Environmental Toxicology and Pharmacology 56 (2017) 383–392

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Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Research Paper

A zinc Schiff base complex inhibits cancer progression both in vivo and in MARK
vitro by inducing apoptosis
Arpita Banerjeea,c,1, Kaushik Banerjeea,1, Abhinaba Sinhaa, Satyajit Dasa, Saikat Majumderb,
⁎
Subrata Majumdarb, Soumitra Kumar Choudhuria,
a
In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata, 700 026, West Bengal, India
b
Division of Molecular Medicine, Bose Institute, P 1/12, C. I. T. Road, Scheme – VIIM, Kolkata, 700054, West Bengal, India
c
Department of Chemistry, Rammohan College, 102/1, Raja Rammohan Sarani, College Street, Kolkata, West Bengal 700009, India

A R T I C L E I N F O A B S T R A C T

Keywords: Cancer chemotherapy suffers from selectivity and undesired toxicity of the drugs. Since zinc is a biocompatible
Zinc complex tracer element and cysteine derivatives are used in cancer chemoprevention, we intend to develop a complex of
Apoptosis zinc and cysteine-derivatives as potent, non-toxic anticancer agents. Herein, we synthesized and characterized
T-Lymphoblastic leukemia cysteine based ligand, 2-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-3-mercapto-propionic acid and its Zn-
Ehrlich ascites carcinoma
complex, which are found to be non-toxic towards normal human PBMC. Data also revealed that only Zn-
Multidrug resistance
complex exhibited remarkable apoptosis in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000
cancer cells as assessed by MTT, Cell cycle and AnnexinV binding assay. Moreover, Zn-complex altered ROS and
GSH level of the respective cell lines. Finally, treatment of Zn-complex in Swiss albino mice did not show any
systemic toxicity in preliminary trials in normal mice and remarkably increased the life-span of EAC bearing
mice. In conclusion, the synthesized Zn-complex may be developed for efficacious, multidrug resistance reversal,
non-toxic chemotherapeutic agents in future.

1. Introduction component of various metalloenzymes and transcription factors (Vallee

The major impediments for the successful treatment of cancer are
the cytotoxicity due to lack of selectivity of different antineoplastic
agents between healthy and cancer cells as well as development of
acquired multidrug resistance (MDR) in cancer. Recent studies disclose
some differences between normal and cancer cells (Dhawan and
Chadha, 2010). For example, the relationship between zinc in-
sufficiency and cancer has been observed in human, animal and cell
culture studies (Sharif et al., 2012). Zn deficiency cause oxidative DNA
damage and chromosomal breaks in animals (Ho et al., 2003; Oteiza
et al., 2008; Oteiza et al., 1995; Golub et al., 1985; O’Halloran, 1993)
and plays cardinal role in DNA synthesis, gene regulation and induction
of apoptosis (Liu et al., 2013; Ott and Gust, 2007). Cancer che-
motherapeutic agents primarily cause DNA damage, impede cancer
cells proliferation and ultimately induce apoptosis (Zuber et al., 1998;
Moucheron, 2009; Maheswari et al., 2007; Clarke et al., 1999; Tan
et al., 2009; Bazzicalupi et al., 2008; Liu and Wang, 2009). Zinc com-
plexes cleaves DNA molecule via hydrolytic pathway (Vallee and Auld,
1990; Bae et al., 2003). Zinc is an essential trace element and necessary
and Auld, 1989; Emami et al., 2007). Zinc complexes plays vital roles in
biological fields as radioprotective agents (Huang et al., 2006), tumor
photosensitizers (Nakayama et al., 2008), antidiabetic insulin mimetic
(Sakurai et al., 2008; Katiyar and Singh, 2008) and antibacterial or
antimicrobic agents (Katiyar and Singh, 2008; Chohan et al., 2007).
Divalent zinc [Zn(II)] ions can act as an effective Lewis acid and at the
same time it has no ligand field stabilization energy and thereby can
easily adapt any coordination geometry to meet the requirement for a
particular reaction. Zinc is also significantly nontoxic (Beraldo and
Gambinob, 2004) compared to other metals viz, iron, copper, mercury
(Vallee and Falchuk, 1993), even at higher doses. Although zinc (II)
ions possess diverse physiological activities its application as cytotoxic
mediator on human cancer cell lines is still obscure (Jiang et al., 2009;
Belicchi et al., 2001; Sheng et al., 2008; Trávníček et al., 2006;
Rodrıguez-Arguelles et al., 2004; Miyamoto et al., 1998). Herein we
report the synthesis and characterization of a novel zinc complex,
Zn2L2(H2O)2 (henceforth will be mentioned as Zn2L2) and its anti-
proliferative activity against human T lymphoblastic leukemia cell lines
(in vitro) and against murine Earlich ascites carcinoma cells (in vivo).

⁎
Corresponding author.
E-mail address: soumitrag10@gmail.com (S.K. Choudhuri).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.etap.2017.11.004
Received 5 April 2017; Received in revised form 3 November 2017; Accepted 4 November 2017
Available online 06 November 2017
1382-6689/ © 2017 Elsevier B.V. All rights reserved.
A. Banerjee et al.
The rationale behind designing the reported cysteine-based zinc com-
plex is multi-dimensional. The thiol group in cysteine can act as a nu-
cleophile in enzymatic reactions. Although the role of different cysteine
derivatives in chemoprevention and cure has been reported (Lee et al.,
2011; Casini et al., 2002; Li et al., 2008), the role of cysteine as a metal
chelator of a non-toxic metal like zinc has not yet been explored. Schiffs
bases and their metal complexes as cytotoxic agent have been reported
in recent years (Ganguly et al., 2014; Kumar et al., 2010; Tarafder et al.,
2000) but cysteine containing Schiff base ligand with zinc has not
previously been reported.
2. Material and methods
2.1. Chemicals and instruments
Cysteine.HCl, MTT dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide], propidium iodide and Histopaque 1077 were
purchased from Sigma Chemical Company (St. Louis, MO). RPMI 1640
medium and foetal bovine serum were procured from GIBCO Invitrogen
Corp. (Carlsbad, California). Penicillin and streptomycin were bought
from Himedia (Mumbai, India). FITC-labeled Annexin V and 2′,7′-di-
hydrodichlorofluorescein diacetate (H2-DCFDA) were obtained from
Molecular Probes TM, Invitrogen, (Carlsbad, California). o-vanillin
(Acros Organics, New Jersey, USA), Zn(CH3COO)2. 2H2O (Merck,
Mumbai, India), RNase A (Genei, Bangalore, India), were also pur-
chased from the manufacturer. All other reagents were of AR grade and
used without further purification. Elemental analyses (carbon, hy-
drogen, and nitrogen) were performed using a Perkin Elmer 2400 Series
II CHN analyzer. Infrared spectra (4000–400 cm−1) were recorded at
28 °C on a Perkin-Elmer RX 1 FT spectrophotometer using KBr as
medium. UV–vis spectra were recorded in Shimadzu UV 160 A and in
Varian Cary 100 Scan in the range of 800–200 nm. Mass spectrum was
recorded in a Waters Micromass Quattro micro API mass spectrometer
mass spectrometer. Cell survival was evaluated by ELISA reader (Tecan
200). The cell cycle analyses and annexin-V binding assay were per-
formed using a FACS calibur flow cytometer (Becton Dickinson, USA).
Fluorescence was measured in Varian Cary eclipse spectrofluorimeter.
Haematological parameters were analyzed by automated haematology
analyzer (Sysmex, KX-21) and haematologic biochemical analyses
(blood urea, creatinin, alkaline phosphatase, SGPT, SGOT) were de-
termined by automated clinical chemistry analyzer (Olympus, AU400).
2.2. Synthesis of the ligand (H2L) and zinc complex (Zn2L2)
Ligand (H2L): Cysteine.HCl (1.57 g, 10 m mol) was dissolved in 0.01
(M) NaOH. An ethanolic solution of o-vanillin (1.52 g, 10 m mol) was
added slowly drop wise to the cysteine solution. Resulting mixture was
stirred in cold environment (4 °C) and after 30 mins a light precipitate
deposited. Again 10 mL ethanol and 0.01(M) NaOH was added to the
mixture and stirred for 2 h. A pale yellow precipitate of 2-[(2-Hydroxy-
3-methoxy-benzylidene)-amino]-3-mercapto-propionic acid (H2L) was
deposited. The compound filtered and recrystallised from MeOH and
dried in vacuum. Yield 1.1 g, 43.3%; m.p.133 °C; Anal. Found (%); C
51.69; H 4.87; N 5.56. Calcd for C11H13NO4S (%): C 50.74; H 4.92; N
5.65.
Zinc complex (Zn2L2): According to the above mentioned method
H2L was prepared; the pale yellow precipitate was dissolved in NaOH
(0.01 M) and stirred well until a clear solution was obtained. Aqueous
solution of Zn(CH3COO)2. 2H2O (2.19 g, 1 m mol) was added in situ and
stirred again for 2 h. The resulting zinc complex, Zn2L2(H2O)2 (yellow
precipitate) was filtered off and washed with EtOH. Yield 3.6 g,
73.73%; m.p. > 300 °C; Anal. Found (%);C 41.40; H 3.52; N 4.39; S
9.95. Calcd for C22H24N2O8S2Zn2: C 41.50; H 3.45; N, 4.40; S 10.06.
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Environmental Toxicology and Pharmacology 56 (2017) 383–392
2.3. Preparation of doses for both in vivo and in vitro treatments
A stock solution of Zn2L2 (10−2 M) was prepared by dissolving it in
acid-water just before the in vitro experiments. Then the stock solution
was serially diluted with RPMI medium supplemented with 10% FBS
for studies; for in vivo experiments, the complex, Zn2L2 (25 mg) was
dissolved in 500 μL 1(N) HCl and diluted to 5 mL by adding PBS to
prepare a 5 mg/mL stock solution. This stock solution was serially di-
luted with PBS and was injected intraperitoneally (i.p) to achieve the
desired concentrations. As for vehicle controls, equal volumes of
medium or PBS were added to untreated cells or injected i.p for in vitro
or in vivo experiments respectively.
2.4. Animal study
Swiss albino mice, originally obtained from National Institute of
Nutrition (Hyderabad, India) and reared in the institute animal facil-
ities, were used for all the experimental purpose with prior approval of
the institutional animal ethics committee (IAEC). The experimental
protocols (Approval ID: IAEC-1.2/SKC-7/2007/4 dated 22.01.2008)
described herein were approved by the IAEC (Registration No.: 175/99/
CPCSEA, dated 28.01.2000) in accordance with the ethical guidelines
laid down by the committee for the purpose of Control and Supervision
of Experiments on Animals (CPCSEA) by the Ministry of Social Justice
and Empowerment, Government of India. Adult female Swiss albino
mice (weighing 18–20 gm, aged between 4 and 6 weeks old) were ac-
climatized to the experimental room having temperature 25 ± 2 °C,
controlled humidity conditions, and with photo cycle of 12 h light/12 h
dark. The mice were housed in sterile polypropylene cages containing
sterile paddy husk as bedding material, fed on autoclaved standard
mice food pellets and water ad libitum.
2.5. In vivo cell line, tumor transplantation and experimental protocol
EAC cell was maintained as an ascitic tumor in female Swiss albino
mice. Six groups (each group containing six mice) of animals were
taken for animal survival studies. Each mouse was inoculated with 106
EAC cells (six groups) intra-peritoneally. Following 9 days of inoculums
(i.p) Zn2L2 of various doses (20 mg kg−1, 25 mg kg−1, 30 mg kg−1 and
50 mg kg−1 of body weight) were injected (i.p) to EAC bearing mice
while one group kept untreated (control) and another was kept as ve-
hicle control. Animals were daily inspected for the assessment of ascitic
load, and body weights were measured. Time of death was recorded for
calculation of mean survival time (MST). The MSTs were recorded for
each group. Mouse survival periods in different groups were also
compared as treated/control (T/C) ratio (percent), i.e., the ratio of the
survival time (in days) for treated mice to control (untreated) mice.
According to standard National Cancer Institute protocols for screening
new anticancer drugs, it is considered that the increase in survival time
corresponding to T/C ratios around 120% to be “marginal”, T/C ratios
between 120 and 150% to be “clear” and T/C ratios equal or superior to
150% to be “marked”.
2.6. In vivo toxicity testing
To evaluate preliminary sub acute toxicity of Zn2L2, seven experi-
mental groups (six mice per group) of normal female Swiss albino mice
were taken. All the experiments were performed in triplicate. The Zn2L2
(25 mg) was dissolved in 500 μL ∼ 1(N) HCl and diluted to 5 mL by
adding PBS to prepare a 5 mg/mL stock solution. This stock solution
was serially diluted with PBS and was injected intra-peritoneally to
achieve a final concentration of 20, 25, 30, 50 and 100 mg per kg body
weight. During the experimental period, clinical appearance of the mice
was daily evaluated. Blood was collected from treated, untreated and
vehicle control mice via closed cardiac puncture with the help of a 22-
guage hypodermic needle by subxiphoid approach after 14 days of
A. Banerjee et al.
treatment to evaluate toxicological parameters. Blood from each group
was pooled into separate glass tubes and treated with heparin (antic-
oagulant) or left untreated for serum collection.
2.7. Isolation of bone marrow cells
In order to find out preliminary bone marrow toxicity following
treatment, untreated and Zn2L2 treated female Swiss albino mice were
anaesthetized and the femur bone was cut with the help of a vertebrate
scissor. Bone marrow was flushed with 0.56% KCl solution and cen-
trifuged at 3000 rpm for 15 min at 37 °C. Cell viability was tested by
0.4% trypan blue dye solution and cells were counted in a phase con-
trast microscope. The experiment was repeated thrice. The average
value of percentage of viable bone marrow cells was calculated from
three independent experiments.
2.8. Isolation of spleen cells
Zn2L2 treated and untreated female Swiss mice were anesthetized
by chloroform and 70% alcohol was sprayed on abdominal region.
Spleen was removed aseptically and small amount of PBS was injected
to it; a small portion of it was rubbed against an opaque frosted glass
slide. The cell suspension formed is spinned at 1000–1500 rpm for
5–10 min. The supernatant was discarded and the cells were washed by
spinning in PBS twice at room temperature. Cell viability was tested by
0.4% trypan blue solution and cells were counted in a phase contrast
microscope. The experiments were performed in triplicate.
2.9. In vitro cell lines and culture conditions
The doxorubicin resistant and sensitive human acute T-lympho-
blastic leukaemia cell lines CEM/ADR5000 and CCRF-CEM respectively
were kindly provided by Prof. T. Efferth, University of Mainz, Germany.
The doxorubicin resistant CEM/ADR5000 cell line was generated by
treating CCRF-CEM cells with doxorubicin doses up to a final con-
centration of 5000 ng ml−1 doxorubicin (Kimmig et al., 1990). Due to
the doxorubicin selection pressure CEM/ADR5000 specifically over-
expresses P-glycoprotein without concomitant over-expression of MRP1
or BCRP (Gillet et al., 2004). Furthermore, the cross-resistance profile
of CEM/ADR5000 cells to a broad range of established anti-cancer
drugs and investigative novel compounds have been analyzed (Efferth
et al., 2008). Therefore, to maintain the doxorubicin resistance property
in CEM/ADR5000, the cells requires undergoing a continuous selection
pressure of doxorubicin (5000 ng ml−1 doxorubicin) in the growth
culture media. Thus, both the cell lines were sub cultured at every 72 h
and CEM/ADR5000 cells were maintained on every 15th day in the
presence of 5000 ng ml−1 doxorubicin for the period of 72 h. After that,
the doxorubicin containing medium was replaced with fresh RPMI
medium and cultured for subsequent generations. In all experiments,
CEM/ADR 5000 cells were used that were cultured without doxorubicin
and doxorubicin treatment of the next immediate generation was also
avoided. These cell lines were cultured in RPMI medium supplemented
with 10% heat inactivated foetal bovine serum (FBS), glutamine
(0.15%), HEPES (25 mM) 66.67 mg/L penicillin and 100 mg/L strep-
tomycin. Cells were grown in plastic tissue culture flasks with a hu-
midified atmosphere of 5% CO2 in incubator at 37 °C. Cells from ex-
ponentially growing cultures of passage 15 or less were used for all
experiments. All experiments were repeated three times.
2.10. Cytotoxicity assay
For the proliferative assay, 4 × 104 cells were cultured in 96 mi-
crowell plates for 24 h at 37 °C, 5% CO2. Then H2L and its Zn2L2 were
added in microwells containing the cell at final concentrations of 10−8
M–10−4 M. Following incubation for 48 h and 72 h the cells were in-
cubated further with 5 mg per ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-
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Environmental Toxicology and Pharmacology 56 (2017) 383–392
diphenyl-2H-tetrazolium bromide (MTT) for 4 h at 37 °C, 5% CO2
(Mossmann, 1983). Cell survival was evaluated by measuring the ab-
sorbance at 540 nm by ELISA reader. The control value corresponding
to untreated cells was taken as 100% and the viability of treated sam-
ples were expressed as a percentage of the control. All experiments
were performed in triplicate. The data were used for the construction of
response curves using the Graph Pad Prism software.
2.11. Cell cycle analysis
For the cell cycle analysis cells were seeded in 60 mm tissue culture
plate at a density of 5 × 105/plate and allowed to settle for 24 h prior
to treatment with complex, Zn2L2. At various time points (12 h, 24 h,
48 h, 72 h and 96 h), cells were recovered, washed twice in PBS, fixed
in cold 70% ethanol, and stored at 4 °C until analyzed. Then the cells
were washed twice in PBS, incubated for 1 h at room temperature with
250 μg/mL RNase A and for 20 min at 4 °C with 20 μg/mL propidium
iodide (PI) in dark. The cell cycle distribution and percentage of
apoptotic cells were determined using a FACS calibur flow cytometer.
Ten thousand events were analyzed for each sample. Appropriate gating
was used to select the single-cell population. The same gate was used on
all samples, ensuring that the measurements were made on a standar-
dized cell population. The apoptotic cells were considered to constitute
the sub G0/G1 cell population and percentage of cells in each phase of
cell cycle was calculated (Pozarowski and Darzynkiewicz, 2004).
2.12. Apoptosis assay
Cells (5 × 105) were cultured in 60 mm tissue culture plate and
allowed to settle for 24 h before treatment with Zn2L2. Cells were then
incubated with FITC labeled Annexin V and propidium iodide (PI) at
room temperature for 15 min in the dark and analyzed using a FACS
Calibur flow cytometer (Vermes et al., 1995).
2.13. Intracellular ROS accumulation study
Cells (5 × 105) were cultured in 35 mm tissue culture plate and
allowed to settle for 24 h and then the cells were treated with complex
Zn2L2 and incubated at 37 °C, 5% CO2 for 1h–6 h or left untreated. The
cells were then washed with PBS and further incubated with H2-DCFDA
(2′, 7′ dihydrodichlorofluorescin diacetate) for 30 min at 37 °C in dark.
After incubation, cells were washed twice in PBS at room temperature
for 5 min each time. The fluorescence was measured at excitation and
emission wavelengths of the oxidized form at 504 nm and 529 nm re-
spectively (Ganguly et al., 2011).
2.14. Determination of intracellular GSH contents
Determination of cellular GSH content was performed by a mod-
ification of a previously reported method (Sedlak and Lindsay, 1968).
Cells (5 × 105) were cultured (as stated earlier in the section of ‘cell
cycle analysis’) and then Zn2L2 treated and untreated cells were washed
three times with cold washing buffer (0.1 M sodium phosphate, 5 mM
ethylenediaminetetraacetate [EDTA], pH 7.5) and immediately lysed in
100 mL lysis buffer (0.1% Triton-X, 0.1 M sodium phosphate, 5 mM
EDTA, pH 7.5). Five minutes later, the cell lysates were acidified with
15 μL of 0.1 N HCl, and protein was precipitated with 15 μL of 50%
sulfosalicylic acid. Following centrifugation at 12,000g for 15 min, su-
pernatants were collected for GSH measurement. The supernatants
were assayed for GSH content by spectrophotometric determination of
the reduction of DTNB to 5′- thio-2-nitrobenzonic acid at 412 nm wave
length.
A. Banerjee et al.
2.15. Peripheral blood mononuclear cell (PBMC) isolation and culture
conditions
Heparinized peripheral blood of human was collected from healthy
volunteers and diluted with equal volume of RPMI 1640. Blood drawn
from healthy volunteers for the isolation of PBMC are done with prior
approval of the Institutional Ethics Committee (IEC). The experimental
protocols [IEC Ref: CNCI/IEC-June-2011(3) dated 24/06/2011] de-
scribed herein were approved by the IEC (certified by the Department
of Clinical Biochemistry, Hospital Unit, Chittaranjan National Cancer
Institute) in accordance with the ethical guidelines laid down by the
committee for the purpose of Control and Supervision of Experiments
with human subjects by the Ministry of Social Justice and
Empowerment, Government of India. Informed consent for participa-
tion in the study was obtained from participants. The participants
stated that, ‘they have been fully explained about the nature and pur-
pose of the study by the supervisor and they agreed upon to participate
in the present study’. They have also been given the opportunity to
question about various aspects of the present study (Volunteer’s profile
given in Supplementary Table 1). Lymphocyte-enriched mononuclear
cells were isolated by Histopaque 1077 density gradient centrifugation
of diluted blood which was washed and finally re-suspended in cold
RPMI 1640 medium and supplemented with 10% heat inactivated FBS.
2.16. Statistical analysis
All data reported in this communication are the arithmetic mean
from three independent experiments performed in triplicate
mean ± S.D. unless stated otherwise. The unpaired Student’s t-test was
used to evaluate the significance of differences between groups, ac-
cepting P < 0.05 as a level of significance. Data analyses were per-
formed using the Prism software (GraphPad, San Diego, CA).
3. Results
3.1. Chemical characterization
The Schiff-base ligand (H2L) and its Zn2L2 were synthesized in situ
and characterized through routine physicochemical techniques. UV
spectral bands of ligand H2L (Supplementary Figure S1) and zinc
complex Zn2L2 (Supplementary Figure S2) was recorded in dimethyl
sulpoxide (DMSO) and acid-water respectively. No absorbance in the
visible region was observed for both H2L and Zn2L2. Important UV
bands were observed at 236, 255, 279 and 338 nm. The λ values in the
UV region were due to π-π or n-π* transition of electrons in the com-
pound. For Zn2L2 UV bands were observed at 270 nm (π → π*) and 340
(S → Zn) nm and shifted to towards higher wave length i.e lower energy
indicating complex formation. The sharp peak in the IR spectrum of the
ligand H2L at 1637 cm−1 (Supplementary Figure S3) is characteristic of
the azomethine functionality and the band at 1484 cm−1 is due to
skeletal vibration of the aromatic moiety. A broad weak band at
2550 cm−1 corroborates with the presence of thiol group. A band at
1279 cm−1 is observed due to stretching of phenolic CeO bond. In the
IR spectrum of Zn2L2 (Supplementary Figure S4) the νC]N character-
istic stretching band of the ligand at 1637 cm−1 shifted to a lower
1626 cm−1 possibly due to zinc complex formation. Skeletal vibrations
observed at1445 cm−1. The broad weak band due to presence of thiol
group disappeared in complex may be indicative of coordination of
thiolate sulphur with zinc. Band observed at 3390 cm−1 indicates the
existence of water molecule in the complex (Nakamoto, 2009). Mass
spectral study of H2L dissolved in DMSO showed formation of peak at
256.3 (calculated m/z: 256 amu) which corroborate well with the re-
spective mono-positive species of formulation [LH]+ (Supplementary
Figure S5). The Mass spectral study of Zn2L2 was performed in acid-
water. Formation of the peak at 637 (calculated m/z: 636 amu), cor-
roborate well with the mono-positive species of formulation [M+1] +
386
Environmental Toxicology and Pharmacology 56 (2017) 383–392
Fig. 1. Plausible structure of the ligand H2L and its zinc complex Zn2L2(H2O)2.
(Supplementary Figure S6). The plausible structure of the ligand and its
zinc complex is given in Fig. 1.
3.2. 1H NMR spectrum
The ligand, 2-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-3-mer-
capto-propionic acid (H2L) contains only 12 protons [3 non-equivalent
aromatic protons; 2 equivalent methelene protons; 3 non-equivalent
methoxy protons; 2 equivalent acetylene protons; 1 phenolic-OH
proton; 1 thiol proton & 1 carboxylic proton] (Fig. 1). However, proton
NMR peaks for the ligand records only 12 protons instead of 13 protons
[3 non-equivalent aromatic protons; 2 methelene protons; 3 non-
equivalent methoxy protons; 2 equivalent HCN protons; 1 phenolic-OH
proton & 1 thiol proton] (Supplementary Figure S7). The carboxylic
acid proton remains unascertained. Proton NMR peak of the ligand in
DMSO-d6 recorded at δ 6.70–6.95 (s, 3H) for 3 non-equivalent aromatic
protons. Two equivalent eCH2 protons recorded at δ 3.55 (s, 2H). Three
non-equivalent eOCH3 protons recorded at 3.10–3.20 (m, 3H). One
phenolic-OH proton recorded at δ 5.64 (s, H). Furthermore, two
equivalent HCN protons recorded at δ 2.50–2.57 (s, 2H) and one thiol
(eSH) proton recorded at δ 2.26 (m, H) following experimental ana-
lysis. On the other hand, in the spectrum of the ligand, we could not
ascertain the carboxylic acid (normal range δ 10.1–11.3) proton peaks.
However, based on other elemental analysis (Mass, IR etc.), we have
established the presence of eCOOH group in the ligand. Proton NMR
peak of Zinc complex of the ligand (Zn2L2) in DMSO-d6 (Supplementary
Figure S8) recorded at δ 6.3–6.53 (m, 3H) for 3 non-equivalent aro-
matic protons. Two equivalents eCH2 protons recorded at δ 3.43.
eOCH3 protons recorded at δ 3.6–3.8 (m, 3H). Furthermore, the HCN
protons in the complex recorded at δ 2.45–2.53 (s, 2H) and carboxylic
acid proton at δ 10.26 (m, H). In NMR spectrum of the complex we
observe a shift of electron density from the ligand to metal moiety.
Aromatic protons shift from the range of δ 6.70–6.95 in the ligand to δ
6.30–6.53 in the complex. Methelene protons shift from δ 3.55 in the
ligand to δ 3.43 in the complex. This is an indication of considerable
drift of electrons from two neighbouring groups viz. eN]C and
eCOOH to the metal moiety. The shielding of the aromatic and me-
thelene protons indicates the coordination of the ligand to metal
moiety, i.e. formation of Zn2L2 complex. However, the methoxy protons
A. Banerjee et al. Environmental Toxicology and Pharmacology 56 (2017) 383–392

(eOCH3) in the ligand shifted to lower field; from ligand 3.10–3.20 to
3.6–3.8 in the complex due to deshielding of protons. This indicates the
participation of (CH3eC]Ne) group in coordination with zinc atom.
Probably methoxy (eOCH3) protons were not pulled from ligand to
metal moiety. Furthermore, data revealed that one phenolic-OH proton
peak and one thiol (eSH) proton peak of the ligand are disappeared in
the complex. This indicates the involvement of phenolic-OH group and
eSH group in the bond formation with metal moiety. In addition to the
DMSO-d6 solvent peak, another peak at 1.89 was observed. This free
proton at 1.89 (d ppm) may be assigned as protons peak of water of
crystallization.
On the basis of these assignments we possibly can infer that the
phenolic-OH group and eSH group are involved in bond formation with
metal moiety as a part of a rigid cyclic system and one water molecule
attached to the rigid structure of Zn2L2 in such a manner that the
protons of the water molecule remain a bit shielded.
3.3. Cytotoxicity (MTT) assay
Cytotoxicity of the ligand H2L and its zinc complex Zn2L2 on human
acute T-lymphoblastic leukaemia cell lines (CEM/ADR5000 and CCRF-
CEM) was performed by using MTT-assay (Fig. 2). Both H2L and Zn2L2
were added in microwells containing the cell culture to attain final
concentrations of 10−8M–10−4M. At different hours (48 h and 72 h)
the cell survival was evaluated by measuring the absorbance in the
visible region. The IC50 (i.e., the concentration of the complex that re-
stricts cell growth to 50%, compared with that of the control experi-
ment) values for CEM/ADR5000 and CCRF-CEM were calculated from
the curves constructed by plotting the cell survival (%) versus the
compound concentration (M). The same MTT experiment was also
performed on normal human PBMC taking the same concentration and
time range. The data indicates that H2L and Zn2L2 did not impart any
significant toxicity on the normal cells at 10−4M or lower doses but
only Zn2L2 (and not H2L) induces death to the malignant cells (Fig. 2).
For this reason, only the Zn2L2 was used for biological evaluation. IC50
values for CEM/ADR5000 and CCRF-CEM were found to be 5 × 10−5M
and 10−4M respectively and were used for further in vitro studies. It is
interesting to note that Zn2L2 induces death to the resistant cell line at a
lower concentration.
3.4. Cell cycle analysis
Flow cytometric assay using propidium iodide (PI) has been widely
used for the evaluation of apoptosis in different experimental models.
The flow cytometric assay of PI stained CCRF-CEM and CEM/ADR5000
cell lines was performed to assess the effect of the zinc complex on the
DNA quantification to identify the cell cycle position. Both cell lines
were treated with concentration corresponds to IC50 values of Zn2L2.
The percentage of cells in each phase of the cell cycle was determined.
It was found that in CCRF-CEM (Fig. 3) the counts of sub diploidal (sub
G1/G0; M1) cells increase (1.53%, 7.73%, 8.69%, 8.39% and 15.43%
for 12 h, 24 h, 48 h, 72 h and 96 h respectively) with respect to un-
treated control (0.55%). The increase in sub G0/G1 (M1) population
represents cells with significant DNA fragmentation pointed towards a
late apoptotic stage. Flow cytometry of CEM/ADR5000 (Fig. 4) re-
vealed an enhanced number of cells in sub G1/G0 population in a time
dependent manner (5.65%, 8.28%, 22.79%, 41.67% and 27.88% for
12 h, 24 h, 48 h, 72 h and 96 h respectively) with respect to untreated

Fig. 2. Cytotoxic effects of ligang H2L and omplex Zn2L2 on (a) CCRF-CEM, (b) CEM/ADR5000 and (c) human PBMC cell lines. Dose response curves for H2L and Zn2L2 were assessed by
MTT assay. Cells were seeded into 96-well plates (4 × 104 cells/well) and allowed to incubate overnight at 37 °C in a 5% CO2 incubator. On next day, cells were treated with increasing
concentrations of H2L and Zn2L2 and incubated for 48 and/72 h. Results were expressed as percentage viability of untreated cells. Value represents the mean ± SD of three independent
experiments with four replicates in each. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test.

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Fig. 3. Cell cycle distribution of CCRF-CEM cells after treatment. CCRF-CEM cells treated with Zn2L2 (IC50) for indicated times were harvested and fixed in 70% ethanol. After staining
with propidium iodide they were analyzed using a flow cytometer.

control (2.26%). A sudden reduction in sub G1/G0 population at 96 h Zn2L2 may have the ability to suppress proliferation of drug resistance
may be attributed to formation cell debris (due to large number of cell cancer cells by blocking cell cycle progression and causing apoptosis.
death) that did not fall within the gated population. Moreover a sig-
nificant increase in number of cells in the G1/G0 (M2) phase of the cell
3.5. AnnexinV-FITC binding assay
cycle from 40.74% (untreated) to 47.24% (12 h) and eventually 54.13%
(24 h) was noted which is indicative of G1/S arrest of cell cycle. Thus
To further confirm and evaluate the induction of apoptosis, we

Fig. 4. Cell cycle distribution of CEM/ADR5000 cells after treatment. CEM/ADR5000 cells treated with Zn2L2 (IC50) for indicated times were harvested and fixed in 70% ethanol. After
staining with propidium iodide they were analyzed using a flow cytometer.

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Fig. 5. Cell cycle distribution of (A) CCRF-CEM and (B) CEM/ADR5000 cells following treatment. Cells were treated with Zn2L2 (IC50) for indicated times were harvested and fixed in
70% ethanol. After staining with propidium iodide they were analyzed using a flow cytometer.
stained cells with annexin V-FITC and PI. Flow cytometric analysis of
dual stained (annexin V-FITC and PI) CCRF-CEM (Fig. 5a) treated with
Zn2L2 at different time intervals (12 h, 24 h and 48 h) exhibited a
progressive decrease (13.10%, 10.31% and 7.89%) in the annexin V-
FITC positive population of cells (untreated control;2.32%) and also
showed a gradual increase in dual positive population (86.90%,
89.64%, 92.01% for 12 h, 24 h and 48 h respectively) as compared to
untreated control (0.83%). These results suggest that the complex set
CCRF-CEM cells to death through induction of apoptosis. Same ex-
periment when performed on the resistant counterpart CEM/ADR5000
(Fig. 5b) showed same trend that is, annexin V-FITC positive population
diminishes gradually (53.02%, 42.56%, 28.55% for 12 h, 24 h and 48 h
respectively) and there is a steady raise in dual positive population
(44.59%, 55.49%, 72.85% for 12 h, 24 h and 48 h respectively) with
compared to untreated control (1.45%). Thus the raise in the above said
population depicted that Zn2L2 induces apoptosis also to CEM/
ADR5000 cells. In both cases the drop in annexin-FITC positivity value
indicates that the cells were entered into late apoptotic stage.
3.6. Intracellular ROS accumulation study
In cancer cells excess ROS generation can serve as an endogenous
source of DNA-damaging agents that can promote apoptosis. This ob-
servation encouraged us to enquire whether the intra cellular ROS
generation is involved in Zn2L2 mediated apoptosis. We measured in-
tracellular ROS (viz, hydroxidyl, peroxyl) using oxidation sensitive
fluorescence dye DCFDA in CCRF-CEM and CEM/ADR 5000 cell lines
(Fig. 6). The synthesized zinc complex is capable of producing ROS in
cancerous cells. It was found that the complex increases ROS within the
cells. Accumulation of ROS in experimental cell lines was observed and
sustained up to the 6th h as compared to untreated control. The results
suggested that these elevated levels of ROS (at a given experimental
time frame) may be involved in promotion of apoptosis.
3.7. Determination of intracellular GSH content
We measured intracellular GSH in CCRF-CEM and CEM/ADR 5000
cell lines, following drug treatment (Fig. 7). Zn2L2 increases GSH level
significantly within the CEM/ADR 5000 cells. In its sensitive counter-
part CCRF-CEM it down regulates GSH level in early hours whereas in
late hours non-significant increase of GSH level is observed (up to the
6th h) as compared to untreated control.
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Environmental Toxicology and Pharmacology 56 (2017) 383–392
3.8. In vivo toxicity
A standardized study on the sub acute systemic toxicity (14 days) of
Zn2L2 was performed on female Swiss albino mice taking as a model
system. In preliminary trials the complex administrated at different
doses intra-peritoneally in mice. Results indicated that the animals at
different dose groups (up to 100 mg kg−1 of body weight) did not ex-
hibit any pathological changes as compared to untreated (control)
group of animals. Any changes in biochemical parameters such as, le-
vels of serum glutamate ortho-transferase (SGOT), serum glutamine
pyruvate transaminase (SGPT), alkaline phosphatase (ALP) (indicative
of hepatocellular injury) or creatinine and urea (indicative of impaired
renal function) are indicative of hepatic and/or renal toxicity. No
mortality or toxic symptoms were observed in treated groups with re-
spect to control groups of animals. No significant differences were ob-
served in the body weight gain/loss pattern, organ weight, hematolo-
gical (Table 1) or biochemical parameters (Table 2). Additionally there
was no change in spleen and bone marrow cell count after the treatment
for 14 days (Table 3). The results suggested that the selected doses of
Zn2L2 at concentrations of 20 mg kg−1 bw, 25 mg kg−1 bw,
30 mg kg−1 bw, 50 mg kg−1 bw and 100 mg kg−1 bw are non-toxic.
Although the 100 mg kg−1 bw concentration is non toxic we did not
proceed beyond 50 mg kg−1 bw as at 100 mg kg−1 bw the drug solu-
tion became acidic.
Zn complex caused an increase in life span of Ehrlich ascites carcinoma
bearing mice. The potential of any anti-cancer drug can be judged by its
ability to increase life span. For this purpose female Swiss albino mice
were inoculated through intra peritoneal route with the Ehrlich ascites
carcinoma (EAC). 10 days after development of tumor, the animals
were treated with 20 mg kg−1 of bw, 25 mg kg−1 of bw, 30 mg kg−1 of
bw and 50 mg kg−1 of bw of Zn2L2 and animals were monitored daily
for any discomfort or pain. The results presented in Table 4 showed that
Zn2L2 is able to enhance the survival of each group of treated mice
compared to untreated controls. But among the given doses only
30 mg kg−1 of bw showed an increase in T/C values upto 150%.
Moreover, total volume of ascitic fluid and total packed cell volumes
were also reduced when measured following 14 days treatment
(Table 4).
4. Discussion
The present work is based on our proposition that a coordination
A. Banerjee et al. Environmental Toxicology and Pharmacology 56 (2017) 383–392

Fig. 6. Measurement of Intra-cellular reactive oxygen species (ROS) generation treated with Zn2L2 on (A) CEM/ADR5000 and (B) CCRF-CEM cells. Cells were either kept untreated or
treated with Zn2L2 (IC50) and intra cellular ROS generation was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)], at different time intervals. Data are
expressed as a percentage of the control and are presented as mean ± SD from 3 independent experiments. Differences between the control and treated cells are significant at
*P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test.

Fig. 7. Measurement of Intra-cellular glutathione (GSH) generation of (A) CEM/ADR5000 and (B) CCRF-CEM cells after treatment with Zn2L2. Cells were either kept untreated or treated
with Zn2L2 (IC50) and intra cellular GSH generation was measured at indicated time points. Data are expressed as a percentage of the control and are presented as mean ± SD from 3
independent experiments. Differences between the control and treated cells are significant at *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test.

Table 1
Effect of Zn2L2 on haematological parameters of normal female Swiss albino mice.

Parameters Untreated Vehicle Control 20 mg/Kg 25 mg/Kg 30 mg/Kg 50 mg/Kg 100 mg/Kg

3
WBC (x10 /μL) 6.1 ± 0.7 6.9 ± 0.77ns 6.5 ± 0.72ns 8.1 ± 0.8ns 14.0 ± 3.0* 6.2 ± 0.7ns 8.5 ± 0.82ns
RBC (x106/μL) 10.3 ± 1.1 9.5 ± 0.85ns 9.92 ± 0.89ns 9.91 ± 0.89ns 10.41 ± 1.2ns 9.08 ± 0.9ns 9.56 ± 1.0ns
HGB (g/dL) 15.8 ± 3.1 14.4 ± 3.0ns 14.4 ± 2.9ns 13.5 ± 2.8ns 15.5 ± 2.9ns 14.6 ± 2.6ns 14.6 ± 2.7ns
HCT (%) 52.1 ± 10.2 48.6 ± 8.8ns 50.3 ± 9.7ns 48.1 ± 9.8ns 55.0 ± 11.2ns 47.5 ± 8.8ns 49.5 ± 9.3ns
MCV (fL) 50.8 ± 9.9 51.2 ± 10.0ns 50.7 ± 9.9ns 48.5 ± 9.1ns 52.8 ± 10.4ns 52.3 ± 10.1ns 51.8 ± 10.2ns
MCH (pg) 15.4 ± 2.9 15.2 ± 3.0ns 14.5 ± 2.4ns 13.6 ± 2.0ns 14.9 ± 2.7ns 16.1 ± 3.5ns 15.3 ± 2.9ns
PLT (x103/μL) 897 ± 89 954 ± 93* 868 ± 52.2ns 1057 ± 112* 656 ± 72** 350 ± 31*** 1262 ± 132***
LYM# (x103/μL) 5.2 ± 0.52 5.6 ± 0.53ns 5.8 ± 0.56ns 6.9 ± 0.78ns 8.6 ± 0.86* 5.5 ± 0.56ns 7.0 ± 0.72ns
RDW (fL) 29.9 ± 5.7 27.4 ± 5.2ns 28.7 ± 5.6ns 31.6 ± 6.0ns 29.1 ± 5.5ns 27.3 ± 5.1ns 28.3 ± 5.4ns
PDW (fL) 7.4 ± 0.8 8.0 ± 0.86ns 6.8 ± 0.75ns 7.5 ± 0.83ns 7.4 ± 0.80ns 7.2 ± 0.82ns 7.2 ± 0.83ns
MPV (fL) 6.1 ± 0.62 6.6 ± 0.68ns 6.0 ± 0.65ns 6.2 ± 0.70ns 6.5 ± 0.68ns 6.3 ± 0.64ns 6.3 ± 0.67ns
P-LCR (%) 2.7 ± 0.3 4.7 ± 0.57* 4.0 ± 0.51* 3.3 ± 0.43ns 5.9 ± 0.60* 5.2 ± 0.56* 4.6 ± 0.54*

Data are expressed as a percentage of the control and are presented as mean ± SD from 3 independent experiments. Differences between the control and treated cells are significant at
*P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test, ns: not significant.

Table 2
Effect of Zn2L2 on biochemical parameters (hepatic and renal functions) of normal female Swiss albino mice.

Parameters Untreated Vehicle Control 20 mg/Kg 25 mg/Kg 30 mg/Kg 50 mg/Kg 100 mg/Kg

Urea (mg/dL) 75 ± 6.9 84 ± 8.0ns 62 ± 5.8ns 60 ± 5.8* 41 ± 3.8* 82 ± 7.9ns 108 ± 11.0*
Creatinine (mg/dL) 0.3 ± 0.02 0.4 ± 0.05ns 0.3 ± 0.04ns 0.4 ± 0.03ns 0.4 ± 0.03ns 0.3 ± 0.02ns 0.4 ± 0.032ns
Alkaline Phosphatase (U/L) 179 ± 18.8 282 ± 29.3* 299 ± 30.1** 324 ± 31.4** 217 ± 22.5* 140 ± 13.7ns 200 ± 19.8*
SGOT (U/L) 212 ± 22.4 261 ± 25.8* 164 ± 17.5ns 206 ± 19.2ns 278 ± 26.9* 176 ± 16.6* 57 ± 6.2***
SGPT (U/L) 75 ± 7.8 100 ± 10.7* 71 ± 6.9ns 80 ± 7.8ns 76 ± 7.4ns 259 ± 24.7*** 79 ± 7.7ns

Data are expressed as a percentage of the control and are presented as mean ± SD from 3 independent experiments. Differences between the control and treated cells are significant at
*P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test, ns: not significant.

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Table 3
Effect of Zn2L2 on spleen and bone marrow count of normal female Swiss albino mice.

Parameters Untreated Vehicle Control 20 mg/Kg 25 mg/Kg 30 mg/Kg 50 mg/Kg 100 mg/Kg

Spleen Cells (% of Viable Cells) 93.7 ± 1.56 93.9 ± 2.6ns 93.1 ± 2.6ns 93.9 ± 2.58ns 93.3 ± 2.6ns 94.5 ± 2.52ns 94.4 ± 2.5ns
Bone Cells (% of Viable Cells) 97.7 ± 2.3 97.9 ± 2.1ns 97.8 ± 3.4ns 98.0 ± 2.23ns 98.1 ± 3.21ns 98.2 ± 3.15ns 98.1 ± 3.2ns

Data are expressed as a percentage of the control and are presented as mean ± SD from 3 independent experiments. Differences between the control and treated cells are significant at
*P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test, ns: not significant.

Table 4
Effect of Zn2L2 on survival of EAC bearing female Swiss albino mice.

Parameters Untreated 20 mg/Kg 25 mg/Kg 30 mg/Kg 50 mg/Kg

Total volume (mL) 11.04 ± 1.1 9.01 ± 0.80ns 7.99 ± 0.81* 8.97 ± 0.74** 13.2 ± 0.12ns
PCV (mL) 6.5 ± 0.56 5.33 ± 0.46ns 4.73 ± 0.41* 5.30 ± 0.53** 7.81 ± 0.82*
MST (d) 28 38* 37* 42** 26ns
T/C (%) 100 136* 132* 150** 92.9ns

Data are expressed as a percentage of the control and are presented as mean ± SD from 3 independent experiments. Differences between the control and treated cells are significant at
*P < 0.05, **P < 0.01, ***P < 0.001, by unpaired Student’s t-test, ns: not significant.

complex of zinc may be less toxic and potent anticancer agent if a
suitable cysteine-based moiety can be attached to zinc atom [34–36].
Under this backdrop we selected the non-toxic ligand, 2-[(2-Hydroxy-3-
methoxy-benzylidene)-amino]-3-mercapto-propionic acid that does not
shows any significant cytotoxicity towards malignant cells. The Schiff-
base ligand and its zinc complex have been synthesized as well as
characterized through details spectroscopic studies from which we
found that the assigned ligand to metal charge transfer (S → Zn LMCT)
band may be responsible for the yellow colour of the complex
(Supplementary Figure S1–S8). Their anti cancer properties have been
evaluated. Cytotoxicity assay (MTT assay) of H2L and Zn2L2 on both
CCRF-CEM and CEM/ADR 5000 cell lines shows that Zn2L2 but not H2L
has the anti-proliferative effect against these malignant cell lines. Zn2L2
proves to be non toxic (MTT assay) towards non-malignant PBMC.
Zn2L2 is found to be more effective at a comparatively lower con-
centration on CEM/ADR 5000 and therefore can be used as potent re-
sistant modifying agent (RMA). Cell cycle analysis and annexin V
binding assays revealed that Zn2L2 induces apoptosis in both the cell
lines. In CEM/ADR5000, the Zn2L2 induces apoptosis via G1/S block as
revealed by Cell cycle analysis. Zn2L2 significantly alters cellular ROS
and GSH level in CCRF-CEM and CEM/ADR 5000 with respect to un-
treated control. Therefore we assume that in CCRF-CEM apoptosis is
triggered by generation of oxidative stress that elevates the cellular ROS
level and simultaneously depletes the cellular GSH level. In doxorubicin
resistant CEM/ADR5000, Zn2L2 increases both cellular ROS and GSH.
The elevated ROS level subsequently causes oxidative stress and en-
hances GSH level, which consequently sensitizes the cells towards drug
and ultimately induces apoptosis (Chen et al., 2008). Finally the results
of in vivo sub acute systemic toxicity studies performed on female Swiss
albino mice suggests that Zn2L2 does not impart any significant toxic
symptoms up to a dose of 100 mg/kg of body weight. It is also worth
noting that the Zn2L2 at a dose of 30 mg kg−1, increase the life span of
Ehrlich ascites carcinoma (EAC) bearing mice up to T/C (%) value
150% when compared to untreated control. All these doses showed
“marked activity” according to standard National Cancer Institute
protocols for screening new anticancer drugs. Moreover, Zn2L2 treat-
ment up to 50 mg/kg of body weight exhibited no significant toxicity on
normal female Swiss albino mice. Furthermore, we are trying to deci-
pher the underlying molecular mechanisms of Zn2L2-induced apoptosis.
Additionally, detailed animal experimentation on the reported complex
will provide clinically relevant information in developing possible po-
tent anticancer agent.
5. Conclusions
This newly synthesized Zn-complex appears to be a promising
chemotherapeutic agent that specifically targets malignant cells and
overcomes multidrug resistance in cancer without inducing any sig-
nificant undesired side effects in normal cells as well as in normal mice
model.
Conflicts of interest
None.
Acknowledgements
This work was supported by grants from Council of scientific and
industrial research, India (Award No. 09/030/(0061)/2011 EMR-I) and
Indian Council of Medical Research (ICMR), New Delhi, India [Award
No. 74/10/2014-PERS. (EMS)] for financial support. The funder had no
role in the study design, data collection and analysis, decision to pub-
lish or preparation of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.etap.2017.11.004.
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