ToxSci Advance Access originally published online on December 14, 2006
Toxicological Sciences 2007 96(1):145-153; doi:10.1093/toxsci/kfl185
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In Vitro and In Vivo Evaluation of the Estrogenic, Androgenic, and Progestagenic Potential of Two Cyclic Siloxanes
Dow Corning Corporation, Midland, Michigan 48686
1 To whom correspondence should be addressed at Dow Corning Corporation, 2200 West Salzburg Road, Mail Stop: CO3101, Midland, Michigan 48686-0994. Fax: (989) 496-5595. E-mail: anne.quinn{at}dowcorning.com.
Received September 13, 2006; accepted November 21, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The purpose of these experiments was to determine the potential estrogenic, androgenic, and progestagenic activity of two cyclic siloxanes, octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5). Receptor-binding experiments and a luciferase reporter gene assay were used to determine if the materials were able to bind and activate either the estrogen receptors (ERs) or progesterone receptors (PRs)—
or ß. The rat uterotrophic assay (RUA) for estrogenic activity and the Hershberger assay for androgenic activity were utilized as the in vivo assays. For the ER-binding studies, D4 was shown to bind to ER but not to ERß. D5 did not bind to either of the two receptors. D4 activated the reporter gene at 10µM, while D5 was considered negative in the estrogen reporter gene assay. Neither material was a ligand for the PRs. Both the RUA and Hershberger assays were conducted using whole-body inhalation of the two materials for 16 h/day. D4 resulted in a small but significant increase in both wet and blotted uterine weight as well as increases in both luminal and glandular epithelial cell height in both Sprague Dawley and Fischer 344 rats. D5 was negative in both rat strains, indicating that D5 does not possess estrogenic activity. Neither material possessed any significant antiestrogenic activity. Both materials were negative in the Hershberger assay indicating that neither material possesses any significant androgenic activity. Our studies have shown that D4 exhibits a low affinity for ER in vitro and a weakly estrogenic response in vivo.
Key Words: D4; D5; octamethylcyclotetrasiloxane; decamethylcyclopentasiloxane; silicone; siloxane; estrogen receptor and ß; androgen; progesterone; luciferase reporter gene; RUA; estrogenic.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) are clear, odorless cyclic dimethyl siloxanes with molecular weights of 296 and 371, respectively. Both are volatile liquids consisting of alternating silicon-oxygen bonds connected in a ring arrangement with two methyl groups covalently bonded to each silicon atom (Fig. 1). They are used primarily as intermediates in the manufacture of high–molecular weight siloxane polymers, such as polydimethylsiloxanes. These compounds are widely used in industrial and consumer applications (Stark et al., 1982
). Under normal use conditions, humans may be exposed to D4 and D5 by dermal, inhalation or oral routes (Maxim, 1998). The reported experiments focus on the inhalation route of exposure. Physiologically based pharmacokinetic (PBPK) modeling has been carried out for D4 indicating that the inhalation route is the most relevant one for human exposure (Anderson et al., 2001). Additionally, the inhalation route was chosen as the kinetics of an oral administration in rats indicates that D4 and D5 enter the blood stream via the lymphatics within the lipid core of chylomicrons and other lipoproteins, which is a different form than for inhalation and dermal routes of exposure (Plotzke et al., 2000). The dermal route is impractical because both materials are volatile. The kinetics following dermal and inhalation exposure are similar and have allowed for the development of PBPK models that enables accurate predictions of human exposure. The inhalation concentrations used for the in vivo experiments were the highest possible vapor concentrations based on the known vapor pressure for D4 and D5.
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The potential of xenobiotics to effect mammalian reproduction has been the subject of numerous scientific studies in the past years. As a result of this heightened awareness, many materials that have a high probability of human exposure will be evaluated with respect to potential (anti) estrogenic and (anti) androgenic activity. The screening of materials for endocrine activity has been directed by a mandate from the government to address these concerns through the requirements of the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC, 1998
). The assays utilized in this report are in line with the proposed guidelines for Tier I screening of potential estrogenic, androgenic, or progestagenic materials. (EDTA, 2001; Kanno, 2003a,b).
The role of estrogens in biological systems is complex; the ligand-inducible nuclear receptor-mediated action of this hormone results in a plethora of cellular responses. The ability of a material to bind to one of the two well-characterized estrogen receptors (ERs) is the major route by which estrogenic effects are mediated in vivo. Cellular proliferation of the human breast cancer cell line, MCF-7 (Soto et al., 1994, 1995), the expression of estrogenic genes such as pS2 (Burak et al., 1997) and increases in the organ weight of estrogen-sensitive tissues such as uterus (Grunert et al., 1987), and calcium and bone metabolism (Cai et al., 2005) are all examples of the estrogenic effects in vivo elicited through the nuclear receptors.
The ERs and progesterone receptors (PRs), and ß, belong to the nuclear receptor superfamily of ligand-regulated transcription factors, which also include the retinoid, thyroid hormone, and vitamin D receptors as well as many orphan receptors. Classical hormone action involves ligand binding followed by activation of a nuclear response element. More recently, however, alternative mechanisms for the action of estradiol involving association with extranuclear receptors, plasma membrane–associated receptors, and interactions with other signaling pathways, such as ERK 1/2 mitogen-activated protein kinases have been reported. (Cenni and Picard, 1999; Koohi et al., 2005; Marin et al., 2003, 2005).
The receptor-binding assay, reported in this study, utilized the two well-characterized ERs and PRs, and ß (Kuiper et al., 1997, 1998). Both ERs share nearly identical homology in the DNA-binding and dimerization regions but have areas of differences in the ligand-binding region (Paech et al., 1997). Thus, materials may show preferential binding for one of the two receptors as has been shown with numerous materials (Barkhem et al., 1998). The luciferase reporter gene assay utilized here is an in vitro system to quantitate the transcriptional activation of genes under the regulation of the ER and the estrogen response element (ERE) (Fertuck et al., 2001). The system used was the human epithelial cell line, MCF-7, which was transiently transfected with three separate plasmids, resulting in the ability to quantitate estrogen-mediated gene expression as described elsewhere (Charles et al., 2002).
The rat uterotrophic assay (RUA) is a standard assay for the measurement of estrogenic and antiestrogenic activity. Previously, a study which utilized the immature female model for the RUA had identified D4 as a weak estrogen via the oral route of exposure (McKim et al., 2001b). The McKim study indicated that when compared to ethinyl estradiol (EE), D4 was approximately 0.6 million times less potent in Sprague Dawley (SD) pups and 3.8 million times less potent in F-344 pups. It was important to thoroughly compare D4 and D5 with respect to estrogenic and androgenic potential via the inhalation route, using the highest achievable vapor concentration for the longest exposure (16 h/day) possible, as this is the most likely route by which humans are exposed. The possible (anti) androgenic activity of these materials was evaluated using the 10-day Hershberger assay via the inhalation route under similar conditions.
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MATERIALS AND METHODS |
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Chemicals and Reagents
The test materials, D4 and D5, were obtained from Dow Corning Corporation, Midland, Michigan. The test materials were considered stable when stored at room temperature in sealed containers. Both materials were determined to be > 99.0% pure. [2,4,6,7,16,17-3H]-estradiol (specific activity 118 Ci/mmol) was purchased from DuPont NEN (Boston, MA). Nonradioactive 17ß-estradiol and diethylstilbestrol were purchased from Sigma Chemical Co (St Louis, MO). The ERs were purchased from Panvera Corporation (Madison, WI). The MCF-7 cell line was obtained from the American Type Culture Collection (Manassas, VA) repository and was maintained and cultured using standard aseptic techniques. The transfections were carried out using the Lipofectamine procedure detailed previously by Charles et al. (2002)
. The corn oil, 17-EE, testosterone propionate (TP), and flutamide were purchased from Sigma Chemical Co. The genistein (4',5,7-trihydroxyisoflavone) and ICI 182,780 were purchased from Toronto Research Chemical (Ontario, Canada) and Tocris Cookson (Ellisville, MO), respectively.
Experimental Design
Receptor-Binding Experiments
Estrogen receptors.
The human purified receptors (ER and ERß) were used in the assay following dilution in binding buffer. The reaction mixtures contained either one of the ERs, a constant amount of radiolabeled estradiol and a wide concentration range of a competitor, either DES or estradiol. The reaction was allowed to achieve equilibrium based on the predetermined equilibration time of the radiolabeled test materials. The bound ligand-receptor complex was separated from the unbound radioligand using the hydroxyapatite assay recommended by the PanVera Corporation.
Progesterone receptors.
Progesterone binding (for PR and PRß) and activation (for PRß) were measured using both D4 and D5. The HitHunter (Discover X Corporation, Fremont, CA) kit was used to measure PR binding according to the manufacturer. Classical radioligand-binding assays were used to assess binding of D4 and D5 to recombinant human PRß and to calf uterine PR utilizing [3H]-promogestone and [3H]-progesterone as radioligands. The Progesterone Responsive Chemically Activated Luciferase Expression (PR CALUX) reporter gene assay was used for the progesterone activation assay of PRß. The assay utilized human U2-OS osteosarcoma cells transformed with a full-length human PRß cDNA expression vector and a luciferase reporter gene containing three progesterone response elements for receptor binding and activation.
Gas Chromatography–Mass Spectrometry Analysis
The concentrations of D4 or D5 in solution in aliquots of the incubation samples were determined by extraction into toluene and subsequent gas chromatography–mass spectrometry (GC/MS) analysis. An aliquot was transferred to a vial containing an internal standard solution consisting of 150 ng of tetrakis[trimethylsiloxy]silane (M4Q)/g of toluene. The samples were vortexed followed by centrifugation at 3000 x g for 4 min (Beckman GS-6R.) The toluene extract layers were then dried using magnesium sulfate. Aliquots of each of the dried extracts were transferred to limited volume inserts in GC autosampler vials for analysis by GC/MS.
Quantitation of the D4 or D5 in the extracts was performed by comparison to toluene solvent standards containing M4Q added in the same volume as for the samples and D4 or D5 in various amounts from approximately 10–18,000 ng. Linear response plots were prepared from the results of the analyses of these toluene standards, and amounts of D4 or D5 in the incubation samples were calculated based on comparison to these linear response plots. The nanogram amounts of D4 or D5 calculated in this manner were converted to concentrations (ng/ml of incubation sample) based on the known dilution volumes of the samples.
The in vitro ER-binding experiments were carried out in 2-ml glass vials with caps that contained airtight septa. The reaction was set up in the absence of the test material, and the caps were crimped into place. An 8-l Tedlar bag was used to prepare the test material vapors. A Hamilton syringe was used to deliver the appropriate volume of test material to create the 900 ppm D4 or the 160 ppm D5 vapor required through the septa. The vapor concentration of the Tedlar bag was verified using GC analysis prior to the addition of the material to the reaction mixture. The delivery of the test material to the reaction mixture was carried out in the following way: an outlet needle was placed in the septa and the 1 ml headspace was flushed with the vapor using a gastight syringe. After 9 ml of vapor had passed through the system, the outlet syringe was removed and the last 1 ml was retained in the headspace over the reaction. The tubes were incubated at 37°C with gentle inversion for 4 h. The vials were decrimped, and aliquots were removed for analysis at the completion of the experiment. Prior to running the experiments, a time course was set up to determine the optimal incubation time for the two test materials. 14C-D4 and 14C-D5 were used to prepare 900 and 160 ppm test bags as described previously. The measured radioactivity present in the aqueous reaction mixture at 1, 2, 3, and 4 h was determined, and the corresponding D4 and D5 concentrations were calculated. The reaction had reached equilibrium by 3 h.
ER Reporter Gene Activation Assay
A human epithelial cell line, MCF-7, was used for the transfection experiments. Cells were shipped frozen and maintained in a liquid nitrogen cryofreezer until use. A limit of 10 passages was imposed to keep selective culturing conditions from altering the cell population throughout the study. Vials of cells were plated and maintained in a water-jacketed incubator at 37°C ± 2°C under an atmosphere of 95% air and 5% CO2. All aspects of cell culture handling were carried out using aseptic techniques and standard culturing procedures. The MCF-7 cells, grown in six-well plates, were transiently transfected with three plasmids using the Lipofectamine procedure according to the manufacturer's instructions. The plasmids were obtained and used with permission from the laboratory of Professor Pierre Chambon, at the Université Louis Pasteur, Strasbourg, France, and are described elsewhere (Fertuck, 2001). Breifly, the Gal4-ERdef plasmid is constitutively expressed and codes for the functional ligand-binding/dimerization region and the Gal4 DNA-binding region of the ER. The 17m5-G-Luc plasmid codes for the luciferase enzymes and expression is under control of the Gal4 yeast EREs. The control plasmid used for transcriptional efficiency was the ß-galactosidase enzyme which is constitutively expressed.
For the transcriptional activation experiments, D4 and D5, were prepared as dilutions in ethanol and added directly to the culture media, with the ethanol concentration not exceeding 0.25%. The amount of luciferase activity is quantitated as a direct measure of ligand-ER complex activating the luciferase gene at the end of the incubation. The amount of ß-galactosidase activity is a reflection of the overall transfection efficiency; the luciferase activity is normalized to ß-galactosidase activity for each separate well.
The MCF-7 cells were plated using Dulbecco's modified Eagle medium with 10% fetal bovine serum added, and cells were allowed to attach overnight. The following amounts of maxi-prepped DNA were added to 100 µl/well serum-free medium in a sterile tube: 0.2 µg/well Gal4-ER def, 1.5 µg/well 17m5-G-Luc, 0.15µg/well pCMV-lacZ. In a separate tube, 4 µl of the lipofectamine reagent was added to 100 µl serum-free medium for each well. The transfection was according to the lipofectamine manufacturers instructions.
Rat uterotrophic assay.
Ovariectomized (OVEX) adult Sprague Dawley and Fischer 344 rats were utilized in this assay. Following a 14-day postsurgery regression period, the D4 and D5 were delivered via whole-body inhalation exposure for 16 h/day for 3 days. The conditions of the whole-body inhalation exposure are described elsewhere (McKim, 2001a). For comparison of estrogenic activity, additional groups of rats were given subcutaneous doses of EE (0.3, 1.0, and 3.0 µg/kg/day) and genistein (10, 25, and 50 mg/kg/day) followed by a control inhalation exposure of filtered air to mimic exposure conditions. To evaluate any antiestrogenic activity, EE at 3.0 µg/kg/day was given in combination with the ER antagonist, ICI 182,780, D4, or D5. On day 3 of exposure, the positive control groups were sacrificed 6 h after the subcutaneous dosing. The groups being exposed to D4 or D5 were sacrificed immediately at the end of the 16-h exposure on day 3. A separate corn oil control group was used for the 6-h positive controls and the 16-h exposure groups. Uterine wet and blotted weights were collected at the time of necropsy, and uteri were fixed in Bouin's fixative for histopathologic analysis of glandular and luminal epithelial cell height. After fixation, three cross sections were taken from each uterine horn, processed, and embedded in paraffin. Sections were cut at 4–6 µm and stained with hematoxylin and eosin.
Hershberger assay.
Castrated male Fischer 344 rats were exposed by whole-body inhalation to 700 ppm D4 or 160 ppm D5 for 16 h/day for 10 consecutive days. For comparison of androgenic activity, a dose response of TP was utilized (0.1–1.6 mg/kg/day). The control groups were necropsied 24 h after the last subcutaneous dose, and the D4 and D5 test groups were sacrificed immediately following the end of the 16-h exposure on day 10. A separate corn oil control group was used for the control and the test article exposure to control for the time differences in necropsy. To evaluate the antiandrogenic activity, test groups of TP (0.54 mg/kg/day) in combination with the antiandrogenic compound flutamide (10 mg/kg/day given po) or TP with D4 or D5 were used. The following tissues were dissected: ventral prostate (weighed fresh and fixed), seminal vesicle, glans penis, levator ani/bulbocavernosus (LABC) muscle, Cowper's gland, liver, and brain.
Statistical Analysis
Experiments were run in triplicate on three separate days for both the receptor-binding and the reporter gene assays. The means of daily experiments were used in the mean for the whole experiment. SDs were calculated using Microsoft Office Excel 2003. For the RUA, a comparison between treatment groups of absolute and relative (wet and blotted) uterine weights and luminal and glandular epithelial cell heights was conducted using SAS v.8.2. All body weights were analyzed using the one-way ANOVA, using Dunnett test to compare the dose means to the control means, with the p values being adjusted accordingly. Wet uterus weight, wet uterus weight-to-body weight ratio, and dry uterus weight-to-body weight ratio were analyzed using the Kruskal-Wallis test with Shirley test to compare the dose mean ranks to the control mean ranks, with the p values being adjusted accordingly. Organ weights for corn oil controls were compared to the TP dose response for the Hershberger assay. The D4 or D5 exposure group was compared to the 16-h filtered air control group for all end points evaluated. The antiandrogenic activity of D4 was evaluated using the EC70 from the TP dose-response curve previously calculated. For the comparison of TP dose response to the control group, the body weights (initial, terminal, and change) were analyzed using the one-way ANOVA, using Dunnett test to compare the dose means to the control means, with the p values being adjusted accordingly. All other end points (the liver weight, ventral prostate (fresh) weight, ventral prostate (fixed) weight, seminal vesicle weight, LABC muscle weight, glans penis weight, Cowper's gland weight, liver-to-body weight ratio, ventral prostate- (fresh) to-body weight ratio, ventral prostate- (fixed)-to body weight ratio, seminal vesicle-to-body weight ratio, LABC muscle-to-body weight ratio, glans penis-to-body weight ratio, and the Cowper's gland-to-body weight ratio) either were not normally distributed or the variances were not equal over the groups (determined using Bartlett test). These end points were analyzed using the Kruskal-Wallis test with Shirley test to compare the dose mean ranks to the control mean ranks, with the p values being adjusted accordingly.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Receptor-Binding Experiments
The means of three separate ER-binding experiments are presented in Figures 2 and 3. In all cases, the positive control articles, DES or unlabeled 17ß-estradiol, were able to competitively displace the radiolabeled estradiol in a dose-dependent manner indicating that the test system was functioning as expected. For D4, there was a displacement of the radiolabeled estradiol from the ER
, accounting for a small but significant reduction in binding (approximately 20% of the total binding). There was no effect on the binding of estradiol using ERß. Although the test article was delivered to the test system as a vapor, the concentration of D4 in the aqueous reaction mixture was determined to be 0.45µM for both receptors by GC/MS. D5 did not result in any displacement of estradiol from either ER or ERß (Fig. 3). D5 also was confirmed to be present in the reaction mixture by GC/MS at 0.28µM. These data indicate that D4 has the potential to bind to ER, but not ERß, and D5 does not bind to the active site of either receptor subtype under these in vitro experimental conditions. There was no indication that D4 and D5 were ligands for PR or PRß from the results of the HitHunter (PR) (Fig. 5) or classical radiolabel binding assays (PR and PRß) (data not presented).
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Reporter Gene Assay
Bisphenol A (BPA) and estradiol were utilized in this in vitro experiment as positive controls, and both materials resulted in an expected increase in luciferase activity in a dose-dependent manner. Neat D4 and D5 were added to the cell culture wells at a wide range of concentrations from 0.1nM to 10µM. The only increase in luciferase activity with D4 treatment occurred at the 10µM concentration (Fig. 4). D5 did not result in any activation of the luciferase reporter gene at any of the doses utilized, indicating that this material does not possess any estrogenic activity. There was no indication that D4 or D5 resulted in an activation of the PRß reporter gene assay (data not presented).
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Rat Uterotrophic Assay
The data for the uterine wet and blotted weights following D4 and D5 exposure are summarized in Tables 1 and 3 for the two strains of rat. There were significant increases in uterine wet and blotted weights following 16 h/day exposure to D4 in both rat strains. The positive control groups treated with genistein and EE resulted in the expected significant dose-responsive increase in both wet and blotted uterine weights. In Fischer rats, however, the highest dose of EE was not significantly increased over the second highest dose indicating that the dose response was reaching a plateau. Additionally, uterine fluid was noted at the time of necropsy. All the EE animals and most of the genistein animals had fluid-filled uteri at the time of necropsy. All the D4-treated animals had fluid-filled uteri at necropsy. Exposure to D5 resulted in no increase in uterine wet or blotted weight in either strain of rat. Additionally, none of the D5 uteri were noted as fluid filled at necropsy. The measurements of luminal and glandular epithelial cell height are presented in Tables 2 and 4. The data closely tracks the uterine weight data, in that D4 exposure resulted in an increase in both the luminal and glandular epithelial cell height in both SD and F-344 rats. Exposure to D5 did not result in any increase of epithelial cell height in either rat strain.
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The potential of the materials to act as antiestrogens was also evaluated. The highest dose of EE (3 µg/kg/day) was given in combination with the ER antagonist, ICI 182,789, or D4 and D5. Only D4 resulted in a mild suppression of the EE-induced increase in uterine weight in the Fischer 344 rat. This was seen for both wet and blotted uterine weight indicating that D4 may possess some weak antiestrogenic activity in this strain. This effect, however, was not observed in the measurement of the uterine epithelial cell height. Sprague Dawley rats did not exhibit any antiestrogenic effects in any of the measured end point.
Hershberger Assay
The results of the Hershberger assay were negative for both D4 and D5 (Table 5). Of the end points measured (ventral prostate weighed fresh and fixed, seminal vesicle, glans penis, LABC muscle, Cowper's gland), none resulted in any increase in organ weight. This indicates that neither D4 nor D5 possess any androgenic activity. As expected, treatment with TP resulted in a dose-dependent increase in each end point. The combination of a mid-dose of TP and flutamide were used as a standard to assess antiandrogenic activity. When TP was given in combination with D4 or D5, there was no indication that either material possessed any antiandrogenic activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estrogens are known to have a role in reproductive processes as well as events in the brain related to memory (Rehman and Masson, 2005
), bone metabolism (Cai et al., 2005), and cardiac function (Shearman et al., 2005). Progesterone can play a central role in maintenance of cycle regulation in some species and serves a vital role in maintenance of pregnancy in most species. Androgens play a pivotal role in the development of the male reproductive tract. Spermatogenesis requires high levels of intratesticular testosterone secreted by the Leydig cells. Many compounds elicit their actions by binding directly to the hormone receptor, either mimicking or blocking the actions of endogenous hormones. The ability of a compound to interact with the hormone receptors appears to be independent of the chemical class and, instead, being more closely related to the size, shape, and conformation of the chemical (Kuiper et al., 1997). In the event that a material does possess endocrine activity, it is important to put this effect into context of the whole organism, the potency of the material relative to the background of endogenous hormones, and the disposition, metabolism, and elimination of the material over time.
In this paper, we investigated the possible estrogenic, androgenic, and progestagenic activity of two cyclic siloxanes. Both of the cyclic siloxanes utilized in this report are used in a wide range of consumer applications, and it is important to understand the potential activity of the materials. The experiments reported here are the recommended assays for the Tier I Endocrine Screening Procedure set forth by the Endocrine Disruptors Testing and Assessment commission and the Organization for Economic Co-operation and Development validation process. Previous reports had indicated that D4 had weak estrogenic activity in the immature RUA via the oral route (McKim et al., 2001b). These experiments were conducted to verify those results and to determine if the effects seen in immature rats via the oral route of exposure were also seen with the inhalation route of exposure, which is one of the most relevant routes for human exposure. The rationale for the concentrations used for D4 and D5 was based on the properties of the materials. The highest vapor concentration for the whole-body inhalation experiments that can be consistently generated without aerosol generation is 700 ppm for D4 and 160 ppm for D5. For the in vitro ER-binding experiments, 900 ppm D4 was used, as the short duration, increased temperature (37°C rather than 25°C), and small scale made this vapor concentration possible to maintain. For the reporter gene experiments, the delivery of test material was by direct addition. As with many volatile and hydrophobic materials, the delivery into an aqueous-based assay can lead to rapid loss of material. In the event of reporting negative results, every effort should be made to ensure that the test material was actually available to interact. Previous work using radiolabled materials has indicated that in the presence of a cell culture monolayer, the test article is retained in an aqueous-based system. This is consistent with the positive response that was seen with D4 in the reporter gene assay. For the recombinantly expressed cell-free receptor-binding experiments, direct addition of D4 did not result in any displacement of 3H-estradiol, indicating that the D4 was rapidly lost and was unable to interact with the receptors. For these reasons, the current methods were needed to ensure the highest concentration of the material in the reaction mixture.
The primary receptor subtype present in the rat uterus is the ER. It is consistent with the earlier in vivo studies that D4 was able to bind in vitro to the ER and also that it was able to activate the ER reporter gene system. The data reported here also are consistent with an earlier report carried out in mice where D4 and D5 were utilized in an oral gavage RUA in ovariectomized mice (He et al., 2003). D4 resulted in an increase in uterine weight which was completely blocked by the ER antagonist, ICI 182,780. Additionally, the ovariectomized ER knockout mice model showed no increase in uterine weight when D4 was delivered orally. This supports the mechanism that in mice, D4 can result in estrogenic activity and that this activity is mediated through the ER (He et al., 2003). In the aforementioned mouse study, D5 was negative for any estrogenic activity when dosed orally at 1000 mg/kg. This is consistent with the in vitro and in vivo assays reported here. Overall, D5 was consistently negative in both the in vitro and in vivo assays, indicating that this material does not possess any classical estrogenic activity. In addition, we report here that neither material showed any indication of having (anti) androgenic or progestagenic activity.
D4 has been shown to have some reproductive effects in a two-generation reproductive toxicology study (Stump et al., 1999). Previously reported were a decrease in the number of day 1 corpora lutea and a decrease in the mean live litter size in the F0 and F1 generations at a 700 ppm inhalation exposure (Stump et al., 1999). Although the current report illustrates the weak estrogenic activity of D4 via the receptor-mediated pathway, the effects of D4 in the reproductive studies do not appear to be elicited by this mechanism. For example, there were no effects seen in the estrogen-sensitive developmental end points, such as anogenital distance, or time to either vaginal patency or balanopreputial separation. Additionally, in a more recent one-generation reproductive toxicity study, female rats indirectly exposed to D4 (in utero and during nursing), no adverse reproductive outcomes were observed (Siddiqui et al., 2006). In a recent chronic 2-year bioassay with D4 (Paul A. Jean, Steven D. Crofoot, James W. Crissman, Marina L. Jovanovic, K. Monica Lee, Paul A. Smith, Robert G. Meeks, and Kathleen P. Plotzke, unpublished data), there was no indication of enhanced uterine estrogenic stimulation at 6- or 12-month exposure. The apparent lack of effect calls into question the biological relevance of the weakly estrogenic activity of D4 in adult cycling rats where the endogenous hormone levels are high and orders of magnitude more potent than D4. McKim et al. (2001b) report that D4 is 0.6–3.8 million times less potent that EE in an oral RUA using SD and F-344 rats.
The ability of a compound to disrupt the endocrine system depends not only on the ability of the compound to bind to a receptor but also on the bioavailability of the test material, as well as metabolism, disposition, and elimination. The compound must be bioavailable at a concentration sufficient to influence the overall hormonal background of the animal. Endogenous hormones are generally carried through the bloodstream bound to one of several high-affinity binding proteins which provide a means of controlling cellular uptake and clearance. The vast majority of circulating hormone exists in a bound state and is therefore not free or bioavailable. The primary high-affinity binding protein is sex hormone–binding globulin in humans and -fetoprotein in rodents (Nagel et al., 1998). Albumin provides low-affinity binding for both rodents and humans. Estradiol is effectively carried in the blood and the ratio of steady-state–free versus bound hormone can be affected by other materials. The ability of D4 to activate an estrogenic response in the RUA under conditions of estrogen deprivation (OVEX animals) may not be relevant in a background of higher circulating levels of endogenous hormone. The ability of these materials to bind to any of the carrier proteins is also a relevant, yet currently unknown, parameter in determining the overall effect.
In addition to the reported estrogenic activity, D4 has been shown to possess other activities which are more likely to contribute to the reproductive effects observed in the two-generation reproductive study. McKim et al. (2001a) have reported that D4 possesses significant "phenobarbital-like" activity in that the hepatic profile of induced cytochrome P450 enzymes closely mimics that seen with the barbiturate, phenobarbital. It has been reported that inhalation exposure of adult cycling Sprague Dawley rats to D4, over 3 days, results in a suppression of the lutenizing hormone (LH) surge and a subsequent delay in ovulation (Dalu et al., 2002). Suppression of the LH surge is clearly consistent with the reproductive findings of the two-generation reproductive study. Delayed ovulation would clearly lead to reduced litter size. These reproductive reports point to D4 acting through an indirect mechanism, perhaps, by acting like phenobarbital which can disrupt hypothalamic norepinepherine neurotransmission during critical periods which suppresses the LH surge in rodents.
To summarize, a weak estrogenic effect of D4, which appears to be mediated through the ER, has been demonstrated in vivo and in vitro. D5 has no associated estrogenic activity. Neither material possessed (anti) androgenic nor progestagenic activity in the assays conducted.
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ACKNOWLEDGMENTS |
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The authors would like to gratefully acknowledge the laboratory of Professor Pierre Chambon for permission to use the plasmids for the transfection experiments and also Drs Kirsten Fertuck and Tim Zacharewski, Michigan State University, for assistance in the transfection methodology. This work was supported by the Silicones Environmental Health and Safety Committee of North America.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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