher level in the thyroid than ER␣ mRNA. The ERR1 showed a moderate transcript expression in the pituitary and the uterus and was the dominant receptor in the thyroid. The AhR mRNA was only expressed at a low (pituitary and thyroid) to moderate (uterus) level compared to the other receptors. Some of the “classical” markers of estrogenic action have been studied to evaluate the estrogenic potency of BP2 and BP3 at a few estrogenic end points.
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relative transcript abundancy
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400000
Pituitary Thyroid Uterus
300000 200000 100000 75000
50000
25000
0 AhR
ERR1
ER alpha
ER beta
Fig. 1. Relative expression of nuclear receptor mRNA. Total transcript counts after 40 cycles are shown. Mean plus S.E.M., n = 11. Values are only comparable between different receptors to a certain degree since separate standards were used.
The only data shown here are the uterine weight (Fig. 2). There was a significant increase in uterine weight in both treatment groups of BP2 comparable to the effect of estradiol, while in the BP3 treatment groups, no effect was seen. Other parameters were measured by our laboratory but are not shown here. The parameters were animal weight, food intake, vaginal cytology, C3 and IGF1 expression in the uterus and the LH  subunit expression in the pituitary. All data correlated with the effects seen in the uterine weight. Upon treatment with the high doses of BP2 and BP3, the AhR mRNA expression was significantly decreased in the pituitary (Fig. 3). The same decrease, though not significant, was observed in the uterus. A profound
Fig. 2. Uterine wet weight after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
significant decrease of AhR mRNA expression in the thyroid was induced by the high dosage group of BP2. In tendency also, the low dosage of BP2 and E2 reduced AhR mRNA levels while BP3 did not exert an effect on AhR expression. In the pituitary, there was no measurable effect in all treatment groups on the expression of ERR1 mRNA (Fig. 4). The expression of ERR1 mRNA in the thyroid was decreased slightly but significantly by both high dosage treatment groups of BP2 and BP3 as well as by E2 . The opposite effect, a strong increase of the uterine gene expression of ERR1, was seen in the E2 group and in both BP2 groups. BP3 did not alter uterine ERR1 mRNA levels. The strongest effect on the ER␣ gene expression (Fig. 5) in the pituitary was seen in both BP3 treatment groups where the expression was decreased and in the high dosage group, close to the detection limit. There was no clear effect of the test compounds on the expression of ER␣ mRNA in the thyroid. In the uterus, the significant effect was determined in the high dosage group of BP2, with decreased ER␣ gene expression. E2 had a decreasing effect on the ER mRNA level in the pituitary (Fig. 6), while both BP2 and BP3 were without an effect. The gene expression in the thyroid was increased in the high dosage group of BP2 and, in tendency, in the low dosage BP2 and E2 group. The uterine ER gene expression was decreased by E2 , by the high dosage of BP2 and by the low dosage of BP3.
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Fig. 3. AhR mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
Fig. 4. ERR1 mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
Fig. 5. ER␣ mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
4. Discussion With this experiment, we demonstrate that 17estradiol has a different impact on the expression pattern of some nuclear receptors, which are often the first and, therefore, the key targets of endocrine act-
ing chemicals. Additionally, we show for the first time that benzophenones have an impact on the gene expression of the two estrogen receptors as well as on the receptors ERR1 and AhR. The abundancy of the aryl hydrocarbon receptor was highest in the uterus and significantly lower in the
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Fig. 6. ER mRNA expression after a five-day treatment with BP2, BP3 and E2 . Mean plus S.E.M., n = 11. Statistical differences (∗ ) were determined by student’s t-test with P < 0.05 as the level of significance.
thyroid and in the pituitary gland. Its function in these organs is yet unknown but a role in development is probable, since the AhR knockout mice show a significantly high mortality of their offspring. The dominant expression of the ERR1 in the thyroid may lead to the conclusion that a mediating or at least a modulating role in signal transduction is probable but this has not been reported yet. Hence, a role in energy metabolism and bone development has been postulated for the ERR1 (Gigu`ere, 2002). The abundancy of both estrogen receptors (correlating with Kuiper et al., 1997) in the tested organs suggests that ER␣ may be more important in the uterus and anterior pituitary where this receptor type was highly expressed whereas almost no ER␣ transcripts were detectable in the thyroid gland. An opposite distribution was seen for the ER transcripts which were high in the thyroid and low in the uterus and pituitary gland. There is now growing evidence that ER␣ activation mediates proliferating signals whereas activation of ER causes differentiation of cells (F¨orster et al., 2002). This may shed some light on the functions of the receptor subtypes: in the uterus and pituitary where constant remodelling during estrous cycle and pregnancy is going on, ER␣ may be required for proliferation whereas in the thyroid gland, a high degree of differentiation is required at all times; hence, ER is the dominant ER subtype. Benzophenone and hydroxylated benzophenones like benzophenone 2 and 3 are known to exert E2 -like effects in the uterus, i.e. to stimulate uterine weight (Schlumpf et al., 2001; Yamasaki et al., 2003). The uterus is proposed by the OECD as the most important screening tool with most reliable end points of estro-
genic actions. Our results indicate that according to the OECD protocol BP2 would be classified as a strong estrogenic-acting chemical. The non-hydroxylated diphenylketone benzophenone was shown to have estrogenic activities in reporter gene assays as well as in the uterotrophic assay (Yamasaki et al., 2002) while O-alkylated benzophenone subtypes like BP12 did not exert any activity. Benzophenone was also shown not to bind to ERs of either subtype, which may suggest that the mother substance benzophenone needs to be hydroxylated in vivo before acquiring estrogenic activity and that alkylation prevents binding to ERs because of the unavailability of hydroxyl groups. Hence, the alkylated benzophenones remain inactive. BP3 was devoid of any uterotrophic effect and would therefore not be classified as an endocrine disruptor following the OECD protocol. The lacking effect of BP3 in the uterotrophic assay is in apparent contrast to data published by Schlumpf et al. (2001). However, these authors observed effects only in immature rats at doses of 1500 mg/kg/day and above, which are much higher than the doses used in the present experiments. This may also explain why BP3 with no hydroxyl group in para position but with an O-alkyl group turned out to be non-estrogenic at the applied concentrations. In the pituitary, where E2 reduced ER gene expression, both doses of BP2 proved to be ineffective in modulating ER transcript levels. This indicates that BP2 did not exert an estrogenic activity in the pituitary on ER gene expression. On the other hand, BP2 and BP3 proved to be active to reduce AhR gene expression in the pituitary gland, an effect not seen in the
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E2 -treated animals. Hence, other than ER␣- or ERmediated mechanisms may also be initiated by BP2 and BP3. However, the downregulating effect of BP3 on ER␣ mRNA in the pituitary gland suggests that it has effects in estrogen-receptive organs which are not yet unravelled. A reduced ER␣ gene expression in the pituitary may indicate less proliferation. BP2 and BP3 at the higher dose significantly inhibited AhR gene expression in the pituitary, an effect not observed in the E2 -treated animals. Hence, BP2 and BP3 most likely acted via non-ER-mediated mechanisms. It was interesting to observe a mild stimulation of ER under E2 and the low BP2 dosage but a profound stimulation under the high BP2 exposure in the thyroid. This exaggerated response of ER gene expression to the BP2 stimulus was accompanied by largely increased prolactin levels (data not shown) and points again to the differentiating effects of ER. The suppression of the AhR transcripts in the thyroid gland was seen in both the E2 - and the BP2-treated animals, though statistical significance was only achieved in animals treated with the high dose of BP2. Therefore, any substance addressing the AhR may become less active in the thyroid gland of estrogenized animals. Whether this is a beneficial or an adverse effect cannot be judged at the present time because functions of the AhR in the thyroid gland are unknown. In several cell lines, activation of the AhR was shown to reduce ER␣-mediated estrogenic activity (reviewed in Safe and McDougal, 2002). If this would take place in the thyroid gland, it would not reflect on ER␣ transcripts because these remained unchanged under all treatment regimes. In the thyroid gland, all three test substances E2 , BP2 and BP3 downregulated ERR1 gene expression moderately. The effects mediated via ERR1 in the thyroid gland are unknown at present. Surprisingly, BP3, which was devoid of any uterotrophic effect, inhibited ER␣ transcript expression and also mildly the ER gene expression in the uterus. Whether these effects are of functional significance for the uterus remains unknown at present and deserves further investigation. Reduced ER in the uterus may be indicative for less differentiation and allowing more activity of ER␣-induced proliferation. No significant effects of any of the three treatment compounds were observed on gene expression of the AhR in the uterus. The large upregulation of ERR1 in the uterus by E2 and BP2 suggests a profound role of this transcription factor
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in the regulatory mechanisms of this organ. This might not be an essential role for the ERR1, since knockout mice are fertile (Luo et al., 2003). The investigated chemical substances affected the expression of the four measured nuclear receptors in different ways, showing the possibility for endocrine disruptors to evolve adverse effects in all organs, tissues and cells which express nuclear receptors. Our data suggest that E2 and BP2 have generally similar effects in the three organs tested while BP3 exerts effects which are not covered by the uterotrophic assay but might prove to be adverse. Effects mediated via AhR and ERR1 in these organs are unknown, so the physiological relevance of our findings remains to be established.
Acknowledgement This work was supported by the European Commission (EURISKED, contract no. EVK1-CT200200128).
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