Bisphenol A Accelerates Terminal Differentiation of 3T3-L1 Cells into Adipocytes through the Phosphatidylinositol 3-Kinase Pathway
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《毒物学科学杂志》
Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences, Takooda, Tobe-cho, Iyo-gun, Ehime 791-2101, Japan
Department of Environmental Science for Industry, Ehime University, 3-5-7 Tarumi, Matsuyama, Ekime 790-8566, Japan
Department of Orthopaedic Surgery, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan
ABSTRACT
In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) mRNAs. These findings indicate that BPA was able to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 mRNAs. Western blot analysis revealed that BPA increased the level of phosphorylated Akt kinase. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes. The structure-activity relationship for BPA-related chemicals was examined. Eight derivatives of BPA (three diphenylalkanes with different substituents at the central carbon atom, three diphenylalkanes with ester bonds on hydroxyl groups in the phenolic rings, one bisphenol consisting of a sulphur atom at the central position, one chemical with cyanic groups, instead of hydroxyl groups, in the phenolic rings) accelerated terminal adipocyte differentiation and their potencies to increase TG accumulation were 73–97% of that of BPA. Two diphenylalkanes with ether bonds on hydroxyl groups and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) did not have the ability to accelerate terminal adipocyte differentiation.
Key Words: bisphenols; 3T3-L1 cells; terminal adipocyte differentiation; LY294002; Akt kinase.
INTRODUCTION
Bisphenol A (BPA) is an environmental endocrine disrupting chemical that is present ubiquitously in the environment. For example, BPA is used commercially in products containing polycarbonate plastics such as baby bottles and leaches from them when subjected to simulated use by dishwashing, boiling, and brushing (Brede et al., 2003). This chemical also leaches from polycarbonate culture flasks when subjected to autoclaving (Krishnan et al., 1993). Microgram amounts of BPA are found in the liquid of preserved vegetables in cans (Brotons et al., 1995) and in the saliva of patients treated with dental sealants (Olea et al., 1996). A small amount of BPA also is found in tap water and in human serum (Inoue et al., 2000).
BPA mimics the actions of estrogens (Korach, 1993). Most research to date on BPA has focused on the growth of reproductive organs and tumor cells. For example, administration of BPA to ovariectomized rodents increases uterine weight, stimulates cell proliferation in the uterus and vagina, and induces hypertrophy of the luminal epithelium (Goloubkova et al., 2000; Papaconstantinou et al., 2000; Steinmetz et al., 1998). In cultures, BPA stimulates proliferation of estrogen-responsive human breast cancer cells, MCF-7 cells (Rivas et al., 2002). Recently, BPA was reported to affect non-reproductive organs as well as reproductive organs. In ovariectomized rats, BPA stimulated the growth of the anterior pituitary gland (Goloubkova et al., 2000) and increased the expression of progesterone receptor mRNA in the preoptic area (Funabashi et al., 2001). Moreover, two-generation studies in rats (Rubin et al., 2001) and mice (Howdeshell et al., 1999) showed that perinatal exposure to BPA caused an increase in body weight after birth. However, whether this increased body weight was due to an increased adipose tissue mass remains unclear.
The major cellular component of adipose tissue is adipocytes. 3T3-L1 adipocytes have been widely used to understand adipocyte physiology. Insulin is well known to be a potent adipogenic agent in cultures of 3T3-L1 adipocytes (Cornelius et al., 1994; Gregoire et al., 1998; Rubin et al., 1978; Spooner et al., 1979). The adipogenic actions of insulin are mediated by the insulin signaling pathway (White and Kahn, 1994; Xia and Serrero, 1999). The binding of insulin to its specific receptor at the cell surface causes autophosphorylation and activation of the receptor and this activated receptor phosphorylates insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 activates phosphatidylinositol 3-kinase (PI 3-kinase). The function of PI 3-kinase is implicated in terminal differentiation of 3T3-L1 cells into adipocytes. For example, inhibition of PI 3-kinase by a chemical inhibitor LY294002 blocked insulin-induced lipid accumulation in 3T3-L1 cells (Xia and Serrero 1999).
In the present study, we examined whether BPA and BPA-related chemicals had the ability to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. We used three phenotypic markers for adipocytes as follows: (1) triacylglycerol (TG) accumulation in cells, (2) expression of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) genes, and (3) catalytic activity of LPL. We also explored whether BPA acted through the PI 3-kinase pathway.
MATERIALS AND METHODS
Materials.
Bisphenol A (BPA), bisphenol B (BPB), bisphenol A dicyanate (BPA-DC), bisphenol A bis(chloroformate) (BPA-BCF), bisphenol A diacetate (BPA-DA), bisphenol A O,O-diacetic acid (BPA-DAA), bisphenol A dimethacrylate (BPA-DM) and bisphenol A diglycidyl ether (BPA-DGE), 4-nonylphenol (NP, mixture of compounds with branched sidechain), 4-tert-octylphenol (OP) were obtained from Tokyo Kasei Co., Tokyo, Japan. Bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS), and a kit for TG were obtained from Wako Pure Chemicals Co., Osaka, Japan. A GenElute Mammalian Total RNA kit and LY294002 hydrochloride were obtained from Sigma. An AlkPhos direct labeling kit, an ECL Plus Western Blotting Starter kit, hybridization buffer, blocking reagent, Hyperfilm MP, Hybond-N+, and Hybond-P were obtained from Amersham Pharmacia Biotech, U.K. A mouse monoclonal antibody to Akt1 (B-1) and a rabbit polyclonal antibody to p-Akt1/2/3 (Ser 473) were obtained from Santa Cruz Biotechnology, Inc. A DC Protein Assay was obtained from Bio-Rad Laboratories.
Cell culture and measurements of DNA and TG contents and LPL activity.
3T3-L1 cells were grown to confluence in a standard medium, which consisted of 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B in Dulbecco's modified Eagle's medium, on a 60-mm plate. Confluent cultures were induced to differentiate into adipocytes by treating them for two days in a standard medium supplemented with 10 μg/ml insulin, 1 μM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (MIX). The medium was then replaced with a standard medium supplemented with either 80 μM BPA alone or a combination of 80 μM BPA and LY294002 at the indicated concentrations and changed every two days. Six days later, cells were harvested in 1.2 ml of 50 mM NH4Cl/NH4OH buffer (pH 8.2) containing 20 μg/ml heparin and 2% (w/v) bovine serum albumin and sonicated briefly at 0°C. Aliquots of the homogenate were used for DNA and TG measurements. DNA was measured fluorometrically by the method of Hinegardner (1971) and TG was measured using a kit for TG. Another aliquot of the homogenate was used for preparing an acetone/ether powder for measurement of LPL activity (Masuno et al., 1990). LPL activity in the extract of an acetone/ether powder was measured using phosphatidylcholine-stabilized triolein as a substrate (Masuno et al., 2002). One unit of lipolytic activity was defined as that releasing 1 μmol of fatty acid/min at 37°C.
Lipid staining in cells.
The cultures were fixed with 10% (v/v) formalin in phosphate-buffered saline, and then stained with Oil Red O as described by Kuri-Harcuch and Green (1978). Cells were considered as lipid-positive when droplets were stained red. Four different microscopic fields (x100 magnification) per culture were photographed. The percentage of lipid-positive cells, which was calculated by dividing the number of lipid-positive cells by the number of total cells (at least 250 cells), in each photograph was determined. A value was obtained as an average value for four fields per culture. The results were expressed as the average value for two cultures.
Northern blot.
Total RNA was isolated from the cultures using a GenElute Mammalian Total RNA kit. RNA samples from two plates were combined for each treatment. RNA samples (20 μg/lane) were denatured with formamide and formaldehyde and electrophoresed on a 1.5% agarose gel containing 6.3% formaldehyde. The RNAs were blotted onto a nylon membrane (Hybond-N+) and crosslinked with 0.05 N NaOH. The blotted membrane was prehybridized for 30 min at 55°C in hybridization buffer containing 0.5 M NaCl and 4% (w/v) blocking reagent. A probe labeled using an AlkPhos direct labeling kit was then hybridized for 15–20 h at 55°C. After hybridization, the membrane was washed twice for 10 min at 55°C in the primary wash buffer (50 mM NaH2PO4, 2 M urea, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1 mM MgCl2, 0.2% blocking reagent, pH 7.0) and washed twice for 5 min at room temperature in the secondary wash buffer (50 mM Tris, 2 mM MgCl2, pH 10.0). The blot was left in CDP-Star detection reagent (30–40 μl/cm2) for 3 min and excess detection reagent was drained off. The membrane was exposed to a Hyperfilm MP with an intensifying screen for approximately 60 min. Relative densitometric units were determined using the analysis software Diversity Database.
The probes used for detection of mRNAs of LPL and aP2 were a 542bp EcoRI fragment (nucleotide 194–735) of mouse LPL cDNA clone and a 600bp BamHI fragment of mouse aP2 cDNA clone. The probe for aP2 was kindly donated by Dr. S. A. Kliewer (GlaxoSmithKline, North Carolina).
Western blot.
Cells were lysed in a lysis buffer (20 mM HEPES, 3 mM MgCl2, 2 mM EDTA, 100 mM NaF, 1% Triton X-100, 1 mM PMSF, 100 μg/ml leupeptin, 10 μg/ml aprotinin, pH 7.4) for 20 min at 4°C and centrifuged to remove insoluble materials. The protein concentrations in the cell lysates were measured using a DC Protein Assay. The same amount (10 μg of protein/lane) of proteins were denatured by boiling in Laemmli sample buffer containing 10% 2-mercaptoethanol, separated by SDS-PAGE, and transferred electrophoretically to a PVDF membrane (Hybond-P). The blotted membranes were incubated with indicated primary antibodies (1:500) and horseradish peroxidase-conjugated secondary antibody (1:25,000). Blots were visualized with an ELC Plus Western Blotting Starter kit according to the manufacturer's instructions. The membrane was exposed to a Hyperfilm MP with an intensifying screen for approximately 5–15 min. Relative densitometric units were determined using the analysis software Diversity Database.
Statistical analysis.
Statistical analysis of group means was conducted by ANOVA followed by post hoc comparisons using Fisher's protected least significant difference test. Statistical analysis was conducted using Student's t-test for unpaired samples. For all the statistical analyses, the criterion of significance was p < 0.05. All values were expressed as means ± SD.
RESULTS
Can BPA Accelerate Terminal Differentiation of 3T3-L1 Cells into Adipocytes
Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days with BPA alone. The untreated cultures, in which BPA was absent during the treatment period, contained 30.7 μg/plate of DNA and 1.97 μg/μg DNA of TG. The addition of BPA to the cultures did not cause changes in the DNA content (Fig. 1A) but caused a dose-dependent increase in the TG content (Fig. 1B). BPA at 40 and 80 μM increased the TG content by 110 and 310%, respectively. However, in cultures treated with 160 μM BPA for more than two days, cells began to detach from the bottom of the plates. Therefore, the following experiments were performed using cultures treated with 80 μM BPA, termed the BPA-treated cultures.
Oil Red O staining showed that approximately 25% of cells in the untreated cultures contained discrete lipid droplets (Fig. 2A). The presence of BPA caused an increase in the percentage of lipid-positive cells to approximately 85% (Fig. 2B). These results suggest that BPA may accelerate terminal differentiation of 3T3-L1 cells into adipocytes.
To confirm this, the expression of LPL and aP2 genes was analyzed by Northern blot and the catalytic activity of LPL in the extract of acetone/ether powder of cells was measured. BPA increased the expression of these two genes (Fig. 1C). Densitometrical analysis revealed that BPA increased the levels of LPL and aP2 mRNAs by 293 and 438%, respectively. The catalytic activity of LPL was also higher in the BPA-treated cultures than in the untreated cultures [the former cultures (n = 4), 1.71 ± 0.06 units/mg DNA; the latter cultures (n = 4), 1.29 ± 0.11 units/mg DNA; p < 0.01].
Does BPA Act through the PI 3-Kinase and Akt Kinase Pathway
Since the insulin signaling pathway plays a pivotal role in terminal adipocyte differentiation (Kohn et al., 1996; Xia and Serrero, 1999), we examined using LY294002 whether BPA acted through the PI 3-kinase pathway. Following the hormonal induction of differentiation, 3T3-L1 cells were treated with either BPA alone or a combination of BPA and LY294002. The DNA content of the cultures decreased with an increase in LY294002 concentrations (Fig. 3A). The DNA content of the cultures treated with a combination of BPA and 8 μM LY294002 was 76% of that of the cultures treated with BPA alone. Thus, LY294002 inhibited proliferation of 3T3-L1 cells. This result is compatible with the findings of others that PI 3-kinase plays an important role in mediating the action of insulin on cell growth (Backer et al., 1992; Kapeller et al., 1991). Of course, there is a concern that this inhibition could be due to a toxic effect of LY294002. However, this does not appear to be the case, because cells had not been detached from the bottom of the plates during the experiment period. Moreover, cells attached to the bottom of the plates excluded trypan blue (data not shown).
LY294002 suppressed dose-dependently the increasing effect of BPA on the TG content (Fig. 3B). In the cultures treated with a combination of BPA and 8 μM LY294002, the TG content was decreased to a level similar to that of the untreated cultures. The percentage of lipid-positive cells in these cultures was also similar to that in the untreated cultures (Fig. 2C). In addition, LY294002 suppressed dose-dependently the increasing effect of BPA on expression of LPL and aP2 genes (Fig. 3C). The levels of LPL and aP2 mRNAs in the cultures treated with a combination of BPA and 8 μM LY294002 were similar to those in the untreated cultures.
Since Akt kinase functions downstream of PI 3-kinase (Burgering and Coffer, 1995; Franke et al., 1995), we examined the effect of BPA on the levels of Akt and phosphorylated Akt (phospho-Akt) by Western blot. Treatment with BPA alone decreased the level of Akt by 53% (Fig. 4A) and increased the level of phospho-Akt by 97% (Fig. 4B), compared with those of the untreated cultures. Treatment with LY294002 alone did not affect the level of Akt but decreased the level of phospho-Akt by 26%. In the cultures treated with a combination of BPA and LY294002, the levels of Akt and phospho-Akt were 88 and 106%, respectively, of those of the untreated cultures. Thus, LY294002 reversed the decreasing effect of BPA on the level of Akt and suppressed the increasing effect of BPA on the level of phospho-Akt.
Relationship between the Structures of BPA-Related Chemicals and Terminal Adipocyte Differentiation
The effects of chemicals with different substituents at the central carbon atom on the TG content and the expression of aP2 gene were examined. The chemical structures are shown in Figure 5A. BPA, BPB, BPE, and BPF are chemicals with different lengths of alkyl substituents at the central carbon atom. Like BPA, BPB, BPE, and BPF increased the TG content (Fig. 5B) and the level of aP2 mRNA (Fig. 5C). The potency of BPB to increase TG accumulation was similar to that of BPA, while the potencies of BPE and BPF were 93 and 89%, respectively, of that of BPA. BPS is a bisphenol consisting of a sulphur atom, instead of a carbon atom, at the central position; the whole chemical structure of BPS is shown in Figure 5A. This chemical also increased the TG content and the level of aP2 mRNA. Its potency to increase TG accumulation was 92% of that of BPA.
The effects of chemicals with different substituents on hydroxyl groups in the para position of the phenolic rings on the TG content and the expression of aP2 gene were examined. The chemical structures are shown in Figure 6A. BPA-DA, BPA-BCF, and BPA-DM are ester derivatives of BPA. These chemicals with ester bonds increased the TG content (Fig. 6B) and the level of aP2 mRNA (Fig. 6C). The potencies of BPA-DA, BPA-BCF, and BPA-DM to increase TG accumulation were 93, 85, and 73%, respectively, of that of BPA. BPA-DGE and BPA-DAA are ether derivatives of BPA. These chemicals with ether bonds did not have the ability to increase the TG content and the level of aP2 mRNA. BPA-DC is a chemical with cyanic groups, instead of hydroxyl groups, at the phenolic rings. This chemical increased the TG content and the level of aP2 mRNA. Its potency to increase TG accumulation was 80% of that of BPA.
Effect of Alkylphenols on TG Accumulation and LPL Activity
Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days with either 45 μM 4-nonylphenol (NP) alone or 45 μM 4-tert-octylphenol (OP) alone. Treatment of cells with either NP or OP caused a 28 or 35%, respectively, decrease in the TG content, compared with the untreated cultures [the untreated cultures (n = 3), 2.69 ± 0.08 μg/μg DNA; the NP-treated cultures (n = 3), 1.94 ± 0.20 μg/μg DNA (p < 0.01, vs. the untreated cultures); the OP-treated cultures (n = 3), 1.75 ± 0.30 μg/μg DNA (p < 0.01, vs. the untreated cultures)]. NP and OP did not affect the LPL activity of the cultures (data not shown).
DISCUSSION
Although 3T3-L1 cells are converted spontaneously to adipocytes when maintained in culture with fetal bovine serum alone, this process takes several weeks (Green and Meuth, 1974). A variety of agents that can retract the conversion process by rapidly and irreversibly triggering 3T3-L1 cells to differentiate into adipocytes have been reported. These triggers include insulin (Russell and Ho, 1976; Spooner et al., 1979), DEX (Rubin et al., 1978), MIX (Russell and Ho, 1976; Spooner et al., 1979), and sodium butyrate (Toscani et al., 1990). The most effective means to trigger the differentiation is to treat confluent cultures of 3T3-L1 cells with a combination of insulin, DEX, and MIX (Rubin et al., 1978). Previously, we found that BPA had the ability to trigger the differentiation of 3T3-L1 cells into adipocytes (Masuno et al., 2002). Following the induction of differentiation by BPA, treatment with a combination of BPA and insulin increased markedly TG accumulation in cells but treatment with BPA alone decreased it. Thus, whether BPA by itself had the ability to accelerate terminal differentiation of 3T3-L1 cells into adipocytes was not clear. The present study was conducted to extend our previous study on the effect of BPA on terminal adipocyte differentiation. For this purpose, the confluent cultures of 3T3-L1 cells were treated with a combination of insulin, DEX, and MIX for two days and subsequently treated with BPA alone. The results of TG measurement suggested that BPA might increase TG accumulation in cells. This was confirmed by the finding that there were many more Oil Red O-staining cells in the BPA-treated cultures than in the untreated cultures. Northern blot analysis revealed that BPA increased the expression of LPL and aP2 genes. In addition, the catalytic activity of LPL was higher in the BPA-treated cultures than in the untreated cultures. These findings indicate that BPA by itself had the ability to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. This conclusion raises the question of how BPA accelerates terminal adipocyte differentiation.
Expression of the constitutively active variant of Akt kinase in 3T3-L1 cells resulted in spontaneous differentiation of fibroblasts into adipocytes, associated with the accumulation of increased lipid droplets (Kohn et al., 1996). A study with LY294002 demonstrated that the function of PI 3-kinase was indispensable for lipid accumulation in 3T3-L1 adipocytes (Xia and Serrero, 1999). Thus, the PI 3-kinase and Akt kinase pathway plays a pivotal role in terminal adipocyte differentiation. Therefore, to explore the above-mentioned question, we examined whether BPA increased TG accumulation and expression of adipocyte genes through this pathway. Since LY294002 blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 genes, this indicated that the function of PI 3-kinase was required to accelerate terminal adipocyte differentiation. Treatment with BPA alone increased the level of phospho-Akt, indicating that BPA activated Akt. Moreover, LY294002 blocked the increasing effect of BPA on the level of phospho-Akt. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes.
Recently, Sakurai et al. (2004) reported that BPA affected neither the expression of IRS-1 nor the insulin-induced phosphorylation of IRS-1 in 3T3-F442A adipocytes. In addition, BPA alone was not able to phosphorylate IRS-1. Based on these findings, they concluded that the action of BPA was not mediated by the insulin signaling pathway in 3T3-F442 adipocytes. The discrepancy between our conclusion and theirs may result from different treatment periods. We treated cells with 80 μM BPA for six days but they treated cells with 100 μM BPA for only 26 h. Of course, a possibility that this discrepancy may be due to different cell lines cannot be excluded.
Perez et al. (1998) reported that in cultures of MCF-7 cells, the longer the alkyl groups at the central carbon atom of diphenylalkanes, the stronger the estrogenic potency estimated from relative cell proliferation activity. They also found that a lower polarity at the central carbon atoms enhanced estrogenicity. Thus, estrogenicity of diphenylalkanes was influenced not only by the length of the substituents at the central carbon atom but also by their nature. In the present study, we found that in diphenylalkanes with free hydroxyl groups in the para position of the phenolic rings, there was no relationship between the length of alkyl groups at the central carbon atom and the potency to increase TG accumulation. This raises the question of whether the central carbon atom was required to accelerate terminal adipocyte differentiation. To explore this question, we used BPS, a bisphenol consisting of a sulphur atom at the central position. Since this chemical increased the expression of aP2 gene and its potency to increase TG accumulation was 92% of that of BPA, this indicated that the central carbon atom was not necessarily needed to accelerate terminal adipocyte differentiation. This finding is consistent with that of Hashimoto et al. (2001) that bisphenols consisting of atoms and compounds other than the carbon atom at the central position had a strong/moderate estrogenic potency in cultures of MCF-7 cells.
Since NP and OP, alkylphenols consisting of a single phenolic ring, also stimulated cell proliferation in cultures of MCF-7 cells (Soto et al., 1991; White et al., 1994), we examined whether they accelerated terminal differentiation of 3T3-L1 cells into adipocytes. In contrast to diphenylalkanes, these alkylphenols inhibited TG accumulation in cells. In addition, they were not able to increase the LPL activity of the cultures. Previously, we found that NP and OP both blocked the increasing effect of insulin on TG accumulation and expression of adipocyte genes in culture of 3T3-L1 cells (Masuno et al., 2003). Taken together, the present findings indicate that both phenolic rings of diphenylalkanes are required to accelerate terminal differentiation of 3T3-L1 cells into adipocytes.
Leclercq (1992) reported that the existence of an oxygen functional group in the para position of the phenolic rings conferred estrogenicity. The replacement of the phenolic hydroxyl groups with diethylaminoethoxy substituents reduced the proliferative activity in cultures of MCF-7 cells (Gilbert et al., 1994). Perez et al. (1998) reported that ester derivatives of BPA displayed a high proliferative activity in cultures of MCF-7 cells, while ether derivatives displayed a low or no proliferative activity. In our culture system, all ester derivatives of BPA tested increased the expression of aP2 gene and their potencies to increase TG accumulation were 73–93% of that of BPA, whereas ether derivatives were not able to increase TG accumulation and expression of aP2 gene. Perez et al. (1998) found that bisphenol A ethoxylate diacrylate (BPA-EDA) and BPA-DGE were estrogenic in the proliferation bioassay even though they showed very low (BPA-EDA) and no binding affinity (BPA-DGE) for the estrogen receptor in in vitro competitive binding assay. Based on this finding, they described that estrogenicity of diphenylalkanes with ester and ether bonds on the para hydroxyl groups could be related to the ability of cellular enzymatic systems to break down these bonds and to generate molecules with free hydroxyl groups. Whether 3T3-L1 cells were able to cleave ester bonds to release free hydroxyl groups is unclear. It is likely that functional groups, which can form the hydrogen bonds, were required to accelerate terminal adipocyte differentiation, because BPA-DC, a chemical with cyanic groups in the para position of the phenolic rings, increased the expression of aP2 gene and its potency to increase TG accumulation was 80% of that of BPA.
In conclusion, 9 of 11 bisphenols tested were able to accelerate terminal differentiation of 3T3-L1 cells into adipocyteas. This action of BPA was mediated by the mechanism that involved the PI 3-kinase and Akt kinase pathway.
ACKNOWLEDGMENTS
We thank Dr. Steven A. Kliewer, GlaxoSmithKline, North Carolina, U.S.A., for donating a mouse aP2 probe. This work was supported in part by a Grant-in-Aid for Science Research (C) from the Japan Society for the Promotion of Science (H.M.).
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Department of Environmental Science for Industry, Ehime University, 3-5-7 Tarumi, Matsuyama, Ekime 790-8566, Japan
Department of Orthopaedic Surgery, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan
ABSTRACT
In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) mRNAs. These findings indicate that BPA was able to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 mRNAs. Western blot analysis revealed that BPA increased the level of phosphorylated Akt kinase. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes. The structure-activity relationship for BPA-related chemicals was examined. Eight derivatives of BPA (three diphenylalkanes with different substituents at the central carbon atom, three diphenylalkanes with ester bonds on hydroxyl groups in the phenolic rings, one bisphenol consisting of a sulphur atom at the central position, one chemical with cyanic groups, instead of hydroxyl groups, in the phenolic rings) accelerated terminal adipocyte differentiation and their potencies to increase TG accumulation were 73–97% of that of BPA. Two diphenylalkanes with ether bonds on hydroxyl groups and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) did not have the ability to accelerate terminal adipocyte differentiation.
Key Words: bisphenols; 3T3-L1 cells; terminal adipocyte differentiation; LY294002; Akt kinase.
INTRODUCTION
Bisphenol A (BPA) is an environmental endocrine disrupting chemical that is present ubiquitously in the environment. For example, BPA is used commercially in products containing polycarbonate plastics such as baby bottles and leaches from them when subjected to simulated use by dishwashing, boiling, and brushing (Brede et al., 2003). This chemical also leaches from polycarbonate culture flasks when subjected to autoclaving (Krishnan et al., 1993). Microgram amounts of BPA are found in the liquid of preserved vegetables in cans (Brotons et al., 1995) and in the saliva of patients treated with dental sealants (Olea et al., 1996). A small amount of BPA also is found in tap water and in human serum (Inoue et al., 2000).
BPA mimics the actions of estrogens (Korach, 1993). Most research to date on BPA has focused on the growth of reproductive organs and tumor cells. For example, administration of BPA to ovariectomized rodents increases uterine weight, stimulates cell proliferation in the uterus and vagina, and induces hypertrophy of the luminal epithelium (Goloubkova et al., 2000; Papaconstantinou et al., 2000; Steinmetz et al., 1998). In cultures, BPA stimulates proliferation of estrogen-responsive human breast cancer cells, MCF-7 cells (Rivas et al., 2002). Recently, BPA was reported to affect non-reproductive organs as well as reproductive organs. In ovariectomized rats, BPA stimulated the growth of the anterior pituitary gland (Goloubkova et al., 2000) and increased the expression of progesterone receptor mRNA in the preoptic area (Funabashi et al., 2001). Moreover, two-generation studies in rats (Rubin et al., 2001) and mice (Howdeshell et al., 1999) showed that perinatal exposure to BPA caused an increase in body weight after birth. However, whether this increased body weight was due to an increased adipose tissue mass remains unclear.
The major cellular component of adipose tissue is adipocytes. 3T3-L1 adipocytes have been widely used to understand adipocyte physiology. Insulin is well known to be a potent adipogenic agent in cultures of 3T3-L1 adipocytes (Cornelius et al., 1994; Gregoire et al., 1998; Rubin et al., 1978; Spooner et al., 1979). The adipogenic actions of insulin are mediated by the insulin signaling pathway (White and Kahn, 1994; Xia and Serrero, 1999). The binding of insulin to its specific receptor at the cell surface causes autophosphorylation and activation of the receptor and this activated receptor phosphorylates insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 activates phosphatidylinositol 3-kinase (PI 3-kinase). The function of PI 3-kinase is implicated in terminal differentiation of 3T3-L1 cells into adipocytes. For example, inhibition of PI 3-kinase by a chemical inhibitor LY294002 blocked insulin-induced lipid accumulation in 3T3-L1 cells (Xia and Serrero 1999).
In the present study, we examined whether BPA and BPA-related chemicals had the ability to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. We used three phenotypic markers for adipocytes as follows: (1) triacylglycerol (TG) accumulation in cells, (2) expression of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) genes, and (3) catalytic activity of LPL. We also explored whether BPA acted through the PI 3-kinase pathway.
MATERIALS AND METHODS
Materials.
Bisphenol A (BPA), bisphenol B (BPB), bisphenol A dicyanate (BPA-DC), bisphenol A bis(chloroformate) (BPA-BCF), bisphenol A diacetate (BPA-DA), bisphenol A O,O-diacetic acid (BPA-DAA), bisphenol A dimethacrylate (BPA-DM) and bisphenol A diglycidyl ether (BPA-DGE), 4-nonylphenol (NP, mixture of compounds with branched sidechain), 4-tert-octylphenol (OP) were obtained from Tokyo Kasei Co., Tokyo, Japan. Bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS), and a kit for TG were obtained from Wako Pure Chemicals Co., Osaka, Japan. A GenElute Mammalian Total RNA kit and LY294002 hydrochloride were obtained from Sigma. An AlkPhos direct labeling kit, an ECL Plus Western Blotting Starter kit, hybridization buffer, blocking reagent, Hyperfilm MP, Hybond-N+, and Hybond-P were obtained from Amersham Pharmacia Biotech, U.K. A mouse monoclonal antibody to Akt1 (B-1) and a rabbit polyclonal antibody to p-Akt1/2/3 (Ser 473) were obtained from Santa Cruz Biotechnology, Inc. A DC Protein Assay was obtained from Bio-Rad Laboratories.
Cell culture and measurements of DNA and TG contents and LPL activity.
3T3-L1 cells were grown to confluence in a standard medium, which consisted of 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B in Dulbecco's modified Eagle's medium, on a 60-mm plate. Confluent cultures were induced to differentiate into adipocytes by treating them for two days in a standard medium supplemented with 10 μg/ml insulin, 1 μM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (MIX). The medium was then replaced with a standard medium supplemented with either 80 μM BPA alone or a combination of 80 μM BPA and LY294002 at the indicated concentrations and changed every two days. Six days later, cells were harvested in 1.2 ml of 50 mM NH4Cl/NH4OH buffer (pH 8.2) containing 20 μg/ml heparin and 2% (w/v) bovine serum albumin and sonicated briefly at 0°C. Aliquots of the homogenate were used for DNA and TG measurements. DNA was measured fluorometrically by the method of Hinegardner (1971) and TG was measured using a kit for TG. Another aliquot of the homogenate was used for preparing an acetone/ether powder for measurement of LPL activity (Masuno et al., 1990). LPL activity in the extract of an acetone/ether powder was measured using phosphatidylcholine-stabilized triolein as a substrate (Masuno et al., 2002). One unit of lipolytic activity was defined as that releasing 1 μmol of fatty acid/min at 37°C.
Lipid staining in cells.
The cultures were fixed with 10% (v/v) formalin in phosphate-buffered saline, and then stained with Oil Red O as described by Kuri-Harcuch and Green (1978). Cells were considered as lipid-positive when droplets were stained red. Four different microscopic fields (x100 magnification) per culture were photographed. The percentage of lipid-positive cells, which was calculated by dividing the number of lipid-positive cells by the number of total cells (at least 250 cells), in each photograph was determined. A value was obtained as an average value for four fields per culture. The results were expressed as the average value for two cultures.
Northern blot.
Total RNA was isolated from the cultures using a GenElute Mammalian Total RNA kit. RNA samples from two plates were combined for each treatment. RNA samples (20 μg/lane) were denatured with formamide and formaldehyde and electrophoresed on a 1.5% agarose gel containing 6.3% formaldehyde. The RNAs were blotted onto a nylon membrane (Hybond-N+) and crosslinked with 0.05 N NaOH. The blotted membrane was prehybridized for 30 min at 55°C in hybridization buffer containing 0.5 M NaCl and 4% (w/v) blocking reagent. A probe labeled using an AlkPhos direct labeling kit was then hybridized for 15–20 h at 55°C. After hybridization, the membrane was washed twice for 10 min at 55°C in the primary wash buffer (50 mM NaH2PO4, 2 M urea, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1 mM MgCl2, 0.2% blocking reagent, pH 7.0) and washed twice for 5 min at room temperature in the secondary wash buffer (50 mM Tris, 2 mM MgCl2, pH 10.0). The blot was left in CDP-Star detection reagent (30–40 μl/cm2) for 3 min and excess detection reagent was drained off. The membrane was exposed to a Hyperfilm MP with an intensifying screen for approximately 60 min. Relative densitometric units were determined using the analysis software Diversity Database.
The probes used for detection of mRNAs of LPL and aP2 were a 542bp EcoRI fragment (nucleotide 194–735) of mouse LPL cDNA clone and a 600bp BamHI fragment of mouse aP2 cDNA clone. The probe for aP2 was kindly donated by Dr. S. A. Kliewer (GlaxoSmithKline, North Carolina).
Western blot.
Cells were lysed in a lysis buffer (20 mM HEPES, 3 mM MgCl2, 2 mM EDTA, 100 mM NaF, 1% Triton X-100, 1 mM PMSF, 100 μg/ml leupeptin, 10 μg/ml aprotinin, pH 7.4) for 20 min at 4°C and centrifuged to remove insoluble materials. The protein concentrations in the cell lysates were measured using a DC Protein Assay. The same amount (10 μg of protein/lane) of proteins were denatured by boiling in Laemmli sample buffer containing 10% 2-mercaptoethanol, separated by SDS-PAGE, and transferred electrophoretically to a PVDF membrane (Hybond-P). The blotted membranes were incubated with indicated primary antibodies (1:500) and horseradish peroxidase-conjugated secondary antibody (1:25,000). Blots were visualized with an ELC Plus Western Blotting Starter kit according to the manufacturer's instructions. The membrane was exposed to a Hyperfilm MP with an intensifying screen for approximately 5–15 min. Relative densitometric units were determined using the analysis software Diversity Database.
Statistical analysis.
Statistical analysis of group means was conducted by ANOVA followed by post hoc comparisons using Fisher's protected least significant difference test. Statistical analysis was conducted using Student's t-test for unpaired samples. For all the statistical analyses, the criterion of significance was p < 0.05. All values were expressed as means ± SD.
RESULTS
Can BPA Accelerate Terminal Differentiation of 3T3-L1 Cells into Adipocytes
Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days with BPA alone. The untreated cultures, in which BPA was absent during the treatment period, contained 30.7 μg/plate of DNA and 1.97 μg/μg DNA of TG. The addition of BPA to the cultures did not cause changes in the DNA content (Fig. 1A) but caused a dose-dependent increase in the TG content (Fig. 1B). BPA at 40 and 80 μM increased the TG content by 110 and 310%, respectively. However, in cultures treated with 160 μM BPA for more than two days, cells began to detach from the bottom of the plates. Therefore, the following experiments were performed using cultures treated with 80 μM BPA, termed the BPA-treated cultures.
Oil Red O staining showed that approximately 25% of cells in the untreated cultures contained discrete lipid droplets (Fig. 2A). The presence of BPA caused an increase in the percentage of lipid-positive cells to approximately 85% (Fig. 2B). These results suggest that BPA may accelerate terminal differentiation of 3T3-L1 cells into adipocytes.
To confirm this, the expression of LPL and aP2 genes was analyzed by Northern blot and the catalytic activity of LPL in the extract of acetone/ether powder of cells was measured. BPA increased the expression of these two genes (Fig. 1C). Densitometrical analysis revealed that BPA increased the levels of LPL and aP2 mRNAs by 293 and 438%, respectively. The catalytic activity of LPL was also higher in the BPA-treated cultures than in the untreated cultures [the former cultures (n = 4), 1.71 ± 0.06 units/mg DNA; the latter cultures (n = 4), 1.29 ± 0.11 units/mg DNA; p < 0.01].
Does BPA Act through the PI 3-Kinase and Akt Kinase Pathway
Since the insulin signaling pathway plays a pivotal role in terminal adipocyte differentiation (Kohn et al., 1996; Xia and Serrero, 1999), we examined using LY294002 whether BPA acted through the PI 3-kinase pathway. Following the hormonal induction of differentiation, 3T3-L1 cells were treated with either BPA alone or a combination of BPA and LY294002. The DNA content of the cultures decreased with an increase in LY294002 concentrations (Fig. 3A). The DNA content of the cultures treated with a combination of BPA and 8 μM LY294002 was 76% of that of the cultures treated with BPA alone. Thus, LY294002 inhibited proliferation of 3T3-L1 cells. This result is compatible with the findings of others that PI 3-kinase plays an important role in mediating the action of insulin on cell growth (Backer et al., 1992; Kapeller et al., 1991). Of course, there is a concern that this inhibition could be due to a toxic effect of LY294002. However, this does not appear to be the case, because cells had not been detached from the bottom of the plates during the experiment period. Moreover, cells attached to the bottom of the plates excluded trypan blue (data not shown).
LY294002 suppressed dose-dependently the increasing effect of BPA on the TG content (Fig. 3B). In the cultures treated with a combination of BPA and 8 μM LY294002, the TG content was decreased to a level similar to that of the untreated cultures. The percentage of lipid-positive cells in these cultures was also similar to that in the untreated cultures (Fig. 2C). In addition, LY294002 suppressed dose-dependently the increasing effect of BPA on expression of LPL and aP2 genes (Fig. 3C). The levels of LPL and aP2 mRNAs in the cultures treated with a combination of BPA and 8 μM LY294002 were similar to those in the untreated cultures.
Since Akt kinase functions downstream of PI 3-kinase (Burgering and Coffer, 1995; Franke et al., 1995), we examined the effect of BPA on the levels of Akt and phosphorylated Akt (phospho-Akt) by Western blot. Treatment with BPA alone decreased the level of Akt by 53% (Fig. 4A) and increased the level of phospho-Akt by 97% (Fig. 4B), compared with those of the untreated cultures. Treatment with LY294002 alone did not affect the level of Akt but decreased the level of phospho-Akt by 26%. In the cultures treated with a combination of BPA and LY294002, the levels of Akt and phospho-Akt were 88 and 106%, respectively, of those of the untreated cultures. Thus, LY294002 reversed the decreasing effect of BPA on the level of Akt and suppressed the increasing effect of BPA on the level of phospho-Akt.
Relationship between the Structures of BPA-Related Chemicals and Terminal Adipocyte Differentiation
The effects of chemicals with different substituents at the central carbon atom on the TG content and the expression of aP2 gene were examined. The chemical structures are shown in Figure 5A. BPA, BPB, BPE, and BPF are chemicals with different lengths of alkyl substituents at the central carbon atom. Like BPA, BPB, BPE, and BPF increased the TG content (Fig. 5B) and the level of aP2 mRNA (Fig. 5C). The potency of BPB to increase TG accumulation was similar to that of BPA, while the potencies of BPE and BPF were 93 and 89%, respectively, of that of BPA. BPS is a bisphenol consisting of a sulphur atom, instead of a carbon atom, at the central position; the whole chemical structure of BPS is shown in Figure 5A. This chemical also increased the TG content and the level of aP2 mRNA. Its potency to increase TG accumulation was 92% of that of BPA.
The effects of chemicals with different substituents on hydroxyl groups in the para position of the phenolic rings on the TG content and the expression of aP2 gene were examined. The chemical structures are shown in Figure 6A. BPA-DA, BPA-BCF, and BPA-DM are ester derivatives of BPA. These chemicals with ester bonds increased the TG content (Fig. 6B) and the level of aP2 mRNA (Fig. 6C). The potencies of BPA-DA, BPA-BCF, and BPA-DM to increase TG accumulation were 93, 85, and 73%, respectively, of that of BPA. BPA-DGE and BPA-DAA are ether derivatives of BPA. These chemicals with ether bonds did not have the ability to increase the TG content and the level of aP2 mRNA. BPA-DC is a chemical with cyanic groups, instead of hydroxyl groups, at the phenolic rings. This chemical increased the TG content and the level of aP2 mRNA. Its potency to increase TG accumulation was 80% of that of BPA.
Effect of Alkylphenols on TG Accumulation and LPL Activity
Following the hormonal induction of differentiation, 3T3-L1 cells were treated for six days with either 45 μM 4-nonylphenol (NP) alone or 45 μM 4-tert-octylphenol (OP) alone. Treatment of cells with either NP or OP caused a 28 or 35%, respectively, decrease in the TG content, compared with the untreated cultures [the untreated cultures (n = 3), 2.69 ± 0.08 μg/μg DNA; the NP-treated cultures (n = 3), 1.94 ± 0.20 μg/μg DNA (p < 0.01, vs. the untreated cultures); the OP-treated cultures (n = 3), 1.75 ± 0.30 μg/μg DNA (p < 0.01, vs. the untreated cultures)]. NP and OP did not affect the LPL activity of the cultures (data not shown).
DISCUSSION
Although 3T3-L1 cells are converted spontaneously to adipocytes when maintained in culture with fetal bovine serum alone, this process takes several weeks (Green and Meuth, 1974). A variety of agents that can retract the conversion process by rapidly and irreversibly triggering 3T3-L1 cells to differentiate into adipocytes have been reported. These triggers include insulin (Russell and Ho, 1976; Spooner et al., 1979), DEX (Rubin et al., 1978), MIX (Russell and Ho, 1976; Spooner et al., 1979), and sodium butyrate (Toscani et al., 1990). The most effective means to trigger the differentiation is to treat confluent cultures of 3T3-L1 cells with a combination of insulin, DEX, and MIX (Rubin et al., 1978). Previously, we found that BPA had the ability to trigger the differentiation of 3T3-L1 cells into adipocytes (Masuno et al., 2002). Following the induction of differentiation by BPA, treatment with a combination of BPA and insulin increased markedly TG accumulation in cells but treatment with BPA alone decreased it. Thus, whether BPA by itself had the ability to accelerate terminal differentiation of 3T3-L1 cells into adipocytes was not clear. The present study was conducted to extend our previous study on the effect of BPA on terminal adipocyte differentiation. For this purpose, the confluent cultures of 3T3-L1 cells were treated with a combination of insulin, DEX, and MIX for two days and subsequently treated with BPA alone. The results of TG measurement suggested that BPA might increase TG accumulation in cells. This was confirmed by the finding that there were many more Oil Red O-staining cells in the BPA-treated cultures than in the untreated cultures. Northern blot analysis revealed that BPA increased the expression of LPL and aP2 genes. In addition, the catalytic activity of LPL was higher in the BPA-treated cultures than in the untreated cultures. These findings indicate that BPA by itself had the ability to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. This conclusion raises the question of how BPA accelerates terminal adipocyte differentiation.
Expression of the constitutively active variant of Akt kinase in 3T3-L1 cells resulted in spontaneous differentiation of fibroblasts into adipocytes, associated with the accumulation of increased lipid droplets (Kohn et al., 1996). A study with LY294002 demonstrated that the function of PI 3-kinase was indispensable for lipid accumulation in 3T3-L1 adipocytes (Xia and Serrero, 1999). Thus, the PI 3-kinase and Akt kinase pathway plays a pivotal role in terminal adipocyte differentiation. Therefore, to explore the above-mentioned question, we examined whether BPA increased TG accumulation and expression of adipocyte genes through this pathway. Since LY294002 blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 genes, this indicated that the function of PI 3-kinase was required to accelerate terminal adipocyte differentiation. Treatment with BPA alone increased the level of phospho-Akt, indicating that BPA activated Akt. Moreover, LY294002 blocked the increasing effect of BPA on the level of phospho-Akt. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes.
Recently, Sakurai et al. (2004) reported that BPA affected neither the expression of IRS-1 nor the insulin-induced phosphorylation of IRS-1 in 3T3-F442A adipocytes. In addition, BPA alone was not able to phosphorylate IRS-1. Based on these findings, they concluded that the action of BPA was not mediated by the insulin signaling pathway in 3T3-F442 adipocytes. The discrepancy between our conclusion and theirs may result from different treatment periods. We treated cells with 80 μM BPA for six days but they treated cells with 100 μM BPA for only 26 h. Of course, a possibility that this discrepancy may be due to different cell lines cannot be excluded.
Perez et al. (1998) reported that in cultures of MCF-7 cells, the longer the alkyl groups at the central carbon atom of diphenylalkanes, the stronger the estrogenic potency estimated from relative cell proliferation activity. They also found that a lower polarity at the central carbon atoms enhanced estrogenicity. Thus, estrogenicity of diphenylalkanes was influenced not only by the length of the substituents at the central carbon atom but also by their nature. In the present study, we found that in diphenylalkanes with free hydroxyl groups in the para position of the phenolic rings, there was no relationship between the length of alkyl groups at the central carbon atom and the potency to increase TG accumulation. This raises the question of whether the central carbon atom was required to accelerate terminal adipocyte differentiation. To explore this question, we used BPS, a bisphenol consisting of a sulphur atom at the central position. Since this chemical increased the expression of aP2 gene and its potency to increase TG accumulation was 92% of that of BPA, this indicated that the central carbon atom was not necessarily needed to accelerate terminal adipocyte differentiation. This finding is consistent with that of Hashimoto et al. (2001) that bisphenols consisting of atoms and compounds other than the carbon atom at the central position had a strong/moderate estrogenic potency in cultures of MCF-7 cells.
Since NP and OP, alkylphenols consisting of a single phenolic ring, also stimulated cell proliferation in cultures of MCF-7 cells (Soto et al., 1991; White et al., 1994), we examined whether they accelerated terminal differentiation of 3T3-L1 cells into adipocytes. In contrast to diphenylalkanes, these alkylphenols inhibited TG accumulation in cells. In addition, they were not able to increase the LPL activity of the cultures. Previously, we found that NP and OP both blocked the increasing effect of insulin on TG accumulation and expression of adipocyte genes in culture of 3T3-L1 cells (Masuno et al., 2003). Taken together, the present findings indicate that both phenolic rings of diphenylalkanes are required to accelerate terminal differentiation of 3T3-L1 cells into adipocytes.
Leclercq (1992) reported that the existence of an oxygen functional group in the para position of the phenolic rings conferred estrogenicity. The replacement of the phenolic hydroxyl groups with diethylaminoethoxy substituents reduced the proliferative activity in cultures of MCF-7 cells (Gilbert et al., 1994). Perez et al. (1998) reported that ester derivatives of BPA displayed a high proliferative activity in cultures of MCF-7 cells, while ether derivatives displayed a low or no proliferative activity. In our culture system, all ester derivatives of BPA tested increased the expression of aP2 gene and their potencies to increase TG accumulation were 73–93% of that of BPA, whereas ether derivatives were not able to increase TG accumulation and expression of aP2 gene. Perez et al. (1998) found that bisphenol A ethoxylate diacrylate (BPA-EDA) and BPA-DGE were estrogenic in the proliferation bioassay even though they showed very low (BPA-EDA) and no binding affinity (BPA-DGE) for the estrogen receptor in in vitro competitive binding assay. Based on this finding, they described that estrogenicity of diphenylalkanes with ester and ether bonds on the para hydroxyl groups could be related to the ability of cellular enzymatic systems to break down these bonds and to generate molecules with free hydroxyl groups. Whether 3T3-L1 cells were able to cleave ester bonds to release free hydroxyl groups is unclear. It is likely that functional groups, which can form the hydrogen bonds, were required to accelerate terminal adipocyte differentiation, because BPA-DC, a chemical with cyanic groups in the para position of the phenolic rings, increased the expression of aP2 gene and its potency to increase TG accumulation was 80% of that of BPA.
In conclusion, 9 of 11 bisphenols tested were able to accelerate terminal differentiation of 3T3-L1 cells into adipocyteas. This action of BPA was mediated by the mechanism that involved the PI 3-kinase and Akt kinase pathway.
ACKNOWLEDGMENTS
We thank Dr. Steven A. Kliewer, GlaxoSmithKline, North Carolina, U.S.A., for donating a mouse aP2 probe. This work was supported in part by a Grant-in-Aid for Science Research (C) from the Japan Society for the Promotion of Science (H.M.).
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