Kumasaka,1,3Gaku Ichihara,3 Masashi Kato1,3,4 1 Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-s
Trang 1Human Nontumorigenic Keratinocytes Exposed
to a Low Level of Barium for a Long Time
Nguyen D Thang,1,2 Ichiro Yajima,1,3Shoko Ohnuma,4Nobutaka Ohgami,1,4
Mayuko Y Kumasaka,1,3Gaku Ichihara,3 Masashi Kato1,3,4
1
Unit of Environmental Health Sciences, Department of Biomedical Sciences,
College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, 487-8501, Japan
2
Department of Biochemistry and Plant Biology, Vietnam National University, Hanoi, Vietnam
3
Department of Occupational and Environmental Health, Nagoya University Graduate School
of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan
4
Voluntary Body for International Health Care in Chubu University, Kasugai,
Aichi, 487-8501, Japan
Received 14 January 2013; revised 20 May 2013; accepted 22 May 2013
ABSTRACT: We have recently demonstrated that exposure to barium for a short time (4 days) and at a
low level (5 mM 5 687 mg/L) promotes invasion of human nontumorigenic HaCaT cells, which have
charac-teristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time
enhances malignant characteristics Here we examined the effect of exposure to low level of barium for a
long time, a condition mimicking the exposure to barium through well water, on malignant characteristics
of HaCaT keratinocytes Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and
activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human
epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma
cells were augmented following an increase in malignancy grade of the cells Constitutive invasion
activ-ity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 mM
barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes Taken
together, our results suggest that exposure to a low level of barium for a long time enhances constitutive
malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion.
V C 2013 Wiley Periodicals, Inc Environ Toxicol 30: 161–167, 2015.
Keywords: barium; focal adhesion kinase; invasion; keratinocytes; matrix metalloproteinase; well water
INTRODUCTION
Pollution of well drinking water with toxic elements is a serious public health problem throughout the world Previ-ous studies showed that over 200 million people worldwide face the risk of exposure to highly toxic elements, including arsenic, from drinking well water (Sun et al., 2006; Wang
et al., 2010) More than 25 million patients with arsenicosis
in Bangladesh have been reported (Chowdhury et al., 2000), and about 10 million patients with arsenicosis in the Red
Correspondence to: M Kato; e-mail: katomasa@med.nagoya-u.ac.jp
Supported by: Ministry of Education, Culture, Sports, Science and Technology
(MEXT) (Grants-in-Aid for Scientific Research [B], Nos 20406003, 24390157, and
24406002; Grant-in-Aid for Challenging Exploratory Research, No 23650241;
Grant-in-Aid for Scientific Research on Innovative Areas, No 24108001); MEXT
and Chubu University (COE Project [Health Science Hills] for Private Universities,
No S0801055); Supported by: Japan Society for the Promotion of Science (JSPS
[AA Science Platform Program]); Tokyo Biochemical Research Foundation; the
Naito Foundation Natural Science Scholarship; the Uehara Memorial Foundation;
AEON Environmental Foundation; The Cosmetology Research Foundation.
Published online 26 June 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/tox.21881
Trang 2River Delta and 1 million patients with arsenicosis in the
Mekong River Delta in Vietnam have been reported (Berg
et al., 2007) Moreover, epidemiological studies have
revealed increases in skin, lung, liver, prostate, and bladder
cancers in patients with arsenicosis (Smith et al., 1992)
Since experimental studies have also provided evidence that
arsenic promotes cellular malignant characteristics (Tokar
et al., 2010; Kato et al., 2011b; Singh et al., 2011), arsenic in
well water is believed to be a main cause of tumorigenesis
The skin is thought to be one of target organs of arsenic
because skin melanosis and hyperkeratosis develop in
patients with arsenicosis (Ahsan et al., 2000; Chowdhury
et al., 2000; Pimparkar and Bhave, 2010) Bowen’s disease
(carcinomain situ) and squamous cell carcinoma (SCC) are
known as representative skin cancers derived from chronic
exposure to arsenic (Chowdhury et al., 2000; Pimparkar and
Bhave, 2010) However, the evidence does not rule out the
possibility that another element as well as arsenic in well
water contributes to carcinogenesis of the skin in humans
Multistep carcinogenesis through steps including
initia-tion, promoinitia-tion, transformainitia-tion, and progression has been
proposed for various carcinomas including skin cancer
(Zoumpourlis et al., 2003; Kumasaka et al., 2010, 2013)
Ini-tiation is the first step of tumor development and is
irreversi-ble Promotion is the step of clonal expansion of the initiated
cells, resulting in formation of benign tumors (Zoumpourlis
et al., 2003) Transformation is the step of change from
benign tumors to malignant tumors Transformed cells gain
hyperproliferative and antiapoptotic characteristics, invasion
activity, and anchorage-independent growth capability (Berg
et al., 2007; Tsuji et al., 2008) Progression is the step to
gain further invasive potential for distant metastasis and
sur-vival potential of anchorage-independent growth
(Zoum-pourlis et al., 2003; Mehlen and Puisieux, 2006) The in
vitro invasion assay is a method to evaluate cellular motility
and invasion activity in transformed cells (Thang et al.,
2011) Since increased invasion is a crucial step in the
devel-opment of cancer, invasion activity is one of the hallmarks
of malignant characteristics (Kato et al., 2002; Thang et al.,
2011; Yajima et al., 2012a) Previous studies showed that
focal adhesion kinase (FAK) phosphorylation and matrix
metalloproteinase 14 (MMP14) expression levels play a key
role in invasion (Hahne et al., 2005; Kato et al., 2008; Thang
et al., 2011)
HaCaT keratinocytes, spontaneously transformed human
epithelial cells from adult skin, are immortal but remain
non-tumorigenic HaCaT keratinocytes express
differentiation-specific keratins and other markers (involucrin and filaggrin)
and exhibit normal proliferation, suggesting that HaCaT
keratinocytes are similar to normal keratinocytes (Boukamp
et al., 1988) Therefore, HaCaT keratinocytes are useful for
examining the effects of environmental factors on malignant
characteristics of human keratinocytesin vitro (Thang et al.,
2011) In fact, we showed that exposure to barium alone for
a short time (4 days) promotes invasion activity in human
nontumorigenic HaCaT keratinocytes in addition to murine nontumorigenic melanocytes and fibroblasts (Bennett et al., 1987; Boukamp et al., 1988; Kato et al., 2002; Thang et al., 2011) Moreover, a classical study in humans showed that one external application of barium and dimethyl sulfoxide to the uterine cervix resulted in the development of dysplasia and carcinoma in situ (Ayre and LeGuerrier, 1967) These results suggest that exposure to barium for a short time enhances cellular malignant characteristics in nontumori-genic cells However, there has been very limited study on the influence of exposure to barium for a long time on con-stitutive cellular malignant characteristics, despite the fact that residents of cancer-prone areas suffer form chronic exposure to barium in addition to arsenic
Previous studies showed high levels of barium in arsenic-polluted well water in Bangladesh and Vietnam (Thang
et al., 2011; Winkel et al., 2011) Since previous studies have shown increased malignant characteristics in HaCaT keratinocytes that were exposed to arsenicin vitro for a long time (Achanzar et al., 2002; Benbrahim-Tallaa et al., 2005), this study was carried out to determine the constitutive effect
of exposure to barium for a long time, at a level at which people drinking well water are exposed to, on cellular malig-nant characteristics through invasion activity
MATERIALS AND METHODS Analysis of Barium Concentration
The levels of barium in well water samples from Ho Chi Minh (n 5 33) and Mekong Delta (n 5 67) in Vietnam were measured by using an inductively coupled plasma-mass spectrophotometer (7500cx, Agilent Technologies, CA) fol-lowing the method previously described (Kato et al., 2010)
Cell Lines and Culture Conditions
Human immortalized HaCaT keratinocytes (German Can-cer Research Center “DKFZ,” Germany) (Boukamp et al., 1988; Calay et al., 2010) and human HSC5 (Health Sci-ence Research Resources Bank, Japan) and A431 (Riken Bioresource Center, Japan) SCC cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) on collagen-coated dishes (Kato et al., 1999; Ohshima et al., 2010) Normal human epithelial keratinocytes (NHEK) purchased from Kohjin Bio were cultured following the instructions from the company Barium-treated HaCaT (BT-HaCaT) keratinocytes and barium-untreated control HaCaT (C-HaCaT) keratinocytes were developed through culture in the presence or absence of 5 mM barium (BaCl2, Wako, Osaka, Japan), respectively, for 4 months (37 pas-sages) BT-HaCaT keratinocytes were used for in vitro invasion assay and immunoblot analysis after culturing in the medium without barium for more than 2 weeks to wash out barium from the keratinocytes
Trang 3The In Vitro Invasion Assay
The in vitro invasion assay was performed according to
the method described previously (Thang et al., 2011) To
examine constitutive invasion activity in various kinds of
keratinocytic cells, the assay in this study was performed
in the condition without barium Briefly, 2 3 105 cells in
300 mL culture medium with 0.5% FBS were applied to
the Matrigel-coated upper chamber of 8 mm in diameter
(8 mm in pore size) Then the upper chambers were
placed in 24-well culture plates containing 600 mL
condi-tioned medium with 0.5% FBS to trigger invasion activity
and were incubated for 12 hours Invading cells were
stained with hematoxylin and counted under a
microscope
Immunoblot Analysis
Immunoblot analysis was performed according to the
method described previously (Kato et al., 2002) Rabbit
pol-yclonal primary antibody against anti-phosphorylated
tyro-sine 397 in FAK (Invitrogen, California, USA) and mouse
monoclonal first antibodies against anti-FAK (Millipore,
Massachusetts, USA), MMP14 (Millipore, Massachusetts,
USA) and anti-a-Tubulin (Sigma, Missouri, USA) were
used
Statistical Analysis
Statistical analysis was performed following the method pre-viously described (Kato et al., 2011a) We used the SPSS version 18 software package (SPSS Japan) for the statistical analyses, and the significance level was set atp < 0.05
RESULTS Concentrations of Barium in Well Water in Vietnam
We first measured concentrations (mean 6 SD) of barium in well water in Ho Chi Minh (129 6 62 mg/L) and Mekong Delta (281 6 133 mg/L) in Vietnam (Fig 1) The mean con-centration of barium in well water samples obtained from Mekong Delta was more than twofold higher than that in well water samples obtained from Ho Chi Minh Since the maximum level of barium in well water in Mekong Delta was 970 mg/L (57.1 mM), chronic exposure to 5 mM barium
is possible to expose
Invasion in Various Human Keratinocytic Cells
We next examined invasion activity in four kinds of kerati-nocytic cells with different malignancy grades Invasion activities of NHEK (lane 1), human nontumorigenic HaCaT keratinocytes (lane 2), and human HSC5 (lane 3) and A431 (lane 4) SCC cells are shown in Figure 2 No invading cells were observed in NHEK The ratios of invading cells in HSC5 and A431 SCC cells were about 10-fold and 14-fold higher than those in HaCaT keratinocytes, respectively
Fig 1 Levels of barium in well water samples from Vietnam.
Barium concentrations in well water samples from Vietnam
are shown Value in each sample (lanes 1 and 2) and mean
6 SD (lanes 3 and 4) are shown for samples from Ho Chi
Minh (lanes 1 and 3) and Mekong Delta (lanes 2 and 4) *,
Significantly different (p < 0.05) from the Ho Chi Minh
sam-ple by the Mann-Whitney U test.
Fig 2 Constitutive invasion activities in various human ker-atinocytic cells Ratio of constitutive invasion activities in primary cultured NHEK, nontumorigenic HaCaT keratino-cytes, and HSC5 and A431 SCC cells are shown **, Signifi-cantly different (p < 0.01) from HaCaT keratinocytes by the Mann-Whitney U test.
Trang 4These results indicated that invasion activity of HaCaT
kera-tinocytes was near that of NHEK rather than that of SCC
cells, whereas invasion activity of HaCaT keratinocytes was
intermediate between NHEK and SCC cells
We next examined phosphorylation and/or expression
levels of FAK and MMP14 in NHEK, HaCaT keratinocytes,
and HSC5 and A431 SCC cells As shown in Figure 3, level
of FAK phosphorylation and levels of FAK and MMP14
protein expression in HaCaT keratinocytes were significantly
higher than those in NHEK and were significantly lower
than those in HSC5 and A431 SCC cells Since ratios of
phosphorylated FAK (pY397/FAK) were comparable among
NHEK and HaCaT, HSC5 and A431 cells, higher levels of FAK activity in HSC5 and A431 cells than in HaCaT cells may be due to increased levels of FAK protein expression in HSC5 and A431 cells
Promoted Constitutive Activities of Invasion
in BT-HaCaT Keratinocytes
We then examined constitutive invasion activities in BT-HaCaT and C-BT-HaCaT keratinocytes As shown in Figure 4, invasion activity of BT-HaCaT keratinocytes was significantly increased compared with that of C-HaCaT keratinocytes
We finally examined constitutive levels of phosphoryla-tion and/or expression of FAK and MMP14 in BT-HaCaT and C-HaCaT keratinocytes FAK protein expression levels were comparable in BT-HaCaT and C-HaCaT keratinocytes (Figure 5) However, levels of FAK activity and MMP14 expression in BT-HaCaT keratinocytes were higher than those in C-HaCaT keratinocytes (Fig 5) Since BT-HaCaT keratinocytes were not exposed to barium in either the in vitro invasion assay or immunoblot analysis, our results sug-gest that invasion activities in BT-HaCaT keratinocytes are
Fig 3 Phosphorylation and/or expression levels of FAK and
MMP14 in various human keratinocytic cells Constitutive
tyrosine 397 (Y397) phosphorylation level in FAK
(pY397-FAK) and protein expression levels of FAK, MMP14, and
TUBULIN in primary cultured NHEK, nontumorigenic HaCaT
keratinocytes, and HSC5 and A431 SCC cells are shown.
The levels (mean 6 SD) of pY397/TUBULIN, FAK/TUBULIN,
pY397/FAK, and MMP14/TUBLIN determined by
densito-metric analyses of the bands in three independent
experi-ments are presented as graphs showing relative intensities
(multiple numbers) for HaCaT keratinocytes *, Significantly
different (p < 0.05) from control HaCaT keratinocytes by the
Mann-Whitney U test.
Fig 4 Constitutive invasion activities of BT-HaCaT and C-HaCaT keratinocytes Ratio of constitutive invasion activ-ities of BT-HaCaT and C-HaCaT keratinocytes, which were cultured in the presence or absence of 5 mM barium for 4 months, is shown *, Significantly different (p < 0.05) from C-HaCaT keratinocytes by the Mann-Whitney U test.
Trang 5constitutively increased with increased FAK activity and
MMP expression
DISCUSSION
Some elements, including manganese, iron, and zinc, for
which values were shown in the 3rd edition of WHO
health-based guidelines for drinking water were deleted in the
new-est 4th edition revised in 2011 However, the guideline value
of 700 mg/L (55.1 mM) for barium has been maintained in
the present edition of the guidelines, suggesting that toxicity
of barium is impossible to overlook In fact, increased blood pressure by vasoconstriction has been reported in mice treated with 100,000 mg/L of barium for 16 months (Nish-ioka, 1975) A significant decrease in survival rate with lymphoid depletions in the spleen, thymus, and lymph nodes has been reported in mice treated with 2,500,000 mg/L of barium for 15 months (NTP [National Toxicology Program], 1994) However, 2,000,000 mg/L of barium had no effects
on body weight, renal function, and reproduction in rats and mice (Dietz et al., 1992) Thus, the concentrations of barium used in most previous studies were much higher than the lev-els of barium to which people drinking well water are exposed to, and there has been a lack of studies showing tox-icity of low levels of barium
Recently, we have been performing studies focusing on toxicity of low levels (700 mg/L) of barium Our pervious
in vitro study showed that exposure to barium alone (2.5–100 mM) for 4 days significantly promoted invasion activity in nontumorigenic keratinocytes, melanocytes, and fibroblasts (Thang et al., 2011) Cancer toxicity of 3 mM arsenic was enhanced by coexposure to 1 mM barium for 4 days, 3 mM arsenic and 1 mM barium being about the aver-age concentrations in arsenic-polluted well drinking water in cancer-prone areas of Bangladesh (Yajima et al., 2012b) Our previous results suggest that exposure to barium for a short time (4 days) and at a low level not exceeding the value of WHO health-based guidelines promote malignant characteristics of human HaCaT keratinocytes in vitro Together with the results of another previous study by us showing acceleration of hearing loss in wild-type mice drinking 700 mg/L barium for 1 month (Ohgami et al., 2012), our previous results suggest that 700 mg/L of bar-ium exhibited toxicityin vivo and in vitro In this study, we demonstrated that invasion, FAK protein expression and activity, and MMP14 protein expression were constitutively augmented following an increase in malignancy grade in human keratinocytic cells We further demonstrated increased constitutive invasion activity with enhanced FAK phosphorylation and MMP14 expression in HaCaT keratino-cytes treated with 5 mM barium for 4 months Since FAK and MMP14 are representative signal transduction mole-cules closely linked to invasion activity (Hahne et al., 2005; Kato et al., 2008; Thang et al., 2011), our results suggest that increased FAK activity and MMP14 expression are cor-related with promoted invasion of HaCaT keratinocytes Taken together, our results suggest that malignant character-istics of HaCaT keratinocytes were increased by exposure to barium at a low concentration and for a long time from the analytical points of view of invasion However, there is no evidence showing barium-mediated cancer development in either an epidemiological study or animal experiment, despite a classical study showing development of dysplasia
in humans by external application of barium to the uterine cervix (Ayre, 1966) Further in vivo studies are needed to determine whether chronic exposure to a low level
Fig 5 Levels of constitutive phosphorylation and/or
expres-sion of FAK and MMP14 in BT-HaCaT and C-HaCaT
kerati-nocytes Constitutive tyrosine 397 (pY397) phosphorylation
level in FAK (pY397-FAK) and protein expression levels of
FAK, MMP14, and TUBULIN in BT-HaCaT (BT, lane 2) and
C-HaCaT (C, lane 1) keratinocytes, which were cultured in
the presence or absence of 5 mM barium for 4 months, are
shown The levels (mean 6 SD) of pY397/TUBULIN, FAK/
TUBULIN, pY397/FAK, and MMP14/TUBULIN determined
by densitometric analyses of the bands in three
independ-ent experimindepend-ents are presindepend-ented as graphs showing relative
intensities (multiple numbers) for control C-HaCaT
keratino-cytes *, Significantly different (p < 0.05) from C-HaCaT
keratinocytes by the Mann-Whitney U test.
Trang 6of barium affects development of skin diseases including
cancer
The authors thank Ms Aoi Sato, Ms Rie Shimizu, and Ms
Yoko Kato for technical assistance.
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