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Toxicology and Applied Pharmacology 329 (2017) 158–164

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Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/taap

Curcumin inhibits adipogenesis induced by benzyl butyl phthalate in


3T3-L1 cells
Satoru Sakuma, Maki Sumida, Yukiko Endoh, Ayaka Kurita, Ayana Yamaguchi, Tomoki Watanabe,
Tetsuya Kohda, Yui Tsukiyama, Yohko Fujimoto ⁎
Laboratory of Physiological Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Phthalates are a group of endocrine disrupting chemicals and may have contributed to the recent global obesity
Received 6 March 2017 health crisis. Increased adipogenesis via the peroxisome proliferator-activated receptor γ (PPARγ)-CCAAT-en-
Revised 16 May 2017 hancer binding protein α (C/EBPα) pathway could be one critical mechanism responsible for phthalate-induced
Accepted 30 May 2017
weight gain. On the other hand, curcumin has been shown to inhibit adipogenesis in cells and animal models. The
present study was undertaken to evaluate, for the first time, whether curcumin could reduce adipogenesis in-
Keywords:
duced by benzyl butyl phthalate (BBP) via downregulation of the PPARγ-C/EBPα pathway. 3T3-L1 preadipocytes
Benzyl butyl phthalate were differentiated by treating them with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the pres-
Curcumin ence of BBP, with or without curcumin. Cells that were grown in the presence of BBP alone showed a significant
Adipocyte differentiation increase in triacylglycerol (TG) levels. In addition, the number of Oil Red O-stained cells and the mRNA expres-
3T3-L1 cell sion levels of PPARγ, C/EBPα, adiponectin, and tumor necrosis factor-α (TNFα) were significantly increased.
Tumor necrosis factor-α However, treatment with BBP in combination with curcumin resulted in major reductions in TG levels, the num-
bers of Oil Red O-stained cells, and the mRNA expression levels of the four proteins. These results suggest that
curcumin might be an inhibitor of BBP-induced weight gain and inflammation via stimulation of adipocyte differ-
entiation and TNFα generation. Curcumin may, therefore, be a potential medication for preventing the harmful
effects of phthalates.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction on wildlife and human health, it is essential to elucidate the effects of


the chemicals on other physiological and pathophysiological processes
Substantial evidence has surfaced regarding the sex-hormone-like besides their sex-hormone-like activity. We have previously shown
effects of anthropogenic chemicals released into the environment. For that nonylphenol and bisphenol A, which are endocrine disruptors,
example, the diminution of the propagative power of alligators on modulate homeostasis in the body by inhibiting the biosynthesis of ei-
Lake Apopka (FL, USA) (Guillette et al., 1994), increases in the rates of cosanoids (prostaglandins etc.) (Fujimoto et al., 2002, 2003, 2005) and
hypospadias in British Columbia (Leung et al., 1985) and in the United inducing the generation of reactive oxygen species (Sakuma et al.,
States (Paulozzi et al., 1997), and the fall in semen quality and male fer- 2010a,b).
tility in Danish men (Bostofte et al., 1983) could be linked to the spill of Obesity is a growing health problem and has been found to be close-
anthropogenic chemicals. These so-called endocrine disruptors have ly associated with increased risks of developing various diseases
been thought to mimic the effects of estrogen, antagonize the effects (Allender and Rayner, 2007; Hossain et al., 2007; Hursting and
of androgen, and disrupt the synthesis and metabolism of sex hormones Dunlap, 2012; Swinburn et al., 2011). Accumulating evidence suggests
(Kelce and Wilson, 1997; Kelce et al., 1998; Sonnenschein and Soto, that exposure to environmental chemicals, also termed “obesogens”,
1998). However, to understand the potential impacts of these chemicals may alter human metabolism, predispose some people to gain weight,
and contribute to the development of obesity and metabolic disorders
Abbreviations: BBP, benzyl butyl phthalate; C/EBP, CCAAT-enhancer binding protein; (Janesick and Blumberg, 2011; Newbold et al., 2009).
C/EBPα, CCAAT-enhancer binding protein α; DMEM, Dulbecco's modified Eagle's Phthalates are commonly used as plasticizers in polyvinyl chloride
medium; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; PPARγ, plastics. They are, therefore, found in numerous household products
peroxisome proliferator-activated receptor γ; TG, triacylglycerol; TNF-α, tumor necrosis such as food packaging, furniture, toys, and even in medical devices.
factor-α.
⁎ Corresponding author.
Phthalates can enter the body through inhalation, ingestion, or dermal
E-mail addresses: sakuma@gly.oups.ac.jp (S. Sakuma), fujimoto@gly.oups.ac.jp exposure. They are rapidly degraded to their respective monoesters
(Y. Fujimoto). and eliminated in urine. Although phthalates have been identified as

http://dx.doi.org/10.1016/j.taap.2017.05.036
0041-008X/© 2017 Elsevier Inc. All rights reserved.
S. Sakuma et al. / Toxicology and Applied Pharmacology 329 (2017) 158–164 159

endocrine disruptors (Foster, 2006; Gray et al., 2000; Wolff et al., 2014), containing 1 μg/mL insulin (Maturation medium). Thereafter, the cells
emerging evidence suggests their potential connection with the devel- were cultured in the Growth medium for 2 days. Treatment with vari-
opment of obesity (Desvergne et al., 2009; Grun and Blumberg, 2007; ous drugs during the differentiation of the 3T3-L1 preadipocytes and
Kim and Park, 2014; Newbold et al., 2009; Stahlhut et al., 2007; measurements of triacylglycerol (TG) levels, the number of Oil Red O-
Teitelbaum et al., 2012). 3T3-L1 preadipocytes are extensively used as stained cells, and the mRNA expression levels of PPARγ, C/EBPα,
an in vitro model for testing adipogenesis. They have been recently pro- adiponectin, and TNFα were performed following methods we have
posed as an alternative in vitro model for screening environmental previously reported (Sakuma et al., 2010a,b, 2012).
obesogens (Pereira-Fernandes et al., 2013, 2014). Benzyl butyl phthal-
ate (BBP) has recently been shown to promote adipogenesis in 3T3-L1 2.3. Treatment with BBP and curcumin
preadipocytes via the peroxisome proliferator-activated receptor γ
(PPARγ)-CCAAT-enhancer binding protein α (C/EBPα) pathway, BBP and curcumin were prepared in Me2SO and added to the
which is major pathway for adipogenesis (Pereira-Fernandes et al., Growth, Initiation, and Maturation media from day 3 (time of addition
2014; Yin et al., 2016). of dexamethasone, IBMX, and insulin) to day 9 (the end of the experi-
Many phytochemical compounds have over the years been found to ment). The Me2SO concentration was maintained up to 0.25% of the
possess a high capacity to act against various disorders including meta- total volume of the medium. Preliminary experiments demonstrated
bolic syndrome. Of these substances, curcumin is positioned as an inter- no significant effects of 0.25% v/v Me2SO on cell differentiation.
esting nutraceutical. Curcumin is an active ingredient of turmeric, a
well-known Indian spice that is derived from the dried roots of the 2.4. Oil Red O staining
Curcuma longa plant. We have previously reported that curcumin in-
hibits the proliferation of the human colorectal cancer cell line Caco-2 The cells were fixed in 4% formaldehyde-phosphate buffer (pH 7.4)
by apoptosis and G2/M cell cycle arrest (Kohda et al., 2016; Sakuma et for 1 h, rinsed with water, and stained with 0.3% Oil Red O dye for 1 h.
al., 2014). Besides our previous in vitro study, several clinical trials After washing again with water, the cells were visually monitored by
have tested the effects of curcumin in metabolic syndrome. In those microscopic observation (10 × 20 magnification). Cell number was de-
studies, curcumin improved the lipid profiles of the subjects and modi- termined by counting Oil Red O-stained and -unstained cells in each of 4
fied their cholesterol-related parameters (Panahi et al., 2014; Yang et al., fields.
2014). Furthermore, curcumin can improve anthropometric measure-
ments and the body composition when it is taken as part of the diet to- 2.5. Measurement of TG levels
gether with implementing the appropriate lifestyle interventions (Di
Pierro et al., 2015). Several groups have reported that curcumin sup- The cells were harvested by scraping them from the culture dishes
presses the adipogenic differentiation of 3T3-L1 preadipocytes by into lysis buffer [1% Triton-100, 150 mM NaCl, 4 mM ethylenediamine-
downregulating the PPARγ-C/EBPα pathway (Ahn et al., 2010; Ejaz et tetraacetic acid, and 20 mM Tris-HCl (pH 7.4) containing protease inhib-
al., 2009; Lee et al., 2009). itor cocktail] and lysed completely using a horn-type sonicator. The
The present study was designed to confirm whether BBP increases amount of TG, which is an index of lipid accumulation, was quantitative-
whereas curcumin decreases adipogenesis during the differentiation ly measured using a Triglyceride E-test Wako kit following normaliza-
of 3T3-L1 preadipocytes. More interestingly, the study also evaluated, tion of the protein amounts and expressed as TG content (μg/mg
for the first time, whether curcumin could reduce BBP-induced adipo- protein).
genesis via the downregulation of the PPARγ-C/EBPα pathway.
2.6. Measurement of the mRNA expression levels of β-actin, adiponectin,
2. Materials and methods TNFα, PPARγ, and C/EBPα

2.1. Materials Untreated cells, as well as cells treated with BBP or BBP plus
curcumin up to day 5 were washed with ice-cold phosphate-buffered
Mouse 3T3-L1 preadipocytes were obtained from the European Col- saline. Total cellular RNA was prepared using TRIzol reagent. One micro-
lection of Cell Cultures (Wiltshire, UK). Transcriptor First Strand cDNA gram of total RNA was reverse transcribed into cDNA using a
Synthesis kit and LightCycler FirstStart DNA Masterplus SYBR green re- Transcriptor First Strand cDNA Synthesis kit. Next, the concentration
agent were obtained from Roche Diagnostics (Indianapolis, IN, USA). and quality of the purified total RNA were determined spectrophoto-
TRIzol reagent and the primers for β-actin, adiponectin, tumor necrosis metrically at 260 nm and by the OD260:280 ratio. mRNA expression was
factor-α (TNFα), PPARγ, and C/EBPα were purchased from Invitrogen measured by real-time reverse transcription polymerase chain reaction
(Carlsbad, CA, USA). BBP, curcumin, and protease inhibitor cocktail using LightCycler FirstStart DNA Masterplus SYBR green reagent and a
were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Triglycer- LightCycler instrument. The results were expressed as the amount of
ide E-test Wako was obtained from Wako Pure Chemical Industries, the target mRNA relative to that of β-actin mRNA, and the values ob-
Limited (Osaka, Japan). All the other reagents used were of analytical tained in the presence or absence of the drugs were expressed in rela-
grade. tion to the values obtained in the Differentiation medium (DM) alone.
The primers for β-actin, adiponectin, TNFα, PPARγ, and C/EBPα were as
2.2. Cell culture follows: β-actin, 5′-ACACCCCAGCCATGTACG-3′, 5′-TGGTGGT
GAAGCTGTAGCC-3′; adiponectin, 5′-GGCAGGCATCCCAGGACATC-3′, 5′-
3T3-L1 preadipocytes were cultured at 37 °C in a humidified atmo- TCTCACCCTTAGGACCAAGAAGAC-3′; TNFα, 5′-ACCTTTCCAGATT
sphere of 5% CO2/95% air. The cells were maintained in a growth medi- CTTCCCTGAG-3′, 5′-CCCGGCCTTCCAAATAAATACATT-3′; PPARγ, 5′-
um containing Dulbecco's modified Eagle's medium with 10% fetal GTGAAGCCCATCGAGGACA-3′, 5′-TGGAGCACCTTGGCGAACA-3′; and C/
bovine serum and 1% penicillin-streptomycin (Growth medium). Cell EBPα, 5′-ATGGTTTCGGGTCGCTGGAT-3′, 5′-CCACGGCCTGACTCCCTCAT-3′.
differentiation was induced according to the protocol that came with
the 3T3-L1 preadipocytes obtained from the European Collection of 2.7. Statistical analysis
Cell Cultures. The procedure was initiated 2 days after confluence for
3 days in the Growth medium containing 0.25 μM dexamethasone, The results have been expressed as mean ± standard error of the
0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 1 μg/mL insulin (Ini- mean. Significant differences in data between two groups were assessed
tiation medium). This was followed by 2 days in the Growth medium using the t-test, whereas differences between multiple groups were
160 S. Sakuma et al. / Toxicology and Applied Pharmacology 329 (2017) 158–164

assessed by one-way analysis of variance, followed by Scheffé's multiple adiponectin mRNA ratio resulting from treatment with 20 μM BBP was
range test. P-values b 0.05 were considered statistically significant. approximately 3.2-fold higher than that caused by treatment with 5
μM rosiglitazone (BBP, 0.57; rosiglitazone, 0.18). These results show
3. Results that BBP and rosiglitazone exert different effects on the regulation of
TNFα mRNA levels.
3.1. BBP promotes the differentiation of 3T3-L1 cells
3.2. Curcumin inhibits the maturation of 3T3-L1 preadipocytes
Figs. 1 and 2 show the effects of BBP on the differentiation of 3T3-L1
preadipocytes into mature adipocytes. Curcumin at concentrations ranging from 5 to 20 μM dose-depen-
Treatment of the DM (Initiation and Maturation media) following dently reduced TG accumulation in the cells when the later were incu-
the procedure described in the Materials and methods section resulted bated with the DM (15 and 20 μM curcumin resulted in 48 and 74%
in increased TG levels [Fig. 1, 4.0-fold vs untreated cells (None); Fig. 2A, inhibition of TG accumulation, respectively) (Fig. 3A). As shown in Fig.
3.2-fold vs None]. In addition, it was observed that a lot of the adipo- 3B, after the Oil Red O staining test, it was observed that cells that
cytes were filled with lipid (Fig. 2B, 15.2-fold vs None). The DM-induced were treated with curcumin (15 and 20 μM) were less stained than
differentiation was accompanied by upregulations in the mRNA expres- those treated without curcumin (DM alone) were. The percentages of
sions of PPARγ (5.4-fold), C/EBPα (5.6-fold), adiponectin (29.8-fold), cells showing lipid when cultured in 20 μM curcumin, were significantly
and TNFα (1.7-fold) (Fig. 2C). These observations are in accordance lower compared with DM alone. These findings are in accordance with
with our previous results (Sakuma et al., 2010a,b, 2012) as well as those in previous reports (Ahn et al., 2010; Ejaz et al., 2009; Lee et al.,
those of other investigators (Pereira-Fernandes et al., 2014; Yin et al., 2009). Thus, these results together with the previous reports demon-
2016). strate that curcumin inhibits the differentiation of 3T3-L1 preadipocytes
Under the basal differentiation conditions, BBP at concentrations into mature adipocytes.
ranging of 1–40 μM dose-dependently increased TG levels (1.1–1.7-
fold, Fig. 1). As shown in Fig. 2B, the cells treated with 20 μM BBP 3.3. Curcumin interferes with BBP-induced adipogenesis in 3T3-L1 cells
were dyed red more intensely with Oil Red O than those treated with
DM alone were. The percentage of cells showing lipid accumulation In order to study the potential inhibitory effects of curcumin on BBP-
was 2.1-fold greater in BBP-treated cultures compared with DM alone. induced adipogenesis, 15 or 20 μM curcumin in combination with BBP
Furthermore, BBP significantly increased the expression levels of (20 μM) was added to the DM during culturing of the 3T3-L1 cells. Dif-
PPARγ (1.8-fold, Fig. 2C), C/EBPα (1.5-fold, Fig. 2D), adiponectin (3.0- ferentiation was monitored by the measurements of TG levels (Fig. 4A),
fold, Fig. 2E), and TNFα (1.7-fold, Fig. 2F) more than DM alone did. Oil Red O staining (Fig. 4B), and the mRNA expression levels of PPARγ,
Okuno et al. (1998) and Yamauchi et al. (2001) have shown that the C/EBPα, adiponectin, and TNFα (Fig. 5A–D).
thiazolidinedione compound rosiglitazone upregulates the PPARγ-C/ Similar to the results illustrated in Figs. 1 and 2, cells grown in the
EBPα pathway. It also stimulates the productions of TG and the benign presence of 20 μM BBP showed significant increases in TG levels (1.5-
adipocytes that preferentially secrete adiponectin; however, it does fold, Fig. 4A), the number of differentiated cells (2.1-fold, Fig. 4B), and
not affect TNFα production. When rosiglitazone was used in the present the mRNA expression levels of PPARγ (2.0-fold), C/EBPα (1.5-fold),
study (Fig. 2), the results obtained were similar to those in previous re- adiponectin (2.9-fold), and TNFα (1.8-fold) (Fig. 5A–D). However, the
ports (Okuno et al., 1998; Yamauchi et al., 2001). Treatment of the cells addition of 15 μM curcumin to the medium almost nullified the effects
with rosiglitazone (5 μM) resulted in increases in TG levels (1.6-fold, Fig. of BBP. This is because, TG levels (Fig. 4A), the number of Oil Red O-
1A), the number of Oil Red O-stained cells (2.0-fold, Fig. 2B), and the stained cells (Fig. 4B), and the mRNA expression levels of PPARγ, C/
mRNA expression levels of PPARγ (1.7-fold, Fig. 2C), C/EBPα (2.4-fold, EBPα, adiponectin, and TNFα (Fig. 5A–D) in the presence of 20 μM
Fig. 2D), and adiponectin (4.5-fold, Fig. 2E); however, the mRNA levels BBP and 15 μM curcumin were similar to the respective levels in the
of TNFα tended to decrease (23% inhibition, Fig. 2F). The TNFα mRNA/ presence of only DM.

4. Discussion

Previous studies have suggested that enhanced adipogenesis during


preadipocyte differentiation is a potential mechanism underlying the
weight gain associated with exposure to phthalates (Desvergne et al.,
2009; Grun and Blumberg, 2007; Kim and Park, 2014; Newbold et al.,
2009; Stahlhut et al., 2007; Teitelbaum et al., 2012). Fig. 1 showed that
BBP at concentrations ranging from 1 to 40 μM dose-dependently in-
creased adipogenesis in the 3T3-L1 cells. The results do not only confirm
the previous reports on BBP as an obesogen; however, they also reveal
that the dose of BBP may play a major role in the induction of adipogen-
esis. Exposure to phthalate is ubiquitous in the modern lifestyle. The
level of phthalates in the plasma of a healthy individual has been detect-
ed to be around several micromolar concentrations (Durmaz et al.,
2010; Zhang et al., 2003). However, a large variation in phthalate body
burden (Becker et al., 2009; Weuve et al., 2006), as well as high levels
of phthalates of up to 100 μM in human plasma and adipose samples
(Ellero-Simatos et al., 2011) have been observed. Moreover, clinic
usage of plastics can lead to a maximal phthalate exposure of 300 μM
in patients (Food and Drug Administration, 2001). Based on these
Fig. 1. Alterations in triacylglycerol (TG) levels in 3T3-L1 adipocytes treated with benzyl data, the BBP amounts used in the present study were chosen to be
butyl phthalate (BBP). The data have been presented as mean ± standard error of the
mean (n = 4). a indicates P b 0.01 when compared to the untreated cells, whereas b
comparable with the clinical exposure levels. Importantly, it was ob-
indicates P b 0.05 and c indicates P b 0.01 when compared to the cells cultured in the served that BBP at a concentration as low as 10 μM is enough to cause
differentiation medium (Initiation and Maturation media). significant functional changes in adipocytes (Figs. 1 and 2).
S. Sakuma et al. / Toxicology and Applied Pharmacology 329 (2017) 158–164 161

Fig. 2. Alterations in (A) triacylglycerol (TG) content, (B) Oil Red O staining, and the mRNA expression levels of (C) peroxisome proliferator-activated receptor γ (PPARγ), (D) CCAAT-
enhancer binding protein α (C/EBPα), (E) adiponectin, and (F) tumor necrosis factor-α (TNFα) in 3T3-L1 adipocytes treated with benzyl butyl phthalate (BBP) or rosiglitazone (ROSI).
(A) The data have been presented as mean ± standard error of the mean (S.E.) (n = 8). (B) The photographs shown are representative of the results from three independent
experiments. The data have been presented as mean ± S.E. (n = 3). (C–F) The data have been presented as mean ± S.E. (n = 6). a indicates P b 0.05 and b indicates P b 0.01 when
compared with the untreated cells (None), whereas c indicates P b 0.05 and d indicates P b 0.01 when compared to the cells cultured in the Differentiation medium (DM).

Curcumin is an active ingredient of turmeric, a well-known Indian demonstrated that curcumin can reduce blood TG levels in patients
spice that is derived from the dried roots of the Curcuma longa plant. with type II diabetes (Panahi et al., 2017; Rahimi et al., 2016). In the
The fact that humans are able to consume up to 8 g of curcumin per present study, it was demonstrated that the level of adipogenesis in
day without experiencing toxic effects (Cheng et al., 2001) makes cells treated with 20 μM BBP plus 15 or 20 μM curcumin was almost
curcumin a very interesting preventive agent against various diseases. identical to that in cells that were not treated with BBP (Figs. 4 and 5).
Curcumin has been reported to inhibit adipogenesis, both in cellular These findings strongly suggest that curcumin can prevent BBP-induced
and animal models of obesity, by several research groups (Ahn et al., adipogenesis. This is the first study to show that curcumin is a potent
2010; Di Pierro et al., 2015; Ejaz et al., 2009; Lee et al., 2009; Panahi et naturally occurring compound that can prevent the adipogenesis in-
al., 2014; Yang et al., 2014). In addition, recent clinical trials have duced by phthalates, which are environmental pollutants.
162 S. Sakuma et al. / Toxicology and Applied Pharmacology 329 (2017) 158–164

Fig. 4. Alterations in (A) triacylglycerol (TG) content and (B) Oil Red O staining of 3T3-L1
adipocytes treated with benzyl butyl phthalate (BBP) and curcumin (CUR). (A) The data
have been presented as mean ± standard error of the mean (n = 12). (B) The
photographs shown are representative of the results from three independent
Fig. 3. Alterations in (A) triacylglycerol (TG) content and (B) Oil Red O staining of 3T3-L1 experiments. The data have been presented as mean ± S.E. (n = 3). a indicates P b 0.01
adipocytes treated with curcumin (CUR). (A) The data have been presented as mean ± when compared with the untreated cells (None). b indicates P b 0.05 and c indicates P b
standard error of the mean (n = 9). (B) The photographs shown are representative of 0.01 when compared to the cells cultured in the Differentiation medium (DM). d
the results from three independent experiments. The data have been presented as mean indicates P b 0.05 and e indicates P b 0.01 when compared to the cells treated with BBP
± S.E. (n = 3). a indicates P b 0.01 when compared to the untreated cells (None), (20 μM) alone.
whereas b indicates P b 0.01 when compared to the cells cultured in the Differentiation
medium (DM). activates PPARγ, which in turn stimulates the accumulation of TG and
increases the number of benign adipocytes that preferentially secrete
The differentiation of preadipocytes into mature insulin-responsive adiponectin in white adipose tissues. In order to elucidate the possible
adipocytes involves exposure of a confluent, quiescent population of mechanism by which curcumin inhibits BBP-induced adipogenesis, we
cells to a variety of effectors that activate a cascade of transcription fac- investigated the genes of PPARγ, C/EBPα, adiponectin, and TNFα during
tors commencing with C/EBPβ and C/EBPδ, ultimately inducing the ex- the differentiation of 3T3-L1 cells following treatment with BBP. Fig. 2
pressions of C/EBPα and PPARγ (Morrison and Farmer, 1999; Rosen et showed that treatment of the cells with 20 μM BBP upregulated the
al., 2000). PPARγ is a master regulator of adipocyte differentiation. To- mRNA expressions of PPARγ, C/EBPα, adiponectin, and TNFα. A similar
gether with C/EBPα, PPARγ promotes terminal differentiation by acti- observation was made following treatment with 5 μM rosiglitazone, ex-
vating the expression of downstream adipocyte-specific genes. The cept that there was a tentative downregulation in the mRNA expression
mature adipocytes generated then secrete various bioactive molecules of TNFα. These results suggest that although both BBP and rosiglitazone
called adipocytokines. Of these, TNFα, which increases in amount in enhance adipogenesis via the PPARγ-C/EBPα pathway, only BBP causes
the obese state and is expressed in enlarged adipocytes, has been impli- an enhanced expression of TNFα, which contributes to the development
cated in various metabolic disorders. On the other hand, adiponectin, of insulin resistance and type II diabetes. Further studies are needed to
which is expressed in small adipocytes, is considered to protect against clarify the concerning differences in the effects of the two compounds
diabetes and atherosclerosis, among other diseases (Audrey Nguyen et on TNFα expression; however, one explanation may be that
al., 2005; Yamauchi et al., 2001). Okuno et al. (1998) and Yamauchi et rosiglitazone has efficacy to repress TNFα expression via diverse reac-
al. (2001) have shown that one of the thiazolidinediones, rosiglitazone, tion points (inhibition of free fatty acid release, etc.) (Arner, 2003).
S. Sakuma et al. / Toxicology and Applied Pharmacology 329 (2017) 158–164 163

Fig. 5. Alterations in the mRNA expression levels of (A) peroxisome proliferator-activated receptor γ (PPARγ), (B) CCAAT-enhancer binding protein α (C/EBPα), (C) adiponectin, and (D)
tumor necrosis factor-α (TNFα) in 3T3-L1 adipocytes treated with benzyl butyl phthalate (BBP) in combination with curcumin (CUR). The data have been presented as mean ± standard
error of the mean (n = 6). a indicates P b 0.05 and b indicates P b 0.01 when compared to the untreated cells (None). c indicates P b 0.05 and d indicates P b 0.01 when compared to the cells
cultured in the Differentiation medium (DM). e indicates P b 0.05 and f indicates P b 0.01 when compared to the cells treated with BBP (20 μM) alone.

Nevertheless, the present finding that BBP generates larger amounts of Bostofte, E., Serup, J., Rebbe, H., 1983. Has the fertility of Danish men declined through the
years in terms of semen quality? A comparison of semen qualities between 1952 and
TNFα than rosiglitazone does may be valuable in understanding the 1972. Int. J. Fertil. 28, 91–95.
harmful effects of phthalates in the body. Furthermore, the results Cheng, A.L., Hsu, C.H., Lin, J.K., Hsu, M.M., Ho, Y.F., Shen, T.S., Ko, J.Y., Lin, J.T., Lin, B.R.,
shown in Figs. 4 and 5 demonstrate that curcumin reversed BBP-in- Ming-Shiang, W., Yu, H.S., Jee, S.H., Chen, G.S., Chen, T.M., Chen, C.A., Lai, M.K., Pu,
Y.S., Pan, M.H., Wang, Y.J., Tsai, C.C., Hsieh, C.Y., 2001. Phase 1 clinical trial of
duced increments in the mRNA expression levels of PPARγ, C/EBPα, curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant les-
adiponectin, and TNFα. As a result, the mRNA levels of these four pro- sons. Anticancer Res. 21, 2895–2900.
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hibits BBP-induced adipogenesis through a suppressive effect on BBP- tential role of bioavailable curcumin in weight loss and omental adipose tissue de-
elicited activation of the PPARγ-C/EBPα pathway. crease: preliminary data of a randomized, controlled trial in overweight people
with metabolic syndrome. Preliminary study. Eur. Rev. Med. Pharmacol. Sci. 19,
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