Chemosphere 201 (2018) 667e675

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

A comprehensive study including monitoring, assessment of health

effects and development of a remediation method for chromium
pollution
Masafumi Yoshinaga a, 1, Hiromasa Ninomiya a, b, M.M. Aeorangajeb Al Hossain a, b,
Makoto Sudo a, b, Anwarul Azim Akhand c, Nazmul Ahsan c, Md. Abdul Alim d,
Md. Khalequzzaman e, Machiko Iida a, b, f, Ichiro Yajima a, b, f, Nobutaka Ohgami a, b, f,
Masashi Kato a, b, f, *
a
Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
b
Voluntary Body for International Healthcare in Universities, Nagoya, Aichi, Japan
c
Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka, Bangladesh
d
Institute of Public Health Nutrition, Directorate General of Health Services, Dhaka, Bangladesh
e
Department of Public Health and Informatics, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh
f
Units of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi, Japan

h i g h l i g h t s

Carcinogenicity of Cr(VI) was synergistically promoted in the presence of Cr(III).

Cr(VI) and Cr(III) must be removed to remediate water pollution in tannery areas.

A cheap depurative that efficiently removes both Cr(VI) and Cr(III) is developed.

Our depurative is more effective in removing Cr(III) than other reported depuratives.

A solution for the worldwide environmental Cr pollution by tannery is suggested.

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

Article history: Chromium (Cr) pollution caused by wastewater from tanneries is a worldwide environmental problem.
Received 17 July 2017 To develop a countermeasure, we performed a comprehensive study using Hazaribagh, the tannery area
Received in revised form in Dhaka City, Bangladesh, as a model. Our environmental monitoring indicated that the soluble form of
28 February 2018
Cr, but not barium or arsenic, in Buriganga River is derived from Hazaribagh. Our chemical analysis next
Accepted 4 March 2018
Available online 5 March 2018
showed that Cr, the primary pollutant in canal water at Hazaribagh, consisted of 0.7 mM hexavalent Cr
[Cr(VI)] and 1705 mM trivalent Cr [Cr(III)]. Our biological study then showed that coexposure to Cr(VI)
Handling Editor: Chang-Ping Yu and Cr(III) at possible ratios in canal water at Hazaribagh synergistically promotes transforming activity
of human non-tumorigenic HaCaT keratinocytes with activated MEK/ERK and AKT. Our environmental
Keywords: engineering study finally indicated that a magnesium and iron-based hydrotalcite-like compound (MF-
Water pollution HT), our original depurative, can maximally adsorb 9.0 mg/g Cr(VI) and 1041 mg/g Cr(III). Our results
Carcinogenic toxicity suggested the importance of removal of Cr(III) as well as Cr(VI) by showing that Cr(III), which is generally
Chromium recognized as a chemical with low toxicity, synergistically promoted carcinogenicity of a low level of
Depurative
Cr(VI). Therefore, we propose the use of our original high-efficient and low-cost depurative as a coun-
Tannery waste
termeasure to address the worldwide problem of environmental Cr pollution.
© 2018 Elsevier Ltd. All rights reserved.

Abbreviations: MF-HT, magnesium (Mg)-iron (Fe)-based hydrotalcite-like compounds; HPLC, high pressure liquid chromatography; ICP-MS, inductively coupled plasma
mass spectrometry.
* Corresponding author. Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya,
Aichi, 466-8550, Japan.
E-mail address: katomasa@med.nagoya-u.ac.jp (M. Kato).
1
Current address: Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida 33199, USA.

https://doi.org/10.1016/j.chemosphere.2018.03.026
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
668 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675
1. Introduction
Diseases caused by environmental pollution in Japan include
Itai-Itai disease, which was caused by cadmium (Cd) pollution in
Jinzu River (Maruzeni et al., 2014), and Niigata Minamata disease,
which was caused by mercury (Hg) pollution in Agano River (Hara
et al., 2013). Arsenicosis was caused by arsenic (As) pollution in
Mekong River in Cambodia (Sampson et al., 2008). Various diseases
including cancers and cardiovascular diseases have been suggested
to be caused by lead (Pb) and Cd pollution in Hengshine River in
China (Wang et al., 2011). Thus, the presence of toxic metal pol-
lutants in river water is a worldwide hazard for human health.
More than 120 L of water are required for the production of 1 kg
of leather in tanneries (Farenzena et al., 2005). If toxic elements are
used in tanneries, wastewater from tanneries without appropriate
depuration could cause enormous water pollution. Environmental
pollution caused by wastewater from tannery industries is in fact a
worldwide problem (Belay, 2010; Bharagava and Mishra, 2018;
Saxena et al., 2016).
Environmental pollution in a built-up area of tanneries at
Hazaribagh in Dhaka City, Bangladesh has been reported (Anawar
et al., 2000). It has been suggested that 7.7 million liters of liquid
waste per day from the tanneries (Azom et al., 2012) could be a
pollutant source of Buriganga River and canals in Hazaribagh. Since
health risks of workers in tanneries at Hazaribagh have also been
reported (Tinni et al., 2014), canal water containing wastewater
from the tanneries and Buriganga River water, which is used as a
domestic water supply (Rahman and Al Bakri, 2010), must be a
health hazard for the residents. However, there is a lack of infor-
mation on pollution with toxic elements from Hazaribagh and
pollution with toxic elements from other sources in Buriganga
River.
The World Health Organization (WHO) suggests 50 mg/L of Cr as
the health-based guideline value for drinking water (WHO, 2017).
Chromium (Cr) generally exists in the environment either as hex-
avalent Cr [Cr(VI)] or trivalent Cr [Cr(III)]. Cr(VI) has toxic, geno-
toxic, mutagenic and carcinogenic effects, causing negative health
problems such as skin lesions, ulceration and perforation of the
nasal septum, eardrum perforation, decreased spermatogenesis
and lung carcinoma. (Bharagava and Mishra, 2018; Mishra and
Bharagava, 2016). In contrast, Cr(III) is generally regarded as
nontoxic, although several recent studies have suggested potential
health hazards of Cr(III) (Fan et al., 2015). The International Agency
for Research on Cancer (IARC) categorized Cr(VI) and Cr(III) as a
Group 1 carcinogen (carcinogenic for humans) and a Group 3
carcinogen (not classifiable as to its carcinogenicity to humans),
respectively (Field and Withers, 2012). Thus, carcinogenic toxicity
of sole exposure to Cr(III) is believed to be limited (Biedermann and
Landolph, 1990; IARC, 2016). Therefore, there is more interest in
removal of Cr(VI) than in removal of Cr(III). Moreover, there has
been no study showing the ratio of Cr(VI) and Cr(III) in canal water
from tanneries and in Buriganga River water. If both Cr(VI) and
Cr(III) are detected in the water, the ratio should be considered,
especially when estimating potential health risks due to coex-
posure to Cr(VI) and Cr(III).
Environmental pollution and health risks of arsenic (As) and
barium (Ba) in rural areas in Bangladesh have been reported (Kato
et al., 2013; Kumasaka et al., 2013; Ohgami et al., 2016; Thang et al.,
2011; Yajima et al., 2018, 2015, 2012). Excess exposure to As has
been reported to increase various health risks including risks of
hyperpigmented skin (Yajima et al., 2018, 2017), diabetes (Drobn
a sulfate (Cr(SO4)OH, Yoshida Chemical Industrial Co., Japan) were
et al., 2013), hearing loss (Li et al., 2017) and cancers of the
bladder (Chen et al., 2003), lung (Putila and Guo, 2011) and skin
(Yajima et al., 2015). WHO suggests the health-based guideline
value for As in drinking water as 10 mg/L (WHO, 2017). Increases of
various toxicity-related health problems including hypertension
(Perry et al., 1989), renal dysfunction (Dietz et al., 1992), hearing
loss (Ohgami et al., 2012) and carcinogenesis (Thang et al., 2011,
2015b) caused by excess exposure to Ba have been reported. WHO
suggests the health-based guideline value for Ba in drinking water
as 1300 mg/L (WHO, 2017). In this study, As and Ba as well as Cr
were investigated in canal water containing wastewater from the
tanneries at Hazaribagh and in Buriganga River in Dhaka City,
Bangladesh. After focusing on Cr, the pollutant with the highest
level, our comprehensive study consisted of 1) environmental
monitoring, 2) health risk assessment and 3) development of a
depurative to establish countermeasures for environmental pollu-
tion and human health hazards. Our proposed countermeasure for
Cr pollution caused by wastewater from tanneries at Hazaribagh in
Dhaka City could be a useful model to address the environmental
pollution generated from tanneries in various areas of the world.
2. Materials and methods
2.1. Sampling and analyses of elements
Areas for sampling of Buriganga River water and canal water
containing wastewater from tanneries at Hazaribagh in Dhaka City,
Bangladesh (Anawar et al., 2000) are shown in Fig. 1A. Sampling
bottles were filled with sampled water after rinsing out the inside
of each bottle with sampled water. The bottles were transferred
from Dhaka City, Bangladesh to Nagoya City, Japan after being
stored at room temperature for a few weeks. After the samples had
been stored at 4  C in Nagoya University, Japan, total levels of Cr, As
and Ba were measured by using inductively coupled plasma mass
spectrometry (ICP-MS) (7500cx, Agilent Technologies Inc, CA)
following the method previously described (Kato et al., 2013). The
chemical species of Cr was analyzed by high pressure liquid chro-
matography (HPLC) (SCL-10AVP, Shimazu Corporation, Japan) ICP-
MS using a Cr speciation column (G3268-80001,
30 mm  4.6 mm, Agilent Technologies, Inc.). The level of Cr(VI) in
each sample was quantified by using MassHunter Software (Agilent
Technologies, Inc.). The level of Cr(III) was calculated by subtracting
the Cr(VI) level from the total Cr level.
This study was approved by the Ethical Committee of Nagoya
University (approval no. 2013-0070) in Nagoya City and the Ethical
Committee of Chubu University (approval no. 250007) in Kasugai
City in Japan.
2.2. Cell line and culture conditions
A human immortalized cutaneous HaCaT keratinocyte cell line
(Boukamp et al., 1988; Calay et al., 2010) was supplied by Eppel-
heim, Germany. The method for culture of HaCaT keratinocytes was
described previously (Yajima et al., 2015). Briefly, HaCaT cells were
cultured in Dulbecco's Modified Eagle's Medium (DMEM Low
glucose, Wako) supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin/streptomycin/amphotericin B suspension (Wako)
at 37  C in a 5% CO2 humidified incubator.
2.3. Cell survival assay
Chromium(VI) oxide (CrO3, Wako, Japan) and basic chromium
used as hexavalent chromium and trivalent chromium, respec-
tively. After starvation with 1% fetal bovine serum (FBS) for 16 h,
HaCaT keratinocytes were cultured in the presence or absence of
the indicated concentrations of Cr(VI) or Cr(III). Half of the medium
 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675
Fig. 1. Environmental monitoring for water samples from Buriganga River and
canals at Hazaribagh in Dhaka City, Bangladesh (modified with Google map). (A)
Water samples were collected in the upstream region (a: n ¼ 21) and proximal (d:
n ¼ 20) and distal (e: n ¼ 17) downstream regions of Buriganga River after defining the
confluence (c: n ¼ 11) of Buriganga River and canals from tanneries at Hazaribagh (b:
n ¼ 39) as a basic point. (BeD) Concentrations (mean ± s.d.: mg/L) of soluble forms of Cr
(B), As (C) and Ba (D) in surface water samples from each section are expressed by log
scale (Y-axis). (E) Concentrations (mean ± s.d.: mg/L) of soluble forms of Cr(VI) (closed
bar) and Cr(III) (open bar) in water samples from Hazaribagh area (n ¼ 21) are
expressed by log scale (Y-axis). **Significantly different from the control (**p < 0.01) as
analyzed by the Mann-Whitney U test.
669
was exchanged every day during the culture. After 0, 1, 3, 5 and 7
days of incubation, survival of HaCaT keratinocytes was investi-
gated by a cell survival assay following the method described
previously (Yajima et al., 2012).
2.4. Colony formation assay
The effects of exposure to Cr(VI) and/or Cr(III) on anchorage-
independent growth of HaCaT keratinocytes were investigated by
a colony formation assay as described previously (Yajima et al.,
2015) with slight modification. HaCaT cells that had been starved
for 16 h were pre-incubated in the presence or absence of the
indicated concentrations of Cr(VI) and/or Cr(III) for 2 days. The
keratinocytes were transferred to 1% methylcellulose/Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% FBS and
continuously incubated in the presence or absence of the same
concentrations of Cr(VI) and/or Cr(III) in Costar Ultra-Low attach-
ment 96-well plates (Corning, NY, United States) at a density of
1.6  103 cells per well. The number of colonies with diameters
50 mm was counted.
2.5. Immunoblot analysis
Immunoblot analysis was performed by the method previously
described (Akhand et al., 1999). Mouse monoclonal antibodies to a-
TUBLIN (Sigma-Aldrich, MO, United States) and to MEK1/2 and
phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, MA,
United States), rabbit monoclonal antibodies to phospho-MEK1/2
(Ser217/221) (Cell Signaling Technology) and rabbit polyclonal
antibodies to ERK1/2, AKT and phospho-AKT (Ser473) (Cell
Signaling Technology) were used as first antibodies. Goat poly-
clonal antibodies to mouse IgG and rabbit IgG conjugated with
peroxidase (Merck Millipore) were used as second antibodies.
2.6. Chromium adsorption by HT-MF
Our original magnesium and iron-based hydrotalcite-like com-
pound [Mg(II)4-Fe(III)2(OH)12]2þ[NO3xCO3$zH2O]2- (MF-HT) was
developed according to the previously described method (Kato
et al., 2013; Kumasaka et al., 2013). Briefly, 300 ml of a solution of
0.67 M Mg(NO3)2 and 0.33 mM Fe(NO3)3 was slowly delivered by
drops into 525 ml of 1 M Na2CO3 solution, following which pre-
cipitates were developed. The precipitates were filtered under
reduced pressure and dried for more than 16 h. After the solid
structure had been analyzed by using an X-ray diffractometer, the
adsorption capacity of MF-HT (particle size: <250 mm) for Cr(VI)
and Cr(III) was examined. Solutions containing the indicated con-
centrations of Cr(VI) or Cr(III) were shaken with 1% (w/v) of MF-HT
at 300 rpm for the indicated incubation time. After centrifugation
(21600  g, 10 min), Cr levels in the supernatants diluted by HNO3
up to a 1% final concentration were measured by ICP-MS. Incuba-
tion for 60 min was used to obtain the Langmuir adsorption
isotherm, and incubation for 1 min was used for the water samples
in Hazaribagh for practical application of MF-HT.
2.7. Statistical analysis
Statistical analyses were performed by Dunnett's test and the
Mann-Whitney U test according to the methods previously
described (Kato et al., 2004; Ohgami et al., 2016) by using JMP Pro
software (version 11.0.0) (SAS Institute Inc., NC, USA).
670 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675

Fig. 2. Effects of sole exposure to Cr(VI) and Cr(III) on survival of HaCaT keratinocytes. (A) Ratios (mean ± s.d.; n ¼ 3) of surviving HaCaT keratinocytes cultured for the indicated
days in the presence (0.1e3 mM) or absence of Cr(VI) are presented. (B) Ratios (mean ± s.d.; n ¼ 3) of surviving HaCaT keratinocytes cultured for the indicated days in the presence
(10-1700 mM) or absence of Cr(III) are presented. The results are shown as ratio to the value of nil control on day 7 (Ctrl, open square). *, **Significantly different from the control as
(*p < 0.05, **p < 0.01) analyzed by Dunnett's test on each day.

Fig. 3. Effects of exposure to either Cr(VI) or Cr(III) on transforming activity in HaCaT keratinocytes. (A, B) Graphs (mean ± s.d.; n ¼ 4) showing the ratios of colony number per
well (A) and photographs of colonies (B) of HaCaT keratinocytes in the presence (0.001e0.1 mM) or absence of Cr(VI) are presented. (C, D) Graphs (mean ± s.d.; n ¼ 4) showing the
ratios of colony number per well (C) and photographs of colonies (D) of HaCaT keratinocytes in the presence (0.1e10 mM) or absence of Cr(III) are presented. The results (closed bars)
are shown as ratio to the value of nil control (Ctrl, open bar) (A, C). **Significantly different from the control (**p < 0.01) as analyzed by Dunnett's test.
 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675 671

3. Results Then Cr pollution was focused on because the level of Cr was

3.1. Levels of Cr, As and Ba in water of Buriganga River and canals at
Hazaribagh
Sampling (Fig. 1A) was performed in the upstream region (a)
and proximal (d) and distal (e) downstream regions of Buriganga
River after defining the confluence (c) of Buriganga River and canals
from tanneries at Hazaribagh (b) as a base point. Concentrations
(mg/L) of soluble forms of Cr (Fig. 1B), As (Fig. 1C) and Ba (Fig. 1D) in
water from 5 sections are shown. Mean levels of Cr in canal water at
Hazaribagh and in Buriganga River water from the confluence and
proximal and distal downstream regions were about 5160-fold, 5-
fold, 4-fold and 1-fold higher than the mean level of Cr in Bur-
iganga River water from the upstream region, respectively (Fig. 1B).
However, mean levels of As and Ba in canal water at Hazaribagh and
Buriganga River water from the upstream, confluence, and prox-
imal and distal downstream regions were comparable (Fig. 1C and
D).
252-fold and 2211-fold higher than the levels of Ba and As,
respectively, in canal water at Hazaribagh. Our analysis of chemical
species showed the mean concentration of Cr(VI) [6.7 mg/L (0.1 mM)]
and mean total concentration of Cr [14284.7 mg/L (274.7 mM)] in
canal water at Hazaribagh (Fig. 1E). The maximum concentrations
of Cr(VI) and Cr(III) were 38.1 mg/L (0.7 mM) and 88697.3 mg/L
(1705.7 mM), respectively.
3.2. Effects of exposure to either Cr(VI) or Cr(III) on anchorage-
independent growth
A previous study showed that canal water at Hazaribagh
(UNICEF (The United Nations Children's Fund), 2010; Watch, 2012)
in addition to Buriganga River water (Rahman and Al Bakri, 2010)
was used for washing and bathing by the residents. To examine the
potential health risk of sole exposure to Cr(VI) and Cr(III), a colony
formation assay was performed following a cell survival assay by
using human non-tumorigenic HaCaT keratinocytes (Figs. 2 and 3).

Fig. 4. Effects of coexposure to Cr(VI) and Cr(III) on transforming activity in HaCaT keratinocytes. Graphs (mean ± s.d.; n ¼ 4) showing the ratios of colony number per well (A,
C) and photographs of colonies (B, D) of HaCaT keratinocytes in the presence or absence of 0.001 mM Cr(VI) and/or 10 mM Cr(III) (A, B) or 0.003 mM Cr(VI) and/or 1 mM Cr(III) (C, D)
are presented. The results (closed bars) are shown as ratio to the value of nil control (Ctrl, open bar) (A, C). *Significantly different from the control (*p < 0.05) as analyzed by
Dunnett's test.
672 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675
Fig. 5. Effects of coexposure to Cr(VI) and Cr(III) on MEK/ERK and AKT signaling in
HaCaT keratinocytes. Results of immunoblot analysis in HaCaT keratinocytes with nil
control (lane 1; Ctrl), sole exposure to 0.001 mM Cr(VI) (lane 2) and 10 mM of Cr(III)
(lane 3) and co-exposure to 0.001 mM of Cr(VI) and 10 mM of Cr(III) (lane 4) are pre-
sented. Photographs show levels of phosphorylation of MEK (pMEK), ERK (pERK) and
AKT (pAKT) and expression of MEK (MEK), ERK (ERK), AKT (AKT) and TUBLIN (TUBLIN).
After confirming that 0.3 mM Cr(VI) (Fig. 2A) and 30 mM Cr(III)
(Fig. 2B) had no effect on the survival of HaCaT keratinocytes in our
cell survival assay for 7 days, colony formation assays of 0.1 mM
Cr(VI) (Fig. 3A) and 10 mM Cr(III) (Fig. 3B) were performed. Levels
of anchorage-independent growth were 2.3-fold and 2.2-fold
increased by sole exposure to 0.03 mM and 0.1 mM Cr(VI), respec-
tively (Fig. 3A). On the other hand, levels of anchorage-independent
growth of cells solely exposed to 0.1 mM, 1 mM and 10 mM Cr(III)
were comparable with that of the nil control.
3.3. Effects of coexposure to Cr(VI) and Cr(III) on anchorage-
independent growth
Our results of environmental monitoring in Dhaka City indi-
cated that the skin of residents was possibly exposed to molar ratios
of Cr(VI):Cr(III) ranging from 1:29 to 1:32782. Therefore, the effect
of coexposure to Cr(VI) and Cr(III) in that range of ratios on
anchorage-independent growth of HaCaT keratinocytes was
examined. Unexpectedly, levels of anchorage-independent growth
were 2.5-fold and 2.3-fold increased by coexposure to 0.001 mM
Cr(VI) and 10 mM Cr(III) (Fig. 4A) and coexposure to 0.003 mM Cr(VI)
and 1 mM Cr(III) (Fig. 4B), respectively, compared to that of the nil
control.
3.4. Effects of coexposure to Cr(VI) and Cr(III) on MEK/ERK and AKT
signaling in HaCaT keratinocytes
Phosphorylated levels of MEK/ERK in HaCaT keratinocytes
coexposed to 0.001 mM Cr(VI) and 10 mM Cr(III) were higher than
those in cells solely exposed to 0.001 mM Cr(VI) and 10 mM Cr(III)
and in the nil control (Fig. 5). Phosphorylated levels of AKT in cells
coexposed to 0.001 mM Cr(VI) and 10 mM Cr(III) were also higher
than those in cells solely exposed 0.001 mM Cr(VI) and 10 mM Cr(III)
and in the nil control (Fig. 5).
3.5. Adsorption of Cr(VI) and Cr(III) by MF-HT
Adsorption of Cr(III) and Cr(VI) by MF-HT was examined by a
batch method previously described (Kato et al., 2016). After
investigating plural adsorption models including Freundlich and
Langmuir isotherms, the use of Langmuir isotherms was selected as
a suitable method for examining the equilibrium adsorption results
of Cr in MF-HT. Equilibrium data of Cr(VI) (Fig. 6A) and Cr(III)
(Fig. 6C) and Langmuir isotherms for Cr(VI) (R2 > 0.95; Fig. 6B) and
Cr(III) (R2 > 0.95; Fig. 6D) adsorption by MF-HT are shown.
Maximum adsorption capacities of MF-HT for Cr(VI) and Cr(III)
calculated from the Langmuir isotherms were 9.0 mg/g and
1041 mg/g, respectively, in theory.
An adsorption test for Cr by MF-HT was finally performed in
canal water containing wastewater from tanneries at Hazaribagh.
The mean concentration of Cr (1213 mg/L) in canal water samples
(n ¼ 10) treated with 1% weight of MF-HT for 1 min was decreased
to 142 mg/L (Fig. 6E and F).
4. Discussion
Our environmental monitoring in wide areas of Buriganga River
and canals at Hazaribagh provided more direct evidence of
Hazaribagh-dependent pollution with Cr and Hazaribagh-
independent pollution with As and Ba in Buriganga River. Since
the level of Cr is > 2000-fold and >200-fold higher than the levels
of As and Ba, respectively, in canal water at Hazaribagh, we focused
on Cr pollution. There has been no study showing the biological
effects of coexposure to Cr(VI) and Cr(III). Therefore, the biological
effects of coexposure to the range of molar ratios of Cr(VI) and
Cr(III) in canal water at Hazaribagh were examined using human
non-tumorigenic skin keratinocytes. Our colony formation assay
demonstrated for the first time that coexposure to Cr(VI) and Cr(III)
synergistically promoted transforming activity of HaCaT keratino-
cytes. MEK/ERK and AKT, which regulate transforming activity
(Thang et al., 2015a; Yajima et al., 2015), were synergistically acti-
vated by coexposure to Cr(VI) and Cr(III). Synergistic promotion of
transforming activity by coexposure to 0.001 mM Cr(VI) and 1 mM
Cr(III), which are in the range of ratios in the water of Dhaka City,
was also shown in our colony formation assay using human non-
tumorigenic lung epithelial Beas-2B cells (see Supplementary
Fig. S1 online). Our results using plural human cells provided
more solid evidence of the synergistic promotion of transforming
activity by coexposure to Cr(VI) and Cr(III). Our results also partially
revealed the molecular mechanism of the synergistic promotion.
Since our results showed a synergistic effect when low-toxicity
Cr(III) was present in conjunction with high-toxicity Cr(VI), both
Cr(VI) and Cr(III) must be effectively removed. Our analysis of
environmental engineering by using Langmuir isotherms showed
that our original MF-HT could maximally adsorb 9.0 mg/g Cr(VI)
and 1041 mg/g Cr(III) in theory. To our knowledge, the Cr(III)
removal ability of MF-HT is greater than that of any other previ-
ously reported depuratives evaluated by the Langmuir isotherm
(see Supplementary Table S1 online). Since the cost of MF-HT was
calculated to be 0.7 cents per kg from previous reports (Gillman,
2006; Kato et al., 2016), MF-HT, a depurative of low cost and high
efficacy, could be a strong tool for countermeasures for environ-
mental pollution and health risks caused by wastewater from
tanneries. Since Cr-polluted canal water at Hazaribagh could
actually be remediated by MF-HT, we propose that MF-HT be
 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675 673

Fig. 6. Capability of MF-HT for chromium adsorption. (AeD) Equilibrium data (A, C) and Langmuir isotherms (B, D) for MF-HT-mediated adsorption of Cr(VI) (A, B) and Cr(III) (C,
D) are presented. Ce and qe are the concentration of chromium in solution at equilibrium and the amount of chromium absorbed by MF-HT at equilibrium, respectively. (E, F)
Chromium levels of each sample (E) and mean ± s.d. (n ¼ 10) (F) in canal water at Hazaribagh with (lane 2 in E and F) or without (lane 1 in E and F) treatment with 1% weight of MF-
HT for 1-min incubation. **Significantly different from the control (**p < 0.01) as analyzed by the Mann-Whitney U test (F).

installed at the sources of chromium pollution (tanneries, in this
case). We anticipate that MF-HT will be applied after the tanning
process in tanneries in the near future. Moreover, it was shown in
our previous studies that MF-HT could remove pentavalent and
trivalent As, Ba, iron (Fe) and uranium (U) from well water in rural
areas of Bangladesh, Vietnam and Afghanistan (Kato et al., 2016,
2013; Kumasaka et al., 2013). Thus, MF-HT can be used for removal
of toxic elements in various countries as well as rural and urban
areas of Bangladesh.
Cr(VI) is increased in the presence of Cr(III), suggesting the
importance of removal of Cr(III) in addition to Cr(VI) from water.
Our study of environmental engineering finally showed that MF-
HT, a low-cost and high-efficacy depurative, can be used for
removal of both Cr(VI) and Cr(III). Thus, our comprehensive study
has provided a realizable countermeasure for Cr pollution in canal
and river water in Dhaka City, Bangladesh. More importantly, our
study suggests an efficient solution to address the worldwide
environmental Cr pollution caused by tannery wastes.

5. Conclusions

Our environmental monitoring showed 38 mg/L Cr(VI) and

88697 mg/L Cr(III) in water of Buriganga River and canals con- Conflicts of interest
taining wastewater from tanneries in Dhaka City, Bangladesh. Our
study of molecular biology then showed that the carcinogenicity of The authors declare that they have no conflict of interest.
674 M. Yoshinaga et al. / Chemosphere 201 (2018) 667e675
Acknowledgements
This study was supported in part by Grants-in-Aid for Scientific
Research (A) (15H01743 and 15H02588), (B) (17KT0033) and (C)
(16K11177, 16K08440, 16K08343 and 17K09156), Grant-in-Aid for
Challenging Exploratory Research (26670525), Grant-in-Aid for
Scientific Research on Innovative Areas (16H01639 and 24108002)
and Grant-in-Aid for Research Activity Start-up (15H06274) from
the Ministry of Education, Culture, Sports, Science and Technology,
the Mitsui & Co., Ltd. Environment Fund (R13-0014), Foundation
from Center for Advanced Medical and Clinical Research of Nagoya
University Hospital, The Mitsubishi Foundation (27310), Ichihara
International Scholarship Foundation (196), KENKO-KAGAKU Zai-
dan (Health Sciences Foundation), Aichi Health Promotion Foun-
dation, AEON Environmental Foundation, Nagono Medical
Foundation, Grant for Environmental Research Projects from the
Sumitomo Foundation (163119) and The Salt Science Research
Foundation.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.chemosphere.2018.03.026.
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