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Molecular Endocrinology 20 (9): 2141-2155
Copyright © 2006 by The Endocrine Society

Endocrine-Disrupting Organotin Compounds Are Potent Inducers of Adipogenesis in Vertebrates

Felix Grün, Hajime Watanabe, Zamaneh Zamanian, Lauren Maeda, Kayo Arima, Ryan Cubacha, David M. Gardiner, Jun Kanno, Taisen Iguchi and Bruce Blumberg

Department of Developmental and Cell Biology (F.G., Z.Z., L.M., K.A., R.C., D.M.G., B.B.), University of California Irvine, Irvine California 92697-2300; National Institutes of Natural Sciences (H.W., T.I.), National Institute for Basic Biology, Okazaki Institute for Integrative Bioscience, Okazaki 444-8787, Japan; and Division of Cellular & Molecular Toxicology (J.K.), Biological Safety Research Center, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan

Address all correspondence and requests for reprints to: Bruce Blumberg, Department of Developmental and Cell Biology, University of California Irvine, 2113 McGaugh Hall, Irvine, California 92697-2300. E-mail: blumberg{at}uci.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dietary and xenobiotic compounds can disrupt endocrine signaling, particularly of steroid receptors and sexual differentiation. Evidence is also mounting that implicates environmental agents in the growing epidemic of obesity. Despite a long-standing interest in such compounds, their identity has remained elusive. Here we show that the persistent and ubiquitous environmental contaminant, tributyltin chloride (TBT), induces the differentiation of adipocytes in vitro and increases adipose mass in vivo. TBT is a dual, nanomolar affinity ligand for both the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). TBT promotes adipogenesis in the murine 3T3-L1 cell model and perturbs key regulators of adipogenesis and lipogenic pathways in vivo. Moreover, in utero exposure to TBT leads to strikingly elevated lipid accumulation in adipose depots, liver, and testis of neonate mice and results in increased epididymal adipose mass in adults. In the amphibian Xenopus laevis, ectopic adipocytes form in and around gonadal tissues after organotin, RXR, or PPAR{gamma} ligand exposure. TBT represents, to our knowledge, the first example of an environmental endocrine disrupter that promotes adipogenesis through RXR and PPAR{gamma} activation. Developmental or chronic lifetime exposure to organotins may therefore act as a chemical stressor for obesity and related disorders.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ORGANOTINS ARE A diverse group of widely distributed environmental pollutants. Tributyltin chloride (TBT) and bis(triphenyltin) oxide (TPTO), have pleiotropic adverse effects on both invertebrate and vertebrate endocrine systems. Organotins were first used in the 1960s as antifouling agents in marine shipping paints, although such use has been restricted in recent years. Organotins persist as prevalent contaminants in dietary sources, such as fish and shellfish, and through pesticide use on high-value food crops (1, 2). Additional human exposure to organotins may occur through their use as antifungal agents in wood treatments, industrial water systems, and textiles. Mono- and diorganotins are prevalently used as stabilizers in the manufacture of polyolefin plastics (polyvinyl chloride), which introduces the potential for transfer by contact with drinking water and foods.

Exposure to organotins such as TBT and TPTO results in imposex, the abnormal induction of male sex characteristics in female gastropod mollusks (3, 4). Bioaccumulation of organotins decreases aromatase activity leading to a rise in testosterone levels that promotes development of male characteristics (5). Imposex results in impaired reproductive fitness or sterility in the affected animals and is one of the clearest examples of environmental endocrine disruption. TBT exposure also leads to masculinization of at least two fish species (6, 7), but TBT is only reported to have modest adverse effects on mammalian male and female reproductive tracts and does not alter sex ratios (8, 9). Instead, hepatic-, neuro-, and immunotoxicity appear to be the predominant effects of organotin exposure (10). Hence, the current mechanistic understanding of the endocrine-disrupting potential of organotins is based on their direct actions on the levels or activity of key steroid-regulatory enzymes such as aromatase and more general toxicity mediated via damage to mitochondrial functions and subsequent cellular stress responses (11, 12, 13, 14, 15).

However, it remains an open question whether in vivo organotins act primarily as protein and enzyme inhibitors, or rather mediate their endocrine-disrupting effects at the transcriptional level. Recent work has shown that aromatase mRNA levels can be down-regulated in human ovarian granulosa cells by treatment with organotins or ligands for the nuclear hormone receptors, retinoid X receptor (RXRs) or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (16, 17, 18). Furthermore, Nishikawa et al. (19) have demonstrated that the gastropod Thais clavigara RXR homolog is responsive to 9-cis-retinoic acid (9-cis-RA) and TBT, and 9-cis RA can also induce imposex, suggesting a conserved transcriptional mechanism for TBT action across phyla. These ligand-dependent transcription factors belong to the nuclear hormone receptor superfamily—a group of approximately 150 members (48 human genes) that includes the estrogen receptor, androgen receptor, glucocorticoid receptor, thyroid hormone receptor, vitamin D receptor (VDR), retinoic acid receptors (RARs and RXRs), PPARs, and numerous orphan receptors. We were therefore intrigued by the similar effects of TBT and RXR/PPAR{gamma} ligands on mammalian aromatase mRNA expression and hypothesized that TBT might be exerting some of its biological effects via transcriptional regulation of gene expression through activation of one or more nuclear hormone receptors.

Our results show that organotins such as TBT are indeed potent and efficacious agonistic ligands of the vertebrate nuclear receptors, retinoid X receptors (RXRs) and PPAR{gamma}. The physiological consequences of receptor activation predict that permissive RXR heterodimer target genes and downstream signaling cascades are sensitive to organotin misregulation. Consistent with this prediction we observe that organotins phenocopy the effects of RXR and PPAR{gamma} ligands using in vitro and in vivo models of adipogenesis. Therefore, TBT and related organotin compounds are the first of a potentially new class of environmental endocrine disrupters that targets adipogenesis by modulating the activity of key regulatory transcription factors in the adipogenic pathway, RXR{alpha} and PPAR{gamma}. The existence of such xenobiotic compounds was previously hypothesized (20, 21). Our results suggest that developmental exposure to TBT and its congeners that activate RXR/PPAR{gamma} might be expected to increase the incidence of obesity in exposed individuals and that chronic lifetime exposure could act as a potential chemical stressor for obesity and obesity-related disorders.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Organotins Are Agonists of Vertebrate RXR and RXR-Permissive Heterodimers
Many known or suspected environmental endocrine-disrupting chemicals mimic natural lipophilic hormones that act through members of the superfamily of nuclear receptor transcription factors (22, 23). In a screen of high-priority endocrine-disrupting chemicals against a bank of vertebrate nuclear receptor ligand-binding domains (LBDs), we observed that organotins, specifically tributyltin chloride (TBT) and bis(triphenyltin) oxide (TPTO), could fully activate an RXR{alpha} LBD construct (GAL4-RXR{alpha}) in transient transfection assays. Both TBT and TPTO were as potent (EC50 ~3–10 nM) as 9-cis retinoic acid, an endogenous RXR ligand and approximately 2- to 5-fold less potent than the synthetic RXR-specific ligands LG100268 (EC50 ~ 2 nM) or AGN195203 (EC50 ~ 0.5 nM) (Fig. 1AGo and see Table 2Go). Maximal activation for TBT reached the same levels as LG100268 or AGN195203.


Figure 1
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  		Fig. 1. Organotins Are Agonist Ligands of RXR{alpha} and PPAR{gamma}

Organotins are high-affinity ligand agonists of RXR{alpha} and PPAR{gamma}. A–D, Activation of GAL4-hRXR{alpha}, -hPPAR{gamma}, -hRAR{alpha}, or -hPPAR{alpha} in transiently transfected Cos7 cells by organotins and receptor-specific ligands. Data represent reporter luciferase activity normalized to ß-galactosidase and plotted as the average fold activation ± SEM (n = 3) relative to solvent-only controls from representative experiments. E and F, Competition binding curves of histidine-tagged RXR{alpha} or PPAR{gamma} LBDs with TBT. Data shown are from a representative experiment analyzed in GraphPad Prism 4.0 and Ki values deduced (Table 3Go). Conc, Concentration; DBT, dibutyltin chloride; TROG, troglitazone.

 

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  		Table 2. Organotin EC50 Values for Nuclear Receptor LBDs

 
We next tested whether activation by TBT was unique to RXR{alpha} only, restricted to RXR heterodimer complexes, or a general nuclear receptor transcriptional response (Fig. 1Go, B–D, and Table 1Go). TBT activated RXR{alpha} and RXR{gamma} from the amphibian Xenopus laevis in addition to human RXRs (Table 1Go). Our results are consistent with recent findings by Nishikawa et al. (19, 24) that organotins promote activation of all three human RXR subtypes in a yeast two-hybrid screen. We also observed significant activation of receptors typically considered to be permissive heterodimeric partners of RXR including human PPAR{gamma} (Fig. 1BGo, ~30% maximal activation of 10 µM troglitazone, but note that activation is compromised by cellular toxicity above 100 nM), PPAR{delta}, liver X receptor (LXR), and the orphan receptor NURR1. In contrast, typical nonpermissive partners such as RARs, thyroid hormone receptor, and VDR failed to show activation by organotins (Fig. 1CGo and Table 1Go). Murine PPAR{alpha} was also not activated by TBT although it was fully activated by its specific synthetic agonist WY-14643 (Fig. 1DGo). The steroid and xenobiotic receptor was likewise unresponsive. The orphan receptor NURR1, which has no discernable ligand pocket and is believed to be ligand independent (25), was nevertheless activated 7- to 10-fold at 100 nM TBT. Similarly, other RXR-specific ligands, e.g. LG100268, activated NURR1 to the same degree, suggesting that this response occurred through NURR1’s heterodimeric partner RXR as has been previously described (25, 26). Like other RXR-specific ligands, tributyltin was also able to promote the ligand-dependent recruitment of nuclear receptor cofactors such as receptor-associated coactivator 3 (ACTR), steroid receptor coactivator-1, and PPAR-binding protein in mammalian two-hybrid interaction assays (data not shown). We infer from these results that nuclear receptor activation by TBT activation is specific to a small subset of receptors and not a consequence of a general effect on the cellular transcriptional machinery.


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  		Table 1. TBT Activates RXRs and RXR-Permissive Heterodimers

 
We next investigated the relationship between the structure of the tin compounds and RXR activation by testing the response of GAL4-RXR{alpha} to mono-, di-, tri-, and tetra-substituted butyltin, branched side chains, variations in the alkyl chain length, and changes in the halide component (Fig. 1AGo and Table 2Go). Overall, trialkyltin compounds were the most effective with nanomolar EC50 values. Monobutyltin gave no significant activation whereas dibutyltin was moderately active in the micromolar range (Fig. 1AGo and Table 2Go). Tetrabutyltin was 20-fold less potent than TBT, whereas the branched side-chain butyltin tris(2-ethylhexanoate) [BT(2-EHA)3] was inactive (Table 2Go). Although activation by dialkyltins is weaker than that of TBT, it is potentially significant due to their widespread use in the manufacture of polyvinyl chloride plastics and greater solubility than TBT.

The effect of the hydrocarbon chain length was very pronounced, suggesting an important structure-activity relationship. A reduction in hydrophobicity from butyl to ethyl side chains raised the EC50 value by almost 1000-fold into the micromolar range. Trimethyltin was weakly active only above 100 µM (Table 2Go). Substitution of the halide component had no significant effect on the EC50 values for TBT, probably due to the lability of the halide atom through exchange in aqueous tissue culture media where chloride ions are prevalent.

TBT Is a Potent Ligand of Both RXR{alpha} and PPAR{gamma}
Many, if not most, natural and synthetic nuclear receptor agonists act as ligands that specifically interact with their cognate receptor LBDs. We therefore performed equilibrium competition binding experiments with purified histidine-tagged human RXR{alpha} (H6-RXR{alpha}) and PPAR{gamma} (H6-PPAR{gamma}) LBDs to determine whether the potent and specific activation of these receptors by TBT was due to direct ligand-receptor interaction (Fig. 1Go, E and F).

The equilibrium binding curves indicate that TBT is a high-affinity, competitive ligand for 9-cis RA-bound RXR{alpha}. The inhibition equilibrium dissociation constant was calculated by the Chang-Prusoff method [inhibition constant (Ki) = dissociation constant (Kd)] as 12.5 nM (10–15 nM; 95% confidence interval) (Table 3Go). By comparison, the value obtained for the synthetic RXR agonist LG100268 was 7.5 nM, which compared favorably with its published value of approximately 3 ± 1 nM (27). Therefore, the identification of TBT as an RXR ligand expands the molecular definition of known rexinoids (agonists able to activate RXR) to include this structurally unique class of organotin compounds.


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  		Table 3. TBT Binding Constants (Kd) for hRXR{alpha} and hPPAR{gamma} LBDs

 
Somewhat surprisingly, we also observed potent specific competitive binding by TBT for rosiglitazone bound to human PPAR{gamma} LBD (Fig. 2BGo). The deduced Ki of 20 nM (17–40 nM; 95% confidence interval) was slightly higher than for RXR{alpha} but significantly better than the Ki for the PPAR{gamma} agonist troglitazone, which yielded a Ki of 300 nM, consistent with its published Kd (28). The Kd values for TBT binding to RXR{alpha} (12.5 nM) and PPAR{gamma} (20 nM) are also in close agreement with EC50 values obtained from transient transfection assays using GAL4-RXR{alpha} and GAL4-PPAR{gamma} constructs (Table 2Go).


Figure 2
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  		Fig. 2. Tributyltin Induces Adipogenesis in 3T3-L1 Cells

Uninduced (A) and MDIT-induced (B) 3T3-L1 cultures grown for 1 wk in the presence of vehicle (DMSO), or ligands were analyzed for mature adipocyte differentiation by Oil Red O staining. Scale bar represents 100 µm. C and D, The percentage area stained was determined by automated analysis of random fields (n = 9) from high-contrast dissecting scope photographs of monolayers analyzed in ImageJ; 1–100 nM of TBT, AGN195203, and TTNPB or 1–10 µM troglitazone. E, Quantitative real-time PCR (QRT-PCR) of adipocyte-specific fatty acid binding protein aP2 (aP2/Fabp4) expression levels in postconfluent 3T3-L1 cells treated with the indicated ligands for 24 h. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase controls and plotted as average fold induction ± SEM (n = 3).TROG, Troglitazone.

 
Taken together, these data show that organotins such as TBT, although structurally distinct from previously described natural or synthetic ligands, can interact with RXR{alpha} and PPAR{gamma}, via direct ligand binding to induce productive receptor-coactivator interactions and promote transcription in a concentration-dependent manner. Organotins are therefore potent nanomolar receptor activators on par with synthetic RXR and PPAR{gamma} ligands such as LG100268, AGN195203, and thiazolidinediones.

TBT Promotes Adipogenesis in the Murine 3T3-L1 Cell (Embryonic Murine Preadipocyte Fibroblast Cell Line) Model
Numerous studies have demonstrated the critical role played by RXR{alpha}:PPAR{gamma} signaling in regulation of mammalian adipogenesis (29, 30, 31). In the murine 3T3-L1 preadipocyte cell model, adipogenic signals induce early key transcriptional regulators such as CCAAT/enhancer binding proteins (C/EBPs) ß and {delta} that lead to mitotic clonal expansion of growth-arrested preadipocytes and induction of the late differentiation factors C/EBP{alpha} and PPAR{gamma} (32, 33, 34). The combination of C/EBP{alpha} expression together with PPAR{gamma} signaling efficiently drives terminal adipocyte differentiation and lipid accumulation. We therefore tested whether TBT signaling through RXR:PPAR{gamma} could promote adipogenesis in the murine 3T3-L1 differentiation assay and compared its effect to other RXR-specific or PPAR{gamma} ligands (Fig. 2Go). Undifferentiated 3T3-L1 cells were cultured for 1 wk in the presence of ligands either with or without a prior 2-d treatment with MDIT (an adipogenic-sensitizing cocktail of 3-isobutyl-1-methylxanthine, dexamethasone, insulin, and T3) (35). Cells were then scored for lipid accumulation using Oil Red O staining to determine the degree of terminal adipocyte differentiation. TBT was as effective as LG100268 or AGN195203 in promoting differentiation in the absence of MDIT treatment, increasing the number of differentiated adipocytes about 7-fold over solvent-only controls (Fig. 2Go, A and C). The PPAR{gamma} agonist troglitazone was a weak inducer in the absence of MDIT. Prior treatment with MDIT increased the response to TBT, LG100268, and AGN195203 a further 3- to 5-fold (Fig. 2Go, B and D). MDIT treatment also boosted the response to troglitazone to equivalent levels as expected from published studies showing that combination treatment with PPAR{gamma} ligands promotes efficient adipocyte differentiation (36, 37, 38). In contrast, the RAR agonist TTNPB inhibited the differentiation of 3T3-L1 cells, consistent with previously published data that showed RAR signaling blocks adipogenesis during the early stages of differentiation in vitro and can modulate adiposity and whole body weight in vivo (39, 40, 41). The differential response of 3T3-L1 cells to receptor-selective retinoids indicates that TBT favors RXR homodimer or permissive RXR-heterodimer rather than RXR:RAR signaling in this cell model.

Adipocyte differentiation by TBT was accompanied by direct transcriptional effects on RXR:PPAR{gamma} targets such as adipocyte-specific fatty acid-binding protein (aP2) mRNA. The aP2 promoter contains response elements sensitive to C/EBP factors and RXR{alpha}:PPAR{gamma} signaling (42). Quantitative real-time PCR analysis showed aP2 levels were elevated by TBT treatment approximately 5-fold at 24 h (Fig. 2EGo) and 45-fold at 72 h (data not shown). LG100268, troglitazone, and MDIT treatment also increased aP2 expression at these time points whereas the RAR agonist TTNPB was inhibitory, consistent with the observed cellular responses.

TBT Induces Adipogenic Regulators and Markers of RXR{alpha}:PPAR{gamma} Signaling in Vivo
The ability of organotins to regulate RXR{alpha}:PPAR{gamma} target genes and key modulators of adipogenesis and lipid homeostasis in vivo has not been previously examined. Therefore, we next asked whether TBT could perturb expression of critical transcriptional mediators of adipogenesis such as RXR{alpha}, PPAR{gamma}, C/EBP{alpha}/ß/{delta}, and sterol regulatory element binding factor 1 (Srebf1) as well as known target genes of RXR{alpha}:PPAR{gamma} signaling from liver, epididymal adipose tissue, and testis of 6-wk-old male mice dosed for 24 h with TBT [0.3 mg/kg body weight (b.w.)], AGN195203 (0.3 mg/kg b.w.), troglitazone (3 mg/kg b.w), or vehicle (corn oil) administered by ip injection. TBT either had no effect or weakly repressed RXR{alpha} and PPAR{gamma} transcription in liver (Fig. 3Go, A and B). A more pronounced decrease was observed for RXR{alpha}, PPAR{gamma}, C/EBP{alpha}, and C/EBP{delta} in adipose tissue and testis (Fig. 3Go, B and C). In contrast, TBT, AGN195203, and troglitazone significantly induced expression of the early adipogenic transcription factor C/EBPß in liver and testis, whereas it was more weakly induced in adipose tissue. Induction was strongest in testis where TBT and troglitazone increased expression greater than 10-fold and AGN195203 increased expression 60-fold compared with vehicle controls (Fig. 3CGo). In addition to C/EBPß, the proadipogenic transcription factor Srebf1 was also significantly increased in adipose tissue by all three receptor ligands and weakly induced in liver.


Figure 3
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  		Fig. 3. In Vivo Induction of Adipogenic Modulators and RXR:PPAR{gamma} Target Genes

C57BL/6 male mice (three animals per treatment) were dosed with TBT (0.3 mg/kg b.w.), AGN195203 (0.3 mg/kg), troglitazone (3 mg/kg b.w.), or vehicle (corn oil) only by ip injection. Animals were killed after 24 h and dissected and cDNA was prepared from liver, epidydimal fat pad, or testis for quantitative real-time PCR analysis. Expression levels were normalized to histone Hist2h4 and shown as the average fold change ± SEM (n = 3) compared with vehicle (corn oil) controls. Control vs. ligand treatments were analyzed by the unpaired Student’s t test: *, P < 0.1; **, P < 0.05. TROG, Troglitazone.

 
We also observed coordinate changes in several well-characterized direct target genes of RXR:PPAR{gamma} signaling. Fatty acid transport protein (Fatp) acts as a key control point for regulation of cellular fatty acid content. The Fatp promoter contains a functional PPAR response element shown to be sensitive to RXR:PPAR{gamma} signaling in 3T3-L1 adipocytes and white fat (43, 44, 45, 46). Fatp mRNA levels were up regulated 2- to 3-fold in liver and epididymal adipose tissue but not testis by TBT, AGN195203, and troglitazone (see Fig. 5Go, A and B). Similarly, the PPAR{gamma} target phosphoenolpyruvate carboxykinase 1 (PEPCK/Pck1) (47), the rate-limiting step in hepatic gluconeogenesis and adipose glyceroneogenesis, was up-regulated in liver and adipose tissues by TBT or troglitazone treatment.


Figure 5
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  		Fig. 5. In Utero Exposure to TBT Increases Adipose Mass But Not Body Weight in Adult Mice

A, Growth curves of C57BL/6 male and female pups exposed to control (sesame oil) or TBT in utero (E12–18). Data are mean ± SEM (n = 10). B, Epididymal fat pad weights from control or TBT-treated males at 10 wk. *, Epididymal adipose mass from exposed males was approximately 20% greater [control vs. TBT: 0.30 g ± 0.020 (n = 9) vs. 0.36 g ± 0.018 (n = 10); *, P = 0.0374). Data represent mean ± SEM (n = 9–10).

 
Signaling through RXR:PPAR{gamma}, RXR:LXR, and ADD1/Srebf1 in hepatocytes has been shown to modulate fatty acid synthesis through transcriptional control of acetyl-coenzyme A carboxylase (Acac), the rate-limiting step in long-chain fatty acid synthesis (48, 49), as well as fatty acid synthase (Fasn) (50, 51, 52, 53). Hepatic expression of both Acac and Fasn was unregulated between 1.5–2.5-fold by TBT, AGN195203, and troglitazone. Therefore, the coordinate increased expression of Fatp, Pck1, Acac, and Fasn in liver suggests that TBT stimulates fatty acid uptake and triglyceride synthesis. Similar changes have been reported in the induction of hepatic steatosis by overactive PPAR{gamma} signaling (49, 54).

Taken together, these data show that TBT exposure induces lipogenic RXR:PPAR{gamma} target gene expression, in adipose tissue and liver, and modulates associated early adipocyte differentiation factors such as C/EBPß and Srebf1. We inferred from these data that organotins are potential adipogenic agents in vivo.

Developmental Exposure to TBT Disrupts Lipid Homeostasis and Adipogenesis in Vertebrates
Based on its molecular pharmacology, ability to induce 3T3-L1 adipocyte differentiation, and in vivo transcriptional responses, we reasoned that TBT would disrupt normal endocrine control over lipid homeostasis and impact adipogenesis, particularly when exposure occurred during sensitive periods of development. We therefore tested this hypothesis in two vertebrate model systems, mouse and X. laevis, during embryogenesis.

Pregnant C57BL/6 mice were injected daily from gestational d 12–18 with TBT (0.05 or 0.5 mg/kg body weight ip) dissolved in sesame oil or vehicle alone. Pups were then killed at birth, and histological sections were prepared from liver, testis, mammary gland, and inguinal adipose tissue. Sections were stained with Oil Red O to assess changes in total tissue lipid accumulation. TBT exposure caused a disorganization of hepatic (Fig. 4Go, A and B) and gonadal (Fig. 4Go, C and D) architecture and significantly increased Oil Red O staining in treated animals vs. controls. Liver sections exhibited signs of steatosis consistent with the misregulation of fatty acid uptake and synthesis observed using molecular markers. In addition, Oil Red O positive staining in mammary and inguinal adipose (Fig. 4Go, E–H) tissues was dramatically elevated, reflecting either an increase in lipid accumulation or an increase in mature adipocytes.


Figure 4
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  		Fig. 4. In Utero Exposure to TBT Increases Adiposity in Mouse Liver, Testis, and Adipose Depots

Histological sections (12 µm) of newborn mouse liver (A and B), testis (C and D), inguinal adipose (E and F) and mammary adipose (G and H) stained with Oil Red O and counterstained with hematoxylin following in utero exposure to vehicle only (sesame oil) (A, C, E, and G) or 0.5 mg/kg b.w. TBT (B, D, F, and H) given s.c. daily from E12–18. Scale bar, 100 µm.

 
To determine whether exposure induced long-term changes in growth or adipose tissue, we followed mice from birth to adulthood after in utero exposure to TBT as indicated above. At birth, mice were cross-fostered to unexposed dams, and total body weight was recorded until 10 wk of age (Fig. 5AGo). Growth curves for male and female pups showed a slight trend for lower total body weight consistent with published observations (9) but were not statistically significant at 10 wk [control vs. TBT: male, 26.00 g ± 0.70 (n = 9) vs. 25.53 g ± 0.39 (n = 10), P = 0.583; female, 21.22 g ± 0.41 (n =10), vs. 20.24 g ± 0.24 (n =10), P = 0.0529]. Males were killed at 10 wk and epididymal fat pads were weighed (Fig. 5BGo). Adipose mass in TBT-treated males was increased significantly by 20% over controls [control vs. TBT: 0.30 g ± 0.020 (n = 9) vs. 0.36 g ± 0.018 (n =10), P = 0.0374]. These data support the conclusion that TBT can increase body adiposity without overtly increasing total body weight. Similar lipid accumulation and changes in adipose tissue mass have also been observed after TZD or rexinoid treatment (55, 56, 57).

We had previously observed that TBT activated Xenopus RXRs (Table 1Go) and reasoned that the strong conservation in vertebrate nuclear receptor signaling pathways should result in consistent responses to organotin and RXR/PPAR{gamma} ligands across diverse vertebrate species. We therefore tested chronic exposure to environmentally relevant low doses of TBT (1–10 nM), the RXR-specific ligands LG100268 and AGN195203 (10–100 nM), troglitazone (0.1–1 µM), and estradiol (1–10 nM) on developing X. laevis tadpoles from stage 48 to metamorphs. To determine the effectiveness of these doses in X. laevis tadpoles, we used aromatase expression as a molecular marker because activity and expression are sensitive to endocrine disruption by organotins and RXR/PPAR{gamma} ligands in mammals (17, 18). Xenopus aromatase expression was similarly repressed 2- to 3-fold by 10 nM TBT, AGN195203, LG100268, or 1 µM troglitazone at stage 56 tadpoles (Fig. 6AGo) and at all subsequent stages. Despite significant ligand-induced aromatase down-regulation, neither sex ratios nor the time required to reach metamorphosis was altered (data not shown). Xenopus liver and kidney also exhibited no gross structural abnormalities at the doses given.


Figure 6
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  		Fig. 6. Endocrine Disruption of RXR:PPAR{gamma} Signaling and Ectopic Induction of Adipocytes in X. laevis by TBT

A, Expression levels of Xenopus aromatase (XCYP19) were determined in tadpoles (stage 56) by quantitative real-time PCR after 24-h exposure to vehicle only (DMSO) or the indicated ligands. Expression was normalized to Xenopus EF1{alpha} and expressed as average fold change in expression ± SEM (n = 9) relative to vehicle controls. B, X. laevis tadpoles were dosed weekly under static renewal conditions with indicated ligands from stage 48 (before gonadogenesis) until stage 64 (metamorphic climax). Metamorphs (stage 66) were scored for ectopic adipocyte patches on gonads and urogenital ducts. Data are shown as the percentage of metamorphs exhibiting ectopic adipocyte patches posterior to the fat bodies; mean ± SD from triplicate tanks. C–E, Dissecting microscope photographs of kidneys (k), testis (t), and fat bodies (fb) from DMSO control, 10 nM TBT, and 1 µM troglitazone-treated male metamorphs. Multiple ectopic adipocyte patches (red arrows) are present posterior to the fat bodies along the anterior-posterior axis of gonads in TBT (D)- and troglitazone (E)-treated animals but not controls (C). Histological sections of kidneys and gonads from the same control (F) and 10 nM TBT (G)-treated males at the level indicated by the white line in C and D. Gonadal and connective tissue was either completely replaced by, or interspersed with, adipocytes (red arrows) in TBT-treated animals. Sections were developed with Mallory’s trichrome stain. Scale bars, 100 µm.

 
However, consistent with the testis and adipose results from mice presented above, we observed a dose-dependent increase in ectopic adipocyte formation posterior to the fat bodies in and around the gonads of both sexes after TBT or RXR/PPAR{gamma} ligand exposure (Fig. 6BGo). In contrast, estradiol-treated animals did not show increased adipocyte formation compared with controls. At 10 nM TBT, 10 nM AGN195203, or 1 µM troglitazone, ectopic adipocytes were observed in approximately 45–60% of animals. At the highest dose of TBT in males, testicular tissue was interspersed with, or replaced by, adipocytes along the anterior-posterior axis (Fig. 6Go, D, E, and G).

The concordant changes observed in Xenopus aromatase expression, gonadal adipocyte formation, and increased murine adiposity after exposure to TBT, RXR and PPAR{gamma} ligands are therefore consistent with a common mechanism of action through RXR:PPAR{gamma} activation, supporting the conclusion that endocrine disruption via nuclear receptor transcriptional regulation is a novel and key feature of organotin toxicity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have shown above that TBT is a potent inducer of adipogenesis, in vitro and in vivo, by acting as a novel, high-affinity xenobiotic ligand for RXR{alpha} and PPAR{gamma}. The ability of organotins to bind and activate these receptors, in particular the RXRs, which exhibit very restricted ligand specificity, is unexpected given the radically different chemical composition and three-dimensional molecular structure of organotins when compared with known natural and synthetic nuclear receptor ligands. Typically, RXR ligands comprise a carboxylic acid functional group and a three-dimensional molecular shape that mimics 9-cis RA. Structure-activity profiles indicate distinct structural preferences for organotins but also a relatively broad accommodation for agonist activity that is not easily reconciled with the classical ligand-binding model. Organotins may therefore interact somewhat differently than previously described RXR/PPAR{gamma} ligands with receptor LBDs to induce productive conformational changes required for coactivator recruitment. However, the binding data indicate that TBT is a potent and efficacious ligand for both RXRs and PPAR{gamma} that interacts, at least partially, with the same receptor-binding sites of other high-affinity ligands and promotes the necessary cofactor interactions required for agonist activation. In the study of Kanayama et al. (24), TBT was only effective in coactivator recruitment assays with PPAR{gamma} above 10 µM in vitro. However, in accord with our findings, they show that TBT activated PPAR{gamma} significantly at nanomolar concentrations in transfection assays. This may reflect a limitation of preference in the cofactor used in vitro. Alternatively, the lower maximal activation observed with TBT on PPAR{gamma} in cells (~30% at 100 nM TBT cf troglitazone) is consistent with one of two possibilities: either nonspecific cellular toxicity at high levels or activation as a partial agonist.

The ability of TBT to act as a dual ligand for permissive heterodimers such as RXR{alpha}:PPAR{gamma}, which can be activated by specific ligands for either receptor, also raises the possibility for additive or synergistic effects that might increase the potency of these compounds in vivo at low doses for this specific signaling pathway. Of note is that receptor activation is observed at nanomolar concentrations, whereas other mechanisms of toxicity and endocrine disruption, e.g. direct inhibition of aromatase activity, typically occur in the micromolar range. Furthermore, the activation of other permissive RXR heterodimeric partners, e.g. LXR and NURR1, suggests that organotins may act more widely to disrupt multiple nuclear receptor-mediated hormonal signaling pathways.

The biological consequences of organotin activation of the RXR:PPAR{gamma} signaling pathway are predictable and should follow known aspects of RXR/PPAR{gamma} biology. The RXR:PPAR{gamma} pathway plays a key role in adipocyte differentiation and energy storage, and is central to the control of whole-body metabolism (58). PPAR{gamma} activation increases the expression of genes that promote fatty acid storage and represses genes that induce lipolysis in adipocytes in white adipose tissue (59). PPAR{gamma} such as the thiazolidinediones can modulate insulin sensitivity due to these effects on the adipocyte, reversing insulin resistance in the whole body by sensitizing the muscle and liver tissue to insulin (60). However, a consequence of this increase in whole-body insulin sensitivity is that fat mass is increased through the promotion of triglyceride storage in adipocytes. Evidence is also mounting that depot-specific remodeling and adipocyte numbers increase after thiazolidinedione treatment (55, 56, 57). Therefore, PPAR{gamma} agonists comprise a class of pharmaceutical therapies for type 2 diabetes that can also promote obesity by increasing fat storage. Likewise, RXR ligands also act as insulin-sensitizing agonists in rodents (61), underscoring the permissive nature of the PPAR{gamma}:RXR heterodimer and the potential effects on diabetes and obesity of both PPAR{gamma} and RXR agonists.

Our data are consistent with recent studies that organotins can mediate some of their endocrine disruption effects by transcriptional regulation through nuclear receptors, in particular RXR:PPAR{gamma} signaling (17, 18, 19, 24). Consequently, TBT exposure can promote adipocyte differentiation in the same manner as other RXR or PPAR{gamma} ligands in vitro using the standard murine 3T3-L1 cell model and in vivo through increased adiposity after intrauterine organotin exposure in newborn mice. It is currently unknown whether the increased adiposity in vivo results from an increase in adipocyte precursor cell number, enhanced adipocyte differentiation from the same number of precursors, an increase in adipocyte size without an increase in number, or some combination of these.

The prevailing epidemiological data ascribe high-density caloric and/or fatty diets coupled with decreased physical activity as the root causes for the rise in obesity rates in the general population (62). The contribution of genetic components is less clear. Although genetic variation contributes to an individual’s propensity to develop obesity, the rapid worldwide increase in obesity suggests that interaction with the modern environment exposes inherent genetic differences. The Barker hypothesis postulates that in utero fetal nutritional status is a potential risk factor for metabolic syndrome diseases (63, 64, 65, 66, 67). In this view, developmental metabolic programming of a thrifty phenotype limits the range in adaptive responses to the environment, e.g. diet and exercise, in later life (68). Experimental evidence from animal models lends support to this hypothesis (69). Plausible mechanisms include imprinting of obesity-sensitive hormonal pathways or changes in cell type and number, e.g. adipocytes, established during development.

Others, however, argue that the environment plays another role in obesity. Because the increase in obesity rates parallels the rapid growth in the use of industrial chemicals over the past 40 yr, it is plausible and provocative to associate in utero or chronic lifetime exposure to chemical triggers present in the modern environment with this epidemic. Hence, an "obesogen" model predicts the existence of xenobiotic chemicals that inappropriately regulate lipid metabolism and adipogenesis to promote obesity. Several recent studies serve as proof-of-principle for such a hypothesis. Environmental estrogens such as bisphenol A and nonylphenol, for instance, can promote adipocyte differentiation or proliferation in murine cell lines (70, 71). Furthermore, epidemiological studies link maternal smoking during pregnancy to an elevated risk of childhood obesity (72, 73, 74, 75, 76).

Seen in this context, we propose that organotins such as TBT and its congeners are chemical stressors or obesogens that activate RXR:PPAR{gamma} signaling to promote long-term changes in adipocyte number and/or lipid homeostasis after developmental or chronic lifetime exposure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Transfections
pCMX-GAL4 and pCMX-VP16 plasmid fusion constructs to nuclear receptor LBDs and coactivators [GAL4-hRAR{alpha}, hRXR{alpha}, -xRXR{alpha}/{gamma}, -hPPAR{gamma}, -mPPAR{alpha}, -human steroid and xenobiotic receptor (SXR), -NURR1, -VDR, -LXR, -hACTR, -hPPAR-binding protein (PBP), -human steroid receptor coactivator-1 (SRC-1), human transcriptional intermediary factor 2 (TIF2)] have been previously described (77, 78, 79, 80, 81, 82). Transfections were performed in Cos7 cells (transformed green monkey kidney fibroblast cell line) essentially as described elsewhere (83) using MH200-x4-TK-Luc as reporter and normalized to pCMX-ß-galactosidase controls. Briefly, Cos7 cells were seeded at 5000 cells per well in 96-well tissue culture plates in 10% fetal bovine serum/DMEM and transfected for 8 h with 11 µg/plate of DNA/calcium phosphate precipitate mix (MH200x4-TK-Luc-CMX-ß-galactosidase-nuclear receptor/coactivator effector(s) at a ratio of 5:5:1). Cells were washed free of precipitate with PBS and media were replaced with serum-free insulin, transferrin, lipid, bovine serum albumin supplemented (ITLB)/DMEM (84) plus ligands for an additional 24 h before assays for luciferase and ß-galactosidase activity. All transfection data points were performed in triplicate, and all experiments were repeated at least three times.

Quantitative Real-Time PCR Analyses
Total cellular RNA from C57BL/6 mouse and X. laevis tissues was isolated with Trizol reagent and reversed transcribed with oligo dT and Superscript II (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Triplicate cDNA samples (50 ng/reaction) were analyzed by quantitative real-time PCR on a DNA Engine Opticon thermal cycler [MJ Research (Watertown, MA)/Bio-Rad Laboratories (Hercules, CA)] using SYBR Green chemistry (PerkinElmer Life Sciences, Wellesley, MA). Fold changes in expression levels were calculated after normalization to histone Hist2h4 using the {Delta}-{Delta} cycle threshold method (85). Gene-specific primers were as follows. Hist2h4 forward (F): 5'-CCCGTGGTGTGCTGAAGGTGTT-3'; reverse (R), 5'-GAATTGAAGCGGCGGCGTCTA-3'; RXR{alpha} F: 5'-CGGCTGCTCAGGGTACTTGTGTTT-3'; R, 5'-CGGCTGCTCAGGGTACTTGTGTTT-3'; PPAR{gamma} F: 5'-TGGGTGAAACTCTGGGAGATTC-3'; R, 5'-AATTTCTTGTGAAGTGCTCATAGGC-3'; C/EBP{alpha} F: 5'-CCAAGAAGTCGGTGGACAAGA-3'; R, 5'-CGGTCATTGTCACTGGTCAACT-3'; C/EBPß F: 5'-GCCCGCCGCCTTTAGACC-3'; R, 5'-CGCTCGTGCTCGCCAATG-3'; C/EBP{delta} F: 5'-AACCCGCGGCCTTCTACGAG-3'; R, 5'-ACGGCGGCCATGGAGTCAAT-3'; aP2 F: 5'-GAATTCGATGAAATCACCGCA-3'; R, 5'-CTCTTTATTGTGGTCGACTTTCCA-3'; FATP F: 5'-AGCCGCTTCTGGGATGACTGTGT-3'; R, 5'-ACCGAAGCGCTGCGTGAACTC-3'; ACS F: 5'-CCCAGCCAGTCCCCACCAG-3'; R, 5'-CACACCACTCAGGCTCACACTCGT-3'; FASN F: 5'-TCGGGTGTGGTGGGTTTGGTGAAT-3'; R, 5'-ACTTGGGGGCGTGAGATGTGTTGC-3';ACAC F: 5'-G GATGGCAGCTCTGGAGGTGTATG-3'; R, 5'-TGTCCTTAAGCTGGCGGTGTTGTA; Pck1 F: 5'-CTGGCAGCATGGGGTGTTTGTAGG-3'; R, 5'-TGCCGAAGTTGTAGCCGAAGAAGG-3'; Srebf1 F: 5'-GCCCCTGCCCACCTCAAACCT-3'; R, 5'-ACTGGCACGGGCATCCTTCCTC-3'; Xenopus EF1{alpha} F: 5'-GATCCCAGGAAAGCCAATGTGC 3'; R, 5'-CCGGATCCTGCTGCCTTCTTCT-3'; Xenopus CYP19 (aromatase) F: 5'-GTCTGGATTAATGGCGAGGAAACA-3'; R, 5'-CTGATGAAGTATGGCCGAATGACC-3'.

Ligand Binding
Histidine-tagged RXR{alpha} LBD (H6-RXR{alpha} LBDs) was expressed and purified from pET15b(+) vector in BL21(DE3) pLysS bacteria cultures after induction with 1 mM isopropyl-ß-D-thiogalactopyranoside for 3 h at 37 C (86). Purified H6-PPAR{gamma} was purchased from Invitrogen. Proteins were bound to 96-well Nickel Chelate Flashplates (PerkinElmer Life Sciences) at 100 µg/ml overnight at 4 C and washed five times with 200 µl/well Flashplate Assay Buffer (20 mM HEPES, pH 7.9; 100 mM KCl, 0.1% cholamidopropyldimethylammonio-2-hydroxy-1-propanesulfonate, 0.1 mM dithiothreitol). Competition assays typically used 1–5 nM [3H]-9-cis-RA (PerkinElmer Life Sciences) or 10–50 nM [3H]rosiglitazone (American Radiochemicals, Inc., St. Louis, MO) plus cold competitor ligands in Flashplate Assay Buffer at concentrations indicated in the figures. Plates were incubated at room temperature, protected from light, and read after 4 h on a Packard Topcount scintillation counter (Packard Instruments, Meriden, CT). Specific bound counts/min were determined by subtraction of counts/min from uncoated wells at each ligand concentration. Data were analyzed with GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA) using a one-site competition binding equation to determine Ki values for competitor ligands; Kd values of 1.4 and 41 nM for 9-cis-RA and rosiglitazone for their respective receptors were used in the calculations (87, 88).

3T3-L1 Cell Assays
3T3-L1 (American Type Culture Collection, Manassas, VA) cells were maintained as subconfluent cultures by passage every 3 d from cultures seeded at 5000 cells/cm2 in 8% calf serum/DMEM. For differentiation assays, cells were seeded at 15 x 103 cells per well into 24-well tissue culture plates in 8% fetal bovine serum/DMEM, after which cultures were grown for 2 d and then treated with the indicated RXR, RAR, and PPAR ligands either with or without MDIT (100 µM 3-isobutyl-1-methylxanthine, 100 nM dexamethasone, 0.1 ng/ml insulin, and 2 nM T3 thyroid hormone) induction cocktail. Media and ligand treatments were renewed every 2 d. After 1 wk, cells were scored for adipocyte differentiation by Oil Red O staining for lipid droplet accumulation. Cultures were washed with PBS, fixed with 10% formaldehyde for 15 min, washed with distilled water, and stained with filtered Oil Red O solution (4 g/liter, 60% isopropanol) for 15 min. Excess stain was removed by washing three times with distilled water. Three random fields from each well were photographed under phase contrast and analyzed in ImageJ. Images were converted into high-contrast black and white images to visualize lipid droplets and scored as the percentage area per field. Data are shown as the mean ± SEM from three wells per treatment. The method was validated by extraction of Oil Red O from stained cells into 100% isopropanol and quantitated by absorbance at 540 nm on a spectrophotometer.

In Vivo Animal Exposure Experiments
C57BL/6J mice were housed under a 12-h light, 12-h dark cycle. Pregnant mice were dosed by ip injection with TBT [0.05 or 0.5 mg/kg body weight (b.w.)] or vehicle (sesame oil) from embryonic d 12 (E12) every 24 h until the day before delivery. Neonates were killed at the day of delivery and analyzed. The samples were embedded in optimal cutting temperature embedding compound and sectioned (12 mm) using a cryostat. Sections were fixed on slides with 4% paraformaldehyde for 10 min and rinsed in PBS. The slides were then sequentially washed with distilled water and 60% of isopropanol and stained with Oil Red O (4 g/liter, 60% isopropanol). After washing with 60% isopropanol and distilled water, the slides were counterstained with hematoxylin. Sections were evaluated and photographed using a Zeiss microscope (Carl Zeiss, Thornwood, NY).

For long-term growth studies, pups were cross-fostered to unexposed C57BL/6 dams after birth, and litter sizes were kept constant at eight pups per dam (control, two male + two female; TBT treated, two male + two female). Animals were weaned at 3 wk of age and maintained on standard rodent chow. Total body weight was followed until 10 wk of age. Males were then killed, and epididymal fat pads were dissected and weighed.

X. laevis tadpoles were sorted at stage 48 (89) and maintained in 1-liter glass tanks in 20% Holtfreter’s buffered salt solution (90) at a density of 10 tadpoles per tank on a diet of ground Tetramin Fish Flakes and spirulina. Compounds prepared in dimethylsulfoxide (DMSO) as 105-fold stock solutions were tested on triplicate tanks and dosed by static renewal after weekly water changes. Metamorphs at stage 64 were transferred to individual containers and fed frozen brine shrimp for 2 wk until stage 66. Froglets were euthanized with 250 mg/liter MS222 in 20% Holtfreter’s solution and then scored for gonadal abnormalities and interrenal/gonadal adipocyte formation under a dissecting microscope. Kidneys with attached gonads and livers were fixed in 10% formalin-PBS, embedded in paraffin, and sectioned at 15 µm thickness. Sections were developed with Mallory’s trichrome stain.

All animal experiments were approved and performed in accordance with Institutional Animal Care and Use Committee protocols.


   ACKNOWLEDGMENTS
 
We thank Drs. I. Blitz, K. Cho, C. Zhou, and T. Osborne for critical reading and comments on the manuscript, Dr. C. Li (Expression Technologies) for the H6-RXR{alpha} LBD construct, and Dr. R. Chandraratna (Allergan Pharmaceuticals, Irvine, CA) for AGN203 and LG268).


   FOOTNOTES
 
This work was supported by grants from the U.S. Environmental Protection Agency (STAR R830686) and National Institutes of Health (GM-60572) (to B.B.); from the Ministries of Education, Culture, Sports, Science and Technology, Environment and Health Labor and Welfare, Japan (to T.I.); and from the University of California Toxic Substance Research and Training Program (UC-37579) (to F.G.).

F.G., H.W., Z.Z., L.M., K.A., R.C., D.M.G., J.K., T.I. have nothing to declare. B.B. is a named inventor on U.S. patents US 5,861,274, US 6,200,802, and US 6,815,168.

First Published Online April 13, 2006

Abbreviations: Acac, Acetyl-coenzyme A carboxylase; b.w., body weight; C/EBP, CCAAT/enhancer binding protein; 9-cis RA, 9-cis retinoic acid; DMSO, dimethylsulfoxide; F, forward; Fatp, fatty acid transport protein; LBD, ligand-binding domain; LXR, liver X receptor; MDIT, 3-isobutyl-1-methylxanthine, dexamethasone, insulin and T3 adipocyte differentiation mix; PPAR, peroxisome proliferator-activated receptor; R, reverse; RAR, retinoic acid receptor; RXR, retinoid X receptor; Srebf1, sterol-regulatory element binding factor 1; TBT, tributyltin chloride; TPTO, triphenyltin oxide; TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid; VDR, vitamin D receptor.

Received for publication September 8, 2005. Accepted for publication March 30, 2006.


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