Absence of genotoxic effects of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) and its potential chemoprevention against DNA damage using in vitro and in vivo assays

Article abstract of DOI:10.1371/journal.pone.0171224

The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, displays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to investigate the cytotoxic, genotoxic, mutagenic, and protective effects of

Full text of DOI:10.1371/journal.pone.0171224

RESEARCH ARTICLE
Absence of genotoxic effects of the chalcone
(E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-
prop-2-en-1-one) and its potential
chemoprevention against DNA damage using
in vitro and in vivo assays
1☯
Debora Cristina da Silva Lima1☯, Camila Regina do Vale1☯, Jefferson Hollanda Veras
,
´
´
Aline Bernardes2☯, Caridad Noda Perez2☯, Lee Chen-Chen1☯
*
´
´
1 Department of Genetics, Institute of Biological Sciences, Federal University of Goias, Goiania, GO, Brazil,
ˆ
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
´
2 Institute of Chemistry, Federal University of Goias, Goiania, GO, Brazil
ˆ
☯ These authors contributed equally to this work.
* chenleego@yahoo.com.br
Abstract
The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, dis-
plays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to
investigate the cytotoxic, genotoxic, mutagenic, and protective effects of 2HMC using the
Ames mutagenicity test, the mouse bone marrow micronucleus test, and the comet assay in
mice. In the assessment using the Ames test, 2HMC did not increase the number of His+
revertants in Salmonella typhimurium strains, demonstrating lack of mutagenicity. 2HMC
showed no significant increase in micronucleated polychromatic erythrocyte frequency
(MNPCE) in the micronucleus test, or in DNA strand breaks using the comet assay, evi-
dencing absence of genotoxicity. Regarding cytotoxicity, 2HMC exhibited moderate cytotox-
icity in mouse bone marrow cells by micronucleus test. 2HMC showed antimutagenic action
in co-administration with the positive controls, sodium azide (SA) and 4-nitroquinoline-1-
oxide (4NQO), in the Ames test. Co-administered and mainly pre-administered with cyclo-
phosphamide (CPA), 2HMC caused a decrease in the frequency of MNPCE using the
micronucleus test and in DNA strand breaks using the comet assay. Thus, 2HMC exhibited
antimutagenic and antigenotoxic effects, displaying a DNA-protective effect against CPA,
SA, and 4NQO carcinogens. In conclusion, 2HMC presented antimutagenic, antigenotoxic
and moderate cytotoxic effects; therefore it is a promising molecule for cancer prevention.
OPEN ACCESS
´
Citation: Lima DCdS, Vale CRd, Veras JH,
´
Bernardes A, Perez CN, Chen-Chen L (2017)
Absence of genotoxic effects of the chalcone (E)-1-
(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-
1-one) and its potential chemoprevention against
DNA damage using in vitro and in vivo assays.
PLoS ONE 12(2): e0171224. doi:10.1371/journal.
pone.0171224
Editor: David L. McCormick, IIT Research Institute,
UNITED STATES
Received: September 26, 2016
Accepted: January 17, 2017
Published: February 16, 2017
Copyright: © 2017 Lima 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.
Introduction
Data Availability Statement: All relevant data are
within the paper.
Chalcones (1,3-diphenyl-2-propen-1-one) are naturally occurring aromatic ketones, consist-
ing of an α,β-unsaturated carbonyl system joining two aryl rings [1]. Chalcone is a class of
open-chain flavonoids, abundantly found in plants of the families Leguminosae, Compositae,
and Moraceae [2], and present in fruits, vegetables, grains, roots, flowers, tea, and wine, prod-
ucts regularly used for human consumption [3].
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Significant progress has been made over the past few years in the research of chalcones not
only biosynthesized by plants, but also synthetically prepared. 2’-hydroxychalcones, known for
their effectiveness in gene expression and enzyme activity [4,5], are the most important inter-
mediates in the synthesis of flavonoids and flavones [6,7]. This group of chalcones also exhibits
remarkable anticancer [8] and antioxidant [9] activities, besides having potential as a neuro-
protective and analgesic agent, as well as a cognitive enhancer [10]. So far, a large variety of 2’-
hydroxychalcone compounds have been synthesized and studied aiming to obtain promising
therapeutic agents.
The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC,
has already been shown to have potential pharmacological applications as an antileishmanial
and antimalarial agent [11,12]. It has also been considered a promising molecule for anticancer
therapy, since it has been proven to have antiproliferative activity against MCF-7 human breast
cancer cells [13], to inhibit cathepsin B (protease overexpressed in human primary tumors)
[14], and to inhibit the endogenous tumor promoter prostaglandin-E2 (PGE2) in the murine
macrophage cell line RAW 264.7 [15]. Given the pharmacological and therapeutic potential of
2HMC, this compound has attracted great interest. Therefore, it is of great importance to
investigate its mutagenic, genotoxic, and protective effects against DNA damage.
Several in vitro and in vivo tests have been combined to evaluate the mutagenic/genotoxic
activity of numerous compounds. The combination of these tests has been generally employed
due to the possibility of using various target organs in the same animal as well as the feasibility
of evaluating different genetic endpoints [16]. Among the most used tests in genetic toxicol-
ogy, the Ames mutagenicity test (in vitro), the mouse bone marrow micronucleus test, and the
comet assay (in vivo) stand out.
The Ames mutagenicity test uses several different tester strains of Salmonella typhimurium
to measure two classes of gene mutation, namely base pair substitution and frameshifts [17].
The micronucleus test is widely used to detect clastogenicity and aneugenicity of chemicals
[18]. Another recommended test is the comet assay, also called the single cell gel electrophore-
sis assay, which can determine DNA strand breaks after in vivo exposure to the test compound
[19].
Compounds that prevent mutagenic or genotoxic events affecting DNA can act by different
mechanisms. Some compounds reduce the mutagenic or genotoxic effect by directly interact-
ing with the mutagens or by blocking their effects through the inhibition of their metabolic
activation or the enhancement of their detoxifying enzymes (usually detected in pre-treatment
or co-treatment). Other substances act after the damage has already taken place by promoting
DNA repair (which can be detected in post-treatment), increasing the fidelity of DNA replica-
tion, inhibiting error prone replication, or suppressing the growth and replication of cells with
damaged DNA [20]. Thus, depending on the type of treatment, it is possible to suggest differ-
ent mechanisms of action to a test compound.
Considering the promising capacity of 2HMC in cancer therapy and its several pharmaco-
logical activities, the aim of the present study was to evaluate the mutagenic/genotoxic and
antimutagenic/antigenotoxic effects of this compound using the Ames mutagenicity test in S.
typhimurium strains, the micronucleus test, and the comet assay in mice.
Materials and methods
Chemicals
All commercially available reagents were purchased from Sigma-Aldrich (St. Louis, MO,
USA). 1H NMR (Proton Nuclear Magnetic Resonance) and 13C NMR (Carbon-13 Nuclear
Magnetic Resonance) spectra were recorded on a Bruker Avance III (500 MHz) spectrometer
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Potential chemoprevention of chalcone in in vitro and in vivo assays
(Bruker Optik GmbH, Ettlingen, Germany). Infrared (IR) spectra were performed on a
Bomem M102 spectrometer (Bomem Inc., Vanier, Quebec, Canada). The melting point was
measured using a melting point apparatus (Karl Kolb GmbH & Co., Dreieich, Germany). The
purity of the compound was determined by high performance liquid chromatography (HPLC)
using the Waters Alliance–2695 apparatus, XTerra C18 (5 μm, 4.6 mm × 150 mm) column,
and Waters 2998 Photodiode Array (PAD) detector at 330 nm for quantification of chalcone
2HMC. The injector was programmed to inject a volume of 10 μL. The mobile phase was
CH3CN:aqueous buffer containing 0.1% trichloroacetic acid (55:45).
Synthesis of chalcone 2HMC (1)
The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, was
´
synthesized in the Institute of Chemistry, at Federal University of Goias, Goiania, GO, Brazil.
ˆ
The synthesis of the chalcone is outlined in Fig 1.
Chalcone 2HMC was synthesized by Claisen-Schmidt condensation as previously described
in the literature [11,21] with small modifications. Equimolar portions of o-hydroxyacetophe-
none (1.0 mmol) and p-methylbenzaldehyde (1.0 mmol) were added to 30 mL 50% NaOH eth-
anol solution (w/w). The reaction mixture was stirred at room temperature for 12 h, poured
into cold water (6 mL), and acidified with 10% hydrochloric acid (w/v) to pH 3, forming a yel-
low solid compound, which was crystallized with chloroform. The melting point, IR (Infrared),
13C NMR, and H NMR are in agreement with previous reports [11,14].
(E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one
Yellow crystals yielded 67%. Melting point: 112–115˚C. IR (KBr) cm-1: 3436 (ν OH), 1647 (ν
CO), 1564 [ν C = C (sp2)]. H NMR (Hz–DMSO-d6) δ: 12.61 (s; 1H; OH), 8.26 (dd; 1H; J =
8.47; J = 1.75; H6’), 7.99 (d; 1H; J = 15.47; Hβ), 7.83 (d; 1H; J = 15.47; Hα), 7.81 (d; 2H; J = 8.17;
H2, H6), 7.57 (ddd; 1H; J = 8.17; J = 7.30; J = 1.75; H4’), 7.29 (d; 2H; J = 8.17; H3, H5), 7.01 (m;
2H; H5’, H3’). 13C NMR (DMSO-d6) δ: 192.93 (CO), 161.99 (C2’), 145.07 (Cβ), 141.30 (C4),
136.36 (C4’), 131.78 (C1), 129.69 (C6’), 129.30 (C2 and C6), 120.73 (C1’), 120.60 (C5’), 119.23
(Cα), 117.80 (C3’), 21.18 (CH3). Puritiy of 98.86% in HPLC [retention time = 8.604 min;
CH3CN: Cl3CCOOH buffer (55:45)]. A coupling constant of 15.47 Hz for the vinyl H-atoms in
the 1H NMR spectrum confirmed the (E)-configuration.
Doses of 2HMC used in in vitro and in vivo assays
In the Salmonella mutagenicity test (Ames test), the doses of 2HMC used were 1, 10, 50, 100,
500, and 1000 μg/plate. These six doses used in our study cover a range from 1 to 1000 μg/plate
in accordance to Mortelmans and Zeiger [22], which recommend a minimum of five doses
and a range of at least three logs.
The doses of 25 and 50 mg/Kg of 2HMC used in the animal testing were based on previous
studies in mice that used hydroxichalcones for biological activity evaluations [23, 24].
Dimethylsulfoxide (DMSO) was used to dissolve 2HMC and it was used as negative control in
all performed assays.
Fig 1. Synthetic route of chalcone 2HMC (1). Reagents and conditions: (a) 50% NaOH (w/w), EtOH, 12 h
(room temperature).
doi:10.1371/journal.pone.0171224.g001
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Ames test: Salmonella mutagenicity assay
Strains. Salmonella typhimurium tester strains TA98 (hisD3052) (rfa) (uvrB) (AmpR) and
TA100 (hisG46) (rfa) (uvrB) (AmpR) [25] were kindly supplied by Dr. Bruce N. Ames, from
the University of California (Berkeley, CA, United States).
Experimental procedure. The S. typhimurium histidine point mutation assay was fol-
lowed [22]. A 0.1-mL aliquot of bacterial suspension (1–2 × 109 cells/mL) of each strain (TA98
and TA100) was incubated with 1, 10, 50, 100, 500, and 1000 μg/plate 2HMC at 37˚C for 25
min. A 2.0-mL aliquot of top agar [0.6% agar (lot no. 082613209, Kasvi, Curitiba, PR, Brazil),
0.5% NaCl, 50 μM L-histidine, and 50 μΜ biotin, at 45˚C] was added to the test tubes and
poured onto Petri dishes containing minimal agar medium [1.5% agar (lot no. 082613209,
Kasvi, Curitiba, PR, Brazil), 2% glucose, and Vogel-Bonner medium E (MgSO4H2O, lot no.
1109865; C6H8O.7H2O, lot no. 1100795; K2HPO2, lot no. DCBB4100; Na2NH2PO4.H2O, lot
´
no. 1208265, Vetec Quımica Fina Ltda., Duque de Caxias, RJ, Brazil)]. Each assay was per-
formed three times in triplicate and included a negative control [20 μL dimethylsulfoxide
ˆ
´
ˆ
(DMSO, lot no. 77320, Dinamica Quımica Contemporanea Ltda., Diadema, SP, Brazil)] and a
positive control [0.5 μg 4-nitroquinoline-1-oxide (4NQO, lot no. SLBD6960V, Sigma-Aldrich,
São Paulo, SP, Brazil) per plate for TA98 and 3.0 μg sodium azide (lot no. 26628–22–8, Merck,
Cotia, SP, Brazil) for TA100]. For the antimutagenicity evaluation, the same doses of 2HMC
employed in the mutagenic evaluation were co-treated with their respective positive controls.
After incubation at 37˚C for 48 h, His+ revertant colonies were counted.
Statistical analysis. The software Sigma Stat 3.5 was used in all analyses. The results were
expressed as mean standard deviation (SD). Mutagenicity results were evaluated using analy-
sis of variance (ANOVA) and the Tukey’s test for differences among the means. Mutagenicity
induction was measured by the mutagenic index (MI), calculated as the ratio between the
number of colonies in the test treatment and the number of colonies in the negative control
treatment. A substance is considered mutagenic when MI ꢀ 2. In order to evaluate the antimu-
tagenicity, the inhibition percentage of mutagenicity (IP) induced by the mutagen was calcu-
lated using the following formula [26]:
ꢂ
ꢀ
ꢁꢃ
TP À SR
IP ð%Þ ¼ 1 À
X 100
PCP À SR
where:
TP: number of His+ revertants on test plates (plates incubated with mutagen and test
compound)
SR: spontaneous His+ revertants on negative control plates (tester strains incubated in the
absence of test compound and mutagen)
PCP: number of His+ revertants on positive control plates (plates incubated with the muta-
gen alone)
Animal testing. This study was approved by the Animal Research Ethics Committee of
´
the Federal University of Goias (CEUA/UFG no. 013/14). Healthy, young male adult (8–12
weeks) outbred mice (Mus musculus, Swiss Webster), weighing 25–30 g, obtained from the
animal facilities of the same university, were brought to the laboratory five days before the
experiments and housed in plastic cages (40 cm × 30 cm × 16 cm), lined with wood shavings,
kept at 24 2˚C and 55 10% humidity, with a light-dark natural cycle of 12 h. Standard food
pellets (appropriate commercial rodent diet Labina, Ecibra Ltda., Santo Amaro, SP, Brazil) and
water were provided ad libitum.
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Protocols in vivo. The animals were randomized into control and experimental groups,
divided into 10 groups of five animals each group and weighed before the administration of
the chemicals. All the doses of 2HMC were administered orally.
Control. The animals in Group 1 received 0.15 mL DMSO (negative control) adminis-
tered orally, while the animals in Group 2 received 50 mg/kg (body weight) BW cyclophospha-
mide (CPA, lot no. 5L091, Baxter Hospitalar Ltda., São Paulo, SP, Brazil) (positive control) in a
single intraperitoneal administration (ip).
Genotoxicity and cytotoxicity. The animals in Group 3 received a single dose of 50 mg/
kg BW 2HMC (24 h), whereas those in Group 4 received a treatment only with 50 mg/kg BW
2HMC for five consecutive days (120 h).
Co-treatment. The animals in Groups 5 and 6 were respectively treated with 25 and 50
mg/kg BW 2HMC and concomitantly received a single dose of 50 mg/kg BW CPA ip each.
Pre-treatment. The animals in Groups 7 and 8 received 25 and 50 mg/kg BW 2HMC,
respectively, for five consecutive days. On the last day, the animals in both groups received a
single dose of 50 mg/kg BW CPA ip 2 h after the administration of 2HMC.
Post-treatment. The animals in Groups 9 and 10 were treated with a single dose of 50
mg/kg BW CPA ip. After 6 h and 12 h, the animals in Group 9 received 25 mg/kg BW 2HMC,
whereas the animals in Group 10 received 50 mg/kg BW 2HMC.
All the animals were euthanized by cervical dislocation. The ones treated with CPA were
euthanized 24 h after the administration, while those who received 2HMC alone were eutha-
nized 24 h after the last administration of the chalcone. The bone marrow cells from both
femurs of the animals were flushed using fetal calf serum (lot no. 0004, Cultilab Ltda., Campi-
nas, SP, Brazil) and, after centrifugation (300× g, 5 min), the cells pellets were used for prepara-
tion of micronucleus test slides and the comet assay.
Micronucleus test. The micronucleus test was performed according to Von Ledebur and
Schimid [27]. Bone marrow cells prepared as described above were smeared on glass slides,
coded for blind analysis, air-dried, and fixed with absolute methanol (lot no. 1207433 COD:
´
000102.06, Vetec Quımica Fina Ltda., Duque de Caxias, RJ, Brazil) at room temperature for 5
min. The smears were stained with Giemsa (lot no. 13420C, New Prov Ltda., Pinhais, PR, Bra-
zil), dibasic sodium phosphate (lot no. A0339828, Acros Organics, Morristown, NJ, United
´
States), and monobasic sodium phosphate (lot no. 33791/11, Cromoline Quımica Fina, Dia-
dema, SP, Brazil). In the micronucleus test we analyzed 2,000 polychromatic erythrocytes
(PCE) per animal. Five animals were analyzed for each dose, thus a total of 10,000 PCE were
analyzed per dose to determine the frequency of micronucleated polychromatic erythrocytes
(MNPCE) using light microscopy (Olympus BH-2 10 × 100, Tokyo, Japan). Genotoxicity and
antigenotoxicity were assessed by the frequency of MNPCE, whereas cytotoxicity was evalu-
ated by the PCE and normochromatic erythrocytes (NCE) ratio (PCE/NCE), using a total of
10,000 cells for each dose.
Comet assay. The comet assay was performed using the alkaline method with few modifi-
cations [28]. Slides previously coated with normal melting point agarose (1.5%) received a
mixture that contains 15 μl of bone marrow cells and 120 μl of low melting point agarose
(0.75%) at 37˚C. The mixture was spread on the slides with coverslips and taken to a cold
chamber. After gelation, the coverslips were carefully removed. The slides were immersed in
´
lysis solution protected from light [1% triton X-100 (lot no. DCBB3232, Vetec Quımica Fina
ˆ
Ltda., Duque de Caxias, RJ, Brazil), 10% DMSO, 2.5 M NaCl (lot no. 73516, Dinamica Quı-
´
ˆ
mica Contemporanea Ltda., Diadema, SP, Brazil), 100 mM Na2EDTA (lot no. 2965C504, Invi-
trogen by Life Technologies, Itapevi, SP, Brazil), and 10 mM Tris (lot no. 0805008, Vetec
´
Quımica Fina Ltda., Duque de Caxias, RJ, Brazil), pH 10.0] at 4˚C for 12–24 h. Subsequently,
the slides were incubated with freshly made alkaline solution [300 mM NaOH (lot no. 19804,
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Neon Comercial Ltda., São Paulo, SP, Brazil), 1 mM EDTA, pH > 13] at 4˚C for 20 min for
DNA unwinding. The electrophoresis was performed in the same buffer at 300 mA and 25 V/
cm at 4˚C for 30 min. After the electrophoresis, the slides were placed in a staining tray, cov-
ered with a neutralizing buffer (0.4 M Tris-HCl, pH 7.5), stained with ethidium bromide, and
analyzed. For each treated group 500 nucleoids were analyzed,100 nucleoids per animal, using
an epifluorescence Leica DM 2000 Citogen microscope (Leica Microsystems, Wetzlar, Ger-
many), equipped with a Jenoptik ProgRes1 MF camera (Optronics, Goleta, CA, USA), driven
by Lucia CytogeneticsTM version 2.5 software (Laboratory Imaging Ltd, Prague, Czech Repub-
lic), 200 x magnification.
For the assessment of the genomic damage using the comet assay, the OpenCometTM soft-
ware, version 1.3 (Cometbio-OpenComet, Singapore) was employed. Using a novel and robust
method for finding comets based on geometric shape attributes, this open-source software tool
provides automated analysis of comet assay images segmenting the comet heads through
image intensity profile analysis. As a result of automation, it is more accurate, less prone to
human bias, and faster than manual analysis [29]. Since percentage of DNA in the tail (%DNA
in tail) is commonly used in several studies for quantification of DNA damage [30–32], this
parameter was also chosen in our study.
Statistical analysis for the micronucleus test and the comet assay. The genotoxic and
antigenotoxic activities were evaluated using one-way ANOVA followed by the Tukey’s test,
whereas cytotoxicity and anticytotoxicity were analyzed using the chi-square test. For the
comet assay, the average and standard deviation of the %DNA in tail were considered for each
treated group, and the one-way ANOVA test was used to compare the treated groups with
their respective control groups. For genotoxicity and cytotoxicity evaluation, groups 3 and 4
were compared to the negative control (group 1). In the evaluation of antigenotoxicity, groups
5 and 6 (co-treatment), groups 7 and 8 (pre-treatment), groups 9 and 10 (post-treatment),
were compared with the positive control (group 2), in order to evaluate 2HMC effects against
CPA induced damage. The results were considered statistically significant when p < 0.05. The
percentage reduction in CPA-induced genotoxicity was calculated using the following formula
[33,34]:
ꢀ
ꢁ
A À B
A À C
% Reduction ¼
X 100
where:
A: MNPCE mean in CPA treatment (positive control)
B: MNPCE mean in antigenotoxic treatment (chalcone 2HMC + CPA)
C: MNPCE mean in negative control
Results
Ames test: Salmonella mutagenicity assay
The results of the mutagenic and antimutagenic evaluation, from three independent experi-
ments carried out in triplicate, are presented in Table 1. The results obtained for the positive
and negative controls are in agreement with Maron and Ames [25] and Mortelmans and Zei-
ger [22].
In the evaluation of mutagenicity, the doses of 2HMC tested (1, 10, 50, 100, 500, and
1000 μg/plate) did not cause a significant increase in the number of His+ revertant colonies in
either tester strains, TA98 or TA100 (p > 0.05). Furthermore, none of the strains tested
reached MI ꢀ 2 or dose-response effect with 2HMC treatment, with the highest induction
occurring for strain TA98, which reached MI = 0.96 at the dose of 1 mg/plate.
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Table 1. Means ± standard deviation (SD) of histidine revertant colonies (obtained from three independent experiments carried out in triplicate),
mutagenic index (MI), and inhibition percentage of mutagenicity (IP) for two tester strains of Salmonella typhimurium, TA98 and TA100, after treat-
ment with different doses of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) (2HMC).
Treatment
Mutagenicity
Antimutagenicity
TA 98
Mean ± SD
TA 100
TA 98
Mean ± SD
TA 100
MI
Mean ± SD
MI
IP (%)
_
Mean ± SD
IP (%)
_
Negative control1
Positive control2
20.66 ± 1.00A
509.55 ± 18.76
19.88 ± 3.02A
14.77 ± 1.07B
18.99 ± 0.87A
16.21 ± 0.51A
17.10 ± 2.26A
15.33 ± 1.33B
1.00
24.66
0.96
0.71
0.91
0.78
0.82
0.74
143.00 ± 8.71A
2157.66 ± 141.86
129.33 ± 14.64A
86.33 ± 8.96B
78.33 ± 12.22B
84.00 ± 6.08B
77.00 ± 7.55B
68.00 ± 9.64B
1.00
15.08
0.90
0.60
0.54
0.58
0.53
0.47
21.99 ± 0.33
514.10 ± 80.52C
285.33 ± 64.45D
174.10 ± 10.80D
244.55 ± 56.07D
187.66 ± 27.56D
100.88 ± 6.44D
64.99 ± 0.87D
164.66 ± 38.08
2088.33 ± 264.04C
1549.00 ± 25.06D
1508.33 ± 280.57D
1453.00 ± 201.16D
1530.66 ± 112.69D
1443.00 ± 152.67D
1445.33 ± 98.33D
_
_
2HMC 1 μg/plate
2HMC 10 μg/plate
2HMC 50 μg/plate
2HMC 100 μg/plate
2HMC 500 μg/plate
2HMC 1000 μg/plate
46.5
69.1
54.8
66.4
83.9
91.3
28.0
30.2
33.1
29.0
33.6
33.5
1Negative control: 20 μL dimethylsulfoxide (DMSO).
2Positive control: 0.5 μg 4-nitroquinoline-1-oxide (4NQO) per plate for TA98 and 3.0 μg of sodium azide for TA100.
All values are means ± SD of three independent experiments.
Statistical analysis: one-way ANOVA and the Tukey’s test. Mutagenicity:
ANo significant difference in comparison with the negative control (p > 0.05).
BSignificant difference in comparison with the negative control (p < 0.05). Antimutagenicity:
CNo significant difference in comparison with the positive control (p > 0.05).
DSignificant difference in comparison with the positive control (p < 0.05).
doi:10.1371/journal.pone.0171224.t001
The results of the Ames test demonstrated that 2HMC did not exhibit a mutagenic effect.
Tester strains TA98 and TA100 exhibited MI lower than the negative control (MI = 1.00) at all
doses, indicating that this chalcone possibly had a mild cytotoxic effect against S. typhimurium
cells. Based on the results of the antimutagenic evaluation, at all doses tested (1, 10, 50, 100,
500, and 1000 μg/plate) 2HMC showed a significant decrease in the number of His+ revertant
colonies in tester strains TA98 and TA100 compared to the respective positive controls (p <
0.05). At doses of 1, 10, 50, 100, 500, and 1000 μg/plate, the results of IP were 46.5%, 69.1%,
54.8%, 66.4%, 83.9%, and 91.3% for strain TA98, and 28.0%, 30.2%, 33.1%, 29.0%, 33.6%, and
33.5% for strain TA100, respectively. Therefore, 2HMC exhibited significant antimutagenic
action at all doses, mainly in tester strain TA98, with IP 91.3% at the dose of 1000 μg/plate.
Hence, it is possible to affirm that at all doses tested 2HMC was able to significantly protect
DNA from the mutagens sodium azide and 4NQO.
Micronucleus test
The frequencies of MNPCE and PCE/NCE ratio of groups co-treated, pre-treated, or post-
treated with 2HMC and CPA are presented in Table 2. The negative control group (Group 1)
had low MNPCE values, as expected, and the positive control group (Group 2) exhibited a sig-
nificant increase in MNPCE compared to the negative control group (p < 0.05), confirming
the sensitivity of the test.
Regarding the genotoxicity, MNPCE frequency in Group 3, which received only 50 mg/kg
BW 2HMC (24 h), was 4.8 (per 2000 PCE), whereas in the negative control group it was 4.2.
Given that, this dose of 2HMC did not cause a significant increase in MNPCE frequency at 24
h compared to the negative control group (p > 0.05), thus the absence of genotoxic effects in
24 h of exposure to this chalcone was proven. Group 4, which received only 50 mg/kg BW
2HMC for five consecutive days (120 h), did not have a significant increase in MNPCE
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Table 2. Effects of treatments with different doses of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) (2HMC) on the
frequency of micronucleated polychromatic erythrocytes (MNPCE) and polychromatic/normochromatic erythrocyte ratio (PCE/NCE) in bone mar-
row cells of mice.
Group
Control
Treatment
MNPCE/2000 PCE
MNPCE reducion (%)
PCE/NCE
1
2
Negative control (DMSO)1
Positive control (CPA)2
4.2 ± 0.45
27.2 ± 1.3
1.1 ± 0.05
0.71 ± 0.02
Genotoxicity and Cytotoxicity
3
2HMC 50 mg/kg BW (24 h)
4.8 ± 0.84
5.2 ± 1.30
0.87 ± 0.04 §
0.77 ± 0.1 §
4
2HMC 50 mg/kg BW (120 h)
Co-treatment
5
2HMC 25 mg/kg BW (24 h) + CPA
2HMC 50 mg/kg BW (24 h) + CPA
17 ± 1.58*
15.8 ± 3.03*
44.3
49.6
0.78 ± 0.02
0.71 ± 0.02
6
Pre-treatment
7
2HMC 25 mg/kg BW (120 h) + CPA
2HMC 50 mg/kg BW (120 h) + CPA
13.2 ± 2.16*
10.8 ± 1.92*
60.9
71.3
0.63 ± 0.02
0.53 ± 0.02 *
8
Post-treatment
9
CPA + 2HMC 25 mg/kg BW
CPA + 2HMC 50 mg/kg BW
27.4 ± 1.81
26.8 ± 0.84
0.71 ± 0.01
0.70 ± 0.01
10
1Negative control: 0.15 mL dimethylsulfoxide (DMSO) administered orally.
2Positive control: 50 mg/kg BW cyclophosphamide (CPA) diluted in physiological saline and administered ip.
Groups 3 and 4, treated only with 2HMC, were compared to the negative control group.
Groups 5, 6, 7, 8, 9, and 10, co-, pre-, or post-treated were compared to the positive control group.
* Significant compared to the positive control (p < 0.05).
§Significant compared to the negative control (p < 0.05)
The data were analyzed using one-way ANOVA, Tukey’s test, and chi-square test.
doi:10.1371/journal.pone.0171224.t002
frequency (5.2) compared to the negative control group (4.2) (p > 0.05), demonstrating
absence of genotoxic effect even after 120 h of exposure.
The PCE/NCE ratio is used as an indicator of cytotoxicity. In Group 3, treated only with
50 mg/kg BW 2HMC (24 h), the PCE/NCE ratio was 0.87, while in the negative control group
it was 1.1. These results demonstrate that this treatment with 2HMC caused a significant
decrease in the PCE/NCE ratio compared to the negative control group (p < 0.05), suggesting
a mild cytotoxic effect of the chalcone at this dose and 24 h of exposure. Group 4 showed sig-
nificant decrease in PCE/NCE ratio (0.77) compared to the negative control group (1.1)
(p < 0.05), which also suggests a moderate cytotoxic effect of 2HMC at this dose and 120 h of
exposure.
Co-treatment. In the evaluation of 2HMC antigenotoxicity, MNPCE frequencies in
Groups 5 and 6, treated with 25 and 50 mg/kg BW, were 17 and 15.8, respectively, whereas in
the positive control group it was 27.2. At both doses, 2HMC caused a significant decrease in
MNPCE frequencies compared to the positive control group (p < 0.05). Based on these results,
both doses of 2HMC reduced CPA-induced genotoxicity, by 44.3% and 49.6%, respectively,
indicating its antigenotoxic effect.
Regarding to anticytotoxicity assessed in the co-treatment, Groups 5 and 6, treated with 25
and 50 mg/kg BW 2HMC, had PCE/NCE ratios of 0.78 and 0.71, respectively, while in the pos-
itive control group it was 0.71. According to these results, neither of the treatments with
2HMC cause a significant increase in PCE/NCE ratio compared to the positive control group
(p > 0.05), evidencing absence of anticytotoxic effect.
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Pre-treatment. The evaluation of 2HMC antigenotoxicity showed that MNPCE frequen-
cies in Groups 7 and 8, which received 25 and 50 mg/kg BW 2HMC, were 13.2 and 10.8,
respectively. These results are significantly lower than those obtained for the positive control
group (27.2) (p < 0.05). Therefore, both doses of 2HMC reduced the genotoxic activity of
CPA, by 60.9% and 71.3%, respectively, revealing a strong antigenotoxic effect of the 2HMC in
pre-treatment.
For the anticytotoxic assessment in pre-treatment the PCE/NCE ratios in Groups 7 and 8
were 0.63 and 0.53 respectively, whereas in the positive control group was 0.71. At these doses,
no increase was observed in PCE/NCE ratio compared to the positive control group, demon-
strating the 2HMC absence of anticytotoxic effect. Unlike that, it was observed that 50 mg/kg
BW 2HMC dose significantly reduced the PCE/NCE ratio compared to the positive control
group (p < 0.05). Thus, 2HMC enhanced the cytotoxic action of CPA in pre-treatment.
Post-treatment. Groups 9 and 10, treated with 25 and 50 mg/kg BW 2HMC, had
MNPCE frequencies of 27.4 and 26.8 and PCE/NCE ratios of 0.71 and 0.70, respectively, not
presenting a significant difference compared to the positive control group (p > 0.05). Accord-
ing to these results, none of the doses of 2HMC exhibited antigenotoxic or anticytotoxic effects
in post-treatment.
Comet assay
Bone marrow cells from all the groups of mice treated were used to evaluate DNA damage
using the comet assay. The measurement of the parameter %DNA in tail is presented in Fig 2.
Examining the results of the comet assay, 2HMC did not induce significant DNA damage
at the dose of 50 mg/kg BW at 24 h and 120 h. This dose did not cause significant difference in
the parameter %DNA in tail compared to the negative control group (p > 0.05) (Fig 2), dem-
onstrating absence of genotoxic effects at 24 h and 120 h.
Also, Fig 2 shows the extent of DNA damage in mice bone marrow cells simultaneously
exposed to 2HMC and CPA (co-treatment). The two doses tested, 25 and 50 mg/kg BW,
promoted a significant reduction in the extent of DNA damage compared to Group 2 (posi-
tive control), treated with CPA alone (p < 0.05), exhibiting antigenotoxic effect in the co-
treatment.
Fig 2. Assessment of the genotoxic and antigenotoxic activities of the chalcone (E)-1-(2-hydroxyphenyl)-
3-(4-methylphenyl)-prop-2-en-1-one) (2HMC) at different doses in mice bone marrow cells using the
comet assay estimated by the parameter %DNA in tail. DMSO, dimethylsulfoxide (negative control); CPA,
cyclophosphamide (50 mg/kg BW) (positive control). ANOVA and the Tukey’s test: *significant compared to the
positive control (p < 0.05). Groups 3 and 4, treated only with 2HMC, were compared to the negative control. Groups
5, 6, 7, 8, 9, and 10, co-, pre-, or post-treated were compared to the positive control.
doi:10.1371/journal.pone.0171224.g002
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Potential chemoprevention of chalcone in in vitro and in vivo assays
Mice bone marrow cells exposed to both doses of 2HMC for five consecutive days (120 h)
followed by CPA showed the highest reductions of DNA damage compared to the positive
control group (p < 0.05). Based on these results, it is possible to affirm that 2HMC clearly pro-
tected DNA against CPA-induced damage.
The post-treatment with 25 and 50 mg/kg BW 2HMC did not statistically differ from the
CPA positive control group regarding %DNA in tail. These results show that neither of the
doses of 2HMC tested decreased DNA damage in post-treatment.
Discussion
The aim of the present study was to evaluate the genotoxic, cytotoxic, and protective effects of
2HMC using in vitro and in vivo assays. The use of the in vitro mutagenicity test (Ames test)
combined with the in vivo mouse bone marrow micronucleus test has been recommended in
several international guidelines to test the genotoxic/antigenotoxic potential of various types
of substances [35]. In addition to these tests, the comet assay also has been used to assess DNA
damage [36,37].
In our study, 2HMC did not exhibit mutagenic effect by Ames test, since none of the doses
reached MI ꢀ 2 on either tester strains (Table 1). Several studies have reported the lack of
mutagenic action for many classes of chalconas [38,39]. Furthemore, in a study using diferent
hydroxychalcones, 4’5’-dimethyl-2’,3,4-trihydroxylchalcone, 6 methyl-2’,3,4-trihydroxychal-
cone, and 3,3’,4’,7-tetrahydroxyflavone, none of them presented mutagenic effect by the Ames
test [40]. Also in our study, all the doses of 2HMC exhibited MI lower than the negative con-
trol (MI = 1.00) in both tester strains, indicating a possible toxic effect of this chalcone in S.
typhimurium. This mild toxicity is in accordance with an early study using 2HMC, which pre-
sented moderate antibacterial effect against Bacillus megaterium and Klebsiella sp. [41].
The mutagenic agents 4NQO and sodium azide were employed in the present study to eval-
uate the antimutagenic effect of 2HMC. 4NQO induces intracellular oxidative stress, generat-
ing reactive oxygen species (ROS) and metabolic products that can bind to DNA, mainly to
guanine residues, causing mutations [42]. Sodium azide produce reactive metabolites and
induces ROS generation that can bind to DNA, causing point mutations [43,44].
In the antimutagenicity evaluation using the Ames test, 2HMC exhibited significant anti-
mutagenic activity against both mutagens (4NQO and sodium azide) at all doses tested. Tester
strain TA98 showed the higher antimutagenic effect, reaching an IP of 91.3% against mutation
induced by 4NQO and protected DNA mainly against frameshift mutations (Table 1). This
significant antimutagenicity of 2HMC in our study could be related to the presence of an α,β-
unsaturation in its structure, which creates a nucleophilic center at β-carbon [45], interacting
with 4NQO and sodium azide metabolites, reducing their mutagenic effects.
In addition to the Ames mutagenicity test, the micronucleus test and the comet assay in
mice were performed in our study, since both tests display different mechanisms of DNA dam-
age [46–48]. In the present study, neither the administration of 2HMC alone at 24 h nor its
usage for 120 h exhibited genotoxic effects using the micronucleus test (Table 2) and comet
assay (Fig 2). These results are similar to recent findings involving two main chalcones,
4-hydroxyderricin and xanthoangelol, which did not display genotoxic effects in bone marrow
erythrocytes of mice using the chromosome aberration assay and the in vivo micronucleus test
[49].
Several authors have reported that structural, geometrical and physico-chemical patterns of
several compounds are essentially related to their biological activities [50]. Previous studies of
Density Functional Theory (DFT) and 1H NMR shifts demonstrated that 2HMC exists prefer-
ably in the trans-conformation, which is most thermodynamically stable [51,52]. This trans-
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Potential chemoprevention of chalcone in in vitro and in vivo assays
conformation allows a hydrogen bond between the C = O and the 2’-OH, in accordance with
the crystallographic characterization of 2HMC, thus facilitating the electron transfer mecha-
nism [53].
Donating electrons, these compounds generate aryloxyl (ArO.) radicals that could interact
with free radicals, exerting an antioxidant effect [54]. Studies have attributed DNA damage
preventive activity to ROS scavenging capacity [55]. The antigenotoxicity evaluation showed
that co-treatment and pre-treatment with 2HMC were able to significantly reduce the fre-
quency of micronucleus in the micronucleus test and decreased the number of DNA breaks in
the comet assay.
CPA is rapidly absorbed by the organism and converted by P450-enzymes into active
metabolites, resulting in increased ROS generation and lipid peroxidation [56,57]. These ROS
induce DNA damage by promoting DNA cross-links and breaks, which are crucial in geno-
toxic and carcinogenic process [58–60]. In recent studies, 2HMC revealed antioxidant activity
against superoxides and it was also able to inhibit lipid peroxidation [61–63]. Structure-activity
relationship studies showed that ArO. radical formed in the electron transfer mechanism of
2’-hydroxychalcones, binds to ROS, deactivating these groups and displaying an antioxidant
activity [54]. Also, a study using in vitro DPPH assay reported that 2HMC presented 10-fold
greater free radical scavenging activity when compared to other B ring non-substituted 2’-
hydroxychalcones [63–65]. Therefore, the antimutagenic activity of 2HMC observed in the
micronucleus test and comet assay could be partially attributed to its high free radical scaveng-
ing capacity, which protected DNA from the harmful actions of CP metabolites.
From all tested groups, the pre-treatment was the most effective in reducing the frequency
of MNPCE and DNA breaks, indicating a greater chemopreventive action of 2HMC against
CPA damaging effects. However, animals post-treated with 2HMC did not present a signifi-
cant reduction of MNPCE and DNA breaks, revealing that this compound was not able to
induce DNA repair system.
It’s important emphasize that the ArO. radical displays a significant role in the ROS scav-
enging mechanisms, but when local concentrations of ArO. increase, it also participates in
pro-oxidant reactions [54]. This dual characterist of ArO. radical could explain the moderate
cytotoxic effect shown at groups treated only with 2HMC (groups 3 and 4), mainly at 120 h
treatment. The ability of polyphenols to generate free radical species has been reported to
interfer the mitochondrial membrane potential, thus inducing apoptosis [66].
In our results, animals pre-treated with 2HMC (groups 7 and 8) presented a decrease in the
PCE/NCE ratio compared to the positive control, revealing a synergistic action of 2HMC and
CPA. However, even 2HMC presenting a cytotoxic effect at pre-treatment, this treatment pre-
sented the lowest MNPCE frequencies. This fact may indicate that these cells might undergo
an apoptosis process, which could have reduced the genotoxicity promoted by CPA.
Therefore, 2HMC showed antimutagenic effect by Ames test and antigenotoxic activity in
the micronucleus test and comet assay. The capacity of protecting DNA against damage
induced by different mutagens indicates that 2HMC presents an interesting profile for the
development of chemopreventive drugs.
Conclusion
The results obtained in this study using the Ames test, micronucleus test, and comet assay
showed that 2HMC did not exhibit mutagenic and genotoxic effects. 2HMC presented a mod-
erate cytotoxicity in mouse bone marrow cells in micronucleus test, mainly at pre-treatment.
Regarding the protective effects of 2HMC, this chalcone reduced DNA damage promoted by
genotoxic agents sodium azide, 4NQO, and CPA (in co-treatment and pre-treatment). The
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Potential chemoprevention of chalcone in in vitro and in vivo assays
absence of effect in post-treatment indicated that this compound did not act on DNA repair
processes. We suggested that protective activity of 2HMC found in the present study could be
due to an antioxidant activity and its capacity to form complexes with molecules, preventing
DNA damage. Therefore, this protective effect against different mutagens suggests that 2HMC
could be a probable candidate for the development of chemopreventive agents.
Acknowledgments
This study was financially supported by the Brazilian agency Coordenac¸ão de Aperfeic¸oa-
´
mento de Pessoal de Nıvel Superior (CAPES) under the auspices of the Federal University of
´
Goias (UFG).
Author Contributions
Conceptualization: DCSL CNP LCC.
Formal analysis: DCSL LCC.
Investigation: DCSL CRV AB CNP LCC.
Methodology: DCSL CNP LCC.
Resources: DCSL CNP LCC.
Supervision: DCSL CRV AB.
Writing – original draft: DCSL LCC.
Writing – review & editing: DCSL CRV AB JHV CNP LCC.
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