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Endocrinology Vol. 143, No. 12 4544-4551
Copyright © 2002 by The Endocrine Society

ARTICLE

Isomer-Specific Activity of Dichlorodyphenyl-trichloroethane with Estrogen Receptor in Adult and Suckling Estrogen Reporter Mice

D. Di Lorenzo, R. Villa, G. Biasiotto, S. Belloli, G. Ruggeri, A. Albertini, P. Apostoli, M. Raviscioni, P. Ciana and A. Maggi

3rd Laboratory/Biotechnology (D.D.L., R.V., G.R., A.A.), Civic Hospital of Brescia, and Institute of Chemistry (R.V., G.B., S.B., A.A.), and Institute of Occupational Health and Industrial Hygiene (P.A.), University of Brescia, 25123 Brescia, Italy; and Department of Pharmacological Sciences (M.R., P.C., A.M.), University of Milan, 20133 Milan, Italy

Address all correspondence and requests for reprints to: Diego Di Lorenzo, Laboratory of Biotechnology, Civic Hospital of Brescia, 25123 Brescia, Italy. E-mail: dilorenz{at}master.cci.unibs.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the tissue-specific effects of dichlorodyphenyltrichloroethane (DDT) isomers in adult and suckling newborn mice, using a novel mouse line engineered to express a reporter of estrogen receptor transcriptional activity (ERE-tkLUC mouse). The DDT isomers p,p’-DDT [1,1,1-trichloro2,2-bis(p-chlorophenyl) ethane] and o,p’-DDT [1,1,1-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl) ethane] were specifically selected as a weak and a strong estrogen, respectively. In adult male mice, p,p’-DDT induced luciferase activity in liver, brain, thymus, and prostate but not in heart and lung. The effect of p,p’-DDT was dose-dependent, maximal at 16 h after sc treatment, and completely blocked by the estrogen receptor antagonist ICI-182,780. In all the organs analyzed, except the liver, administration of o,p’-DDT showed a pattern of luciferase induction superimposable to that of its isomer p,p’-DDT. In liver, o,p’-DDT significantly decreased basal luciferase activity and blocked the reporter induction by 17ß-estradiol. These data lead us to hypothesize that a modulation of ER activity may be involved in the toxic effects of DDT demonstrated by epidemiological and experimental studies.

Luciferase activity was also studied in 4-d-old mice lactating from a mother injected with either p,p’-DDT or o,p’-DDT. Both isomers induced a 2-fold increase in the newborn brain. An opposite effect was observed in liver, where p,p’-DDT increased and o,p’-DDT decreased luciferase, thus indicating that these compounds modulate ER activity in adult and newborn tissues by use of a similar mechanism.

The ERE-tkLUC mouse proves to be a suitable tool to functionally assess the tissue specificity of estrogenic/antiestrogenic compounds in adult (as well as in suckling) mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ORGANOCHLORINE PESTICIDES COMPOSE a large family of chemicals that are toxic for endocrine functions (1, 2, 3, 4, 5, 6). The use of these compounds has been banned or severely restricted in most industrialized countries (7). However, because of their persistence in the environment and in living organisms, they still represent a largely unsolved problem for human health (8, 9, 10, 11, 12). Studies carried out over the last decade documented that these xenohormones bind to and activate the estrogen receptors (ERs) and, consequently, induce estrogenic effects in different animals. It is now clear that these compounds, also named endocrine disrupters (EDs), are able to evocate an endocrine response at the level of tissues target of sex steroids, thyroid, and adrenal hormones (13, 14, 15). This has been shown in vitro using cell lines engineered to overexpress specific receptors and reporter genes (16, 17) and in vivo with specific animal models in which some of the aspects of the pathophysiological action of these compounds have been illustrated (18, 19, 20). The recent recognition of the multifaceted activities of estrogen and its receptors reveals new potential targets for the activity of EDs. This, together with the novel acquired knowledge that, in the context of different tissues, a given ligand may have even opposite activity (e.g. agonist or antagonist) on ERs, imposes evaluation of the potential hazard of ER-interacting chemicals by the analysis of their effects on the transcriptional activity of ER in each specific target organ. The animal models applied to date to the study of the in vivo effects of organochlorine pesticides (21, 22, 23) do not allow us to investigate the toxicodynamic and toxicokinetic properties of these compounds in the whole organism and, in particular, in the nonreproductive organs. The availability of a novel line of mice reporter of ER transcriptional activation (ERE-tkLUC; Ref. 24) prompted us to prove its applicability to the study of EDs by evaluating the activity of the two isomers composing dichlorodyphenyltrichloroethane (DDT), a well-known weak environmental estrogen. We here provide a map of ER activation by the two DDT isomers, p,p’-DDT and o,p’-DDT in nonreproductive tissues and prostate and demonstrate that these two compounds may either block or activate this receptor transcriptional activity, depending on the tissue targeted. In addition, because of the high concern regarding the effective exposure of the newborn to chemicals through the mother’s milk (25), we used our system to assess the extent of ER activation in tissues of suckling newborns breast-fed by DDT-exposed mothers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
The procedures involving the animals and their care were conducted in accord with 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 ed. Washington, D.C.; National Academy Press, National Research Council Guide, www.nap.edu/readingroom/books/labrats).

ERE-tkLUC 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 littermates obtained by mating our founders with C57BL/6 wild-type mice. Heterozygous transgenic male mice were screened by PCR analysis for the presence of the transgenic cluster.

Heterozygous male mice (2 months old) were injected ip with 100 µl 17ß-E2, p,p’-DDT or o,p’-DDT at the needed concentration or with 100 µl of vehicle (vegetal oil) as control. At the time of death of the animals, by cervical dislocation, the tissues were dissected and immediately frozen on dry ice. Tissue extracts were prepared by homogenization in 500 µl 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 Bradford’s assay (26).

Chemicals
We purchased 17ß-estradiol (17ß-E2) from Sigma (Pomezia, Italy). Radioligands for progesterone receptors (PRs) were purchased from Amersham (Milan, Italy). Organochlorine compounds p,p’-DDT [1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane] and o,p’-DDT [1,1,1,-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl)ethane] were purchased from Superchrom (Milan, Italy). ICI 182,780 was a gift from Zeneca Pharmaceuticals (Cheshire, UK).

Enzymatic assay
Luciferase enzymatic activity was measured, as reported by de Vet et al. (27), in tissue extracts at a protein concentration of 1 mg/ml. The light intensity was measured with a luminometer (Murex Diagnostici, Rome, Italy) over 10 sec and expressed as relative light units per microgram proteins.

Ligand-binding assays
The PR concentration in the cytosols from liver was measured by the charcoal-dextran method, at a protein concentration between 1 and 2 mg/ml, and carried out by means of the multiple-saturation analysis (range of 3H-ORG2058: 0.75–12 nM) in the presence or absence of a 100-fold excess of cold progesterone as competitor. The values were plotted by the method of Scatchard (28). Proteins in cytosol were determined by the method of Bradford (26).

Gas chromatography-mass spectrometry (GC-MS) analysis
Sample extraction.
One hundred microliters of serum or 40 mg of milk were extracted with n-hexane/diethylether, 1:1 (vol/vol), mixture 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 µl).

GC-MS analysis.
Two microliters of the extracts were injected in GC-MS. Then, p,p’-DDT was separated by chromatography on a PONA fused silica capillary column (Hewlett-Packard, Palo Alto, CA) with helium as carrier gas (flow-rate, 1 ml/min constant flow) and by temperature programming. The instrument used was a GC HP 6890 MS HP 5972-A (Hewlett-Packard); p,p’-DDT was identified by 235 and 237 ions and quantified by the standard addition method (10- and 100-ng/ml solutions). The concentrations are expressed as ng/ml or ng/g•milk fat.

Statistical analysis
P values were calculated with ANOVA followed by the Scheffé test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue-specific modulation of transgene expression by p,p’-DDT
To determine the activity of the DDT isomer p,p’-DDT on ER, in the context of specific tissues, male mice were treated with p,p’-DDT using a route of administration (ip) and a dosage known, by preceding studies carried out in vivo, to be active on ER. In parallel, mice were also treated with 17ß-E2 as a reference compound. Figure 1Go shows that p,p’-DDT and 17ß-E2 induced luciferase activity in liver, brain, prostate, and thymus but not in heart. The p,p’-DDT was also unable to induce luciferase in lung, an organ clearly responsive to the treatment with estradiol. With p,p’-DDT, the strongest luciferase induction was observed in liver (10-fold induction). In the other responsive organs, luciferase content was generally doubled, with respect to time 0 and to controls (brain and thymus, 2-fold induction; prostate, 1.6-fold induction). Interestingly, with p,p’-DDT, the peak of luciferase accumulation was observed 16 h after treatment. With 17ß-E2, this effect was obtained 6 h after injection. Therefore, the ER response to p,p’-DDT seemed to be delayed, with respect to 17ß-E2. Measurement of luciferase content at 6, 16, and 24 h of animals treated with vehicle demonstrated that the basal activity of the promoter is very stable in time.



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  		Figure 1. 17ß-E2 and p,p’-DDT effects on luciferase expression in ERE-tkLUC mice. Two-month-old mice were fed an estrogen-free diet for 2 d before treatments. Vegetal oil (shaded bars) or an oil solution of p,p’-DDT (5000 µg/kg) (A, clear bars) or 17ß-E2 (50 µg/kg) (B, clear bars) were administered ip at time 0. Controls (vehicle-treated animals) at defined time points are indicated as shaded bars. Animals were killed at the indicated time-points, and tissues were dissected and stored at -80 C until assayed. Luciferase activity, measured in tissue extracts, was expressed as relative light units normalized on protein concentration. The experiments were repeated five times, with a total of nine animals per group. Bars represent the average ± SEM. *, P < 0.05, as compared with the control.

 
At the dosage selected for the study, the extent of luciferase accumulation was always lower with p,p’-DDT than with estradiol administration. To investigate whether this was attributable to a different intrinsic activity of the two compounds or to the dosage used, we proceeded to compare the effects of estradiol and p,p’-DDT using a large range of concentrations. In all the tissues investigated, p,p’-DDT was maximally active at a dosage 100 times higher (5000 µg/kg) than that of 17ß-E2 (50 µg/kg) (Fig. 2Go, A and B). Thus, in agreement with the previous observations, in all the tissues assayed, perhaps with the exception of brain, the intrinsic activity of p,p’-DDT never approached that of 17ß-E2.



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  		Figure 2. Dose response analysis. ERE-tkLUC mice were treated ip with oil; with p,p’-DDT at the doses of 50, 500, 5,000, 50,000, and 500,000 µg/kg; or 17ß-E2 at the doses of 0.5, 5, 50, and 500 µg/kg for 16 or 6 h, respectively. Luciferase activity was determined as specified in the previous figure. The experiments were repeated three times, with a total of six animals per group; data represent the mean of all the experiments done. Values are plotted on a logarithmic scale.

 
Luciferase activity reflects the effects of 17ß-E2 and p,p’-DDT on an endogenous target gene, PR
To verify whether the modulation of the transgenic marker by 17ß-E2 and p,p’-DDT mirrored that of an endogenous estrogen target gene, we measured PR binding in liver after the dose- and time-dependency treatments (Fig. 3Go). PR content, as measured by radioligand assay, was maximal at 6 and 16 h after 17ß-E2 or p,p’-DDT injection, respectively. The doses determining the maximal response were 50 and 5000 µg/kg for the two compounds (Fig. 3AGo). In agreement with the observations on the effects of the two compounds on luciferase, 17ß-E2 intrinsic activity was significantly higher, with an induction of PR of 20-fold vs. the 12-fold observed with p,p’-DDT. The basal levels of PR were undetectable at different time points (not shown). The measurement by immunoenzymatic assay of PR content in liver of mice treated for 0, 6, and 16 h with p,p’-DDT or 17ß-E2 confirmed the results obtained by binding (not shown).



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  		Figure 3. Effect of p,p’-DDT administration on progesterone receptors in ERE-tkLUC mice liver. Mice were treated as specified in Figs. 1Go and 2Go; doses of 17ß-E2 (open bars) or p,p’-DDT (shaded bars) are plotted on a logarithmic scale. PR binding was measured in cytosol of mouse liver, by the charcoal-dextran method, using [3H]ORG2058 as radioligand and progesterone as cold competitor. Values are expressed as femtomoles per milligram of cytosolic protein. Bars represent the average ± SEM of two individual experiments, each performed in triplicate. *, P < 0.05, as compared with the control. prot., Protein.

 
p,p’-DDT activity requires ER
Previous studies on DDT isomers and metabolites have questioned whether the less estrogenic DDT isomer p,p’-DDT was really acting through ERs (17, 29), or better via aromatase induction of its major metabolite p,p’-DDE (30) or by stimulating growth factor receptor tyrosine kinase activity (31). Thus, to assess whether the described effects of p,p’-DDT on the ERE-luciferase transgene were mediated by the ER, 2-month-old male mice were treated with the compound together with the selective ER antagonist ICI-182,780. To ensure the blockade of ER, ICI-182,780 was injected at the dose of 20 mg/kg, 1 h before (10 mg/kg) and 6 h after (10 mg/kg) p,p’-DDT (at the dose of 5000 µg/kg). Luciferase activity was measured in liver, brain, prostate, and thymus, 16 h after injection of p,p’-DDT. As shown in Fig. 4Go, the induction of the reporter was efficiently blocked by ICI-182,780 in all the tissues analyzed. ICI-182,780 alone did not elicit any significant effect, suggesting its lack of toxicity on the system. These data indicate that p,p’-DDT activity in these tissues requires ER.



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  		Figure 4. p.p’-DDT induction of luciferase expression is blocked by the ER-specific antagonist ICI-182,780. A total of 10 mg/kg ICI-182,780 was injected ip, 1 h before and 6 h after a single dose of p,p’-DDT (5000 µg/kg). Mice were killed 16 h after p,p’-DDT administration. Luciferase activity was normalized for protein content; fold induction was calculated with respect to values measured in control mice (oil-injected). The graph represents the data average of three separate experiments, each performed on groups of two mice each. *, P < 0.05 vs. p,p’-DDT-treated mice.

 
o,p’-DDT isomer displays an antagonist activity on the liver ERs
p,p’-DDT represents the most abundant isomer (80%) of the technical grade DDT. We then tested also the activity of the less abundant, but more estrogenic o,p’-DDT. In all the tissues tested, except the liver, o,p’-DDT induced maximal luciferase accumulation 16 h after treatment (Fig. 5AGo). As observed with the p,p’-DDT isomer, the maximal effect was at the dose of 5000 µg/kg (not shown). Interestingly, at the same concentrations used to activate the reporter in the brain, thymus, and prostate, o,p’-DDT inhibited luciferase accumulation in the liver. To assess whether the decreased transgene expression observed in liver was attributable to trans-repression or blockade of ER activity, we treated adult male mice with either a fixed amount of 17ß-E2 (50 µg/kg) alone or with increasing doses of o,p’-DDT or p,p’-DDT for 16 h. Figure 5BGo shows that the coadministration of 17ß-E2 and o,p’-DDT resulted in a significant decreased luciferase activity in liver, but not in brain, where the presence of the o,p’-DDT isomer increased the natural hormone effect.



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  		Figure 5. o,p’-DDT activates ER transcriptional activity in all the tissues but not in liver. A, Adult mice were injected with oil or an oil solution of o,p’-DDT at increasing concentrations (50–500 to 5,000–50,000 µg/kg). Mice were killed 16 h after treatment. B, Competition experiments of the DDT isomers with 17ß-E2. Two-month-old mice, fed with an estrogen-free diet for 2 d before treatments, were injected ip with 17ß-E2 (50 µg/kg) alone or with four concentrations of p,p’-DDT or o,p’-DDT (50–500 to 5,000–50,000 µg/kg). Controls were injected with vegetal oil. Luciferase activity was measured in liver and brain of mice killed 16 h after treatment. Doses of p,p’-DDT and o,p’-DDT are plotted on a logarithmic scale. The experiments were repeated three times, with a total of six animals per group. Bars represent the average ± SEM of two individual experiments, each performed in triplicate. *, P < 0.05, as compared with the control.

 
Activity of ER in 4-d-old mice nursed by p,p’-DDT- and o,p’-DDT-treated mothers
Because exposure to DDT has been shown to be particularly critical during perinatal life (23, 32, 33) and because DDT contaminates milk (5, 25, 34, 35), we used the ERE-tkLUC model to investigate whether DDT was activating ERs in newborns breast-fed by mothers exposed to this compound. Lactating transgenic mothers were given a single ip injection of p,p’-DDT or o,p’-DDT (50 mg/kg body weight) at d 4 after delivery. Suckling pups were killed 16 h after treatment of the mothers, and luciferase activity was measured in their livers and brains. The amount of milk the pups had sucked was hypothesized by weighing the stomachs. In pups nursed by the DDT-treated mothers, the stomach weighed 40% less than controls. Despite that, the amount of DDT in the milk was sufficient to modulate ERs, as shown by luciferase enzymatic activity (Fig. 6Go). Treatment of the lactating mothers with both DDT isomers resulted in an increase of luciferase activity in the pups’ brains. In liver, the effect of the two isomers reflected what was already described in adult mice: p,p’-DDT caused an increase, and o,p’-DDT caused a decrease, of luciferase activity, although with a different magnitude than with the adults. To assess whether these differences in reporter induction could be ascribed to lower levels of circulating DDT in pups, p,p’-DDT was measured in serum of lactating mothers and suckling mice and in milk, by GC-MS, at the time point showing maximal induction. In animals treated with vehicle, p,p’-DDT was undetectable. In the serum of lactating mice, we measured an average of 1,460 ± 266 ng/ml, 16 h after injection. Interestingly, at the same time-point, the concentration of p,p’-DDT in milk was significantly higher than in serum (53,790 ± 3,550 ng/g milk fat). In the serum of pups, p,p’-DDT content was of 208 ± 19 ng/ml (Table 1Go). As shown in Fig. 6Go, this p,p’-DDT concentration was sufficient to activate the reporter, both in liver and brain. These results show that, in suckling newborns, reporter activity is regulated by concentrations of p,p’-DDT similar to those often reported in serum of humans in exposed populations (36).



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  		Figure 6. Effects of DDT isomers in the suckling mice. Lactating mothers, maintained on a normal diet, were injected with 50 mg/kg body weight of p,p’-DDT, o,p’-DDT or vehicle as control. Mothers were treated at d 4 after delivery, and pups were killed 16 h later. Luciferase activity was normalized to protein content and monitored in liver and brain of the newborn. Each column is the mean ± SEM of five mice/group from two separate experiments. *, P < 0,05, compared with the appropriate control.

 

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  		Table 1. Levels of p,p'-DDT in milk and serum of adult and suckling mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By the use of a novel reporter mouse, we here provide evidence of the activity of p,p’-DDT and o,p’-DDT in mouse nonreproductive tissues and prostate. Our data demonstrate a differential isomer-specific activity in liver but not in all the other organs tested. We also show that this isomer-specific effect can be observed in newborn mice lactating from a mother exposed to DDT.

The present study demonstrates, for the first time, that DDT isomers modulate ER activity in nonreproductive organs, such as liver, brain, and thymus. So far, little is known with regard to the effects of these compounds in nonreproductive organs. Previous epidemiological studies have shown effects of DDT on liver and the central nervous system (CNS) (8, 10). Toxicity in liver has also been experimentally reproduced by Takayama et al. (11) in nonhuman primates. The experimental data on the effects on the CNS in vivo are few (33, 37) and relate to the altered behavior of the exposed animals. Our data provide the basis for the understanding of the mechanism of toxicity of these compounds in liver and CNS. Furthermore, by showing an effect in prostate, and particularly in thymus, our findings suggest novel sites for potential toxic effects of DDT. This observation is worth pursuing in more detailed experimental or epidemiological studies.

In this initial systematic analysis of the effects of DDT isomers on ERs in vivo, two observations need to be underlined. First, both isomers were inactive in lung, a tissue known to express ERß and clearly responsive to 17ß-E2. Second, in liver, the two isomers display opposite effects, p,p’-DDT being an ER agonist, and o,p’-DDT an ER antagonist. Liver is known to express predominantly ER{alpha}, whereas lung expresses ERß. These observations lead us to hypothesize that DDT is inactive in tissues expressing only ERß and may have a differential effect when only ER{alpha} is expressed. Supporting this hypothesis is the fact that also in prostate, an organ with predominant expression of ERß, the luciferase induction by DDT is weaker than in the other responsive tissues. This might have important implications for the evaluation of the toxicity profile of different organochlorine pesticides. Remaining unclear is the significance of the delayed response of ERs to DDT; the maximal luciferase and PR response was in fact observed at 6 and 16 h after injection of 17ß-E2 and DDT, respectively. This could be attributable to a slower kinetic of distribution of DDT isomers from the site of injection.

The data here presented demonstrate that the reporter mouse we used is a very promising system for acquiring a global perspective of ED activity in a mammalian organism. As described in the previous publication (24), and here with the analysis of estrogen and DDT effects on an endogenous target for estrogens (PR), the measure of luciferase seems to fully reflect the activity of ERs in vivo. The present ERE-tkLUC mouse can only allow monitoring of the sum of ER{alpha} and ERß transcriptional activities. Novel models obtained by breeding the ERE-tkLUC mice into ER{alpha} and ERß knockout mice, will enable the study of the effects on each ER receptor subtype.

Recently, Nagel et al. (38) also proposed the use of reporter mice in the study of the effects of xenobiotics. This model, however, has several advantages, with respect to Nagel’s. In fact, the presence of insulator sequences surrounding the reporter element prevents position effects and ensures the expression and full responsiveness of the reporter in all the mouse cells. Furthermore, the choice of luciferase as reporter grants a dynamic analysis of the state of ER activity. This is because the wild-type luciferase has proven to have a fast turnover when expressed in mammalian tissues (half-life of 2–3 h). Finally, luciferase activity can be monitored in vivo by photon imaging. This enables one to obtain a global, whole-body view of the state of ER activity in time, within the same animal.

When using a reporter mouse, the major concern regards the limited sensitivity of the animal system. Indeed, in the ERE-tkLUC mouse, the induction of luciferase was triggered at DDT isomer concentrations almost one order of magnitude lower than those necessary to induce the uterotropic effect in prepuberal female mice (18, 39). We also show that o,p’-DDT, at a dose known to interfere with animal fertility (50 µg/kg), significantly affects ER response. In female mice with a single injection of 50,000 µg/kg DDT, we reached serum concentrations similar to those reported in exposed populations (25, 36, 40); and at that dosage, the effect of the toxic compound on luciferase is very strong.

Most interesting are the data on suckling mice. In these mice, breast fed by DDT-exposed mothers, we measured a blood concentration of the toxic agent (208 ng/ml) similar to values of DDT and other organochlorine compounds [polychlorinated biphenyls (PCBs)] found in blood of peoples living in industrialized areas (25, 36). This would indicate that the model is adequate for studies on the activity of xenobiotics concentrations comparable with those found in environmentally exposed individuals. The possibility of following the effects of DDT during development and lactation in all the mouse tissues will finally provide functional data on the effects of pesticides on reproductive and nonreproductive organs, still incomplete for newborn and juvenile mammals. Therefore, different from the uterotropic assay, which is the most used in vivo model so far used for the study of toxic compounds active on ERs, the model system we generated has the necessary sensitivity and target specificity to provide information on the tissue-selective agonist/antagonist effect of EDs.


   Acknowledgments
 
We are grateful to Alessandro Bulla and Ron Risley for their helpful English writing and Francesca Piazza for secretarial assistance.


   Footnotes
 
This work was partially supported by European Union Grant BMH 4-97 2286 and by Ministero dell’Università e della Ricerca Scientifica e Technologica 40% (to A.M.) and Associazione Italiana per la Ricerca sul Cancro (to A.M.).

Abbreviations: CNS, Central nervous system; DDT, dichlorodyphenyltrichloroethane; 17ß-E2, 17ß-estradiol; ED, endocrine disrupter; ER, estrogen receptor; ERE, estrogen-responsive element; GC, gas chromatography; LUC, luciferase; MS, mass spectrometry analysis; PR, progesterone receptor; tk, thymidine kinase.

Received April 25, 2002.

Accepted for publication August 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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