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

Substantial evidence has surfaced regarding the sex-hormone-like
effects of anthropogenic chemicals released into the environment. For
example, the diminution of the propagative power of alligators on
Lake Apopka (FL, USA) (Guillette et al., 1994), increases in the rates of
hypospadias in British Columbia (Leung et al., 1985) and in the United
States (Paulozzi et al., 1997), and the fall in semen quality and male fer-
tility in Danish men (Bostofte et al., 1983) could be linked to the spill of
anthropogenic chemicals. These so-called endocrine disruptors have
been thought to mimic the effects of estrogen, antagonize the effects
of androgen, and disrupt the synthesis and metabolism of sex hormones
(Kelce and Wilson, 1997; Kelce et al., 1998; Sonnenschein and Soto,
1998). However, to understand the potential impacts of these chemicals
Abbreviations: BBP, benzyl butyl phthalate; C/EBP, CCAAT-enhancer binding protein;
C/EBPα, CCAAT-enhancer binding protein α; DMEM, Dulbecco's modified Eagle's
medium; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; PPARγ,
peroxisome proliferator-activated receptor γ; TG, triacylglycerol; TNF-α, tumor necrosis
factor-α.
⁎ Corresponding author.
E-mail addresses: sakuma@gly.oups.ac.jp (S. Sakuma), fujimoto@gly.oups.ac.jp
(Y. Fujimoto).
the chemicals on other physiological and pathophysiological processes
besides their sex-hormone-like activity. We have previously shown
that nonylphenol and bisphenol A, which are endocrine disruptors,
modulate homeostasis in the body by inhibiting the biosynthesis of ei-
cosanoids (prostaglandins etc.) (Fujimoto et al., 2002, 2003, 2005) and
inducing the generation of reactive oxygen species (Sakuma et al.,
2010a,b).
Obesity is a growing health problem and has been found to be close-
ly associated with increased risks of developing various diseases
(Allender and Rayner, 2007; Hossain et al., 2007; Hursting and
Dunlap, 2012; Swinburn et al., 2011). Accumulating evidence suggests
that exposure to environmental chemicals, also termed “obesogens”,
may alter human metabolism, predispose some people to gain weight,
and contribute to the development of obesity and metabolic disorders
(Janesick and Blumberg, 2011; Newbold et al., 2009).
Phthalates are commonly used as plasticizers in polyvinyl chloride
plastics. They are, therefore, found in numerous household products
such as food packaging, furniture, toys, and even in medical devices.
Phthalates can enter the body through inhalation, ingestion, or dermal
exposure. They are rapidly degraded to their respective monoesters
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
endocrine disruptors (Foster, 2006; Gray et al., 2000; Wolff et al., 2014),
emerging evidence suggests their potential connection with the devel-
opment of obesity (Desvergne et al., 2009; Grun and Blumberg, 2007;
Kim and Park, 2014; Newbold et al., 2009; Stahlhut et al., 2007;
Teitelbaum et al., 2012). 3T3-L1 preadipocytes are extensively used as
an in vitro model for testing adipogenesis. They have been recently pro-
posed as an alternative in vitro model for screening environmental
obesogens (Pereira-Fernandes et al., 2013, 2014). Benzyl butyl phthal-
ate (BBP) has recently been shown to promote adipogenesis in 3T3-L1
preadipocytes via the peroxisome proliferator-activated receptor γ
(PPARγ)-CCAAT-enhancer binding protein α (C/EBPα) pathway,
which is major pathway for adipogenesis (Pereira-Fernandes et al.,
2014; Yin et al., 2016).
Many phytochemical compounds have over the years been found to
possess a high capacity to act against various disorders including meta-
bolic syndrome. Of these substances, curcumin is positioned as an inter-
esting nutraceutical. Curcumin is an active ingredient of turmeric, a
well-known Indian spice that is derived from the dried roots of the
Curcuma longa plant. We have previously reported that curcumin in-
hibits the proliferation of the human colorectal cancer cell line Caco-2
by apoptosis and G2/M cell cycle arrest (Kohda et al., 2016; Sakuma et
al., 2014). Besides our previous in vitro study, several clinical trials
have tested the effects of curcumin in metabolic syndrome. In those
studies, curcumin improved the lipid profiles of the subjects and modi-
fied their cholesterol-related parameters (Panahi et al., 2014; Yang et al.,
2014). Furthermore, curcumin can improve anthropometric measure-
ments and the body composition when it is taken as part of the diet to-
gether with implementing the appropriate lifestyle interventions (Di
Pierro et al., 2015). Several groups have reported that curcumin sup-
presses the adipogenic differentiation of 3T3-L1 preadipocytes by
downregulating the PPARγ-C/EBPα pathway (Ahn et al., 2010; Ejaz et
al., 2009; Lee et al., 2009).
The present study was designed to confirm whether BBP increases
whereas curcumin decreases adipogenesis during the differentiation
of 3T3-L1 preadipocytes. More interestingly, the study also evaluated,
for the first time, whether curcumin could reduce BBP-induced adipo-
genesis via the downregulation of the PPARγ-C/EBPα pathway.
2. Materials and methods
2.1. Materials
Mouse 3T3-L1 preadipocytes were obtained from the European Col-
lection of Cell Cultures (Wiltshire, UK). Transcriptor First Strand cDNA
Synthesis kit and LightCycler FirstStart DNA Masterplus SYBR green re-
agent were obtained from Roche Diagnostics (Indianapolis, IN, USA).
TRIzol reagent and the primers for β-actin, adiponectin, tumor necrosis
factor-α (TNFα), PPARγ, and C/EBPα were purchased from Invitrogen
(Carlsbad, CA, USA). BBP, curcumin, and protease inhibitor cocktail
were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Triglycer-
ide E-test Wako was obtained from Wako Pure Chemical Industries,
Limited (Osaka, Japan). All the other reagents used were of analytical
grade.
2.2. Cell culture
3T3-L1 preadipocytes were cultured at 37 °C in a humidified atmo-
sphere of 5% CO2/95% air. The cells were maintained in a growth medi-
um containing Dulbecco's modified Eagle's medium with 10% fetal
bovine serum and 1% penicillin-streptomycin (Growth medium). Cell
differentiation was induced according to the protocol that came with
the 3T3-L1 preadipocytes obtained from the European Collection of
Cell Cultures. The procedure was initiated 2 days after confluence for
3 days in the Growth medium containing 0.25 μM dexamethasone,
0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 1 μg/mL insulin (Ini-
tiation medium). This was followed by 2 days in the Growth medium
159
containing 1 μg/mL insulin (Maturation medium). Thereafter, the cells
were cultured in the Growth medium for 2 days. Treatment with vari-
ous drugs during the differentiation of the 3T3-L1 preadipocytes and
measurements of triacylglycerol (TG) levels, the number of Oil Red O-
stained cells, and the mRNA expression levels of PPARγ, C/EBPα,
adiponectin, and TNFα were performed following methods we have
previously reported (Sakuma et al., 2010a,b, 2012).
2.3. Treatment with BBP and curcumin
BBP and curcumin were prepared in Me2SO and added to the
Growth, Initiation, and Maturation media from day 3 (time of addition
of dexamethasone, IBMX, and insulin) to day 9 (the end of the experi-
ment). The Me2SO concentration was maintained up to 0.25% of the
total volume of the medium. Preliminary experiments demonstrated
no significant effects of 0.25% v/v Me2SO on cell differentiation.
2.4. Oil Red O staining
The cells were fixed in 4% formaldehyde-phosphate buffer (pH 7.4)
for 1 h, rinsed with water, and stained with 0.3% Oil Red O dye for 1 h.
After washing again with water, the cells were visually monitored by
microscopic observation (10 × 20 magnification). Cell number was de-
termined by counting Oil Red O-stained and -unstained cells in each of 4
fields.
2.5. Measurement of TG levels
The cells were harvested by scraping them from the culture dishes
into lysis buffer [1% Triton-100, 150 mM NaCl, 4 mM ethylenediamine-
tetraacetic acid, and 20 mM Tris-HCl (pH 7.4) containing protease inhib-
itor cocktail] and lysed completely using a horn-type sonicator. The
amount of TG, which is an index of lipid accumulation, was quantitative-
ly measured using a Triglyceride E-test Wako kit following normaliza-
tion of the protein amounts and expressed as TG content (μg/mg
protein).
2.6. Measurement of the mRNA expression levels of β-actin, adiponectin,
TNFα, PPARγ, and C/EBPα
Untreated cells, as well as cells treated with BBP or BBP plus
curcumin up to day 5 were washed with ice-cold phosphate-buffered
saline. Total cellular RNA was prepared using TRIzol reagent. One micro-
gram of total RNA was reverse transcribed into cDNA using a
Transcriptor First Strand cDNA Synthesis kit. Next, the concentration
and quality of the purified total RNA were determined spectrophoto-
metrically at 260 nm and by the OD260:280 ratio. mRNA expression was
measured by real-time reverse transcription polymerase chain reaction
using LightCycler FirstStart DNA Masterplus SYBR green reagent and a
LightCycler instrument. The results were expressed as the amount of
the target mRNA relative to that of β-actin mRNA, and the values ob-
tained in the presence or absence of the drugs were expressed in rela-
tion to the values obtained in the Differentiation medium (DM) alone.
The primers for β-actin, adiponectin, TNFα, PPARγ, and C/EBPα were as
follows: β-actin, 5′-ACACCCCAGCCATGTACG-3′, 5′-TGGTGGT
GAAGCTGTAGCC-3′; adiponectin, 5′-GGCAGGCATCCCAGGACATC-3′, 5′-
TCTCACCCTTAGGACCAAGAAGAC-3′; TNFα, 5′-ACCTTTCCAGATT
CTTCCCTGAG-3′, 5′-CCCGGCCTTCCAAATAAATACATT-3′; PPARγ, 5′-
GTGAAGCCCATCGAGGACA-3′, 5′-TGGAGCACCTTGGCGAACA-3′; and C/
EBPα, 5′-ATGGTTTCGGGTCGCTGGAT-3′, 5′-CCACGGCCTGACTCCCTCAT-3′.
2.7. Statistical analysis
The results have been expressed as mean ± standard error of the
mean. Significant differences in data between two groups were assessed
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
range test. P-values b 0.05 were considered statistically significant.
3. Results
3.1. BBP promotes the differentiation of 3T3-L1 cells
Figs. 1 and 2 show the effects of BBP on the differentiation of 3T3-L1
preadipocytes into mature adipocytes.
Treatment of the DM (Initiation and Maturation media) following
the procedure described in the Materials and methods section resulted
in increased TG levels [Fig. 1, 4.0-fold vs untreated cells (None); Fig. 2A,
3.2-fold vs None]. In addition, it was observed that a lot of the adipo-
cytes were filled with lipid (Fig. 2B, 15.2-fold vs None). The DM-induced
differentiation was accompanied by upregulations in the mRNA expres-
sions of PPARγ (5.4-fold), C/EBPα (5.6-fold), adiponectin (29.8-fold),
and TNFα (1.7-fold) (Fig. 2C). These observations are in accordance
with our previous results (Sakuma et al., 2010a,b, 2012) as well as
those of other investigators (Pereira-Fernandes et al., 2014; Yin et al.,
2016).
Under the basal differentiation conditions, BBP at concentrations
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
were dyed red more intensely with Oil Red O than those treated with
DM alone were. The percentage of cells showing lipid accumulation
was 2.1-fold greater in BBP-treated cultures compared with DM alone.
Furthermore, BBP significantly increased the expression levels of
PPARγ (1.8-fold, Fig. 2C), C/EBPα (1.5-fold, Fig. 2D), adiponectin (3.0-
fold, Fig. 2E), and TNFα (1.7-fold, Fig. 2F) more than DM alone did.
Okuno et al. (1998) and Yamauchi et al. (2001) have shown that the
thiazolidinedione compound rosiglitazone upregulates the PPARγ-C/
EBPα pathway. It also stimulates the productions of TG and the benign
adipocytes that preferentially secrete adiponectin; however, it does
not affect TNFα production. When rosiglitazone was used in the present
study (Fig. 2), the results obtained were similar to those in previous re-
ports (Okuno et al., 1998; Yamauchi et al., 2001). Treatment of the cells
with rosiglitazone (5 μM) resulted in increases in TG levels (1.6-fold, Fig.
1A), the number of Oil Red O-stained cells (2.0-fold, Fig. 2B), and the
mRNA expression levels of PPARγ (1.7-fold, Fig. 2C), C/EBPα (2.4-fold,
Fig. 2D), and adiponectin (4.5-fold, Fig. 2E); however, the mRNA levels
of TNFα tended to decrease (23% inhibition, Fig. 2F). The TNFα mRNA/
Fig. 1. Alterations in triacylglycerol (TG) levels in 3T3-L1 adipocytes treated with benzyl
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
indicates P b 0.05 and c indicates P b 0.01 when compared to the cells cultured in the
differentiation medium (Initiation and Maturation media).
adiponectin mRNA ratio resulting from treatment with 20 μM BBP was
approximately 3.2-fold higher than that caused by treatment with 5
μM rosiglitazone (BBP, 0.57; rosiglitazone, 0.18). These results show
that BBP and rosiglitazone exert different effects on the regulation of
TNFα mRNA levels.
3.2. Curcumin inhibits the maturation of 3T3-L1 preadipocytes
Curcumin at concentrations ranging from 5 to 20 μM dose-depen-
dently reduced TG accumulation in the cells when the later were incu-
bated with the DM (15 and 20 μM curcumin resulted in 48 and 74%
inhibition of TG accumulation, respectively) (Fig. 3A). As shown in Fig.
3B, after the Oil Red O staining test, it was observed that cells that
were treated with curcumin (15 and 20 μM) were less stained than
those treated without curcumin (DM alone) were. The percentages of
cells showing lipid when cultured in 20 μM curcumin, were significantly
lower compared with DM alone. These findings are in accordance with
those in previous reports (Ahn et al., 2010; Ejaz et al., 2009; Lee et al.,
2009). Thus, these results together with the previous reports demon-
strate that curcumin inhibits the differentiation of 3T3-L1 preadipocytes
into mature adipocytes.
3.3. Curcumin interferes with BBP-induced adipogenesis in 3T3-L1 cells
In order to study the potential inhibitory effects of curcumin on BBP-
induced adipogenesis, 15 or 20 μM curcumin in combination with BBP
(20 μM) was added to the DM during culturing of the 3T3-L1 cells. Dif-
ferentiation was monitored by the measurements of TG levels (Fig. 4A),
Oil Red O staining (Fig. 4B), and the mRNA expression levels of PPARγ,
C/EBPα, adiponectin, and TNFα (Fig. 5A–D).
Similar to the results illustrated in Figs. 1 and 2, cells grown in the
presence of 20 μM BBP showed significant increases in TG levels (1.5-
fold, Fig. 4A), the number of differentiated cells (2.1-fold, Fig. 4B), and
the mRNA expression levels of PPARγ (2.0-fold), C/EBPα (1.5-fold),
adiponectin (2.9-fold), and TNFα (1.8-fold) (Fig. 5A–D). However, the
addition of 15 μM curcumin to the medium almost nullified the effects
of BBP. This is because, TG levels (Fig. 4A), the number of Oil Red O-
stained cells (Fig. 4B), and the mRNA expression levels of PPARγ, C/
EBPα, adiponectin, and TNFα (Fig. 5A–D) in the presence of 20 μM
BBP and 15 μM curcumin were similar to the respective levels in the
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
data, the BBP amounts used in the present study were chosen to be
comparable with the clinical exposure levels. Importantly, it was ob-
served that BBP at a concentration as low as 10 μM is enough to cause
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
spice that is derived from the dried roots of the Curcuma longa plant.
The fact that humans are able to consume up to 8 g of curcumin per
day without experiencing toxic effects (Cheng et al., 2001) makes
curcumin a very interesting preventive agent against various diseases.
Curcumin has been reported to inhibit adipogenesis, both in cellular
and animal models of obesity, by several research groups (Ahn et al.,
2010; Di Pierro et al., 2015; Ejaz et al., 2009; Lee et al., 2009; Panahi et
al., 2014; Yang et al., 2014). In addition, recent clinical trials have
demonstrated that curcumin can reduce blood TG levels in patients
with type II diabetes (Panahi et al., 2017; Rahimi et al., 2016). In the
present study, it was demonstrated that the level of adipogenesis in
cells treated with 20 μM BBP plus 15 or 20 μM curcumin was almost
identical to that in cells that were not treated with BBP (Figs. 4 and 5).
These findings strongly suggest that curcumin can prevent BBP-induced
adipogenesis. This is the first study to show that curcumin is a potent
naturally occurring compound that can prevent the adipogenesis in-
duced by phthalates, which are environmental pollutants.
162 S. Sakuma et al. / Toxicology and Applied Pharmacology 329 (2017) 158–164
Fig. 3. Alterations in (A) triacylglycerol (TG) content and (B) Oil Red O staining of 3T3-L1
adipocytes treated with curcumin (CUR). (A) The data have been presented as mean ±
standard error of the mean (n = 9). (B) The photographs shown are representative of
the results from three independent experiments. The data have been presented as mean
± S.E. (n = 3). a indicates P b 0.01 when compared to the untreated cells (None),
whereas b indicates P b 0.01 when compared to the cells cultured in the Differentiation
medium (DM).
The differentiation of preadipocytes into mature insulin-responsive
adipocytes involves exposure of a confluent, quiescent population of
cells to a variety of effectors that activate a cascade of transcription fac-
tors commencing with C/EBPβ and C/EBPδ, ultimately inducing the ex-
pressions of C/EBPα and PPARγ (Morrison and Farmer, 1999; Rosen et
al., 2000). PPARγ is a master regulator of adipocyte differentiation. To-
gether with C/EBPα, PPARγ promotes terminal differentiation by acti-
vating the expression of downstream adipocyte-specific genes. The
mature adipocytes generated then secrete various bioactive molecules
called adipocytokines. Of these, TNFα, which increases in amount in
the obese state and is expressed in enlarged adipocytes, has been impli-
cated in various metabolic disorders. On the other hand, adiponectin,
which is expressed in small adipocytes, is considered to protect against
diabetes and atherosclerosis, among other diseases (Audrey Nguyen et
al., 2005; Yamauchi et al., 2001). Okuno et al. (1998) and Yamauchi et
al. (2001) have shown that one of the thiazolidinediones, rosiglitazone,
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
experiments. The data have been presented as mean ± S.E. (n = 3). a indicates P b 0.01
when compared with the untreated cells (None). b indicates P b 0.05 and c indicates P b
0.01 when compared to the cells cultured in the Differentiation medium (DM). d
indicates P b 0.05 and e indicates P b 0.01 when compared to the cells treated with BBP
(20 μM) alone.
activates PPARγ, which in turn stimulates the accumulation of TG and
increases the number of benign adipocytes that preferentially secrete
adiponectin in white adipose tissues. In order to elucidate the possible
mechanism by which curcumin inhibits BBP-induced adipogenesis, we
investigated the genes of PPARγ, C/EBPα, adiponectin, and TNFα during
the differentiation of 3T3-L1 cells following treatment with BBP. Fig. 2
showed that treatment of the cells with 20 μM BBP upregulated the
mRNA expressions of PPARγ, C/EBPα, adiponectin, and TNFα. A similar
observation was made following treatment with 5 μM rosiglitazone, ex-
cept that there was a tentative downregulation in the mRNA expression
of TNFα. These results suggest that although both BBP and rosiglitazone
enhance adipogenesis via the PPARγ-C/EBPα pathway, only BBP causes
an enhanced expression of TNFα, which contributes to the development
of insulin resistance and type II diabetes. Further studies are needed to
clarify the concerning differences in the effects of the two compounds
on TNFα expression; however, one explanation may be that
rosiglitazone has efficacy to repress TNFα expression via diverse reac-
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
TNFα than rosiglitazone does may be valuable in understanding the
harmful effects of phthalates in the body. Furthermore, the results
shown in Figs. 4 and 5 demonstrate that curcumin reversed BBP-in-
duced increments in the mRNA expression levels of PPARγ, C/EBPα,
adiponectin, and TNFα. As a result, the mRNA levels of these four pro-
teins were fully expected to be similar to their respective levels in
cells that were not treated with BBP. These indicate that curcumin in-
hibits BBP-induced adipogenesis through a suppressive effect on BBP-
elicited activation of the PPARγ-C/EBPα pathway.
In conclusion, the present study is the first to show that curcumin
might be an inhibitor of BBP-induced weight gain and inflammation
via its stimulations of adipocyte differentiation and TNFα generation.
These suggest that curcumin is a potential medication for preventing
the harmful effects of phthalates.
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