Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 879884
doi:10.1111/j.1745-7270.2007.00351.x
Microarray Analysis of
Bisphenol A-induced Changes in Gene Expression in Human Oral Epithelial Cells
Keisuke SEKI1,2,
Ryosuke KOSHI3, Naoyuki SUGANO2,3*, Shigeyuki MASUTANI1,
Naoto YOSHINUMA2,3, and Koichi ITO2,3
1
General Practice Residency, Nihon University School of Dentistry Dental
Hospital, Tokyo 101-8310, Japan;
2
Division of Advanced Dental Treatment, Dental Research Center, Nihon University
School of Dentistry, Tokyo 101-8310, Japan;
3
Department of Periodontology, Nihon University School of Dentistry, Tokyo
101-8310, Japan
Received: March 31,
2007
Accepted: August 2,
2007
This work was supported
by a Grant-in-Aid for Technology to Promote Multidisciplinary Research
Projects from the Ministry of Education, Culture, Sports, Science and
Technology of Japan
*Corresponding
author: Tel, 81-3-32198107; Fax, 81-3-32198349; E-mail, [email protected]
Abstract Bisphenol A (BPA) is a common ingredient in dental
materials. However, its potential adverse effects on the oral cavity are
unknown. The purpose of this study is to identify the genes responding to BPA in
a human oral epithelial cell line using DNA microarray. Of the 10,368 genes
examined, changes in mRNA levels were detected in seven genes: five were
up-regulated and two were down-regulated. The expression levels of the calcium
channel, voltage-dependent, L-type, alpha 1C subunit (CACNA1C), cell
death activator CIDE-3 (CIDE-3), haptoglobin-related protein (HPR),
importin 4 (IPO4), and POU domain, class 2 and transcription factor 3 (POU2F3)
were significantly up-regulated in the cells exposed to 100 mM BPA. The spermatogenesis-associated,
serine-rich 2 (SPATS2) and HSPC049 protein (HSPC049) were
significantly down-regulated. The detailed knowledge of the changes in gene
expression obtained using microarray technology will provide a basis for
further elucidating the molecular mechanisms of the toxic effects of BPA in the
oral cavity.
Keywords bisphenol A; human oral epithelial cell line; DNA
microarray
2,2-Bis(4-hydroxyphenyl)propane
(bisphenol A; BPA), an alkylphenol derivative, is a high production-volume
chemical that is used in the manufacture of polycarbonate plastics [1,2]. BPA
binds to estrogen receptors and induces estrogenic activity in a number of
biological systems, suggesting that it acts as an environmental estrogen [3–5]. Human exposure occurs when BPA
leaches from plastic-lined food and beverage cans [6]. Some uncertainty exists
concerning the level and risk of exposure to BPA, but evidence suggests that
BPA disrupts normal reproductive tract development in male and female rodents
[7]. Furthermore, BPA exposure during the prenatal period inhibits
testosterone synthesis in adult rats [8].
BPA is also a
common ingredient in the resin-based restorative composites and sealants used
in dentistry [9]. The resin matrix is initially a fluid containing a monomer
that is cured or converted into a rigid polymer by a chemical or
photo-initiated polymerization reaction [10]. Unpolymerized BPA can leach from
the dental composite or sealant [11–15] or be degraded chemically or mechanically
[16] and become absorbed systemically by the patient [17]. BPA exposure
resulting from the placement of dental sealants or composites has been
reported [10,18–20].
However, the magnitude of these exposures, the long-term potential for sealant
leaching, and the potential for adverse effects are controversial [21–23]. Although
many pharmacological findings indicate male reproductive toxicity induced by
BPA, the adverse effects of BPA on oral tissues remain unclear. Therefore, it
is critical to clarify the global gene regulation of oral epithelial cells by
BPA.
This study
identified the genes that respond to BPA in a human oral epithelial cell line
using DNA microarray.
Materials and Methods
Cell characteristics and cell
culture
Ca9-22, a human oral
epithelial cell line, was used in this study. The cells were maintained in
Eagle’s modified minimum essential medium (EMEM; Asahi Techno Glass, Shizuoka,
Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS;
BioSource, Rockville, USA), 1% (V/V) penicillin-streptomycin
solution (100 U/ml penicillin and 0.1 mg/ml
streptomycin; Sigma, St. Louis, USA), and L-glutamine
(2 mM), in 75 cm3 tissue culture flasks (Falcon; Becton
Dickinson, Lincoln Park, USA) at 37 ?C in 5% CO2 in highly humidified air.
Subconfluent cells were detached with trypsin-EDTA (Gibco BRL Life
Technologies, Grand Island, USA) for subculturing. The cells were maintained
until they reached 70% to 80% confluence, at which time they were harvested and
used in the experiments. The total cell number and cell viability were
determined by counting living and dead cells determined by Trypan Blue dye
exclusion under a microscope.
Cell proliferation assay
Ca9-22 cells were seeded into
96-well plates (Falcon; Becton Dickinson) at a density of 1?103 cells/well in EMEM containing
10% FBS and 1% antibiotics, and cultured at 37 ?C in 5% CO2 in
highly humidified air. After 24 h, the cells were incubated with EMEM
supplemented with 10% FBS and 1% antibiotics containing various concentrations
of BPA (0, 10, 50, 100, 200, and 500 mM) for 1, 3, 6, 12, and 24 h. At each
point, the attached cell number was counted after Trypan Blue staining.
Incubation of cells with BPA
The Ca9-22 cells were cultured
in 75 cm3
tissue culture flasks at a density of 1.6?104 cells/cm3. BPA
(purity 95.0%; Wako Pure Chemical Industries, Osaka, Japan) was dissolved in
ethanol, and added to the cells to a final concentration of 100 mM. The cells were incubated for 6 h at 37
?C in the presence or absence of BPA. Total RNA was purified using an RNeasy
Mini Kit (Qiagen, Valencia, USA). The total RNA in each sample was determined
by spectrophotometry, and the RNA quality was monitored by agarose gel
electrophoresis.
DNA microarray gene expression
analysis
Gene expression was analyzed
with the AceGene Human Oligo Chip 30K SubsetA (Hitachi Software Engineering,
Kanagawa, Japan). The microarray experiments were carried out according to the
manufacturer’s instructions.
Labeled cDNA targets for
hybridizations were synthesized by reverse transcription from total RNA samples
in the presence of Cy5-dUTP and Cy3-dUTP (Amersham Biosciences,
Buckinghamshire,
England). For each reverse transcription reaction, 30 mg
total RNA was mixed with 2 ml oligo(dT)12–18 primer (Invitrogen, Carlsbad, USA) in a
total volume of 15.5 ml, heated to 70 ?C for 10 min,
and cooled on ice for 5 min. To this mixture, we added 3 ml nucleotide cocktail (5 mM each dATP,
dGTP, dCTP, 3 mM dTTP, and 2 mM aminoallyl-dUTP), 3 ml
of 0.1 M dithiothreitol, 0.5 ml of 40 U/ml RNase inhibitor (Toyobo, Osaka, Japan),
and 6 ml of 5?first-strand buffer and
incubated the mixture at 42 ?C for 2 min. We also added 2 ml SuperScript II reverse transcriptase
(Invitrogen). After a 90 min incubation at 42 ?C, we added 5 ml of 0.5 M EDTA and 10 ml of 1 M NaOH, and incubated the mixture
at 70 ?C for 20 min. Then the mixture was neutralized by adding 12 ml of 1 M HCl. The synthesized
aminoallyl-dUTP-labeled cDNA was purified using a QIAquick PCR Purification Kit
(Qiagen) and subjected to ethanol precipitation. The cDNA samples were
dissolved in 9 ml of 0.2 M sodium bicarbonate
buffer, and 1 ml CyDye solution containing
Cy3 or Cy5 Mono-Reactive Dye (Amersham Biosciences) dissolved in 45 ml dimethyl sulfoxide was added. In this
study, the control sample was labeled with Cy3, and the test sample was labeled
with Cy5. After 1 h incubation at 40 ?C, we added 40 ml
distilled water, and the samples were purified using Micro Bio-Spin Columns
with Bio-Gel P-30 (Bio-Rad Laboratories, Tokyo, Japan). We then mixed in 15 ml of 1 mg/ml Human Cot-1 DNA
(Invitrogen), 1 ml of 1 mg/ml poly A
(Invitrogen), and 0.5 ml of 10 mg/ml transfer RNA
yeast solution, and carried out ethanol precipitation. The two kinds of labeled
cDNA sample, the control and test samples, were dissolved in distilled water
and mixed in a total volume of 15.5 ml. We
added 12.5 ml of 20?alin-sodium citrate
(SSC), 2.5 ml of 10% sodium dodecylsulfate (SDS), 4 ml of 50?Denhardt’s solution, and 10 ml hybridization solution. The sample was
heated to 95 ?C for 2 min, and cooled on ice. Then we added 0.5 ml of 10 mg/ml salmon sperm DNA and 5 ml formamide, and incubated the sample at
42 ?C for 5 min. This target mixture was applied to the microarray, which was
covered with a 24 mm?60 mm NEO cover glass (Matsunami, Osaka,
Japan). The microarray was incubated for hybridization at 42 ?C for 16 h in a
humidified chamber. After hybridization, the microarray was washed sequentially
in 2?SSC
with 0.1% SDS for 10 min at 30 ?C, 2?SSC for 5 min at 30 ?C, 1?SSC for 5 min at 30 ?C, and
0.1?SSC
at room temperature to remove the SDS. The washed microarray was scanned in
both the Cy3 and Cy5 channels using ScanArray Lite (GSI Lumonics, Billerica,
USA). The data were analyzed using QuantArray software (GSI Lumonics),
converting the signal intensity of each spot into text format. Data analyses
were carried out with GeneSpring 4.2.1 (Silicon Genetics, Redwood City, USA)
bioinformatics algorithms. Per-spot normalization was carried out with
glyceraldehyde-3-phosphate dehydrogenase in intensity and detection efficiency
between spots. The data presented are the average of three separate
experiments.
Real-time polymerase chain
reaction (PCR) analysis
Total RNA (3 mg per reaction) was reverse transcribed
at 42 ?C for 60 min using a first-strand synthesis kit (Amersham Pharmacia
Biotech, Piscataway, USA). Following cDNA synthesis, 1 ml each reaction was used as the template
for PCR. Real-time PCR was conducted using primers and probe sets from
Assay-on-Demand Gene Expression Products (Applied Biosystems, Foster City,
USA). PCR amplification of the target genes and internal control (glyceraldehyde-3-phosphate
dehydrogenase) was carried out in capped 96-well optical plates. The reaction
conditions were as follows: 5 min at 50 ?C, 10 min at 95 ?C, and 40 cycles of
15 s at 95 ?C and 1 min at 60 ?C. The gene-specific PCR products were measured
continuously using an ABI PRISM 7700 detection system (Applied Biosystems). The
sample results were normalized to the internal control (Ct values were approximately
15), and expressed as the relative fold increase. The results shown are the
mean value of two independent experiments, with the samples in each experiment
run in triplicate.
Statistical analysis
The data were analyzed
statistically using spss software
10.0.5J (SPSS, Chicago, USA). All the data are presented as the mean±SD.
Statistical significance was determined using Student? t-test with P<0.05 considered statistically significant.
Results
The effects of BPA on the
proliferation of Ca9-22 cells were examined. Although 10 to 100 mM BPA slightly affected cell
proliferation, higher concentrations (200 and 500 mM)
significantly decreased attached cell numbers (Fig. 1). We monitored the
differentially expressed genes responsive to BPA in Ca9-22 cells using glass
microarrays hybridized with Cy3 and Cy5 Mono-Reactive Dye-labeled cDNA probes
synthesized from the mRNA of cells that had been treated or not treated with
100 mM BPA. Fig. 2 shows the
overall genetic profile in a scatterplot. Genes were considered up- or
down-regulated if the average fold change in expression was 2.0 or above in
three different experiments. Of the 10,368 genes examined, changes in mRNA
levels were detected in seven genes: five were up-regulated and two were
down-regulated. The differentially expressed genes are listed in Table 1.
Of these genes, the calcium channel, voltage-dependent, L-type, alpha 1C
subunit (CACNA1C), cell death activator CIDE-3 (CIDE-3),
haptoglobin-related protein (HPR), importin 4 (IPO4), and POU
domain, class 2 and transcription factor 3 (POU2F3) genes were up-regulated
in the cells exposed to BPA, with their respective levels being 4.05, 3.49,
2.93, 4.40, and 3.55 times higher than that of the control group. The
spermatogenesis-associated, serine-rich 2 (SPATS2) and HSPC049 protein (HSPC049)
genes were down-regulated in the cells exposed to BPA, and their respective
levels were 0.48 and 0.43 times lower than that of the control. To verify the
microarray results, we carried out real-time PCR. Again, the expression levels
of CACNA1C, CIDE–3, HPR, IPO4, and POU2F3
were significantly up-regulated in the cells exposed to 100 mM BPA, to levels that were 2.53, 3.24,
1.64, 2.02, and 2.25 times higher, respectively, than those in control cultures
(P<0.05; Table 1). SPATS2 and HSPC049 were
significantly down-regulated in the cells exposed to BPA, and the respective
levels were 0.49 and 0.44 times lower than those of the control cultures (P<0.05; Table 1).
Discussion
Sasaki et al. reported
that several tens to 100 ng/ml (approximately 400 mM)
BPA was present in saliva after filling cavities with composite resin [20].
From this observation and considering the effects of a subtoxic dose of BPA
based on cell proliferation data, 100 mM BPA
was chosen for this study.
To evaluate
gene expression with microarray technology, one must use alternative
quantitative methods that can be used as references. Here we used real-time PCR
to verify the data. Real-time PCR has been used in recent studies to validate
microarray data [24].
Apoptosis is a form of programmed cell death that plays an
important role in tissue homeostasis [25]. DNA fragmentation is often
considered as a hallmark of apoptosis, reflecting the activation of
endonucleases that cleave DNA between nucleosomes [26]. These nuclear changes
are triggered mainly by the DNA fragmentation factor (DFF) [27,28]. The DFF
complex consists of two subunits: (1) a 40 kDa endonuclease called DFFB, also
known as caspase-activated DNase, CPAN, or DFF40; and (2) its 45 kDa inhibitor
named DFFA, also known as DFF45 or inhibitor of caspase-activated DNase [28].
Inohara et al. described a novel family of cell death-inducing proteins
structurally related to DFFs that they named cell death-inducing DFF45-like
effector (CIDE). The CIDE family has three members: CIDE-A; CIDE-B; and Fsp27
[29]. CIDE-3 is a human homolog of mouse FSP27 and it can induce cell
apoptosis. CIDE-3 is located on chromosome 3p25, a region that is
deleted in many tumor tissues at high frequency and might be important in the
pathogenesis of various cancers [30]. CIDE-3 induced by BPA might affect
oral epithelial cell growth.
It has been suggested that BPA
disturbs the growth of the male comb and testes through a possible
endocrine-disrupting mechanism. SPATS2 plays a critical role in testicular
germ cell development. SPATS2 is predominantly expressed in the adult
testis, although weak expression occurs in the brain, thymus, kidney, lung,
heart, stomach, and skeletal muscle [31]. However, the role of this gene in
oral epithelial cells is unknown. The roles of CACNA1C, HPR, IPO4,
POU2F3, and HSPC049 in epithelial cells are also not known. In
this study, we identified seven genes that were differentially expressed in
response to a subtoxic dose of BPA in vitro, suggesting that these genes
could play a role in BPA-induced toxicity.
The main function of ovarian
hormones is to control the development and function of female genitalia and
secondary sex organs. However, alterations in the levels of these hormones that
occur during pregnancy and puberty, and at menopause, have been implicated as a
modifying factor in the pathogenesis of oral diseases, such as pregnancy
gingivitis [32,33]. Receptors for estrogen have been reported in the gingival
tissues, providing evidence that this tissue can be a target organ for sex
hormones [34]. It has been reported that oral mucosa of premenopausal woman was
significantly more sensitive to sodium lauryl sulphate in toothpastes than that
of postmenopausal woman. This might indicate a sex hormone influence on the
oral epithelium reactivity to chemical challenge [35]. These observations
indicate that BPA might affect oral tissues.
To our knowledge, this is the first
observation of broad-scale gene expression in oral cells treated with BPA using
a microarray. Using this type of analysis can aid in the identification of
genetic targets for further study, such
as animal models and human samples. The detailed knowledge of the changes in
gene expression obtained using microarray technology will provide a basis for
further elucidating the molecular mechanisms of the toxic effects of BPA in the
oral cavity.
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