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Whole body action of xenoestrogens with different chemical structures in estrogen reporter male mice
Toxicology 205 (2004) 65–73
Whole body action of xenoestrogens with different chemical structures in estrogen reporter male mice M. Penzaa , E. Bonettia , R. Villaa , S. Ganzerlaa , R. Bergonzic , G. Biasiottob , L. Caimid , P. Apostolic , P. Cianae , A. Maggie , D. Di Lorenzoa,∗ a
d
3rd Laboratory/Biotechnology, (D.D.L., R.V., M.P., E.B., S.G.), Civic Hospital of Brescia, Brescia 25123, Italy b Institute of Chemistry (R.V., G.B.), Brescia 25123, Italy c Institute of Occupational Health and Industrial Hygiene (P.A., R.B.), Brescia 25123, Italy Department of Diagnostics, Civic Hospital and Department of Biomedicine and Biotechnology, (L.C.) University of Brescia, Brescia 25123, Italy e Centre of Excellence on Neurodegenerative Diseases (P.C., A.M.), University of Milan, Milan 20133, Italy Available online 7 August 2004
Abstract The present work tested the estrogenic activity of three weak environmental estrogens p,p DDT [1,1,1-trichloro-2,2-bis(pchlorophenyl) ethane], p,p DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] and BHC [-benzene-hexachloride] in the transgenic estrogen-reporter mouse model (ERE-tK-LUC). By a time dependent analysis of the transgenic reporter expression (luciferase), we showed that all these chemicals modulated the estrogen receptors (ERs) in the whole body, although with a different efficacy and depending upon the tissue analyzed. Peak activity was registered at 16 h of treatment with 5000 g/kg of each compound. Organochlorines are lipophylic molecules that accumulate in fat. During weight loss they are mobilized and their concentration increases in blood. We tested whether after experimental accumulation in fat tissue, followed by a 48 h period of fasting, these compounds could be modulated to reach sufficient levels to activate the ERs in target tissues. This experimental setting produced results that were different from those obtained following acute treatments. In loaded mice, fasting induced BHC mobilization resulted in strong ER activation in the liver, lung, eye, cerebellum, hypothalamus and cortex. p,p DDT mobilization had no effect in these tissues, but efficiently acted in the testis, where, on the contrary, BHC inhibited reporter expression. During fasting, BHC, p,p DDT and the metabolite p,p DDE increased in blood concentration, from 2.7 ± 0.36, 0.65 ± 0.01 and 0.48 ± 0.06 g/ml to 9.51 ± 1.1, 4.98 ± 0.77 and 6.0 ± 0.71 g/ml, respectively. We conclude that these organochlorines modulate differently the expression of estrogen regulated genes in a tissue- and compound-specific manner and that their action
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Corresponding author. Tel.: +39 030 3995408 7 6; fax: +39 030 307251. E-mail address: [email protected] (D.D. Lorenzo).
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.06.038
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is dependent on the energy balance. Moreover, we show that this mouse model is suitable to detect the estrogenic activity of chemicals with variable structures such as alkyl phenols and polychlorobiphenyls. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Estrogen receptors; Estrogen reporter mice; Endocrine disruptors; In vivo imaging; p,p DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane]; p,p DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene]; BHC [-Benzene-hexachloride].
2. Materials and methods
edu/readingroom/books/labrats]. ERE-tk-LUC transgenic mice were kept in animal rooms maintained at a temperature of 23 ◦ C, with natural light/dark cycles. For the experiments, we used heterozygous litter mates obtained by mating our founders with C57BL/6 wildtype mice. Heterozygous transgenic male mice were screened by PCR analysis for the presence of the transgenic cluster. Before treatments mice were alwayes put for 7 days on an estrogen-free diet (Mucedola, Milan). For the acute treatments, heterozygous male mice (2-months old) were injected i.p. with 50 g/kg of 17-estradiol, 5000 g/kg of p,p -DDT or o,p -DDT or with 100 l of vehicle (vegetal oil) as control. For loading of compounds the animals were treated for three consecutive days with 100000 g/kg or 50 g/kg of BHC, p,p DDT or 17-estradiol, respectively. The mice were than put into groups which were either fasted for 48 h or fed ad libitum. The animals were sacrificed by cervical dislocation and the tissues were dissected and immediately frozen on dry ice. Tissue extracts were prepared by homogenization in 500 l of 100 mM K2PO4 lysis buffer (pH 7.8) containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, and 0.7 mM phenylmethylsulfonylfluoride, with three cycles of freezing-thawing and 30 min of microfuge centrifugation at maximum speed. Supernatants, containing luciferase, were collected and protein concentration was determined by the Bradford’s assay (Bradford, 1976).
2.1. Experimental animals
2.2. Chemicals
The procedures involving the animals and their care were conducted in accord to institutional guidelines, which comply with national and international laws and policies [National Institutes of Health, Guide for the Care and Use of Laboratory Animals, 1996 (7th edition) (Washington, D.C.); National Academy Press, National Research Council Guide, htpp://www.nap.
We purchased 17-estradiol (17-E2) from Sigma (Pomezia, Italy). Organochlorine compounds p,p DDT [1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane] and p,p -DDE [1,1,1,-trichloro-2(p-chlorophenyl)-2(o-chlorophenyl)ethane], BHC (-benzene-hexachloride), nonylphenol, 4-tert-octylphenol and 4-OHPCB were purchased from Superchrom (Milan, Italy).
1. Introduction The actual concern on the activity of environmental estrogens regards in part their action as ER modulators in the whole body (Di Lorenzo et al., 2002; Palanza et al., 2001; Nagel et al., 2001; Den Hond et al., 2001; Hoyer, 2001). They may interfere with ER-ligands such as endogenous estrogens, therapeutics and with beneficial estrogens present in food. Recent acquisitions allow to hyphotesize that negative effects on normal endocrine function and on hormone dependent pathologies may be caused by blood concentration of estrogenic chemicals as low as those released from fat depots during diets or diseases (Pelletier et al., 2003). The availability of reporter mice in which the state of transcriptonal activity of estrogen receptors can be investigated in all the estrogen target organs (ERE-tKLUC mice) (Ciana et al., 2001), provides an opportunity to measure the tissue specific activity of xenoestrogens at relatively low doses and after mobilization out of fat tissue induced by dietary restriction. Here we demonstrate that three compounds BHC, p,p DDT and p,p -DDE are mobilized differently during weight loss and exert compound-specific effects in ERs expressing tissues of male mice.
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ICI-182780 was a gift of Zeneca Pharmaceuticals (Cheshire, UK). 2.3. Enzymatic assay Luciferase enzymatic activity was measured, as reported by de Vet et al. (1987), in tissue extracts and in vivo, as reported by Ciana et al. (2003). 2.4. Gas chromatography–mass spectrometry (GC–MS) analysis 2.4.1. Sample extraction One milliliter of serum was added to 2 ml of methanol and shaked for 30 min. The mix was than added with 6 mls of n-hexane/diethyl ether, shaked for 30 min and centrifuged for 10 min at 2000 rpm. Four milliliter of the solution were concentrated to 1 ml in a vacuum evaporator and eluted on a Florisil cartridge (6 ml, 1 g; Supelco, Bellefonte, PA) and a silica cartridge (6 ml, 1 g; Supelco) sequentially. The extracts were brought to dryness, and the derivatives were dissolved in n-hexane (100 l). As internal standard, a solution of 2,4,6-trichlorobiphenyl (1 g/ml) was used. 2.4.2. GC–MS analysis Two microliters of the extracts were injected in GC–MS and separated by chromatography on a PONA fused silica capillary column (HP, CA) with helium as carrier gas (flow-rate, 1 ml/min constant flow) and by temperature programming (injector 240 ◦ C, detector 320 ◦ C). The instrument used was a GC HP 6890 MS HP 5972-A (HP, CA). p,p -DDT, p,p -DDE and BHC were identified by single ion monitoring methods (SIM), by the following ions: 217/219, 235/237, 246/248. They were quantified by the standard addition method (10-and 100-ng/ml solutions). Concentrations are expressed as g/ml. 2.5. Statistical analysis Data are reported as mean ± S.D. of 8–10 mice per treatment group. P-values were calculated with ANOVA followed by the Scheffe’ test.
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3. Results 3.1. Time dependent ER activation by BHC, p,p DDT and p,p DDE in mouse organs A time dependent analysis was determined in several tissues of transgenic ERE-iK-LUC reporter male mice (Ciana et al., 2003) injected i.p. with 5000 g/kg of BHC, p,p DDT and p,p DDE separately. The response was quantified by assaying the enzymatic activity of the transgenic marker, luciferase. In parallel, mice were also treated with 17-estradiol (50 g/kg) as a reference compound. As reported in Fig. 1, all the compounds showed ability to modulate the ERs, time dependently and in a tissue preferential manner. Maximal increase in ER action was almost always evident at 16 h treatment. For all these compounds, peak activity was previously registered at 5000 g/kg, in all the tissues tested. The response to these chemicals was always delayed with respect to 17-estradiol which peaks at 6 h. Measurement of luciferase in tissues of animals treated with vehicle alone, showed a stable basal activity of the transgenic marker. 3.2. Whole-body imaging of organochlorine estrogenic action The results on tissue homogenates were confirmed by in vivo imaging in those organs where luciferase activity was measurable by this technique. A detailed analysis of reporter regulation was possible in single alive animals by using an intensified Peltier cooled charge-couple device (CCD) camera. Imaging was performed on vehicle, 17E2 and organochlorine-treated ERE-tK-LUC mice (Fig. 2). Mice were analyzed on whole body before (time 0) and at 16 h after acute injection of BHC, p,p DDT or after 6 h of 17E2 treatment. The substrate luciferine was injected i.p. 15 min before beginning photon recording. The activity of the reporter in areas of vehicle-treated mice at time 0, allowed also to monitor negative modulation. In the liver, the most responsive tissue, a strong induction was detectable with all the compounds. The activity of 17E2 resulted much stronger compared to the organochlorines. In testicles, imaging confirmed a signal increase in p,p DDT-treated mice and a decrease in BHC treated animals.
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Fig. 1. Time dependent regulation of the estrogen driven reporter after a single acute injection of BHC, p,p DDT and p,p DDE in ERE-tK-LUC male mice. Two months old male mice were put on an estrogen-free diet for 1 week before treatments. Vehicle (vegetal oil) or an oil solution of 50 g/kg of 17E2 or 5000 g/kg of BHC, p,p DDT and p,p DDE were administered i.p. Mice were sacrificed after 0, 6, 16, 24 and 48 h. Serum and tissues were collected and stored at −80 ◦ C until assayed. Luciferase activity in tissue extracts was expressed as relative light units normalized on protein concentration. The experiments were repeated three times, with a total of 10 animals per group. Results are expressed as means of 10 determination for each treatment group. Values are plotted on a logarithmic scale. Bars represent the average ± SEM. ∗ P < 0.01 as compared with the relative controls.
3.3. BHC, p,p DDT and p,p DDE released from fat depots reach estrogenic levels in the organism Organochlorines accumulate in adipose tissues and are released during lipid mobilization. To assess the estrogenic activity of these compounds under fasting induced energy unbalance, we set up the following experimental protocol (Fig. 3A). Thirty ERE-tK-LUC mice per group were loaded with 100,000 g/kg/day of BHC or p,p DDT for three consecutive days (accumulation phase). One group was treated with ve-
hicle alone (control) and a group with 17-estradiol (50 g/kg/day). Compound-loaded and control mice were left untreated for 15 days on an estrogen-free diet and water ad libitum. After 15 days (t0) we assessed the presence of residual estrogenic activity by quantification of the luciferase enzyme. Luciferase was slightly above control levels in mice loaded with BHC and p,p DDT, indicating the presence of residual amounts of chemicals after 15 days from the treatment (white bars) (Fig. 2B). We than verified if fasting could induce a further increase in
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Fig. 2. Analysis of ERE-tK mediated luciferase activity by in vivo imaging. Imaging data were registered in vehicle (control), BHC and p,p DDT treated ERE-tK-LUC mice (5000 g/kg) at 16 h and 17E2 treated mice (50 g/kg) at 6 h. Luminescence measurements are expressed as the integration of the average brightness/pixel unit. The mice on the picture represents a single experiment. Numbers represent the average ± SEM of two experiments with four mice per group. ∗ P < 0.05; as compared with the relative controls.
compound bioavailability and if the amount released from fat stores could affect ERE-dependent gene expression. At time 0, 20 mice of each group were fasted for 48 h. Of these, 10 mice were administered with 100 g/kg of ICI-182780 at the following times with respect to time 0: −18, −6, 0, +12, +24, +36 h. Fasting itself slightly activated the ERs in most of the tissues, while it caused a dramatic reduction in reporter activity in the liver (<80%) (black bars). In the BHC treated group, compound mobilization strongly reverted fasting-induced reporter down regulation in the liver (four-fold induction) and induced ER activity in the lung (two-fold). In these tissues induction was inhibited by ICI-182780 indicating an ER mediated action (gray bars). Also in this experimental setting, BHC was inhibitory in the testicle, where it caused a decrease in reporter level down to 30% of control values (<70%). Different results were obtained in mice loaded with p,p DDT. A significant induction was observed in the testicles (2.3-fold induction), while the chemical was completely unable to activate the reporter in the liver and lung. No differences in ER activity were detected in 17estradiol treated mice before or after 48 h fasting, compared to control vehicle treated mice, indicating the clearance of the estrogen shortly after loading (not shown).
3.4. Serum levels of BHC, p,p DDT, and p,p DDE mobilized during fasting To assess whether the observed differences in reporter induction in loaded and fasted mice were due to a mobilization of the chemicals, a quantitative evaluation of the circulating levels of BHC, p,p DDT and p,p DDE was performed after 48 h fasting. Fed mice were used as controls. The metabolite p,p DDE was assayed in serum of p,p DDT-loaded mice since this compound is expected to appear shortly after p,p DDT treatment. As shown in Fig. 4, all compounds were mobilized and increased in serum levels. -Benzenehexachloride increased from 2.7 ± 0.36 g/ml to 9.51 ± 1.1 g/ml during the 48 h fasting. p,p DDT and its metabolite p,p DDE, increased from 0.65 ± 0.01 g/ml to 4.98 ± 0.77 g/ml and from 0.48 ± 0.06 g/ml to 6.0 ± 0.71 g/ml, respectively. Fasting induced a decrease in body weight of about 3 g per mouse (from 18.1 ± 1.6 to 15.2 ± 1.2 g/mouse) in all treated and untreated groups. 3.5. Environmental chemicals with different chemical structures show tissue specific estrogenicity in vivo To assess whether this model could detect the ER-mediated activity of xenoestrogens with different
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Fig. 3. Effect of fasting on reporter regulation in transgenic mice loaded or unloaded with BHC and p,p DDT. (A) Experimental protocol. Mice were put on an estrogen-free diet for 1 week before any treatment and for the entire course of the experiments. Mice were loaded with 300,000 g/kg of BHC or p,p DDT and 150 g/kg of 17-estradiol or vehicle (30 mice per group), during the three days treatment and left without any other treatment for the following 15 days. Twenty mice for each group were than put on complete food deprivation with water ad libitum (Time 0). Of these 20 mice, 10 were treated with the antiestrogen ICI-182780 (100 g/kg) in vegetal oil at the following times: −18, −6, 0, +12, +24, +36 h, with respect to time 0. Ten mice were left without further treatments for 48 h. At the end of the 48 h fasting, all the mice were sacrificed and the tissues collected and stored at −80 ◦ C untill assayed. (B) Luciferase values of the BHC, p,p DDT and 17-estradiol treated mice (control, fasted and fasted plus ICI-182780) in different tissues. Luciferase activity was expressed as relative light units normalized on protein concentration. The experiments were repeated twice. Bars represent the average ± SEM. ∗ P < 0.05; ∗∗ P < 0.001, as compared with the relative controls. For the fasted plus ICI-182780 group (gray bars), the relative control is represented by fasted mice (black bars).
chemical structures, we also treated the ERE-tK-LUC mice with two alkyl phenols (nonylphenol and 4tert-octylphenol) and one polychlorobiphenyl (4-OHPCB). As shown in Fig. 5, all these compounds modulated the ERE-dependent reporter in mouse tissues. A significant induction however was reached only in those tissues where induction was more than 1.5-fold. The highest relative induction was reached by 4-OHPCB in two non reproductive tissues, the lung and kidney and in the testicle and prostate.
4. Discussion The organochlorines BHC, p,p DDT and its main metabolite p,p DDE, have in common a low toxicity, the ability to concentrate in the food chain and to be extremely long-lived in the adipose tissues of animals and humans (Jaga and Dharmani, 2003). Previous reports have shown a variable mobilization capacity of environmental chemicals (Diel et al., 2000) from sites of accumulation and a consequential
M. Penza et al. / Toxicology 205 (2004) 65–73
Fig. 4. Serum levels of BHC, p,p DDT, and p,p DDE during fasting. Controls indicate the concentration of BHC, p,p DDT and p,p DDE in serum of compound-loaded mice before fasting (day 15). Mice were analyzed for changes in chemicls levels at 48 h of fasting. Values are expressed as g of substance per ml of mouse serum. The experiments were repeated twice with five mice for each group. Bars represent the average ± SEM. ∗ P < 0.01; as compared with the relative controls.
different biological action in the uterus (Bigsby et al., 1997). Here we have shown, that the blood levels reached by these compounds following fasting induced weight loss, is sufficient to modulate gene expression
Fig. 5. Estrogenic activity of endocrine disruptors with different chemical stucture. Estrogen dependent luciferase activity after a single acute injection (5000 g/kg for 16 h) of alkylphenols (nonylphenol and 4-tert-octylphenol) and the polychlorobiphenyl 4-OH-PCB, in ERE-tK-LUC male mice. Two months old male mice were put on an estrogen-free diet for 1 week before treatments. Mice were sacrificed after 16 h and tissues were collected and stored at −80 ◦ C untill assayed. Luciferase activity in tissue extracts was expressed as relative light units normalized on protein concentration. The experiments were repeated three times. Results are expressed as means of six determination for each treatment group. Bars represent the average ± SEM. ∗ P < 0.05 as compared with the relative controls.
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in the whole body. Moreover, their diverse estrogenic action, is not only due to a different mobilization capacity, but to a tissue and compound-specific action, which is dependent also on energy balance. Acute treatment of ERE-tK-LUC mice indicated that both DDT molecules are strong inducers of the transgenic reporter in all the ER expressing tissues we tested. In some cases they showed a distinct pattern of activation, depending on the tissue analyzed. Interestingly, both p,p DDT and p,p DDE showed similar activity in tissues where ER␣ is predominantly expressed (liver, spleen, kidney and, to a certain extent the hyppocampus), while p,p DDE was almost always more efficient where ER predominates or both ER␣ and ER were expressed (lung, cerebellum, heart, thymus, and eye). At the same doses, BHC appeared to be a weak inducer in most of the tissues, while it acted as an inhibitor in testicles and spleen in a time-dependent manner. The results obtained in ERE-tK-LUC-fasted mice, previously loaded with these compounds, showed several differences in the activity of these molecules, which are associated with energy unbalance. Benzene-hexachloride showed high activity in the liver and lung, where it was a very weak estrogen when administered acutely (5000 g/kg for 16 h), while it confirmed its inhibitory action in the testicle. On the contrary p,p DDT, which was weakly efficient in exerting agonistic effects under this experimental setting in most of the tissues, was an efficient inducer in testicles. Although the slight positive effect of fasting observed in unloaded mice, might have partially shadowed the estrogenicity of the chemicals, compound mobilization in loaded mice added a significant increase over fasting alone (i.e. testicle). Not in all tissues the estrogenic effect of these organochlorines was competed by the antiestrogen ICI182780, indicating the possible involvement of pathways alternative to the ERs. A non receptor-mediated action for these chemicals was hypothesize by previous studies (Meegan and Lloyd, 2003). They might infact activate second signals through mechanisms that do not require binding to the ERs (i.e. a protein kinase or the induction of tissue factors). Indirect effects of environmental pollutants may be responsible for an increase in ligand-independent receptor activity through the elevations of the intracellular levels of cAMP
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(Eckert et al., 1984 and Duchiron et al., 2002). Another explanation for the lack of effect of ICI-182780 in some tissues, may also be the specific pharmacodynamic of this compound which could be different with respect to that of p,p DDT. Both BHC and p,p DDT have been shown to be toxic to the male reproductive tract (Chowdhury and Gautam, 1990; Dikshith et al., 1978; Richthoff et al., 2003). Estrogens are important for testicular development (Hess et al., 1997; Eddy et al., 1996), thus, mechanistic studies should be carried out in order to understand how the opposite hormonal action exerted by these compounds may affect testicular function. In this work we have shown that organochlorines released from fat at doses comparable to those found in blood of exposed populations (Simonich and Hites, 1995), efficiently target ERE-containing genes in tissues of male mice, thus exerting an estrogenic action which is compound and tissue specific and is affected by fasting induced energy unbalance. To conclude, this transgenic mouse model is suitable for characterizing the agonist/antagonist action of xenoestrogens on gene promoters targeted by estrogen receptors in vivo and in alive animals.
Acknowledgments We are grateful to Francesca Piazza, Alessandro Bulla and Rosa Di Lorenzo for their helpful English writing and secretarial assistance. This work was partially supported by the European Union “EDERA”, grant no. QLK4-CT-2002-02221, by Istituto Superiore di Sanit`a (ARACNA Project) and by the FIRB grant n.er RBNE0157EH.
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Ciana, P., Raviscioni, M., Mussi, P., Vegeto, E., Que, I., Parker, M.G., Lowik, C., Maggi, A., 2003. In vivo imaging of transcriptionally active estrogen receptors. Nat. Med. 9, 82–86. Chowdhury, A.R., Gautam, A.K., 1990. BHC induced testicular impairments in rats. Ind. J. Physiol. Pharmacol. 34, 215–217. Den Hond, E., Roels, H.A., Hoppenbrouwers, K., Nawrot, T., Thijs, L., Vandermeulen, C., Winneke, G., Vanderschueren, D., Staessen, J.A., 2001. Sexual maturation in relation to poly chlorinated aromatic hydrocarbons: Sharpe and Skakkebaek’s hypothesis revisited. Environ. Health Perspect. 110, 771–776. de Vet, J.R., Wood, K.V., De Luca, M., Helinski, D.R., Subramani, S., 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell Biol. 7, 725–737. Diel, P., Schultz, T., Smolnikar, K., Strunck, E., Vollmer, G., Michna, H., 2000. Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotrophic activity. J. Steroid Biochem. Mol. Biol. 73, 1–10. Dikshith, T.S., Datta, K.K., Kushwah, H.S., Raizada, R.B., 1978. Histopathological and biochemical changes in guinea pigs after repeated dermal exposure to benzene hexachloride. Toxicology 10, 55–66. Di Lorenzo, D., Villa, R., Biasiotto, G., Belloli, S., Ruggeri, G., Albertini, A., Apostoli, P., Raviscioni, M., Ciana, P., Maggi, A., 2002. Isomer-specific activity of dichlorodyphenyltrichloroethane with estrogen receptor in adult and suckling reporter mice. Endocrinology 143, 4544–4551. Duchiron, C., Reynaud, S., Deschaux, P., 2002. Lindane-induced macrophage activating factor (MAF) production by peripheral blood leukocytes (PBLs) of rainbow trout (Oncorhynchus mykiss): involvement of intracellular cAMP mobilization. Aquat. Toxicol. 56, 81–91. Eckert, R.L., Mullick, A., Rorke, E.A., Katzenellenbogen, B.S., 1984. Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique. Endocrinology 114, 629–637. Eddy, E.M., Washburn, T.F., Bunch, D.O., Goulding, E.H., Gladen, B.C., Lubahn, D.B., Korach, K.S., 1996. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 137, 4796–4805. Hess, R.A., Bunick, D., Lee, K.H., Bahr, J., Taylor, J.A., Korach, K.S., Lubahn, D.B., 1997. A role for oestrogens in the male reproductive system. Nature 390, 509–512. Hoyer, P.B., 2001. Reproductive toxicology:current and future directions. Biochem. Pharmacol. 62, 1557–1564. Jaga, K., Dharmani, C., 2003. Global surveillance of DDT and DDE levels in human tissues. Int. J. Occup. Med. Environ. Health. 16, 7–20. Nagel, C.S., Hagelberger, J.L., McDonnell, D.P., 2001. Develoment of an ER action indicator mouse for the study of estrogens selective ER modulators (SERMs) and xenobiotics. Endocrinology 142, 4721–4727. Meegan, M.J., Lloyd, D.G., 2003. Advances in the science of estrogen receptor modulation. Curr. Med. Chem. 10, 181–210. Palanza, P., Parmigiani, S., vom Saal, F.S., 2001. Effects of prenatal exposure to low doses of diethylstilbestrol, o,p DDT and methoxychlor on postnatal growth and neurobehavioural de-
M. Penza et al. / Toxicology 205 (2004) 65–73 velopment in male and female mice. Horm. Behav. 40, 252– 265. Pelletier, C., Imbeault, P., Tremblay, A., 2003. Energy balance and pollution by organochlorines and poly chlorinated biphenyls. Obes. Rev. 4, 17–24. Richthoff, J., Rylander, L., Jonsson, B.A., Akesson, H., Hagmar, L., Nilsson-Ehle, P., Stridsberg, M., Giwercman, A., 2003. Serum
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Whole body action of xenoestrogens with different chemical structures in estrogen reporter male mice M. Penzaa , E. Bonettia , R. Villaa , S. Ganzerlaa , R. Bergonzic , G. Biasiottob , L. Caimid , P. Apostolic , P. Cianae , A. Maggie , D. Di Lorenzoa,∗ a
d
3rd Laboratory/Biotechnology, (D.D.L., R.V., M.P., E.B., S.G.), Civic Hospital of Brescia, Brescia 25123, Italy b Institute of Chemistry (R.V., G.B.), Brescia 25123, Italy c Institute of Occupational Health and Industrial Hygiene (P.A., R.B.), Brescia 25123, Italy Department of Diagnostics, Civic Hospital and Department of Biomedicine and Biotechnology, (L.C.) University of Brescia, Brescia 25123, Italy e Centre of Excellence on Neurodegenerative Diseases (P.C., A.M.), University of Milan, Milan 20133, Italy Available online 7 August 2004
Abstract The present work tested the estrogenic activity of three weak environmental estrogens p,p DDT [1,1,1-trichloro-2,2-bis(pchlorophenyl) ethane], p,p DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] and BHC [-benzene-hexachloride] in the transgenic estrogen-reporter mouse model (ERE-tK-LUC). By a time dependent analysis of the transgenic reporter expression (luciferase), we showed that all these chemicals modulated the estrogen receptors (ERs) in the whole body, although with a different efficacy and depending upon the tissue analyzed. Peak activity was registered at 16 h of treatment with 5000 g/kg of each compound. Organochlorines are lipophylic molecules that accumulate in fat. During weight loss they are mobilized and their concentration increases in blood. We tested whether after experimental accumulation in fat tissue, followed by a 48 h period of fasting, these compounds could be modulated to reach sufficient levels to activate the ERs in target tissues. This experimental setting produced results that were different from those obtained following acute treatments. In loaded mice, fasting induced BHC mobilization resulted in strong ER activation in the liver, lung, eye, cerebellum, hypothalamus and cortex. p,p DDT mobilization had no effect in these tissues, but efficiently acted in the testis, where, on the contrary, BHC inhibited reporter expression. During fasting, BHC, p,p DDT and the metabolite p,p DDE increased in blood concentration, from 2.7 ± 0.36, 0.65 ± 0.01 and 0.48 ± 0.06 g/ml to 9.51 ± 1.1, 4.98 ± 0.77 and 6.0 ± 0.71 g/ml, respectively. We conclude that these organochlorines modulate differently the expression of estrogen regulated genes in a tissue- and compound-specific manner and that their action
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Corresponding author. Tel.: +39 030 3995408 7 6; fax: +39 030 307251. E-mail address: [email protected] (D.D. Lorenzo).
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is dependent on the energy balance. Moreover, we show that this mouse model is suitable to detect the estrogenic activity of chemicals with variable structures such as alkyl phenols and polychlorobiphenyls. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Estrogen receptors; Estrogen reporter mice; Endocrine disruptors; In vivo imaging; p,p DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane]; p,p DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene]; BHC [-Benzene-hexachloride].
2. Materials and methods
edu/readingroom/books/labrats]. ERE-tk-LUC transgenic mice were kept in animal rooms maintained at a temperature of 23 ◦ C, with natural light/dark cycles. For the experiments, we used heterozygous litter mates obtained by mating our founders with C57BL/6 wildtype mice. Heterozygous transgenic male mice were screened by PCR analysis for the presence of the transgenic cluster. Before treatments mice were alwayes put for 7 days on an estrogen-free diet (Mucedola, Milan). For the acute treatments, heterozygous male mice (2-months old) were injected i.p. with 50 g/kg of 17-estradiol, 5000 g/kg of p,p -DDT or o,p -DDT or with 100 l of vehicle (vegetal oil) as control. For loading of compounds the animals were treated for three consecutive days with 100000 g/kg or 50 g/kg of BHC, p,p DDT or 17-estradiol, respectively. The mice were than put into groups which were either fasted for 48 h or fed ad libitum. The animals were sacrificed by cervical dislocation and the tissues were dissected and immediately frozen on dry ice. Tissue extracts were prepared by homogenization in 500 l of 100 mM K2PO4 lysis buffer (pH 7.8) containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, and 0.7 mM phenylmethylsulfonylfluoride, with three cycles of freezing-thawing and 30 min of microfuge centrifugation at maximum speed. Supernatants, containing luciferase, were collected and protein concentration was determined by the Bradford’s assay (Bradford, 1976).
2.1. Experimental animals
2.2. Chemicals
The procedures involving the animals and their care were conducted in accord to institutional guidelines, which comply with national and international laws and policies [National Institutes of Health, Guide for the Care and Use of Laboratory Animals, 1996 (7th edition) (Washington, D.C.); National Academy Press, National Research Council Guide, htpp://www.nap.
We purchased 17-estradiol (17-E2) from Sigma (Pomezia, Italy). Organochlorine compounds p,p DDT [1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane] and p,p -DDE [1,1,1,-trichloro-2(p-chlorophenyl)-2(o-chlorophenyl)ethane], BHC (-benzene-hexachloride), nonylphenol, 4-tert-octylphenol and 4-OHPCB were purchased from Superchrom (Milan, Italy).
1. Introduction The actual concern on the activity of environmental estrogens regards in part their action as ER modulators in the whole body (Di Lorenzo et al., 2002; Palanza et al., 2001; Nagel et al., 2001; Den Hond et al., 2001; Hoyer, 2001). They may interfere with ER-ligands such as endogenous estrogens, therapeutics and with beneficial estrogens present in food. Recent acquisitions allow to hyphotesize that negative effects on normal endocrine function and on hormone dependent pathologies may be caused by blood concentration of estrogenic chemicals as low as those released from fat depots during diets or diseases (Pelletier et al., 2003). The availability of reporter mice in which the state of transcriptonal activity of estrogen receptors can be investigated in all the estrogen target organs (ERE-tKLUC mice) (Ciana et al., 2001), provides an opportunity to measure the tissue specific activity of xenoestrogens at relatively low doses and after mobilization out of fat tissue induced by dietary restriction. Here we demonstrate that three compounds BHC, p,p DDT and p,p -DDE are mobilized differently during weight loss and exert compound-specific effects in ERs expressing tissues of male mice.
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ICI-182780 was a gift of Zeneca Pharmaceuticals (Cheshire, UK). 2.3. Enzymatic assay Luciferase enzymatic activity was measured, as reported by de Vet et al. (1987), in tissue extracts and in vivo, as reported by Ciana et al. (2003). 2.4. Gas chromatography–mass spectrometry (GC–MS) analysis 2.4.1. Sample extraction One milliliter of serum was added to 2 ml of methanol and shaked for 30 min. The mix was than added with 6 mls of n-hexane/diethyl ether, shaked for 30 min and centrifuged for 10 min at 2000 rpm. Four milliliter of the solution were concentrated to 1 ml in a vacuum evaporator and eluted on a Florisil cartridge (6 ml, 1 g; Supelco, Bellefonte, PA) and a silica cartridge (6 ml, 1 g; Supelco) sequentially. The extracts were brought to dryness, and the derivatives were dissolved in n-hexane (100 l). As internal standard, a solution of 2,4,6-trichlorobiphenyl (1 g/ml) was used. 2.4.2. GC–MS analysis Two microliters of the extracts were injected in GC–MS and separated by chromatography on a PONA fused silica capillary column (HP, CA) with helium as carrier gas (flow-rate, 1 ml/min constant flow) and by temperature programming (injector 240 ◦ C, detector 320 ◦ C). The instrument used was a GC HP 6890 MS HP 5972-A (HP, CA). p,p -DDT, p,p -DDE and BHC were identified by single ion monitoring methods (SIM), by the following ions: 217/219, 235/237, 246/248. They were quantified by the standard addition method (10-and 100-ng/ml solutions). Concentrations are expressed as g/ml. 2.5. Statistical analysis Data are reported as mean ± S.D. of 8–10 mice per treatment group. P-values were calculated with ANOVA followed by the Scheffe’ test.
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3. Results 3.1. Time dependent ER activation by BHC, p,p DDT and p,p DDE in mouse organs A time dependent analysis was determined in several tissues of transgenic ERE-iK-LUC reporter male mice (Ciana et al., 2003) injected i.p. with 5000 g/kg of BHC, p,p DDT and p,p DDE separately. The response was quantified by assaying the enzymatic activity of the transgenic marker, luciferase. In parallel, mice were also treated with 17-estradiol (50 g/kg) as a reference compound. As reported in Fig. 1, all the compounds showed ability to modulate the ERs, time dependently and in a tissue preferential manner. Maximal increase in ER action was almost always evident at 16 h treatment. For all these compounds, peak activity was previously registered at 5000 g/kg, in all the tissues tested. The response to these chemicals was always delayed with respect to 17-estradiol which peaks at 6 h. Measurement of luciferase in tissues of animals treated with vehicle alone, showed a stable basal activity of the transgenic marker. 3.2. Whole-body imaging of organochlorine estrogenic action The results on tissue homogenates were confirmed by in vivo imaging in those organs where luciferase activity was measurable by this technique. A detailed analysis of reporter regulation was possible in single alive animals by using an intensified Peltier cooled charge-couple device (CCD) camera. Imaging was performed on vehicle, 17E2 and organochlorine-treated ERE-tK-LUC mice (Fig. 2). Mice were analyzed on whole body before (time 0) and at 16 h after acute injection of BHC, p,p DDT or after 6 h of 17E2 treatment. The substrate luciferine was injected i.p. 15 min before beginning photon recording. The activity of the reporter in areas of vehicle-treated mice at time 0, allowed also to monitor negative modulation. In the liver, the most responsive tissue, a strong induction was detectable with all the compounds. The activity of 17E2 resulted much stronger compared to the organochlorines. In testicles, imaging confirmed a signal increase in p,p DDT-treated mice and a decrease in BHC treated animals.
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Fig. 1. Time dependent regulation of the estrogen driven reporter after a single acute injection of BHC, p,p DDT and p,p DDE in ERE-tK-LUC male mice. Two months old male mice were put on an estrogen-free diet for 1 week before treatments. Vehicle (vegetal oil) or an oil solution of 50 g/kg of 17E2 or 5000 g/kg of BHC, p,p DDT and p,p DDE were administered i.p. Mice were sacrificed after 0, 6, 16, 24 and 48 h. Serum and tissues were collected and stored at −80 ◦ C until assayed. Luciferase activity in tissue extracts was expressed as relative light units normalized on protein concentration. The experiments were repeated three times, with a total of 10 animals per group. Results are expressed as means of 10 determination for each treatment group. Values are plotted on a logarithmic scale. Bars represent the average ± SEM. ∗ P < 0.01 as compared with the relative controls.
3.3. BHC, p,p DDT and p,p DDE released from fat depots reach estrogenic levels in the organism Organochlorines accumulate in adipose tissues and are released during lipid mobilization. To assess the estrogenic activity of these compounds under fasting induced energy unbalance, we set up the following experimental protocol (Fig. 3A). Thirty ERE-tK-LUC mice per group were loaded with 100,000 g/kg/day of BHC or p,p DDT for three consecutive days (accumulation phase). One group was treated with ve-
hicle alone (control) and a group with 17-estradiol (50 g/kg/day). Compound-loaded and control mice were left untreated for 15 days on an estrogen-free diet and water ad libitum. After 15 days (t0) we assessed the presence of residual estrogenic activity by quantification of the luciferase enzyme. Luciferase was slightly above control levels in mice loaded with BHC and p,p DDT, indicating the presence of residual amounts of chemicals after 15 days from the treatment (white bars) (Fig. 2B). We than verified if fasting could induce a further increase in
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Fig. 2. Analysis of ERE-tK mediated luciferase activity by in vivo imaging. Imaging data were registered in vehicle (control), BHC and p,p DDT treated ERE-tK-LUC mice (5000 g/kg) at 16 h and 17E2 treated mice (50 g/kg) at 6 h. Luminescence measurements are expressed as the integration of the average brightness/pixel unit. The mice on the picture represents a single experiment. Numbers represent the average ± SEM of two experiments with four mice per group. ∗ P < 0.05; as compared with the relative controls.
compound bioavailability and if the amount released from fat stores could affect ERE-dependent gene expression. At time 0, 20 mice of each group were fasted for 48 h. Of these, 10 mice were administered with 100 g/kg of ICI-182780 at the following times with respect to time 0: −18, −6, 0, +12, +24, +36 h. Fasting itself slightly activated the ERs in most of the tissues, while it caused a dramatic reduction in reporter activity in the liver (<80%) (black bars). In the BHC treated group, compound mobilization strongly reverted fasting-induced reporter down regulation in the liver (four-fold induction) and induced ER activity in the lung (two-fold). In these tissues induction was inhibited by ICI-182780 indicating an ER mediated action (gray bars). Also in this experimental setting, BHC was inhibitory in the testicle, where it caused a decrease in reporter level down to 30% of control values (<70%). Different results were obtained in mice loaded with p,p DDT. A significant induction was observed in the testicles (2.3-fold induction), while the chemical was completely unable to activate the reporter in the liver and lung. No differences in ER activity were detected in 17estradiol treated mice before or after 48 h fasting, compared to control vehicle treated mice, indicating the clearance of the estrogen shortly after loading (not shown).
3.4. Serum levels of BHC, p,p DDT, and p,p DDE mobilized during fasting To assess whether the observed differences in reporter induction in loaded and fasted mice were due to a mobilization of the chemicals, a quantitative evaluation of the circulating levels of BHC, p,p DDT and p,p DDE was performed after 48 h fasting. Fed mice were used as controls. The metabolite p,p DDE was assayed in serum of p,p DDT-loaded mice since this compound is expected to appear shortly after p,p DDT treatment. As shown in Fig. 4, all compounds were mobilized and increased in serum levels. -Benzenehexachloride increased from 2.7 ± 0.36 g/ml to 9.51 ± 1.1 g/ml during the 48 h fasting. p,p DDT and its metabolite p,p DDE, increased from 0.65 ± 0.01 g/ml to 4.98 ± 0.77 g/ml and from 0.48 ± 0.06 g/ml to 6.0 ± 0.71 g/ml, respectively. Fasting induced a decrease in body weight of about 3 g per mouse (from 18.1 ± 1.6 to 15.2 ± 1.2 g/mouse) in all treated and untreated groups. 3.5. Environmental chemicals with different chemical structures show tissue specific estrogenicity in vivo To assess whether this model could detect the ER-mediated activity of xenoestrogens with different
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Fig. 3. Effect of fasting on reporter regulation in transgenic mice loaded or unloaded with BHC and p,p DDT. (A) Experimental protocol. Mice were put on an estrogen-free diet for 1 week before any treatment and for the entire course of the experiments. Mice were loaded with 300,000 g/kg of BHC or p,p DDT and 150 g/kg of 17-estradiol or vehicle (30 mice per group), during the three days treatment and left without any other treatment for the following 15 days. Twenty mice for each group were than put on complete food deprivation with water ad libitum (Time 0). Of these 20 mice, 10 were treated with the antiestrogen ICI-182780 (100 g/kg) in vegetal oil at the following times: −18, −6, 0, +12, +24, +36 h, with respect to time 0. Ten mice were left without further treatments for 48 h. At the end of the 48 h fasting, all the mice were sacrificed and the tissues collected and stored at −80 ◦ C untill assayed. (B) Luciferase values of the BHC, p,p DDT and 17-estradiol treated mice (control, fasted and fasted plus ICI-182780) in different tissues. Luciferase activity was expressed as relative light units normalized on protein concentration. The experiments were repeated twice. Bars represent the average ± SEM. ∗ P < 0.05; ∗∗ P < 0.001, as compared with the relative controls. For the fasted plus ICI-182780 group (gray bars), the relative control is represented by fasted mice (black bars).
chemical structures, we also treated the ERE-tK-LUC mice with two alkyl phenols (nonylphenol and 4tert-octylphenol) and one polychlorobiphenyl (4-OHPCB). As shown in Fig. 5, all these compounds modulated the ERE-dependent reporter in mouse tissues. A significant induction however was reached only in those tissues where induction was more than 1.5-fold. The highest relative induction was reached by 4-OHPCB in two non reproductive tissues, the lung and kidney and in the testicle and prostate.
4. Discussion The organochlorines BHC, p,p DDT and its main metabolite p,p DDE, have in common a low toxicity, the ability to concentrate in the food chain and to be extremely long-lived in the adipose tissues of animals and humans (Jaga and Dharmani, 2003). Previous reports have shown a variable mobilization capacity of environmental chemicals (Diel et al., 2000) from sites of accumulation and a consequential
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Fig. 4. Serum levels of BHC, p,p DDT, and p,p DDE during fasting. Controls indicate the concentration of BHC, p,p DDT and p,p DDE in serum of compound-loaded mice before fasting (day 15). Mice were analyzed for changes in chemicls levels at 48 h of fasting. Values are expressed as g of substance per ml of mouse serum. The experiments were repeated twice with five mice for each group. Bars represent the average ± SEM. ∗ P < 0.01; as compared with the relative controls.
different biological action in the uterus (Bigsby et al., 1997). Here we have shown, that the blood levels reached by these compounds following fasting induced weight loss, is sufficient to modulate gene expression
Fig. 5. Estrogenic activity of endocrine disruptors with different chemical stucture. Estrogen dependent luciferase activity after a single acute injection (5000 g/kg for 16 h) of alkylphenols (nonylphenol and 4-tert-octylphenol) and the polychlorobiphenyl 4-OH-PCB, in ERE-tK-LUC male mice. Two months old male mice were put on an estrogen-free diet for 1 week before treatments. Mice were sacrificed after 16 h and tissues were collected and stored at −80 ◦ C untill assayed. Luciferase activity in tissue extracts was expressed as relative light units normalized on protein concentration. The experiments were repeated three times. Results are expressed as means of six determination for each treatment group. Bars represent the average ± SEM. ∗ P < 0.05 as compared with the relative controls.
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in the whole body. Moreover, their diverse estrogenic action, is not only due to a different mobilization capacity, but to a tissue and compound-specific action, which is dependent also on energy balance. Acute treatment of ERE-tK-LUC mice indicated that both DDT molecules are strong inducers of the transgenic reporter in all the ER expressing tissues we tested. In some cases they showed a distinct pattern of activation, depending on the tissue analyzed. Interestingly, both p,p DDT and p,p DDE showed similar activity in tissues where ER␣ is predominantly expressed (liver, spleen, kidney and, to a certain extent the hyppocampus), while p,p DDE was almost always more efficient where ER predominates or both ER␣ and ER were expressed (lung, cerebellum, heart, thymus, and eye). At the same doses, BHC appeared to be a weak inducer in most of the tissues, while it acted as an inhibitor in testicles and spleen in a time-dependent manner. The results obtained in ERE-tK-LUC-fasted mice, previously loaded with these compounds, showed several differences in the activity of these molecules, which are associated with energy unbalance. Benzene-hexachloride showed high activity in the liver and lung, where it was a very weak estrogen when administered acutely (5000 g/kg for 16 h), while it confirmed its inhibitory action in the testicle. On the contrary p,p DDT, which was weakly efficient in exerting agonistic effects under this experimental setting in most of the tissues, was an efficient inducer in testicles. Although the slight positive effect of fasting observed in unloaded mice, might have partially shadowed the estrogenicity of the chemicals, compound mobilization in loaded mice added a significant increase over fasting alone (i.e. testicle). Not in all tissues the estrogenic effect of these organochlorines was competed by the antiestrogen ICI182780, indicating the possible involvement of pathways alternative to the ERs. A non receptor-mediated action for these chemicals was hypothesize by previous studies (Meegan and Lloyd, 2003). They might infact activate second signals through mechanisms that do not require binding to the ERs (i.e. a protein kinase or the induction of tissue factors). Indirect effects of environmental pollutants may be responsible for an increase in ligand-independent receptor activity through the elevations of the intracellular levels of cAMP
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(Eckert et al., 1984 and Duchiron et al., 2002). Another explanation for the lack of effect of ICI-182780 in some tissues, may also be the specific pharmacodynamic of this compound which could be different with respect to that of p,p DDT. Both BHC and p,p DDT have been shown to be toxic to the male reproductive tract (Chowdhury and Gautam, 1990; Dikshith et al., 1978; Richthoff et al., 2003). Estrogens are important for testicular development (Hess et al., 1997; Eddy et al., 1996), thus, mechanistic studies should be carried out in order to understand how the opposite hormonal action exerted by these compounds may affect testicular function. In this work we have shown that organochlorines released from fat at doses comparable to those found in blood of exposed populations (Simonich and Hites, 1995), efficiently target ERE-containing genes in tissues of male mice, thus exerting an estrogenic action which is compound and tissue specific and is affected by fasting induced energy unbalance. To conclude, this transgenic mouse model is suitable for characterizing the agonist/antagonist action of xenoestrogens on gene promoters targeted by estrogen receptors in vivo and in alive animals.
Acknowledgments We are grateful to Francesca Piazza, Alessandro Bulla and Rosa Di Lorenzo for their helpful English writing and secretarial assistance. This work was partially supported by the European Union “EDERA”, grant no. QLK4-CT-2002-02221, by Istituto Superiore di Sanit`a (ARACNA Project) and by the FIRB grant n.er RBNE0157EH.
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