Original Paper |
|
|||
|
Acta Biochim Biophys |
||||
|
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 ºC 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 ºC 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
ºC 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 ºC 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 ºC for 2 min. We also added 2 ml SuperScript II reverse transcriptase
(Invitrogen). After a 90 min incubation at 42 ºC, we added 5 ml of 0.5 M EDTA and 10 ml of 1 M NaOH, and incubated the mixture
at 70 ºC 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 ºC, 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磗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 ºC 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 ºC 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 ºC 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 ºC, 2´SSC for 5 min at 30 ºC, 1´SSC for 5 min at 30 ºC, 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 ºC 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 ºC, 10 min at 95 ºC, and 40 cycles of
15 s at 95 ºC and 1 min at 60 ºC. 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抯 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.
References
1 Colborn T, vom Saal FS, Soto AM.
Developmental effects of endocrine-disrupting chemicals in wildlife and humans.
Environ Health Perspect 1993, 101: 378–384
2 Krishnan AV, Stathis P, Permuth SF, Tokes L,
Feldman D. Bisphenol-A: An estrogenic substance is released from polycarbonate
flasks during autoclaving. Endocrinology 1993, 132: 2279–2286
3 Gaido KW, Leonard LS, Lovell S, Gould JC,
Babai D, Portier CJ, McDonnell DP. Evaluation of chemicals with endocrine
modulating activity in a yeast-based steroid hormone receptor gene
transcription assay. Toxicol Appl Pharmacol 1997, 143: 205–212
4 Tabuchi Y, Kondo T. cDNA microarray analysis reveals
chop-10 plays a key role in Sertoli cell injury induced by bisphenol A. Biochem
Biophys Res Commun 2003, 305: 54–61
5 Tabuchi Y, Takasaki I, Kondo T.
Identification of genetic networks involved in the cell injury accompanying
endoplasmic reticulum stress induced by bisphenol A in testicular Sertoli
cells. Biochem Biophys Res Commun 2006, 345: 1044–1050
6 Brotons JA, Olea-Serrano MF, Villalobos M,
Pedraza V, Olea N. Xenoestrogens released from lacquer coatings in food cans.
Environ Health Perspect 1995, 103: 608–612
7 Singleton DW, Feng Y, Yang J, Puga A, Lee AV,
Khan SA. Gene expression profiling reveals novel regulation by bisphenol-A in
estrogen receptor-alpha-positive human cells. Environ Res 2006, 100: 86–92
8 Akingbemi BT, Sottas CM, Koulova AI,
Klinefelte GR, Hardy MP. Inhibition of testicular steroidogenesis by the
xenoestrogen bisphenol A is associated with reduced pituitary luteinizing
hormone secretion and decreased steroidogenic enzyme gene expression in rat
Leydig cells. Endocrinology 2004, 145: 592–603
9 Schmalz G, Preiss A, Arenholt-Bindslev D.
Bisphenol-A content of resin monomers and related degradation products. Clin
Oral Investig 1999, 3: 114–119
10 Olea N, Pulgar R, Perez P, Olea-Serrano F,
Rivas A, Novillo-Fertrell A, Pedraza V et al. Estrogenicity of
resin-based composites and sealants used in dentistry. Environ Health Perspect
1996, 104: 298–305
11 Thompson LR, Miller EG, Bowles WH. Leaching of
unpolymerized materials from orthodontic bonding resin. J Dent Res 1982, 61:
989–992
12 Eliades T, Eliades G, Brantley WA, Johnston
WM. Residual monomer leaching from chemically cured and visible light-cured
orthodontic adhesives. Am J Orthod Dentofacial Orthop 1995, 108: 316–321
13 Lee SY, Greener EH, Menis DL. Detection of
leached moieties from dental composites in fluids simulating food and saliva.
Dent Mater 1995, 11: 348–353
14 Lee SY, Huang HM, Lin CY, Shih YH. Leached
components from dental composites in oral simulating fluids and the resultant
composite strengths. J Oral Rehabil 1998, 25: 575–588
15 Moon HJ, Lee YK, Lim BS, Kim CW. Effects of
various light curing methods on the leachability of uncured substances and
hardness of a composite resin. J Oral Rehabil 2004, 31: 258–264
16 Larsen IB, Freund M, Munksgaard EC. Change in
surface hardness of BisGMA/TEGDMA polymer due to enzymatic action. J Dent Res
1992, 71: 1851–1853
17 Joskow R, Barr DB, Barr JR, Calafat AM,
Needham LL, Rubin C. Exposure to bisphenol A from bis-glycidyl
dimethacrylate-based dental sealants. J Am Dent Assoc 2006, 137: 353–362
18 Arenholt-Bindslev D, Breinholt V, Preiss A,
Schmalz G. Time-related bisphenol-A content and estrogenic activity in saliva
samples collected in relation to placement of fissure sealants. Clin Oral
Investig 1999, 3: 120–125
19 Fung EY, Ewoldsen NO, St Germain HA Jr, Marx
DB, Miaw CL, Siew C, Chou HN et al. Pharmacokinetics of bisphenol A
released from a dental sealant. J Am Dent Assoc 2000, 131: 51–58
20 Sasaki N, Okuda K, Kato T, Kakishima H, Okuma
H, Abe K, Tachino H et al. Salivary bisphenol-A levels detected by ELISA
after restoration with composite resin. J Mater Sci Mater Med 2005, 16: 297–300
21 Ashby J. Bisphenol-A dental sealants: The
inappropriateness of continued reference to a single female patient. Environ
Health Perspect 1997, 105: 362
22 Imai Y. Comments on “Determination of
bisphenol A and related aromatic compounds released from bis-GMA-based
composites and sealants by high performance liquid chromatography”. Environ
Health Perspect 2000, 108: A545–546
23 Olea N. Bisphenol A and dental sealants: Olea’s
response. Environ Health Perspect 2000, 108: A546
24 Canales RD, Luo Y, Willey JC, Austermiller B,
Barbacioru CC, Boysen C, Hunkapiller K et al. Evaluation of DNA
microarray results with quantitative gene expression platforms. Nat Biotechnol
2006, 24: 1115–1122
25 Metzstein MM, Stanfield GM, Horvitz HR.
Genetics of programmed cell death in C. elegans: Past, present and
future. Trends Genet 1998, 14: 410–416
26 Reed JC, Doctor K, Rojas A, Zapata JM, Stehlik
C, Fiorentino L, Damiano J et al. Comparative analysis of apoptosis and
inflammation genes of mice and humans. Genome Res 2003, 13: 1376–1388
27 Mukae N, Enari M, Sakahira H, Fukuda Y,
Inazawa J, Toh H, Nagata S. Molecular cloning and characterization of human
caspase-activated DNase. Proc Natl Acad
Sci USA 1998, 95: 9123–9128
28 Bayascas JR, Yuste VJ, Sole C, Sanchez-Lopez
I, Segura MF, Perera R, Comella JX. Characterization of splice variants of
human caspase-activated DNase with CIDE-N structure and function. FEBS Lett 2004, 566: 234–240
29 Inohara N, Koseki T, Chen S, Wu X, Nunez G.
CIDE, a novel family of cell death
activators with homology to the 45 kDa subunit of the DNA fragmentation
factor. EMBO J 1998, 17: 2526–2533
30 Liang L, Zhao M, Xu Z, Yokoyama KK, Li T.
Molecular cloning and characterization of CIDE-3, a novel member of the
cell-death-inducing DNA-fragmentation- factor (DFF45)-like effector family.
Biochem J 2003, 370: 195–203
31 Senoo M, Hoshino S, Mochida N, Matsumura Y,
Habu S. Identification of a novel protein p59(scr), which is expressed at
specific stages of mouse spermatogenesis. Biochem Biophys Res Commun 2002, 292:
992–998
32 Deasy MJ, Vogel RI. Female sex hormonal
factors in periodontal disease. Ann Dent 1976, 35: 42–46
33 Mascarenhas P, Gapski R, Al-Shammari K, Wang
HL. Influence of sex hormones on the periodontium. J Clin Periodontol 2003, 30:
671–681
34 Vittek J, Hernandez MR, Wenk EJ, Rappaport SC,
Southren AL. Specific estrogen receptors in human gingiva. J Clin Endocrinol
Metab 1982, 54: 608–612
35 Herlofson BB, Barkvoll P. Oral mucosal
desquamation of pre- and post-menopausal women. A comparison of response to
sodium lauryl sulphate in toothpastes. J Clin Periodontol 1996, 23: 567–571