ToxSci Advance Access originally published online on July 25, 2003
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Toxicological Sciences 75, 314-320 (2003)
Copyright © 2003 by the Society of Toxicology
ENVIRONMENTAL TOXICOLOGY |
Effect of 4-Nonylphenol on Cell Proliferation and Adipocyte Formation in Cultures of Fully Differentiated 3T3-L1 Cells
* Department of Medical Laboratory Technology, Ehime College of Health Science, Takooda, Tobe-cho, Iyo-gun, Ehime 791-2101, Japan;
Department of Environmental Science for Industry, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan; and
Department of Orthopaedic Surgery, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-0295, Japan
Received May 22, 2003; accepted July 16, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of 4-nonylphenol (NP) on cell proliferation and adipocyte formation was examined in cultures of fully differentiated 3T3-L1 cells. Following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for 8 days with or without NP. NP at 5 and 10 µg/ml increased the DNA content by 32% and 68%, respectively, compared with that of the untreated cultures, in which NP was absent during the treatment period. There were many more bromodeoxyuridine (BrdU)-positive cells in the NP-treated cultures, in which NP was present at a concentration of 10 µg/ml during the treatment period, compared to the untreated cultures. These results indicate that NP had the ability to stimulate the proliferation of fully differentiated 3T3-L1 cells. NP at 5 and 10 µg/ml decreased the triacylglycerol (TG) content by 26% and 58%, respectively, and decreased the lipoprotein lipase (LPL) activity by 51% and 71%, respectively. The lipid droplets in individual cells of the NP-treated cultures were smaller than those of the untreated cultures. The mRNA levels of LPL and adipocyte-specific fatty acid binding protein (aP2) were considerably lower in the NP-treated cultures than in the untreated cultures. Thus, NP also had the ability to inhibit adipocyte formation in cultures of fully differentiated 3T3-L1 cells. A study using an antiestrogen ICI 182,780 showed that the NP-stimulated cell proliferation was mediated partly by the estrogen receptor, while the NP-induced inhibition of adipocyte formation was mediated by a mechanism other than the estrogen receptor.
Key Words: 4-nonylphenol; adipocyte formation; fully differentiated 3T3-L1 cells; cell proliferation; ICI 182,780; lipoprotein lipase; adipocyte-specific fatty acid binding protein; bromodeoxyuridine.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alkylphenol ethoxylates have been widely used as plastic additives and components of surfactants, paints, herbicides, and insecticides (Naylor, 1995
; Platt, 1978; Reed, 1978). Approximately 80% of these chemicals are reported to be nonylphenol ethoxylate (NPE) (Naylor, 1995). NPE enters the aquatic environment after use, and the polyethoxylate side chain is cleaved during anaerobic wastewater treatment, leaving nonylphenol (NP) (Nimrod and Benson, 1996). NP was found in water and fish from rivers in Japan (Tsuda et al., 2000). NP also contaminated water flowing through polyvinyl chloride tubing (Junk et al., 1974) and was detected in drinking water (Clark et al., 1992). Moreover, NP leached from plastics used in food processing and packing (Gilbert et al., 1992) and from commercially available polystyrene tubes (Soto et al., 1991). Thus, NP is present ubiquitously in the environment.
NP mimics weakly estrogenic actions both in vivo and in vitro. For example, administration of NP to female rats increased uterine weight and accelerated vaginal opening (Chapin et al., 1999; Laws et al., 2000; Lee and Lee, 1996; Nagao et al., 2001; Odum et al., 1999). The in vitro studies showed that NP stimulated the proliferation of human estrogen-sensitive MCF-7 breast tumor cells (Soto et al., 1991; White et al., 1994) and increased vitellogenin production in cultured trout hepatocytes (Jobling and Sumpter, 1993; White et al., 1994). Since adipose tissue is also an estrogen-dependent organ (Benoit et al., 1982; Wade and Gray, 1979), this study was designated to elucidate whether NP possessed deleterious effects on adipose tissue.
Adipose tissue consists of adipocytes, which store triacylglycerol (TG) as a fuel for the body. Approximately 80–90% of the wet weight of adipocytes consists of TG. To understand adipocyte physiology, a cell line of mouse fibroblasts (3T3-L1 cells) has been widely used. 3T3-L1 cells are necessary to enter the differentiation process for optimal adipocyte formation (Cornelius et al., 1994; Gregoire et al., 1998; Mandrup and Lane, 1997; Rubin et al., 1978). The most efficient means to enter the differentiation process is to treat the confluent cultures of 3T3-L1 cells with a hormone mixture (insulin, dexamethasone, and 1-methyl-3-isobutylxanthine) for 2 days (Rubin et al., 1978). Upon the differentiation, the estrogen receptor has been reported to become demonstrable in 3T3-L1 cells (Somjen et al., 1997). In the present study, we used fully differentiated 3T3-L1 cells to examine whether NP affected adipocyte formation. We also described the effect of NP on the proliferation of fully differentiated 3T3-L1 cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
4-NP (mixture of compounds with branched sidechain, Lot # FGE01) and 4-tert-octylphenol (OP) were from Tokyo Kasei Kogyo Co., Tokyo, Japan. ICI 182,780 was from Tocris Cookson, UK. A fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG was from Zymed, CA. A GenElute Mammalian Total RNA kit was from Sigma. An AlkPhos direct labeling kit, CDP-Star detection reagent, hybridization buffer, HyperfilmTM MP and Hybond-N+ were from Amersham Pharmacia Biotech, U.K. 5-Bromo-2'-deoxyuridine (BrdU) and a kit for TG were from Wako Pure Chemicals Co., Osaka, Japan. A mouse monoclonal anti-BrdU was from Monosan, The Netherlands.
Cell culture.
3T3-L1 cells were grown to confluence in standard medium on a 60-mm plate. Confluent cultures were induced to differentiate into adipocytes by treating them for 2 days in standard medium containing 10 µg/ml insulin, 1 µM dexamethasone, and 0.5 mM 1-methyl-3-isobutylxanthine. The medium was then replaced with complete medium containing NP at the indicated concentrations and changed every 2 days. Eight 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 (BSA) and sonicated briefly at 0°C. Aliquots of the homogenate were used for TG and DNA measurements. TG was measured using a kit for TG, and DNA was measured fluorometrically by the method of Hinegardner (1971). Another aliquot was used for preparing an acetone/ether powder as described previously (Masuno et al., 1990).
Standard medium contained 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. Complete medium contained 5 µg/ml insulin in standard medium.
Lipid staining of cells.
The cultures were fixed with 10% (v/v) formalin in phosphate-buffered saline (PBS), and then stained with Oil Red O as described by Kuri-Harcuch and Green (1978). The cells were considered as lipid-positive when droplets were stained red.
Labeling of DNA with BrdU.
3T3-L1 cells were grown to confluence on a chamber slide (Nalge Nunc International, Osaka, Japan). Following the hormonal induction of differentiation, cells were treated for 3 days with 10 µg/ml NP, and incubated with 30 µM BrdU during the last 2 h of the 3-day treatment period. The slides were washed twice with PBS, fixed in 70% ethanol for 30 min, and then incubated in 100% ethanol for 10 min at room temperature. Ethanol-fixed slides were washed once with PBS, treated with 1.5 N HCl for 30 min, and blocked with 0.5% Tween 20 in PBS for 5 min. After washing twice with PBS, the slides were incubated for 1 h with a mouse monoclonal anti-BrdU, diluted 1:10, in PBS containing 1% BSA. After washing twice with PBS, the slides were finally incubated for 1 h in the dark with an FITC-labeled anti-mouse IgG, diluted 1:20, in PBS containing 1% BSA. The slides were washed twice for 5 min with PBS and mounted for immunofluorescence microscopy analysis (BHS-RFK, Olympus, Tokyo, Japan).
Assay of lipoprotein lipase (LPL) activity.
A stock emulsion containing 1.13 mmol triolein, 60 mg phosphatidylcholine, and 9 ml glycerol was prepared (Masuno et al., 1990). A mixture of 1 volume of the stock emulsion, 19 volumes of 3% (w/v) BSA in 0.2 M Tris/HCl buffer (pH 8.2), and 5 volumes of heat inactivated (56°C, 10 min) serum from starved rats was incubated at 37°C for 15–30 min and used as the activated substrate mixture. LPL was extracted from acetone/ether powders using ice-cold 50 mM NH4Cl/NH4OH buffer (pH 8.2) containing 20 µg/ml heparin as described previously (Masuno et al., 1990). For assay, 100 µl of the activated substrate mixture was added to 100 µl of the powder extract, and the mixture was incubated for 30 min at 37°C. The amount of oleic acid released was measured as described previously (Masuno et al., 2002). One unit of lipolytic activity was defined as that releasing 1 µmol of fatty acid/min at 37°C.
Northern blot.
Total RNA was isolated from the cultures using a GenElute Mammalian Total RNA kit. 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. Then, a probe labeled using an AlkPhos direct labeling kit was 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 NaH2PO42H2O, 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 and 100 mM NaCl, 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 HyperfilmTM MP with an intensifying screen for approximately 60 min.
The probes used for detection of mRNAs of LPL and adipocyte-specific fatty acid binding protein (aP2) were a 542-bp EcoRI fragment (nucleotide 194–735) of the mouse LPL cDNA clone and a 600-bp BamHI fragment of the mouse aP2 cDNA clone. The aP2 probe was kindly donated by Dr. S. A. Kliewer (GlaxoSmithKline, NC).
Statistical analysis.
Comparisons between experimental groups were made by Student’s t test with Bonferroni p values. For all the analyses, the criterion of significance was p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of NP on Cell Proliferation and Adipocyte Formation
Following the hormonal induction of differentiation, 3T3-L1 cells were treated with NP at the indicated concentrations. The untreated cultures, in which NP was absent during the treatment period, contained 42.1 µg/plate of DNA, 13.5 µg/µg DNA of TG, and 4.39 units/mg DNA of LPL activity (Fig. 1
). Most cells of these cultures contained large and discrete lipid droplets (Fig. 2A). The presence of NP in the cultures caused a dose-dependent increase in the DNA content and a dose-dependent decrease in the TG content and LPL activity (Fig. 1). NP at 5 and 10 µg/ml increased the DNA content by 32% and 68%, decreased the TG content by 26% and 58%, and decreased the LPL activity by 51% and 71%, respectively. Even when the values were expressed as per plate, NP decreased the TG content and LPL activity in a dose-dependent manner (data not shown). The lipid droplets in individual cells of the NP-treated cultures, in which NP at a concentration of 10 µg/ml was present during the treatment period, became smaller than those of the untreated cultures (Fig. 2B). NP at the concentrations of >15 µg/ml was toxic to these cells (data not shown).
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The results of DNA measurement suggested that NP may stimulate cell proliferation. To confirm this, we used BrdU to label DNA synthesis. In the untreated cultures, a small percentage of cells were BrdU-positive (Fig. 2D
). Treatment with NP resulted in a marked increase in the production of BrdU-positive cells (Fig. 2E). This result indicates that 3T3-L1 cells were proliferating more actively in the NP-treated cultures than in the untreated cultures.
The results of Oil Red O staining and measurements of TG and LPL activity suggested that NP may inhibit adipocyte formation. To confirm this, the effect of NP on the expressions of LPL and aP2 genes was examined by Northern blot. The mRNA levels of LPL and aP2 in the NP-treated cultures were markedly lower than those in the untreated cultures (Fig. 3).
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Effect of ICI 182,780 on NP-Induced Changes in Cell Proliferation and Adipocyte Formation
To evaluate whether NP acted through the estrogen receptor, the experiment was performed using the antiestrogen ICI 182,780 at 1 µM (Hyder et al., 1997
). Following the hormonal induction of differentiation, 3T3-L1 cells were treated with ICI 182,780 alone. No differences in the DNA content and LPL activity were observed between the untreated cultures and the cultures treated with ICI 182,780 alone (Figs. 4A and 4C). Surprisingly, the TG content was higher in the latter cultures than in the former cultures (Fig. 4B).
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Next, 3T3-L1 cells were treated with either NP alone or a combination of NP and ICI 182,780. The DNA content of the cultures treated with a combination of NP and ICI 182,780 was lower than that of the cultures treated with NP alone but still higher than that of the untreated cultures (Fig. 4A
). ICI 182,780 at higher concentrations (>2 µM) was toxic to these cells when it was added together with NP into the cultures (data not shown). There were fewer BrdU-positive cells in the cultures treated with a combination of NP and ICI 182,780 than in the cultures treated with NP alone but more than in the untreated cultures (Fig. 2F). Thus, ICI 182,780 partially inhibited the NP-stimulated cell proliferation. Surprisingly, ICI 182,780 enhanced the inhibitory effect of NP on the TG content and LPL activity (Figs. 4B and 4C). The TG content and LPL activity of the cultures treated with a combination of NP and ICI 182,780 were 67 and 58%, respectively, of those of the cultures treated with NP alone. The former cultures also contained fewer lipid-positive cells than the latter cultures (Fig. 2C).
Effect of OP on DNA and TG Contents and LPL Activity
The potency of OP to affect three parameters was compared with that of NP. Following the hormonal induction of differentiation, 3T3-L1 cells were treated with either OP or NP. The presence of either OP or NP caused a 22% or 68%, respectively, increase in the DNA content, a 48% or 58%, respectively, decrease in the TG content, and a 38% or 71%, respectively, decrease in the LPL activity, compared with those of the untreated cultures (Fig. 5). Thus, OP was less potent than NP in cultures of fully differentiated 3T3-L1 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most research to date on NP has focused on the growth of reproductive organs in rats (Laws et al., 2000
; Lee and Lee, 1996; Odum et al., 1999) and fish (Ashfield et al., 1998). The multigeneration studies in rats showed that NP affected not only reproductive organs but also nonreproductive organs such as kidney and liver (Chapin et al., 1999; Nagao et al., 2001). Recently, treatment of rats with NP was found to induce hydroxyl radical formation in the striatum, which might make a major contribution at certain stages in the progression of brain injury (Obata and Kubota, 2000). In rat offspring exposed to NP, behavioral alterations in open-field activity were observed (Ferguson et al., 2000). These findings suggest that NP may affect the central nervous system. Thus, deleterious effects of NP on several organs have been reported, but there has been no report on the effect of NP on adipose tissue and adipocytes. The purpose of this study is to determine the novel action of NP in adipocytes.
In the first set of experiments, we examined the effect of NP on cell proliferation in cultures of fully differentiated 3T3-L1 cells, because NP has been known to stimulate breast tumor cell growth in culture (Soto et al., 1991; White et al., 1994). We used two criteria for cell proliferation: (1) the DNA content of the cultures and (2) labeling of DNA with BrdU in the cultures. The DNA content of the NP-treated cultures was considerably higher than that of the untreated cultures. In addition, there were many more BrdU-positive cells in the NP-treated cultures than in the untreated cultures. Based on these results, we concluded that NP had the ability to stimulate the proliferation of fully differentiated 3T3-L1 cells. The results of DNA measurement suggested that OP had a similar effect but was less potent than NP. This is contrary to the finding that OP was more potent than NP in MCF-7 cells (White et al., 1994).
A study with ICI 182,780 in immature rats showed that the NP-induced uterine growth was mediated by the estrogen receptor (Lee and Lee, 1999). Similarly, it was demonstrated using ICI 182,780 that in MCF-7 cells alkylphenols acted through the estrogen receptor (White et al., 1994). However, the present finding that ICI 182,780 was not able to completely reverse the increasing effects of NP on the DNA content and production of BrdU-positive cells suggests that the NP-stimulated proliferation of fully differentiated 3T3-L1 cells was mediated at least partly by an alternative mechanism that does not involve the estrogen receptor. This is similar to the finding that in rat white adipocytes, ICI 182,780 partially inhibited the estradiol-induced expressions of c-fos and c-jun genes (Garcia et al., 2000). Thus, the mechanism by which the action of alkylphenols was mediated differed depending on the origin of cells.
In the second set of experiments, we examined whether NP affected adipocyte formation in cultures of fully differentiated 3T3-L1 cells. Following the hormonal induction of differentiation, 3T3-L1 cells reenter a period of the cell cycle called mitotic clonal expansion, become growth arrested again, and then express adipocyte-specific gene products including LPL and aP2 and accumulate TG (Cook et al., 1985; Cornelius et al., 1994; Gregoire et al., 1998; Mandrup and Lane, 1997). Therefore, we used three criteria for adipocyte formation: (1) TG accumulation in cells, (2) expressions of LPL and aP2 genes, and (3) catalytic activity of LPL. Northern blot analysis revealed that the levels of LPL and aP2 mRNAs were much lower in the NP-treated cultures than in the untreated cultures. The LPL activity of the former cultures also was considerably lower than that of the latter cultures. Moreover, the lipid droplets in individual cells of the NP-treated cultures were smaller than those of the untreated cultures, resulting in a low TG content of the former cultures. Based on these results, we concluded that NP had the ability to inhibit the adipocyte formation in cultures of fully differentiated 3T3-L1 cells. There are some reports on the effect of other nonsteroidal estrogenic compounds on adipocyte formation in cultures of 3T3-L1 cells. For example, bisphenol A diglycidyl ether has been reported to inhibit the differentiation of 3T3-L1 cells into adipocytes (Wright et al., 2000). Recently, we demonstrated that bisphenol A (BPA) by itself had the ability to induce the differentiation of 3T3-L1 cells into adipocytes (Masuno et al., 2002). In addition, BPA in combination with insulin was found to accelerate adipocyte formation in cultures of BPA-differentiated cells. Thus, nonsteroidal estrogenic compounds present in the environment appear to affect lipid metabolism in adipocytes.
The mechanism by which NP inhibited adipocyte formation remains unclear. It is likely that the inhibitory action of NP on adipocyte formation was mediated by mechanisms other than the estrogen receptor, because ICI 182,780 was not able to reverse the NP-induced decreases in TG accumulation and expression of LPL activity. Since growth arrest after mitotic clonal expansion is requisite for adipocyte formation, the inhibitory effect of NP may be due to its proliferative activity. This interpretation may be supported by the finding that growth factors including epidermal growth factor and transforming growth factor-ß acted as inhibitors of adipocyte formation (Gregoire et al., 1998). However, we should note that transforming growth factor-ß was ineffective in fully differentiated 3T3 T cells (Sparks et al., 1992).
Chylomicrons and very low-density lipoproteins are partly hydrolyzed through the action of LPL located on the luminal surfaces of capillaries in extrahepatic tissues, becoming smaller and denser lipoproteins, known as remnant particles. The resulting remnants are taken up and quickly catabolized by the liver (Casarali-Marano et al., 1998; Mahley and Ji, 1999). LPL synthesized in extrahepatic tissues circulates in blood associated with lipoproteins (Vilella et al., 1993) and is taken up by the liver (Vilaro et al., 1988), where it acts as a recognition signal for remnant uptake (Casarali-Marano et al., 1998; Chang et al., 1996; Felts et al., 1975). Thus, LPL involves not only hydrolysis of TG-rich lipoproteins in the circulation but also uptake of the resulting lipoprotein remnants by the liver. Chylomicron remnants have been reported to be implicated in the progression of coronary artery disease (Karpe et al., 1994). In the present study, we found that NP down-regulated the expression of LPL at the transcriptional level, resulting in decreased enzyme activity in fully differentiated 3T3-L1 cells. Taken together, it will be important to examine the causative relationship between chronic exposure to alkylphenols and the formation of coronary atherosclerosis.
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ACKNOWLEDGMENTS |
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We thank Dr. Steven A. Kliewer, GlaxoSmithKline, North Carolina, U.S.A., for donating 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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NOTES |
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1 To whom correspondence should be addressed at Department of Medical Laboratory Technology, Ehime College of Health Science, Takooda, Tobe-cho, Iyo-gun, Ehime 791-2101, Japan. Fax: +81-89-958-2177. E-mail: hmasuno{at}ehime-chs.ac.jp.
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