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Disruption of Skin Stem Cell Homeostasis following Transplacental Arsenicosis; Alleviation by Combined Intake of Selenium and Curcumin.

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RESEARCH ARTICLE
Disruption of Skin Stem Cell Homeostasis
following Transplacental Arsenicosis;
Alleviation by Combined Intake of Selenium
and Curcumin
Shiv Poojan
1
, Sushil Kumar
1¤
*, Vikas Verma
1
, Anupam Dhasmana
2
, Mohtashim Lohani
3
,
Mukesh K. Verma
1
1 Environmental Carcinogenesis Division, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi
Marg, P Box 80, Lucknow-226001, India, 2 Environmental Carcinogenesis & Toxicoinformatics Laboratory,
Department of Bioengineering, Integral University, Lucknow-226026, India, 3 Environmental Carcinogenesis
& Toxicoinformatics Laboratory, Department of Biosciences, Integral University, Lucknow-226026, India
¤ Current address: Central Research Laboratory and Department of Biochemistry, Hind Institute of Medical
Sciences, Safedabad, Barabanki, UP, India
* sushilkumar.iitr@gmail.com
Abstract
Of late, a consirable interest has grown in literatur e on early development of arsenicosis
and untimely death in humans after exposure to iAs in drinking water in utero or during the
childhood. The mechanism of this kind of intrauterine arsenic poisoning is not known; how-
ever it is often suggested to involve stem cells. We looked into this possibility by investigat-
ing in mice the influence of chronic in utero exposure to arsenical drinking water
preliminarily on multipotent adult stem cell and progenitor cell counts at the beginning of
neonatal age. We found that repeated intake of 42.5 or 85ppm iAs in drinking water by preg-
nant BALB/c mice substantially changed the counts of EpASCs, the progenitor cells, and
the differentiated cells in epidermis of their zero day old neonates. EpASCs counts
decreased considerably and the differentiated / apoptosed cell counts increased exten-
sively whereas the cou nts of progenitor cell displayed a biphasic effect. The observed trend
of response was dose-dependent and statistically significant. These observations signified
a disruption in stem cell homeostasis. The disorder was in parallel with changes in expres-
sion of biomarkers of stem cell and progenitor (TA) cell besides changes in expression of
pro-inflammatory and antioxidant molecules namely Nrf2, NFkB, TNF-α, and GSH. The bio-
logical monitoring of exposure to iAs and the ensuing transplacental toxicity was verifiable
correspondingly by the increase in iAs burden in hair, kidney, skin, liver of nulliparous
female mice and the onset of chromosomal aberrations in neonate bone marrow cells. The
combined intake of selenite and curcumin in utero was found to prevent the disruption of
homeostasis and associated biochemical changes to a great extent. The mechanism of pre-
vention seemed possibly to involve (a) curcumin and Kea p-1 interaction, (b) consequent
escalated de novo GSH biosynthesis, and (c) the resultant toxicant disposition. These
observations are important with respect to the development of vulnerability to arsenicosis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 1/17
OPEN ACCESS
Citation: Poojan S, Kumar S, Verma V, Dhasmana A,
Lohani M, Verma MK (2015) Disruption of Skin Stem
Cell Homeostasis following Transplacental
Arsenicosis; Alleviation by Combined Intake of
Selenium and Curcumin. PLoS ONE 10(12):
e0142818. doi:10.1371/journal.pone.0142818
Editor: Zhuo Zhang, University of Kentucky, UNITED
STATES
Received: May 12, 2015
Accepted: October 27, 2015
Published: December 1, 2015
Copyright: © 2015 Poojan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Authors are grateful to the Council of
Scientific and Industrial Research, New Delhi for
providing financial assistance (grant BSC-0302), a
Senior Project Fellowship to VV, and a Senior
Research Fellowship to SP. Acknowledgments are
due also to the Indian Council of Medical Research
New Delhi for providing a Senior Research
Fellowship to MKV.
and other morbidities later in life after repeated in utero or postnatal exposure to iAs in drink-
ing water that may occur speculatively through impairment of adult stem cell dependent
innate tissue repair mechanism.
Highlights
Chronic exposure to arsenite in utero disrupted adult stem cell homeostasis.
Counts of adult stem cell and progenitor cell changed in neonatal mouse epidermis.
Levels of stem cell and differentiated cell marker s modulated correspondingly.
TNF, Nrf2, NFkB, GSH, and tissue iAs load modulation were key events .
In utero exposure to a combination of selenite and curcumin mitigated these effects.
Introduction
Of late, a considerable interest has grown in literature on early development of arsenicosis as
well as untim ely mortality later in human life after repeated exposure to arsenical drinking
water in utero and during the childhood. The underlying mechanism is not known; however,
stem cells are speculated to be involved. It is based on the premise that, like somatic cells, the
multipotent adult stem cells may also get affected during chronic intrauterine exposure to Inor-
ganic arsenic (iAs).
iAs is a multisite transplacental toxicant, carcinogen, as well as deliberate homicidal toxicant
[110]. The clinical manifestations of arsenic poisoning in humans include stillbirth, infant-
deaths, impairment in childrens lung and intellectual function, neuro-toxicity, increased can-
cer incidence in adults and an increased mortality from cancer and bronchiectasis in adoles-
cents [1115].
In embryology, it is understood that after establishment of the germ cell layers in embryo-
genesis, an optimal pool of adult stem cells and progenitor cell count is apportioned for normal
organogenesis. The optimum numbers are considered to be necessary for the tissue growth
during embryogenesis as well as in wound repair both in utero and in postnatal stages [16]. In
view of this tacit information, it is reasonable to believe that the manipulation of stem cell
numbers by chronic exposure to iAs in utero or in postnatal age could disorder the optimal
dynamics of EpASC homeostasis in tissues and eventually the organ growth. This plausibility
however has seldom been explored [1720].
In current study, we have investigated this apparently valid issue using an enriched popula-
tion of adu lt stem cell isolated from neonate mouse skin i.e. the EpASCs. Lesions in skin are
the hallmarks of iAs toxicity observed after chronic exposure to arsenic-contaminated drinking
water [1]; hence, EpASCs from epidermis have been deployed for test of hypothesis. The cul-
tured mouse putative epidermal stem cells are proposed as a potential tool to study stem cell
biology [ 21]. We have investigated experimentally the potential of chronic intrauterine iAs
exposure to manipulate EpASCs pool size in zero day old neonate mouse skin; we determined
the manipulating potential by measuring alterations in EpASCs counts in the tissue. To charac-
terize iAs toxicity in adult stem cell, we have studied changes in levels of oxidative stress and
inflammation molecular mediators and also the changes in expression levels of stem cell and
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 2/17
Competing Interests: The authors have declared
that no competing interests exist.
Abbreviations: AAS, Atomic Absorption
Spectrophotometer; EpASCs, epidermis adult stem
cells; BrdU, 5-bromo-2'-deoxyuridine; CA,
chromosomal aberrations; CB, chromosomal breaks;
CF, chromosomal fragment; CG, chromosomal gap;
CK10, cytokeratin 10; CK14, cytokeratin 14; CMFDA,
5-chloromethylfluorescein di-acetate; CPCSEA,
Committee for the Purpose of Control and
Supervision on Experiments on Animals; DMSO,
dimethyl sulfoxide; EpASCs, Epidermal adult stem
cells; GS-AsH-SG, arseno-diglutathione complex;
GS-Se-SG, seleno-diglutathione complex; iAs,
inorganic Arsenic; IkB, Inhibitor of NF-κB activity;
Keap1, Kelch like-ECH-associated protein 1; LRKs,
label (BrdU)-retaining keratinocytes; MAPK, Mitogen
activated protein kinase; MTT, 3-(4, 5-dimethylthiazol-
2-yl)-2, 5-diphenyl tetrazolium bromide; NFkB,
nuclear factor kappa-light-chain-enhancer of
activated B cells; Nrf2, Nuclear factor (erythroid-
derived 2)-like 2; PO, per os; PCNA, proliferating cell
nuclear antigen; RF, ring formation; TA, transiently
amplifying; TNF-α, tumor necrosis factor alpha.
differentiated cell biomarkers ex vivo. For biological monitoring of transplacental iAs toxicity,
status of chromosomal aberrations has been determined in bone marrow cells of neonates
exposed to arsenical drinking water in utero. This study is an in-utero repeat dose toxicity
study and not a long term follow-up.
We have also attempted the chemoprevention of toxic effects in stem cells using essential
micronutrient selenite and the food additive curcumin both in vivo and in vitro [20, 22]. Possi-
ble interaction of selenite and curcumin with key regulators of oxidative stress and inflamma-
tion and their potential to regenerate antioxidant GSH has been evaluated using in silico studies.
Materials and Methods
The in vitro studies
EpASCs culture and dose selection. The multipotent adult stem cells were isolated from
epidermis of neonate BALB/c mouse skin, and were cultured as described earlier [23,24]. In
brief, the excised epidermis was digested enzymatically; keratinocytes were isolated and cul-
tured in growth-promoting medium in a CO
2
incubator set at 5% CO
2
and 37°C. Putative stem
cells were seeded in collagen-fibronectin pre-coated flasks and placed in CO
2
incubator for 10
min to allow adhering [25]. Culture medium was first changed after 4 days and thereafter on
alternate days to culture EpASCs. The EC
50
value of arsenite, selenite, and curcumin in EpASCs
was determined using MTT-based cell viability assay.
EC
50
determination. EpASCs from neonates born to mothers in control group were
employed for the assay. Cells were seeded (2
10
4
cells/well) in 96-well plates and allowed to
adhere for 24 hours in a CO
2
incubator. iAs and selenite were dissolved in MilliQ water; curcu-
min was dissolved in DMSO. Final concentration of DMSO in the assay was 0.05%; flasks
from the control group received only the vehicle. Cells were exposed (in triplicate, for 24
hours) to serial dilutions (40 to 1.25 μM) of arsenite (NaAsO
2
, Sigma catalogue #S7400) or sele-
nite (Na
2
SeO
3
, Sigma catalogue #S5261) or curcumin (Sigma catalogue #C1386). After 24h
exposure, MTT (10μl of 5mg/ml) was added and cells were further incubated for 3-4hrs. The
formazan crystals formed in viable cells were dissolved in DMSO. A
540
was determined using a
microplate reader (Microquant, Bio-Tek, USA) to establish EC
50
value of arsenite, selenite and
curcumin (S1a Fig).
CMFDA-reactive GSH. Changes in intracellular level of GSH were studie d in EpASCs
using CMFDA (Molecular Probes, Eugene OR USA). EpASCs were seeded on chamber slides.
After exposure to test items, cells were incubated in CO
2
incubator with pre-warmed (37°C)
10μM CMFDA (prepared in culture medium). Following incubation for 30 min, CMFDA-
media was replaced with culture medium without CMFDA (fresh and pre-warme d). The cells
were incubated additionally for 30 min and washed with PBS to fix subsequently in 3.7% para-
formaldehyde. CMFDA-stained cells were viewed under fluorescence microscope and photo-
graphed for documentation.
Immunoassay. EpASCs proteins were extracted by sonication using the Lysis buffer
(Sigma-Aldrich) containing a cocktail of protease and phosphatase inhibitors. Cell-lysates were
centrifuged (13,000g, 4°C, 15 min) and the supernatant was saved. The protein concentration
was determined by the Bradford method. Aliquots (40 μg protein) from each sample were first
heat-inactivated (95°C, 10 min) in denaturing buffer [10% glycerol, 1% SDS, 1% β-mercapto-
ethanol, 0.01% bromophenol blue, 10 mMTris-HCl (pH 6.8)], and then electrophoresed on 12%
polyacrylamide gel. Protein bands were blotted onto Immobilon-P (Millipore) already pre-
blocked for unspecific protein binding (1 hour with 5% skimmed milk in TBS-T buffer, pH 7.6).
Blots were washed extensively three times in 0.1% Tween-20 in TBS for subsequent incubation
with primary antibodies (overnight, 4°C). The primary antibody for Nrf2 (Sigma), NFkB
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 3/17
(Invitrogen), CK10, CK14, (Santa Cruz Biotechnology), PCNA, P38, p63, TNF-α (Cell Signal-
ing)was used in 1:5,000 dilution and the HRP conjugated secondary antibody (Cell Signaling)
was used in 1:10,000 dilution. The probed membranes were incubated with substrate (5 min,
RT) and developed with Enhanced Chemiluminescence (ECL) kit (Thermo, USA). Immuno-
blots were stripped and re-probed with β-actin antibody (Sigma-Aldrich) for loading correction.
The in vivo studies
Animals and treatment. Pregnant BALB/c mice (20-25g) carrying 6- day old embryos
were employed in the study. Animals were randomized into seven groups of five pregnant mice
in each, and housed at 25°C with 12-h light-dark cycle period. For randomization, the random
numbers were allotted to animals for the treatment conditions from the random number table.
Mice were fed arsenic-free standard pellet diet and safe drinking water ad lib (Group 1). The
experimental group of mice received sodium arsenite (42.5ppm or 85ppm) in drinking water
(i.e. arsenical drinking water) ad libitum during the gestation period of 818 days (Group 2 &
3). Doses showing transplacental carcinogenic potential in mice were selected from the study
of Waalkeset al [25] so as to ensure the in utero toxicit y. For chemoprevention of toxicity,
sodium selenite (2.8 or 5.6 mg/kg b wt [26] or curcumin (50 or 100mg/kg b wt [27] were asepti-
cally administered by oral gavage in a total volume of 0.2 ml either alone in parallel to iAs expo-
sure (Group 4 & 5) or in combination (Group 6 & 7); curcumin was mixed homogeneously
with an aqueous solution of gum acacia. Control animals received n-saline in place of chemo-
preventive test items.
After iAs exposures, the zero day old neonates were processed as earlier for stem cell isola-
tion and FACS based characterization [21, 2324]. All experiments were approved by the
CSIR-IITR IAEC (Institutional Animal Ethics Committee) according to CPCSEA (Committee
for the Purpose of Control and Supervision of Experiments on Animals) Guidelines of Govern-
ment of India.
EpASCs labeling and FACS based characterization. BrdU (50mg/ kg b. wt., Sigma cata-
logue #B5002) was administered to female mice twice a day for four days before mating. The
LRKs were examined in neonatal epidermis keratinocyte isolates after a chase period of 28 days
[24]. After excising skin and stripping off epidermis, EpASCs were isolated and cultured ex
vivo as described before. Before use, cells were washed twice with cold PBS. One million cells
were transferred into a polystyrene tube and pelleted. After washing with 0.5% Tween-20, the
pelleted cells were mixed with 10 ml FITC-conjugated anti-BrdU ant ibody. Contents were vor-
texed and left at RT for 30 min. Cells were finally washed again twice with PBS and resus-
pended in 1 ml PBS before BrdU label determination using a BD-FACS-LSR II flowcytometer.
Neonatal keratinocytes from the mice of control or the experimenta l group were pooled before
analyses, and levels of BrdU label determined in triplicate.
Chromosomal aberration in bone marrow cells. Neonates (0 day old), borne to the
mother mice receiving treatment as described above (six groups in total), were sacrificed.
Group 1 (control) received normal drinking water; Groups 2 to 6 received arsenite (85ppm) in
drinking water and the additives. The selenite dose formulation (5.6 mg/kg b wt) was prepared
in distilled water and curcumin (100mg/kg b wt, PO) in aqueous suspension of gum acacia.
Only clear solutions of selenite and arsenite or homogeneous suspensions of curcumin were
administered by oral gavage; and dose of each test item was administered in a total volume of
0.2 ml (per day) for 30 days. Animals in control group were administered n-saline in place of
chemopreventive test items.
Each neonate mouse was injected with 0.04% colchicine 1 mg/100g b wt. i.p. (BW, Sigma,
USA) ninty minutes prior to the sacrifice. Femurs were excised and skeletal muscles were
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 4/17
stripped off; the bones were crushed to collect marrow cells in 75mM KCl (hypotonic solution).
After incubation for 20 min at 37°C, cells from 6 pups were pooled and fixed in 3:1 methanol-
glacial acetic acid. Chromosome preparations were made using the standard procedure of air
drying, and were stained with 7% Giemsa solution (Merck, India). Slides were coded and
blind-scored.
A set of 300 cells was examined in each group. Normal cells showed clear metaphase with
normal chromosomes. The aberration types were identified as per the standard guidelines for
evaluation of genetic toxicity. Cells with one or more aberrations were counted and scored as a
percent of cells with CA with respect to control group. Number of CB, CG, RF and CF were
listed as percentages of total % CA. Data are presented as mean ± standard deviation.
Tissue iAs load determination. Briefly, adult nulliparous female mice (20g b wt) were dis-
tributed into 6 groups of 10 animals in each as described above in the chromosomal aberration
study and exposed to iAs in drinking water for 30 days. Hair, liver, kidney, and skin specimens
were excised from control and experimental group of mice; and were stored frozen in acid-free
vials in liquid nitrogen until analysis. For analyses, the specime ns were thawed and 100 mg tis-
sue samples were acid-digested with 2 ml concentrated nitric acid using a microwave oven.
The mineralized contents were made up to 10 ml using de-ionized water. iAs content was
determined using an Atomic Absorption Spectrophotometer (AAnalyst 300, Perkin Elmer,
USA) equipped with a Flow Injectio n system (FIAS-100). The iAs was determined using a
four-point standard curve prepared from a standard reference solution for arsenic, and quality
control standards were run to calibrate the AAS.
The in silico study
It was performed to explore possibility of curcumin, or selenite influencing the Nrf2-Keap1
dependent antioxidant regeneration pathway. Docking of ligand Keap-1 with curcumin or
GS-AsH-SG or GS-Se-SG (S2aS2c Fig) was simulated using PatchDock. The operation of pro-
tein-protein docking of Nrf2 with natural ligand Keap-1 and with Keap1-Curcumin complex
was also simulated and compared using ZDOCK v2.5 module of Discovery Studio.
Data Analysis and Statistics. All the in vitro and ex vivo studies were performed in tripli-
cate and were repeated three times. Data are average of three mean values. The data from in
vivo studies represent an average of five pregnant (n = 5) or ten nulliparous mice (n = 10). The
results were analyzed statistically using one-way analysis of variance control (ANOVA-non
parametric) using the Prism Graph Pad software; p values <0.05 were considered as signifi-
cant [ 28]. Students t-test [29] was also applied to compare the results of experimental group
with the control group. Bone marrow cell recovery was expressed as the mean number of bone
marrow cells obtained from a pool of a neonates femurs.
Results
FACS based analyses of LRK pool size
The profile of EpASCs pool size in the neonate epidermis is displayed in Fig 1. In the control
group, the total yield of LRKs was found to be 72.17% with a unique dispersion pattern. The
pattern and total yield of LRKs changed remarkably in the group receiving chronic in utero
exposure to iAs in drinking water (Fig 1B1G). At 42.5ppm iAs dose (Fig 1B), total yield of
LRKs increased to 80.78% whereas at 85ppm iAs dose, the yield decreased to 42.24% (Fig 1C).
In 42.5ppm iAs exposure group, the marginal increase in LRKs yield over the control value
accompanied with a distinctive change in dispersion pattern. In 85ppm iAs exposure group,
the significant loss (>40%) portrayed a radical change in dispersion pattern. The observed
trend in response was dose dependent.
In Utero Arsenite Exposures and Stem Cell Homeostasis
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This inconsistency in LRKs yield and the dispersion pattern was scrutinized further. The
counts of EpASCs, progenitor TA cells, and differentiated / apoptosed cell per se were scored
(Fig 2) by plotting the FL1-H value against count of LRKs retaining different amounts of BrdU
label; details of numerical workout are described in the legends.
In 42.5ppm iAs exposed group, a notable inc rease wasseen in counts of LRKs retaining the
least amount of BrdU label (see FL1-H 13 values in 3D line diagram in Fig 2A); and it was evi-
dent in their sum value also (see FL1-H 13 values summarized in 100% stacked column dia-
gram in Fig 2B, and the attached respective numerical data sheets). These cells, retaining least
BrdU content, represented group of differentiated / apoptosed cells. Count of LRKs, retaining a
somewhat greater quantity of BrdU label, showed marginal increase in total yield. This was evi-
dent from FL1-H 48 values in 3D line diagram in Fig 2A and from the sum values FL1-H
48 shown in 100% stacked column diagram in Fig 2B (see also the respective supplement data
sheets of Fig 2). These cells represented TA cell (progenitor / unipotent stem cell) population.
Counts of LRKs keeping most of BrdU label and representing EpASCsshowed marginal
decrease (see FL1-H 913 values in 3D line diagram of Fig 2a) as evident also in sum value
(FL1-H 913 values displayed in 100% stacked column dia gram in Fig 2b) or the attached
numerical data sheet.
A similar effect albeit with greater order of magnitude was observed in 85ppm iAs exposed
group; counts of differentiated / apoptosed cells (i.e. LRKs retaining the least amount of BrdU
label) increased considerably (Fig 2A and 2B). Counts of TA (progenitor / unipotent stem cells
i.e. LRKs retaining comparatively greater amounts of BrdU content) decreased notably. A
major loss was observed in the counts of EpASCs retaining the most amount of BrdU label (Fig
2A and 2B). The observed trend of changes at cell count level showed dose responsiveness.
In real meaning, the increase in percent yield of LRKs at 42.5 ppm iAs dose signified a boost
in counts of differentiated / apoptosed cells, marginal rise in counts of TA cells, and drop in
Fig 1. Yields of LRK in neonate epidermis after in utero exposure to iAs and the additives. (A) control, (B) 42.5ppm iAs, (C) 85ppm iAs, (D) 85ppm iAs
+ selenite (5.6mg mg/kg b wt), (E) 85ppm iAs + curcumin (100mg/kg b wt), (F) 85ppm iAs + selenite + curcumin, (G) 85ppm iAs + ½ dose (selenite
+ curcumin); Note: exhibit is a FACS generated sketch of cell counts vs. amounts of retained BrdU label displaying unique dispersion patterns of LRK counts.
It was visible in framework of the relative fluorescence value outlined on y- axis and the keratinocytes count showing the respective FL1-H counts of
fluorescent BrdU-LRKs charted on x-axis. Cells with lowest concentration of BrdU were found to be large in counts, and cells with highest concentration of the
fluorescent label were found to be less in counts. The latter group of cells represented the stem cell population in LRK isolates [16, 57]. LRKs were a
heterogeneous population of cells as these were isolated from a cluster of neonates born to a group of mother mice.
doi:10.1371/journal.pone.0142818.g001
In Utero Arsenite Exposures and Stem Cell Homeostasis
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EpASCs count. Decrease in percent yield of LRKs at 85 ppm iAs dose signified the preponder-
ant loss of TA cell and EpASCs along with considerable increase in differentiated / apoptosed
cells. The dose of 85ppm was selected for use in further studies.
Biological monitoring of transplacental iAs exposure
Transplacental exposure to iAs was confirmed biologically by monitoring CA in bone marrow
cells of zero day old neonates born to mice drinking iAs containing water. The aberrations are
shown in Fig 3; these included CB, CG, CF and RF. Alterations were statistically significant
(Fig 3A 3C).
The internal exposure to iAs was revealed by the increase of toxicants burden in target tis-
sues namely liver, kidney, hair and skin in adult nulliparous female mice (Fig 3D). Mice of all
the groups receiving arsenic and / or chemopreventive agents for 30 days showed insignificant
changes in body weight (S1b Fig).
Characterization of transplacental molecular toxicity
Repeated in utero exposure to arsenical drinking water was found to induce alterations in
expression of the molecular markers related to EpASCs and the toxicity in neonate epidermis.
iAs increased the levels of cytokeratin-10 (differentiated cell marker), TNF-α and PCNA
(inflammation and hyper proliferation marker), but decreased the levels of cytokeratin-14 and
p63 (the stem cell markers) as summarized in Fig 4A; the effect was dose dependent. The trans-
placental exposure increased the contents of cytoprotective Nrf2 and pro-inflammatory NFkB
also (Fig 4B ) revealing the onset of cellular oxidative stress and the subsequent pro-inflamma-
tory molecular change.
Acute exposure of normal EpASCs to 55μM iAs (the EC
50
value) resulted in a substantial
loss of stem cell viability and the manifestation of cytotoxicity (Fig 5A5F). Dysregulation in
Fig 2. EpASCs, TA, and differentiated cell count profile in LRK pool from neonate epidermis after in utero exposures to iAs and the additives. (A)
the change in cell counts per 1/10
th
change in FL1-H value were plotted; numerical values represent maximum cell counts per 1/10
th
of change in label
density shown in Fig 1A1G; (B) the cell counts showing approximately similar degree of change [ FL1-H 13, FL1-H 48, FL1-H 913] showcased
three sub-populations of cells [16, 57] namely keratinocytes retaining (i) greatest quantity of BrdU label i.e. adult stem cells (EpASCs), (ii) least amount of
BrdUlabel i.e. differentiated or apoptosed cells, and (iii) intermediate amount of BrdU label i.e. the progenitor TA cells on the way to differentiation.
doi:10.1371/journal.pone.0142818.g002
In Utero Arsenite Exposures and Stem Cell Homeostasis
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levels of Nrf2, NFkB, IkB protein products and GSH were observed in vitro (Fig 5B5F). Nrf2
and NFkB protein product level were found to be more. These in vitro observations were in
consonance with in vivo findings described earlier in this study. The time course of toxicant
induced change is shown in Fig 5B and 5C. The level of Nrf2 protein was found to decrease ini-
tially in first 6h of toxicant exposure and to increase thereafter up to 24hrs (Fig 5C). The
increase in the Nrf2 protein level was in parallel to in vivo results (see Fig 4b).
Time course study of NFkB expression showed an initial increase in first 6h of toxicant
exposure followed by a gradual decrease up to 24hrs (Fig 5D). This observation was a deviation
from the in vivo results, and seemed to be a function of possibly different exposure conditions
and the test dose. The time course study of IkB expression showed dysregulation after iAs
exposure, and was found to be complimentary to the expression pattern of NFkB. As evident in
Fig 5E, after initial decrease in first 6h, IkB expression increased linearly up to 24hrs.
iAs inhibited the phosphorylation of MAPK family member p38 in stem cells (Fig 5F); how-
ever, retention of partial activity was observed for next 24hrs. The attenuation of p38-phos-
phorylation indicated inhibition of redox regulated MAPK in stem cells and validated the
stress inducing potential of iAs. It was in line with the observed increase in the counts of differ-
entiating cell populations in vitro.
Fig 3. iAs in drinking water induced CA in 0 day old neonate mouse bone marrow cells after 818 day in utero exposure (A-C) and toxicant burden
in hair, skin, liver and kidney of nulliparous female mice after 30 day oral administration (D) and rescue by PO administration of selenium and/or
curcumin. Display of RF, CB, CG [A], CF [B], significant changes in formation of CB, CF and RF [C]; data are mean of three different experiments; ± SEM, p
values are ***<0.001, **<0.01; [D] significant reduction in iAs burden after PO selenium and/or curcumin administration, data are expressed as mean of
three different experiments; ± SEM, p values are ***<0.001, **<0.01.
doi:10.1371/journal.pone.0142818.g003
In Utero Arsenite Exposures and Stem Cell Homeostasis
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Rescue of iAs induced disorder in LRKs counts and changes at
molecular level
Co-administration of selenite and curcumin combine countered iAs induced transplacental
changes both at cellular and molecular level; the preventive effect was evident both in vivo (Fig
1D1G) and in vitro (Fig 5A5F). The EC
50
valuefor arsenite, selenite, and curcumin were
determined for use in in-vitro experiments and are summarized in S1 Fig. At the cellular level,
selenite and curcumin counteracted the changes in counts of EpASCs and progenitor cells in
vivo. The preventive agents restricted the decrease in EpASCs count by approximately 50%
compared to the decrease observed in their absence (Fig 2A and 2B). Interestingly, selenite and
curcumin combine hampered the toxicant induced increase in formation of differentiated and
progenitor TA cells. It was reflected in counts of differentiated / apoptosed and TA cells as
shown in the numerical datasheet (Fig 2A and 2B). Individually, selenite or curcumin rescued
the loss in counts of EpASCs counts inefficiently (Fig 2A).
At the molecular level, selenite and curcumin foiled iAs induced changes in content of the
cytoprotective Nrf2, antioxidant GSH, inflammation mediator NFkB in adult stem cell (Fig
4B). Selenium and curcumin combine was comparatively more efficient than each individually
to prevent the iAs induced descend in adult stem cell viability, ascend in CMFDA-reactive
GSH content, and change in expression of the toxicity markers (Fig 5A5F). The time course
Fig 4. In utero exposure to iAs and/or additives induced changes in levels of (a) LRKs biomarkers, and (b) Nrf2 and NF-kB expression in neonate
EpASCs. Data are mean of three different experiments; ± SEM, p values are
a
<0.001,
b
<0.01,
c
<0.05 and
d
>0.05 vs. control and
e
<0.001,
f
<0.01,
g
<0.05
and
h
>0.05 vs. arsenic.
doi:10.1371/journal.pone.0142818.g004
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 9/17
Fig 5. In vitro acute exposure to iAs and/or additives induced (A) cytotoxicity and viability, (B) levels
of GSH, (C) time course of Nrf2 expression, (D) time course of NFkB expression, (E) time course of IkB
expression, (F) time course of phosphorylated-P38 expression in neonate EpASCs. Data are
expressed as mean of three different experiments; ± SEM, p values are
a
<0.001,
b
<0.01,
c
<0.05,
d
>0.05
vs. control and
e
<0.001,
f
<0.01,
g
<0.05,
h
>0.05 vs. arsenic.
doi:10.1371/journal.pone.0142818.g005
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 10 / 17
of molecular changes in EpASCs was prevented to near normal values by selenite and curcumin
combine.
The additives counterbalanced other features of iAs toxicity as well, namely (i) inc rease in
CA in bone marrow cells (Fig 3A3C); (ii) the toxicant burden in tissues (Fig 3D) (iii) increase
in de novo synthesis of Nrf2, NFkB in vivo (Fig 4B); and (iv) the cytotoxicity, the altered bio-
chemical parameters like increase in IkB expression and p38 phosphorylation, and the decrease
in expression of Nrf2, NFkB, IkB and GSH biosynthesis in vitro (Fig 5B5F).
A strong induction in the levels of CMFDA-reactive GSH was seen in stem cells after expo-
sure to iAs (Fig 5B). A similar response was seen again after exposure to a combination of sele-
nite and iAs (Fig 5C) or curcumin and iAs (Fig 5D). These observations vindicated the finding
of iAs induced NFkB, IkB, Nrf2 gene over expressions. Induction in levels of CMFDA-reactive
GSH in stem cells might however be confounded by the delay in repression of demand-driven
GSH biosynthesis.
In essence, the combination of selen ite and curcuminproved to be comparatively more
effective for regulation of the iAs induced increase in intracellular levels of GSH in adult stem
cells to near-normal values ( Fig 5B5F) in addition to increase in (a) expressions of NFkB, p38
(Fig 5D and 5F) and (b) formation of TA cells (Fig 2A and 2B).
In silico study of molecular interactions between curcumin, selenite and
Keap1
Results are summarized in S1 & S2 Tables. Individually,curcumin and GS-AsH-SG (the stable
metabolite of arsenite) were found to interact with Keap-1 registering a higher PatchDock
Score of 5022 and 5174 respectively. GS-Se-SG, the stable metabolite of selenite, also interacted
with Keap-1 however on a relatively low score of 4858 (S1 Table).
Investigations revealed that after binding with curcumin (S2a Fig), ZDock score of Keap-1
decreased from 11.68 to 8.58 (S2 Table) suggesting relatively better potential of curcumin-
Keap-1 complex for interacting with Nrf2 and activating it as well. Ligand Keap-1 was found to
interact with natural receptor Nrf2 (S2d Fig) normally with a ZDock Score of 11.68 (S2 Table).
Discussion
This study demonstrated that repeated in utero exposure to arsenical drinking water influenced
adult stem cell homeostasis in neonate epidermis at the time of birth by inducing a significant
change in counts of EpASCs, unipotent / progenitor TA cells, and differentiated cells along
with the alterations in levels of Nrf2, NFkB, IkB, TNF-α protein products and GSH. The results
signified the acquisition of disorder in neonate EpASCs homeostasis in vivo after in utero
exposure to iAs in drinking water. The ultimate foci of damage were the counts of adult stem
cells and progenitor cells, though in different proportions and dimensio ns.
The mechanism of aberration in EpASCscounts seemed to couple with a loss in stemness
and wi th a surge of differentiation. These observations are in line with hazardous effects of
iAs described in literature since long [30]. Changes in skin with respe ct to stem cell homeo-
stasis disorder, such as hyperkerat osis, acan thos is are still being reported both i n vivo in
humans [31] and in vitro in human skin equivalent system [32].Theincreaseinincidences
of an array of adverse health effects as well as rise in mortali ty (from cancer and card iores pi-
ratory diseases) are described both in childhood and adulthood i n populations transpla -
cently exposed to iAs [1115,3340]. Early life exp osure to arsenic and the resultant
acquisition of acute / long-term impairment in l ung func tion and tissue mechani cs, postn atal
development, and behavioural changes is reported in laboratory animals also. The gaining of
predisposition to disease is explained also by gene expression manipulations triggered by
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 11 / 17
transplacental iA s exposure [4144]. Chronic exposure to iAs is inevita ble in the early par t
of life due to contamination of the food chain beyond permissible limit whether in drinking
waterand/orstaplefoodworldwide[4547].
The trigger of change in EpASCs homeo stasis is accompanied with oxidant stress and resul-
tant inflammation mediators via over-expression of rapidly act ing and resident transcription
factor Nrf2 [4851] plus pro-inflammatory molecules TNF-α, NFkB [49]. The activation of the
Nrf2-Keap1 antioxidant pathway by transplacental iAs exposure has been reported in humans
as reviewed recently [4950]; nevertheless, there is a paucity of information in the literature on
this issue in adult stem cell. The present study provides this evidence for the first time.
This study further demonstrated the role of cytokine regulated inflammation and MAPK
pathway in the mechanism of action of iAs in adult stem cells. Both the toxicant stress and the
related biochemical changes seemed to trigger ultimately the de novo biosynthesis of GSH and
other antioxidant molecules in stem cells as evident from the data on CMFDA-reactive GSH
levels. p38 kinase is a part of MAPK that is activated by dual kinases (MKKS), and responds to
extracellular stress stimuli for cell differentiation and apoptosis. Although iAs partially inhib-
ited p38 phosphorylation in our study, the remnant activity of phosphorylated p38 seemed to
support the surge of differentiation, the inconsistencies in counts of EpASCs and progenitor
cells, and the ensuing paradigm shift in dynamics of homeostasis in vivo [5254].
EpASCs homeostasis disorder may impair vital functions like wound repair in tissues,
which eventually could render vulnerability to diseases and disorders of multiple organ systems
in humans. The hypothesis on persistent suboptimal counts of EpASCs after prolonged in
utero exposure to iAs could plausibly be applicable in other ectoderm tissues as well. However,
more studies are required to strengthen the exte nsion of this hypothesis to other tissues.
In current study, the stem cell homeostatic disorder and the exper imental aesenicosis were
effectively countered by repeated simultaneous intake of combined selenite and curcuminin
utero. The rescue seemed to operate through de novo GSH biosynthesis triggered both by oxi-
dant stress and Nrf2 & NFkB over expression (Fig 6).
Data on expressions of NFkB, IkB, Nrf2, and CMFDA-reactive GSH in iAs exposed EpASCs
both in vivo and in vitro corroborates this view and also vindicates the in silico prediction for
(i) Nrf2 release by curcumin, (ii) the subsequent de novo GSH biosynthesis, and (iii) the change
in status of oxidativ e stress. The in silico study has allowed an insight into the contribution of
arsenite, selenite, and curcumin in activation of Nrf2 for inducing de novo GSH biosynthesis.
In silico although arsenite, selenite, curcumin made complex with Keap-1 (the natural ligand
for Nrf2) as illustrated in S2aS2c Fig and S1 & S2 Tables, curcumin-Keap-1 complex, how-
ever, emerged to be a more potent ligand for Nrf2 than the natural ligand Keap-1 alone (S2d
Fig, S1 & S2 Tables); it appeared to spoil the affinity of Keap-1 alone for Nrf2. These possib ili-
ties suggested the contribution of curcumin to induce the de novo GSH biosynthesis via cyto-
protective Nrf2 activation in addition to the pro-inflammatory NFkB pathway. Arsenite and
selenite contribute to augmentation in de novo biosynthesis of GSH through over-expressio n
of both Nrf2 as well as NFkB. These observations corroborate with the findings of CMFDA-
reactive GSH status in EpASCs. The other parallel contributes to prevention of stem cell
homeostasis disorder could be the (i) rapid mobilization of accumulated iAs from hair, skin,
liver attributable to its increased metabolism and disposal in GSH enriched cells finally reduc-
ing the toxic burden in tissues, and (ii) prevention of iAs induced DNA damage and loss of cell
viability. These observations may help explain the wound healing property of curcumin
observed traditionally as well as described in the literature [55,56].
The present study provides a lead for experimental in utero chemoprevention of transpla-
cental iAs toxicity using essential micronutrient and food additives and regenerative-naturopa-
thy in infants born to iAs-exposed mothers that are presumably predisposed to ailments,
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 12 / 17
diseases, and disorders developing later in adulthood. The bio-remedial measures, available in
the literature, are somatic cell specific and are transient. Engineering the removal of arsenic
from potable water is a Herculean task, besides being cost-intensive, toxic sludge waste generat-
ing, and failing the challenge of removing metalloids from food chain. The use of essential
micronutrient and dietary supplements to contain and / or remove iAs-induced disorders in
stem cell homeostasis and to detoxify iAs exposed subjects could be a potentially effective, logi-
cal, non-toxic, and health- improving strategy.
Conclusion
This study evidently demonstrated the acquisition of adult stem cell homeostasis disorder and
critical molecular changes after chronic in utero exposure to iAs early in life and their efficient
chemoprevention by selenite and curcumin combine. Manipulation in counts of EpASCs and
the unipotent / progenitor TA cells, and the differentiated cells in the neonate epidermis at the
beginning of neonatal age is hypothesized to be detrimental to the functions of skin and form a
cellular basis to vulnerability for arsenicosis later in life. After in utero iAs exposures, the adult
stem cell loss, accumulation of stem cell count aberrations, and homeostasis disorder together
with continual insufficient antioxidant activity and inadequate toxicant disposition can possibly
be the crucial contribute to the increases in disease susceptibility and disease burden observed in
neonatal and adulthood life. In silico studies support the observed chemopreventive potential of
Fig 6. Pathway diagram showing Nrf2 activation and enhanced GSH biosynthesis through curcumin and release of arsenic via selenium and GSH
complex.
doi:10.1371/journal.pone.0142818.g006
In Utero Arsenite Exposures and Stem Cell Homeostasis
PLOS ONE | DOI:10.1371/journal.pone.0142818 December 1, 2015 13 / 17
the studied food additives to activate Nrf2-Keap1 dependent regeneration of endogenous anti-
oxidant GSH for the rescue of iAs impaired EpASCs homeostasis.
Supporting Information
S1 Fig. EC
50
value of arsenic, selenite, curcumin in epidermal adult stem cell using cell via-
bility assay (MTT assay) (Fig A). Body weight of mice following 30 day exposure to arsenic,
arsenic with selenium, arsenic with curcumin, arsenic with selenium and curcumin, and arse-
nic with selenium and curcumin in half dose (Fig B).
(DOCX)
S2 Fig. Molecular Interaction Analysis of Keap1-Curcumin (Figure Generated by Discovery
Visualizer) (Fig A). Molecular Interaction Analysis of Keap1-GS-AsH-SG, (Figure Generated
by Discovery Visualizer) (Fig B). Molecular Interaction Analysis of Keap1-GS-Se-SG,
(Figure Generated by Discovery Visualizer) (Fig C). Molecular Interaction Analysis of Keap1 &
Nrf2 (Figure Generated by Discovery Visualizer) (Fig D)
(DOCX)
S1 Table. Molecular Interaction Analysis of Keap-1 with Curcumin, GS-AsH-SG & GS-Se-
SG using PatchDock Server and Discovery Visualizer.
(DOCX)
S2 Table. Molecular Interaction study of Keap1, Keap1-Curcumin, Keap1-[GSH-Se-GSH],
and Keap1-[GS-AsH-SG] with Nrf2 using ZDOCK. S2 Table Footnote. In column No. of
Hydrogen Bond- A, B and UNK indicates aminoacid residues of Keap1, Nrf2 & Curcumin,
[GS-Se-SG], [GS-AsH-SG] respectively.
(DOCX)
Acknowledgments
Authors are grateful to the Council of Scientific and Industrial Research, New Delhi for provid-
ing financial assistance (grant BSC-0302), Senior Project Fellowship to VV, and Senior
Research Fellowship to SP. Acknowledgements are due also to the Indian Council of Medical
Research New Delhi for providing a Senior Resea rch Fellowship to MKV. Creative suggestions
from AK Srivastava MBBS, MD, Vipin Behari MBBS, C Kesavachandran, PhD, Jyoti Sinha,
MBBS, DGO, and Vishal Chandra MSc are gratefully acknowledged.
Author Contributions
Conceived and designed the experiments: SK. Performed the experiments: SP VV MKV AD
ML. Analyzed the data: SK SP AD ML. Contributed reagents/materials/analysis tools: SK AD
ML. Wrote the paper: SK SP VV MKV.
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... The combined intake of selenite (5.6 mg/kg) and curcumin (100 mg/kg) in utero prevented the disruption of homeostasis and associated biochemical changes, e.g., levels of Nrf2, NFkB, IkB, TNF-α protein products, and GSH. The authors suggested that curcumin activates Nrf2 and enhances GSH biosynthesis, and the selenium and GSH complex aids in the release of arsenic [102]. ...
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... Simultaneous application of curcumin and arsenic inhibited arsenic-induced ROS production and prevented the activation of telomerase and caspase-dependent apoptosis. In line with our findings, it has been shown that curcumin alleviates arsenicinduced cell damage in skin stem cell (Poojan et al., 2015), lung cancer cell lines (Hosseinzadeh dehkordi, et al., 2015), hepatic cells (Muthumani and Miltonprabu, 2015), kidney cells (Sankar et al., 2016) and the splenocytes (Khan et al., 2012) , as well as protection against arsenic-induced genotoxicity (Sankar et al., 2014). Arsenic, as a pro-oxidant in different biological systems, can cause oxidative damage in the brain (Ramos et al., 1995). ...
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