An Acad Bras Cienc (2021) 93(Suppl. 3): e20201772 DOI 10.1590/0001-3765202120201772
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal
CELLULAR AND MOLECULAR BIOLOGY
Cytogenotoxicity and protective
effect of piperine and capsaicin on
meristematic cells of Allium cepa L.
MARCONDES S. DIAS, ERASMO P.V. JUNIOR, BIANCA C. DOS SANTOS, FRANCIELLE
A. MARTINS, PEDRO M. DE ALMEIDA & ANA P. PERON
Abstract: Piperine and capsaicin are important molecules with biological and
pharmacological activities. This study aimed to evaluate the cytogenotoxic and protective
effect of piperine and capsaicin on Allium cepa cells. A. cepa roots were exposed to
negative (2% Dimethylsulfoxide) and positive (Methylmethanesulfonate, MMS, 10 µg/
mL) controls, and four concentrations (25-200 µM) of piperine or capsaicin (alone) or
associated before, simultaneously or after with the MMS. Only the lowest concentration
of piperine (25 µM) showed a protective effect because it was not genotoxic. Piperine
and capsaicin were cytotoxic (50, 100 and 200 µM). Piperine (50 to 200 µM) caused a
significant increase in the total average of chromosomal alterations of in A. cepa cells.
For capsaicin, the genotoxic effect was dose-dependent with a significant increase for
all concentrations, highlighting the significant presence of micronuclei and nuclear
buds for the two isolates. In general, bioactive compounds reduced the total average
of chromosomal alterations against damage caused by MMS, mainly micronuclei and/or
nuclear buds. Therefore, the two molecules were cytotoxic and genotoxic at the highest
concentrations, and did not have cytoprotective action, and the lowest concentration of
piperine demonstrated important chemopreventive activity.
Key words: Bioactive compounds, mitotic index, chromosomal alterations, protection
against cell damage.
INTRODUCTION
Peppers comprise groups of plants of the genera
Piper and Capsicum (Salazar et al. 2016) and are
among the main spices appreciated worldwide.
Peppers are functional foods, as they have a
potential medicinal application with protective
and therapeutic action (Antonio et al. 2018,
Rather & Bhagat 2018). The chemoprotective
potential of piperamides has already been
observed in P. nigrum and P. longum (Lai et
al. 2012, Grinevicius et al. 2017) and in some
species of Capsicum there are many biologically
active metabolites, such as capsaicinoids,
capsinoids and carotenoids (Luo et al. 2011,
Bertão et al. 2016). Protection can result from
the interaction of molecules with mutagenic
agents in the intracellular and/or extracellular
(desmutagenic) media and in the damage repair
process (bioantimutagenic) (Santos et al. 2012,
Asita et al. 2015, Andrade et al. 2016).
The detection of phytochemicals capable
of modulating genotoxic, mutagenic and
carcinogenic effects show satisfactory results
for polyphenols, carotenoids, alkaloids, tannins
(Li et al. 2018, Ribeiro et al. 2018, Sá et al.
2019). There are several studies using natural
extracts of Piper and Capsicum for analysis
of genotoxicity and antigenotoxicity, however,
An Acad Bras Cienc (2021) 93(Suppl. 3)
MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
studies evaluating isolated molecules are more
frequent, given the improvement of the methods
for extraction of these compounds and access
to these substances commercially (Bley et al.
2012, Rather & Bhagat 2018).
Piperine (1-piperylpiperidine) is an alkaloid
isolated mainly from P. nigrum, known as
black pepper, but can also be found in other
representatives of the family Piperaceae
(Meghwal & Goswami 2013). The structural
characteristics of piperine are divided into three
parts: (1) an aromatic ring with a methylenedioxy
bridge, associated with the antioxidant and
anti-tumor potential; (2) a conjugated dienone
system, which performs lipophilic interactions
with various molecular residues; and (3) a
piperidine ring that creates an amide bond,
responsible for insecticidal and anti-tumor
actions, in addition to interacting with enzymatic
systems, leading to biotransformation effects
(Qu et al. 2015, Singh & Choudhary 2015).
Capsaicin (8-methyl-N-vanillyl-6nonenamide) is an alkaloid/amide compound
found in Capsicum fruit and can account for
1% of the mass of peppers (Fattori et al. 2016).
Regarding the structure and activity of capsaicin,
stand out the regions: (1) aromatic, responsible
for most of the antioxidant activity; (2) amide
bond, responsible for the effect of analgesia
and antinociceptive activity, and (3) the
aliphatic chain, related to analgesic activity and
with an important role in the total polarity of
the molecule, giving it a hydrophobic character
(Huang et al. 2013).
Piperine and capsaicin also have an
antiparasitic and immunomodulatory effect
(Soutar et al. 2017, Vurmaz et al. 2019). In addition,
studies on the antiproliferative, mutagenic/
genotoxic/protective and anti-apoptotic/proapoptotic effects have been carried out on
different types of tumor cells, but these studies
have contradictory results for both piperine
and capsaicin (Singh & Duggal 2009, Fattori et
al. 2016). Toxicogenic and/or protective effects
depend on the dose and the group of cells
treated. Understanding the modulatory activity,
finding effective concentrations that are safe for
the body is especially important. Although the
evaluation of genotoxicity/antigenotoxicity of
piperine and capsaicin has been carried out in
vitro and in vivo tests (Thiel et al. 2014, FernandezBedmar & Alonso-Moraga 2016), studies that
show their effects using chromosomal changes
such as cytogenetic biomarkers are incipient
and there are no reports on the biological
effects of piperine and capsaicin in a plant
test system. Thus, the present study can show
whether the isolates interact with spindle fibers
and / or with the DNA and whether they have a
modulating effect against the mutagenic agent.
The test system for chromosomal changes in
Allium cepa L. is widely cited in the literature as
a bioindicator for the evaluation of cytotoxicity,
genotoxicity and protective effect of chemical
compounds, as it has rapid cell multiplication,
large and few chromosomes, which allows
better analysis of structural and numerical
changes (Leme & Marin-Morales 2009, Bonciu et
al. 2018). Its low cost and agreement with other
similar tests, which involve the manipulation
and sacrifice of animals, justifies its extensive
use in toxicogenetic bioassays (Eren & Özata
2014). Besides that, it has a good correlation
with cytotoxicity and genotoxicity tests in vitro
or in vivo (Eren & Özata 2014, Sá et al. 2019).
Considering the importance of piperine and
capsaicin as spices used in gastronomy and
popular medicine (Bley et al. 2012, Muhammad
et al. 2018), the present study aimed to
investigate the cytotoxic, genotoxic, protective
or modulating effects against the damages
caused by methylmethanesulfonate, using the
chromosomal alterations test in meristematic
cells of A. cepa.
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MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
MATERIALS AND METHODS
Tested compounds
Piperine ≥ 97% CAS (94-62-2), capsaicin ≥ 95%
CAS (404-86-4) and Methylmethanesulfonate
(MMS, CAS 66-27-3) were obtained from SigmaAldrich (St. Louis, MO, USA).
DNA-damaging agent
MMS was used to induce DNA damage in
meristematic cells of A. cepa. MMS (10 μg/mL) is
an alkylating agent with direct activity, inducing
disturbances such as DNA breaks, bridges and
chromosome loss, which are also expressed
as micronuclei (Bianchi et al. 2016, Couto et al.
2019). MMS was used in A. cepa test because
it has high genotoxicity at low concentrations.
In addition, MMS shows less cytotoxic effect,
allowing cells to continue the cell cycle and
those chromosomal changes can be visualized
in different phases of cell division (Bianchi et
al. 2015).
Allium cepa bioassay
One hundred A. cepa (cv. Vale Ouro IPA-11) seeds
per Petri dish were germinated with distilled
water in an incubator (BOD SL - 224®) under a
12 h photoperiod and temperature of 24ºC for
three days at the Genetics Laboratory (LABGENE)
of the Center for Natural Sciences of UESPI,
Teresina-PI. After germination, seeds with roots
of approximately 1 cm were exposed to different
treatments to assess the cytogenotoxicity and
antigenotoxicity of the bioactive piperine and
capsaicin, according to Nantes et al. (2014) and
Couto et al. (2019).
In the genotoxicity test, seeds were
transferred to the negative controls (NC)
(Dimethylsulfoxide - 2% DMSO in distilled water),
solvent (SC) (distilled water), positive MMS I
(Methylmethanesulfonate, 10 µg/mL dissolved in
DMSO 2%) and MMS II (dissolved only in distilled
water) and for treatments with piperine or
capsaicin in concentrations of 25, 50, 100 and 200
µM for 48 h. The protective effect was performed
by exposing the germinated seeds to piperine
or capsaicin before, simultaneously or after the
MMS, representing the pre, simultaneous and
post treatments, respectively (Rocha et al. 2016).
The pretreatment assesses demutagenic action,
the simultaneous treatment assesses both
demutagenic and bioantimutagenic activity and
the posttreatment indicates bioantimutagenic
action (Fedel-Miyasato et al. 2014, Felicidade et
al. 2014).
The concentrations were previously
determined from studies with the isolates and
this range of concentrations is commonly tested
on molecules with pharmaceutical potential. In
addition, equal or similar concentrations are
tested in chemotherapeutic trials dissolved in
2% DMSO (Greenshields et al. 2015, Siddiqui et
al. 2017).
After the treatments carried out, roots
were fixed in Carnoy (3 ethanol: 1 acetic acid)
for 6-8 h and stored at -20°C until the slide
was prepared. To mount the slides, roots were
washed three times in distilled water for 5 min
each and hydrolyzed at 60°C for 10 min in 1N
HCl. After hydrolysis, roots were again washed
in distilled water and transferred to amber glass
flasks, containing the Schiff Reactive, where they
remained in the dark for 2 hours. Roots were
then washed, until the reagent was completely
removed, transferred to slides, where they
were crushed in a drop of 2% acetic carmine
and mounted with Entellan® (107960; Merck
Millipore) (Almeida et al. 2015).
Cytotoxicity, genotoxicity and antigenotoxicity
were evaluated by counting 5,000 meristematic
cells per treatment (500 cells/slides, with a total
of 10 slides analyzed per treatment) under a
light microscope (Zeiss Primo Star with Axiocam
105 color camera) at 400x magnification in the
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MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Soil Analysis Laboratory (LASO) of the Center for
Agricultural Sciences (CCA) UFPI, Teresina-PI.
For each treatment, the mitotic index (MI,
cytotoxicity) and mean chromosomal alteration
(genotoxicity) resulting from aneugenic action
(metaphase with chromosomal adhesion,
C-metaphase, chromosomal loss, multipolar
anaphase, binucleated cells) were evaluated for
each treatment among others and/or clastogenic
action (chromosomal fragments in metaphase
or anaphase, chromosomal bridges and other
alterations) (Leme & Marin-Morales 2009).
Cytotoxicity was also analyzed by the mean value
of cells and cells in death process (CDP), which
have characteristics such as: heteropicnotic
nucleus and displaced to the periphery of the
cell, vacuolization and swelling of the cytoplasm
(Bianchi et al. 2010). To determine the MI, the
number of cells in different phases of mitosis
was divided by the total number of cells. For
chromosome alterations, the number of
alterations was divided by the total number of
cells.
Protective effect was assessed by analyzing
the percentage of damage reduction (% DR)
for each treatment with piperine or capsaicin,
according to the following formula: % DR
= [(a - b)/(a - c)] x 100 Where: a = average of
MMS chromosomal alterations; b = average of
chromosomal alterations in each treatment and
c = average of chromosomal alterations in the
NC) (Waters 1990).
Data analysis
Data were analyzed using the Kruskal-Wallis
non-parametric test, followed by the StudentNewman-Keuls a posteriori test (p <0.05), in the
BioEstat 5.3 software (Ayres & Ayres 2007), for
comparison between the means of the controls
and groups treated.
RESULTS
Cytogenotoxicity of piperine and capsaicin
Piperine and capsaicin were cytotoxic at
concentrations from 50 to 200 µM, since there
was a significant reduction in the mitotic index
(MI) of the meristematic cells of A. cepa in
relation to the NC, being dose dependent when
exposed to capsaicin. Piperine and capsaicin
significantly reduced the prophases (50 to
200 µM). Additionally, the lower percentage of
prophases in capsaicin resulted in a significant
reduction in the other phases of the A. cepa cell
cycle, mainly in 200 µM (Table I).
A. cepa cells in death process (CDP) were
not significant when compared to the NC,
however there was an increase in CDP in all
concentrations of piperine and at the lowest (25
µM) and higher (200 µM) of capsaicin, which may
have contributed to the cytotoxic effect (Table I,
Figure 1p).
Piperine caused a significant increase in
the total average of chromosomal alterations
(genotoxic effect) at concentrations of 50
to 200 µM in A. cepa cells when compared to
NC. For capsaicin, the genotoxic effect was
dose-dependent with a significant increase
for all concentrations (Table I). Piperine
and capsaicin caused different types of
chromosomal alterations and normal cells:
a) normal interphase; b) normal prophase; c)
normal metaphase; d) normal anaphase; e)
normal telophase; f) micronucleus (arrow); g)
interphase with nuclear bud; h) chromosomal
breaks (arrow); i) chromosomal adherence; j)
C-metaphase; k) chromosomal loss (arrow); l)
chromosomal bridge (arrow) and chromosomal
break (arrow head); m) multipolar anaphase; n)
binucleated cell; o) nuclear alteration; p) cells
under death process (heteropicnotic nucleus
displaced for cell periphery; vacuolization and
cytoplasm swelling) (Table II, Figure 1a-p), but
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MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Figure 1. Chromosomal and nuclear alterations observed by the analysis of meristematic cells from Allium cepa
roots. a) normal interphase. b) normal prophase. c) normal metaphase. d) normal anaphase. e) normal telophase.
f) micronucleus (arrow). g) interphase with nuclear bud. h) chromosomal breaks (arrow). i) chromosomal
adherence. j) C-metaphase. k) chromosomal loss (arrow). l) chromosomal bridge (arrow) and chromosomal break
(arrow head). m) multipolar anaphase. n) binucleated cell. o) nuclear alteration. p) cells under death process
(heteropicnotic nucleus displaced for cell periphery; vacuolization anda cytoplasm swelling). Bar: 10 µm (for all
images). All chromosomal changes were observed for piperine and capsaicin, except multipolar anaphase for
capsaicin.
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MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Table I. Phases of mitosis, mitotic index, cells in death process (CDP) and total chromosomal alterations in
meristematic cells of A. cepa exposed to different concentrations of piperine or capsaicin.
Phases of mitosis (%)
Treatment
Mitotic Index
CDP
Chromosomal
Telophase
(%)
(%)
alteration
1.62 ± 0.60
0.98 ± 0.34
19.86 ± 1.16
0.04 ± 0.12
4.13 ± 2.28
1.09 ± 0.46
0.67 ± 0.50
14.09 ± 1.96**
0.77 ± 1.80
59.52 ± 13.34**
(µM)
Prophase
Metaphase
Anaphase
NC
15.93 ± 0.87
1.33 ± 0.75
MMS I
10.57 ± 1.50**
1.16 ± 0.51
Piperine
25
15.38 ± 0.84
1.36 ± 0.46
0.97 ± 0.39
1.21 ± 0.45
18.93 ± 1.21
0.08 ± 0.24
6.63 ± 3.23
50
14.13 ± 1.05*
1.46 ± 0.49
1.44 ± 0.33
1.08 ± 0.46
18.11 ± 1.06*
0.51 ± 1.29
11.93 ± 2.69**
100
12.90 ± 1.42**
1.31 ± 0.79
1.01 ± 0.52
1.01 ± 0.52
16.22 ± 1.94**
0.27 ± 0.60
11.63 ± 6.44*
200
14.27 ± 0.84*
1.23 ± 0.51
1.26 ± 0.59
1.10 ± 0.67
17.85 ±0.81*
0.47 ± 1.03
15.05 ± 3.79**
Capsaicin
25
14.39 ± 0.61
1.24 ± 0.38
1.02 ± 0.42*
1.43 ± 0.64
18.08 ± 0.97
0.13 ± 0.40
13.31 ± 9.71*
50
13.00 ± 0.91*
1.05 ± 0.39
0.99 ± 0.37*
1.39 ± 0.37
16.42 ± 1.38*
0.02 ± 0.06
14.55 ± 3.46**
100
9.37 ± 1.85**
0.93 ± 0.41
0.81 ± 0.47**
0.79 ± 0.49
11.90 ± 2.12**
0.00 ± 0.00
15.36 ± 4.94**
200
4.64 ± 2.55**
0.15 ± 0.22**
0.17 ± 0.25**
0.24 ± 0.33**
5.21 ± 3.05**
1.74 ± 2.30
16.78 ± 6.73**
Data are means ± SD (Standard Deviation). NC: Negative Control (Dimethylsulfoxide - 2% DMSO in distilled water). MMS I: 10 µg/
mL of Methylmethanesulfonate dissolved in DMSO 2%. *Significant by Kruskal-Wallis test with a posteriori Student-NewmanKeuls test (* p < 0.05; **p <0.01) when compared to NC. Data are for 5,000 cells/treatment. The DMSO 2% was used as a negative
control, but how the results were statistical identical to solvent (distilled water), the data using water were omitted. The MMS I
and MMS II (dissolved only in distilled water) also were statistical identical, the data using MMS II were omitted.
only micronuclei (MN) and nuclear buds (NB)
were significant (50 to 200 µM) in the piperine
treatment. In capsaicin, the same changes
were significant at all concentrations and
chromosomal adherence was significant at
concentrations of 50 and 100 µM.
Modulation of cell damage by piperine and
capsaicin
As for the modulatory effect, there was no
significant difference in MI and CDP in the
three protocols (pre, simultaneous and
posttreatment) exposed to piperine or capsaicin
in relation to MMS I. Further, in capsaicin there
were significant reductions in MI in the pre (100
and 200 µM), simultaneous (25 and 100 µM) and
posttreatment (200 µM) (Table III).
As for the different phases of mitosis, cells
treated with piperine or capsaicin showed no
significant difference in relation to MMS I in
the three protocols. Further, in capsaicin, there
was a reduction in prophases, metaphases
and anaphases in the three protocols, and
telophases only in the posttreatment (Table III).
The observed results showed that piperine or
capsaicin was not able to neutralize the cytotoxic
action of MMS I.
A significant reduction in the total average
of chromosomal alterations in A. cepa when
exposed to piperine was verified in the pre
(57.93 to 85.66%), simultaneous (58.71 to 74.07%)
and posttreatment (54.38 to 64.16%) for all
concentrations in relation to MMS I. In capsaicin,
the reduction of chromosomal alterations
was also observed in the pre (70.39 to 86.21%)
and simultaneous (36.36 to 51.89%) for all
concentrations, while in the posttreatment, only
the highest concentration (200 µM) had the
reduction (35.84%). These results reinforce the
interaction of piperine or capsaicin with MMS,
modulating genotoxic action of MMS, however
there was no protective effect (except for the
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MARCONDES S. DIAS et al.
lowest concentration of piperine), since these
molecules alone were genotoxic (Table III).
The significant reduction in MN and/or NB
was verified in all concentrations of the pre and
simultaneous treatments in relation to MMS I
when exposed to piperine or capsaicin. In the
posttreatment, there was also a reduction in
the same changes in all concentrations with
piperine and for capsaicin occurred only in the
highest concentration (Table IV).
DISCUSSION
The cytotoxic effect of piperine and capsaicin
may be related to a significant reduction in
prophases (50 to 200 µM). The lower percentage
of prophases in capsaicin resulted in a
significant decrease in the other phases of the
A. cepa cell cycle, mainly in 200 µM (Table I).
The observed results are reinforced by previous
studies showing the cytotoxicity of piperine or
capsaicin in concentrations equal to and/or
similar to the present study, which promoted
the arrest of tumor cells in G1 (Ouyang et al.
2013, Fofaria et al. 2014) and/or G2/M (Yaffe et al.
2013, Greenshields et al. 2015, Zhang et al. 2015,
Siddiqui et al. 2017).
An increase in CDP in all concentrations
of piperine and at the lowest and highest
concentrations of capsaicin may also have
contributed to the cytotoxic effect. Similar
results were observed in tumor cells treated
with piperine or capsaicin, which caused
oxidative stress by reactive oxygen species (ROS)
(Yaffe et al. 2013), reactive nitrogen species,
inhibition of NADH-oxidoreductase (an enzyme
that stimulates cell activity and proliferation)
and rupture of the mitochondrial membrane
permeability (Pramanik et al. 2011, Qian et al.
2016, Cho et al. 2017).
An Acad Bras Cienc (2021) 93(Suppl. 3)
Table II. Chromosomal alterations in meristematic cells of A. cepa exposed to different concentrations of piperine or capsaicin.
NC
MMS I
MN
0.84 ± 1.08
33.56 ± 7.66**
NB
2.82 ± 1.62
23.43 ± 5.80**
CB
0.00 ± 0.00
0.46 ± 0.64*
25
50
100
200
3.04 ± 1.74
3.81 ± 3.22*
3.48 ± 4.48*
6.98 ± 2.64**
2.54 ± 2.39
7.55 ± 4.78*
6.77 ± 5.21*
6.65 ± 3.72*
0.00 ± 0.00
0.18 ± 0.37
0.09 ± 0.30
0.10 ± 0.31
25
50
100
200
6.80 ± 6.53*
5.53 ± 2.57*
6.58 ± 2.74**
7.69 ± 3.86**
4.88 ± 3.82*
6.74 ± 6.53*
6.74 ± 2.88*
7.69 ± 3.86*
0.00 ± 0.00
0.09 ± 0.30
0.00 ± 0.00
0.00 ± 0.00
Chromosomal alteration
BC
CA
Cm
0.00 ± 0.00
0.10 ± 0.31
0.10 ± 0.30
0.28 ± 064
0.38 ± 0.50
0.00 ± 0.00
Piperine
0.10 ± 0.31
0.38 ± 0.50
0.09 ± 0.29
0.00 ± 0.00
0.19 ± 0.40
0.00 ± 0.00
0.00 ± 0.00
0.36 ± 0.47
0.09 ± 0.30
0.00 ± 0.00
0.48 ± 0.68
0.09 ± 0.29
Capsaicin
0.00 ± 0.00
1.07 ± 1.63
0.00 ± 0.00
0.20 ± 0.63
1.60 ± 1.41*
0.10 ± 0.31
0.00 ± 0.00
1.55 ± 1.43*
0.00 ± 0.00
0.36 ± 0.75
1.27 ± 1.58
0.00 ± 0.00
CL
0.19 ± 0.39
0.84 ± 1.01
CBr
0.00 ± 0.00
0.28 ± 0.45
MA
0.10 ± 0.31
0.10 ± 0.31
NA
0.00 ± 0.00
0.19 ± 0.39
0.29 ± 0.65
0.10 ± 0.30
0.37 ± 0.47
0.28 ± 0.45
0.10 ± 0.30
0.10 ± 0.31
0.09 ± 0.30
0.19 ± 0.40
0.10 ± 0.31
0.00 ± 0.00
0.09 ± 0.30
0.29 ± 0.46
0.00 ± 0.00
0.00 ± 0.00
0.19 ± 0.40
0.00 ± 0.00
0.19 ± 0.40
0.10 ± 0.31
0.00 ± 0.00
0.09 ± 0.30
0.19 ± 0.40
0.19 ± 0.39
0.38 ± 0.49
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.19 ± 0.39
0.10 ± 0.31
0.10 ± 0.30
0.09 ± 0.28
Data are means ± SD (Standard Deviation). NC: Negative Control (Dimethylsulfoxide - 2% DMSO in distilled water). MMS I: 10 µg/mL of Methylmethanesulfonate dissolved in
DMSO 2%. MN: Micronucleus. NB: Nuclear Bud. CB: Chromosomal Breaking. BC: Binucleated Cell. CA: Chromosomal Adherence. Cm: C-metaphase. CL: Chromosomal Loss. CBr:
Chromosomal Bridge. MA: Multipolar Anaphase. NA: Nuclear Alteration. *Significant by Kruskal-Wallis test with a posteriori Student-Newman-Keuls test (* p < 0.05; **p <0.01)
when compared to NC. Data are for 5,000 cells/treatment. The DMSO 2% was used as a negative control, but how the results were statistical identical to solvent (distilled
water), the data using water were omitted. The MMS I and MMS II (dissolved only in distilled water) also were statistical identical, the data using MMS II were omitted.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
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Treatment (µM)
MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Table III. Phases of mitosis, mitotic index, cells in death process (CDP) and total chromosomal alterations and
percentage of damage reduction (%DR) in meristematic cells of A. cepa exposed to different concentrations of
piperine or capsaicin.
Phases of mitosis (%)
Treatment
(µM)
CDP (%)
Total
chromosomal
alterations
%DR
Prophase
Metaphase
Anaphase
Telophase
Mitotic
index (%)
NC
15.93 ±
0.87++
1.33 ± 0.75
1.62 ± 0.60
0.98 ± 0.34
19.86 ± 1.16++
0.04 ± 0.12
4.13 ± 2.28++
-
MMS I
10.57 ± 1.50
1.16 ± 0.51
1.09 ± 0.46
0.67 ± 0.50
14.09 ± 1.96
0.77 ± 1.80
59.52 ± 13.34
-
11.60 ± 1.08
1.10 ± 0.47
1.00 ± 0.28
1.00 ± 0.44
14.70 ± 1.66
1.03 ± 1.42
27.43 ± 5.29+
57.93
Pre-treatment
Piperine + MMS I
25
50
11.24 ± 1.06
0.75 ± 0.31
+
0.77 ± 0.40
1.01 ± 0.45
13.78 ± 1.33
0.02 ± 0.06
15.06 ± 5.44
++
80.27
++
83.28
100
10.80 ± 0.49
1.18 ± 0.48
0.77 ± 0.28
0.85 ± 0.43
13.60 ± 0.88
0.24 ± 0.43
13.39 ± 4.33
200
10.42 ± 0.85
0.90 ± 0.39
0.66 ± 0.36+
0.56 ± 0.32
12.53 ± 1.06
0.45 ± 0.84
12.07 ± 2.83++
85.66
0.13 ± 0.40
1.03 ± 0.38
0.66 ± 0.32
11.93 ± 2.27
0.22 ± 0.70
18.49 ± 5.68++
74.07
Simultaneous treatment
Piperine + MMS I
25
50
100
200
9.12 ± 2.36
8.14 ± 2.63
9.23 ± 1.40
+
1.15 ± 0.47
0.56 ± 0.39
1.24 ± 0.65
0.62 ± 0.33
+
0.91 ± 0.38
0.90 ± 0.37
10.77 ± 3.21
0.43 ± 0.70
11. 99 ± 1.60
0.00 ± 0.00
++
19.64 ± 22.09
72.00
++
67.57
+
58.71
22.09 ± 6.65
8.13 ± 2.12
0.78 ± 0.38
0.69 ± 0.38
0.85 ± 0.63
10.62 ± 2.28
0.00 ± 0.00
27.00 ± 8.19
25
8.79 ± 1.29
1.27 ± 0.55
1.22 ± 0.39
0.97 ± 0.47
12.25 ± 2.04
0.24 ± 0.77
23.98 ± 7.96++
64.16
50
10.40 ± 0.97
1.47 ± 0.55
1.20 ± 0.50
1.21 ± 0.61
14.26 ± 1.32
0.52 ± 1.10
25.04 ± 6.39++
62.25
100
9.55 ± 1.04
1.13 ± 0.34
1.12 ± 0.45
1.24 ± 0.51
13.03 ± 1.55
0.00 ± 0.00
28.49 ± 8.27++
56.02
++
54.38
86.21
Post-treatment
Piperine + MMS I
200
9.35 ± 1.02
1.36 ± 0.59
0.96 ± 0.50
0.93 ± 0.36
12.59 ± 1.41
0.26 ± 0.82
29.40 ± 7.05
0.82 ± 0.43
0.54 ± 0.33+
0.41 ± 0.29
13.97 ± 1.84
0.00 ± 0.00
11.77 ± 4.95++
Pre-treatment
Capsaicin + MMS I
25
50
12.21 ± 1.36
9.58 ± 1.60
++
0.39 ± 0.25
10.83 ± 1.91
++
0.24 ± 0.29
0.40 ± 0.28
++
7.71 ± 2.10
0.44 ± 0.28++
0.27 ± 0.24++
0.34 ± 0.29
6.54 ± 2.10++
0.82 ± 0.61
0.34 ± 0.19++
0.37 ± 0.29
9.69 ± 1.96+
9.87 ± 1.61
0.90 ± 0.38
+
8.71 ± 1.99
+
+
100
6.15 ± 1.49
200
5.48 ± 1.72++
0.46 ± 0.28
++
0.37 ± 0.39
++
0.39 ± 0.25
0.25 ± 0.55
18.66 ± 4.05
++
73.77
0.08 ± 0.19
++
20.53 ± 8.21
70.39
1.01 ± 1.58
18.64 ± 6.71++
73.80
0.00 ± 0.00
39.38 ± 6.93+
36.36
Simultaneous treatment
Capsaicin + MMS I
25
50
100
200
8.16 ± 1.91+
8.95 ± 1.91
0.71 ± 0.28
0.79 ± 0.28
0.42 ± 0.25
0.24 ± 0.15
++
0.39 ± 0.31
++
0.82 ± 0.36
12.00 ± 1.23
0.47 ± 0.12
+
0.40 ± 0.21
An Acad Bras Cienc (2021) 93(Suppl. 3)
10.19 ± 2.02
10.53 ± 1.95
e20201772
0.00 ± 0.00
0.00 ± 0.00
0.52 ± 1.63
8 | 15
++
44.24
++
51.89
+
40.53
35.01 ± 15.16
30.78 ± 7.63
37.07 ± 11.46
MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Table III. Continuation.
Treatment
(µM)
Phases of mitosis (%)
Prophase
Metaphase
Anaphase
Telophase
Mitotic
index (%)
CDP (%)
Total
chromosomal
alterations
%DR
Post-treatment
Capsaicin + MMS I
25
9.09 ± 1.68
1.50 ± 0.49
0.68 ± 0.24
0.81 ± 0.44
12.09 ± 2.10
0.40 ± 0.86
50.18 ± 11.64
16.86
50
8.76 ± 1.54
1.20 ± 0.48
0.84 ± 0.39
0.81 ± 0.36
11.61 ± 1.80
0.00 ± 0.00
61.60 ± 13.31
-3.75
0.85 ± 0.43
+
11.49 ± 1.76
0.46 ± 0.82
57.46 ± 9.74
3.72
100
200
8.19 ± 1.03
++
4.45 ± 1.54
1.27 ± 0.37
0.46 ± 0.54
+
++
0.21 ± 0.28
0.16 ± 0.55
0.27 ± 0.28
5.38 ± 2.39
++
1.64 ± 3.02
39.67 ± 14.23
+
35.84
Data are means ± SD (Standard Deviation). NC: Negative Control (Dimethylsulfoxide - DMSO 2% in distilled water). MMS I: 10
µg/mL of Methylmethanesulfonate dissolved in 2% DMSO (positive control). Pre-treatment (piperine or capsaicin + MMS).
Simultaneous treatment (piperine or capsaicin added simultaneously with MMS). Post-treatment (MMS + piperine or capsaicin).
+
Significant in the Kruskal-Wallis test with a posteriori Student-Newman-Keuls test (+ p <0.05; ++ p <0.01) when compared to MMS
I. The results refer to the analysis of 5,000 cells per treatment. The DMSO 2% was used as a negative control, but how the results
were statistical identical to solvent (distilled water), the data using water were omitted. The MMS I and MMS II (dissolved only in
distilled water) also were statistical identical, the data using MMS II were omitted.
Pro-oxidant action of piperine or capsaicin
may have resulted in the genotoxic effect, as
ROSs increase the risk of DNA damage, including
the division of cells with unrepaired or poorly
repaired damage, leading to mutations (Kehrer
& Klotz 2015). The genotoxic effect observed in
piperine and capsaicin are, mainly, the result
of significant MN and NB. The MN observed are
due to clastogenic and/or aneugenic damage
not repaired or erroneously repaired in parental
cells, is easily observed in daughter cells as a
structure similar to the main nucleus, but in
a reduced size (Fernandes et al. 2007, Leme &
Marin-Morales 2009). NB may be related to the
formation of MN, through the elimination of
extra genetic material in the main nucleus of
the cell, or it may be due to the aggregation of a
delayed chromosome by the nuclear envelope,
before being fully reincorporated into the main
nucleus (Bianchi et al. 2015). While chromosomal
adherences are a type of abnormality that
involves the protein in the chromatin matrix
and not necessarily the DNA itself; it can also be
irreversible and lead to cell death (Fernandes et
al. 2009).
Piperine and capsaicin are potential
antimutagenic and anticarcinogenic compounds
(Abo-Zeid & Farghaly 2009, Fernandez-Bedmar
& Alonso-Moraga 2016). In this way, we sought
to evaluate the protective or modulatory effect
of both molecules on damage induced by MMS
I in the pre, simultaneous and posttreatment
protocols.
The two molecules are alkaloids, which
have both antioxidant and pro-oxidant action
(capable of generating free radicals) depending
on the dose and the group of treated cells
(Rather & Bhagat 2018, Macáková et al. 2019).
Probably, the molecules acted as pro-oxidants
in the cells of A. cepa, potentiating the cytotoxic
action of MMS I, mainly for capsaicin, which
promoted significant reductions in the MI.
According to Bianchi et al. (2016), ROS may be
associated with decreased MI in A. cepa cells,
as they cause lipid peroxidation, changes in
membrane fluidity and DNA damage. In response
to these damages, there is usually a delay in the
mitotic cycle, mainly in the G1 and/or G2 phases,
to allow the cells to repair the damage induced
before replicating their DNA and starting mitosis
(Feng et al. 2010).
An Acad Bras Cienc (2021) 93(Suppl. 3)
e20201772 9 | 15
MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Table IV. Chromosomal alterations in meristematic cells of A. cepa exposed to different concentrations of piperine
or capsaicin.
Chromosomal alteration
Treatment
(µM)
MN
NB
CB
BC
CA
Cm
CL
CBr
MA
NA
NC
0.84 ± 1.08++ 2.82 ± 1.62++ 0.00 ± 0.00 0.00 ± 0.00 0.10 ± 0.31 0.10 ± 0.30 0.19 ± 0.39 0.00 ± 0.00 0.10 ± 0.31 0.00 ± 0.00
MMS I
33.56 ± 7.66 23.43 ± 5.80 0.46 ± 0.64 0.28 ± 064 0.38 ± 0.50 0.00 ± 0.00 0.84 ± 1.01 0.28 ± 0.45 0.10 ± 0.31 0.19 ± 0.39
Pre-treatment
Piperine + MMS I
12.23 ±
2.88++
25
13.31 ± 4.31++
50
6.09 ± 3.58++ 7.41 ± 4.83++ 0.74 ± 0.74 0.09 ± 0.29 0.18 ± 0.38 0.00 ± 0.00 0.09 ± 0.27 0.46 ± 0.66 0.00 ± 0.00 0.00 ± 0.00
100
4.69 ± 3.05++ 7.56 ± 2.94++ 0.39 ± 0.50 0.00 ± 0.00 0.37 ± 0.65 0.00 ± 0.00 0.19 ± 0.40 0.19 ± 0.40 0.00 ± 0.00 0.00 ± 0.00
200
4.48 ± 2.39++ 6.47 ± 2.92++ 0.48 ± 0.81 0.00 ± 0.00 0.18 ± 0.57 0.00 ± 0.00 0.09 ± 0.30 0.38 ± 0.67 0.00 ± 0.00 0.00 ± 0.00
0.38 ± 0.67 0.00 ± 0.00 0.10 ± 0.31 0.00 ± 0.00 0.85 ± 0.54 0.29 ± 0.46 0.18 ± 0.39 0.10 ± 0.31
Simultaneous treatment
Piperine + MMS I
11.09 ±
4.40++
25
50
6.65 ± 4.38++ 0.19 ± 0.39 0.00 ± 0.00 0.19 ± 0.38 0.10 ± 0.30 0.00 ± 0.00 0.18 ± 0.39 0.10 ± 0.30 0.00 ± 0.00
8.95 ± 4.71++ 9.87 ± 3.57++ 0.28 ± 0.48 0.00 ± 0.00 0.36 ± 0.63 0.00 ± 0.00 0.00 ± 0.00 0.18 ± 0.57 0.00 ± 0.00 0.00 ± 0.00
10.03 ±
2.76++
100
10.55 ±
4.75++
200
12.61 ±
7.05++
13.54 ± 3.76 0.28 ± 0.63 0.00 ± 0.00 0.00 ± 0.00 0.19 ± 0.40 0.28 ± 0.45 0.00 ± 0.00 0.00 ± 0.00 0.09 ± 0.29
25
13.86 ±
6.05++
8.91 ± 3.56+ 0.09 ± 0.29 0.00 ± 0.00 0.66 ± 0.45 0.00 ± 0.00 0.00 ± 0.00 0.47 ± 0.65 0.00 ± 0.00 0.00 ± 0.00
50
13.32 ±
2.82++
11.05 ± 4.62+ 0.00 ± 0.00 0.00 ± 0.00 0.40 ± 0.70 0.08 ± 0.26 0.00 ± 0.00 0.19 ± 0.40 0.00 ± 0.00 0.00 ± 0.00
100
15.88 ±
6.56++
11.67 ±
3.96++
0.00 ± 0.00 0.00 ± 0.00 0.57 ± 0.67 0.00 ± 0.00 0.10 ± 0.31 0.28 ± 0.44 0.00 ± 0.00 0.00 ± 0.00
200
16.24 ±
6.20++
12.63 ±
6.20++
0.00 ± 0.00 0.00 ± 0.00 0.35 ± 0.83 0.00 ± 0.00 0.00 ± 0.00 0.18 ± 0.39 0.00 ± 0.00 0.00 ± 0.00
0.19 ± 0.41 0.00 ± 0.00 0.65 ± 0.77 0.00 ± 0.00 0.19 ± 0.39 0.48 ± 0.82 0.00 ± 0.00 0.00 ± 0.00
Post-treatment
Piperine + MMS I
Pretreatment
Capsaicin + MMS I
25
5.67 ± 2.22++ 5.45 ± 3.63++ 0.10 ± 0.31 0.00 ± 0.00 0.17 ± 0.37 0.00 ± 0.00 0.09 ± 0.29 0.19 ± 0.40 0.00 ± 0.00 0.10 ± 0.31
50
9.76 ± 3.16+ 7.79 ± 2.61++ 0.19 ± 0.40 0.00 ± 0.00 0.73 ± 0.37 0.00 ± 0.00 0.00 ± 0.00 0.19 ± 0.39 0.00 ± 0.00 0.00 ± 0.00
100
8.13 ± 5.64++ 10.22 ± 5.78+ 0.59 ± 1.88 0.00 ± 0.00 0.75 ± 1.08 0.00 ± 0.00 0.28 ± 0.62 0.37 ± 0.48 0.10 ± 0.31 0.09 ± 0.29
200
8.01 ± 3.30++ 9.60 ± 4.30+ 0.00 ± 0.00 0.09 ± 0.28 0.46 ± 0.68 0.00 ± 0.00 0.10 ± 0.31
0.10 ± 0.31 0.09 ± 0.29 0.10 ± 0.31
Simultaneous treatment
Capsaicin + MMS I
25
25.47 ± 4.44 12.31 ± 3.84+ 0.19 ± 0.40 0.00 ± 0.00 0.66 ± 0.65 0.10 ± 0.31 0.37 ± 0.90 0.09 ± 0.29 0.00 ± 0.00 0.19 ± 0.40
50
20.64 ±
8.54+
100
19.07 ±
5.80++
11.89 ± 6.63+ 0.68 ± 0.65 0.09 ± 0.29 0.76 ± 0.89 0.27 ± 0.86 0.39 ± 0.81 0.29 ± 0.46 0.00 ± 0.00 0.00 ± 0.00
10.50 ±
4.24++
0.45 ± 0.65 0.00 ± 0.00 0.46 ± 0.65 0.00 ± 0.00 0.19 ± 0.60 0.00 ± 0.00 0.00 ± 0.00 0.09 ± 0.30
An Acad Bras Cienc (2021) 93(Suppl. 3)
e20201772 10 | 15
MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
Table IV. Continuation.
Chromosomal alteration
Treatment
(µM)
200
MN
NB
CB
BC
CA
Cm
20.59 ± 8.21+ 14.84 ± 4.86 0.39 ± 0.69 0.10 ± 0.32 0.86 ± 1.05 0.10 ± 0.31
CL
0.10 ± 0.31
CBr
MA
NA
0.10 ± 0.31 0.00 ± 0.00 0.00 ± 0.00
Post-treatment
Capsaicin + MMS I
25
31.53 ± 8.44 17.16 ± 4.47 0.10 ± 0.58 0.09 ± 0.30 0.74 ± 0.84 0.20 ± 0.63 0.00 ± 0.00 0.18 ± 0.38 0.09 ± 0.30 0.00 ± 0.00
50
42.56 ± 11.74 16.50 ± 5.00 0.10 ± 0.31 0.00 ± 0.00 0.81 ± 0.67 0.09 ± 0.28 0.91 ± 1.03 0.37 ± 0.48 0.09 ± 0.28 0.17 ± 0.36
100
37.85 ± 7.86 17.35 ± 2.11 0.10 ± 0.31 0.10 ± 0.32 0.86 ± 0.83 0.18 ± 0.38 0.48 ± 0.69 0.55 ± 0.63 0.00 ± 0.00 0.00 ± 0.00
200
26.70 ±
10.25
11.82 ±
6.16++
0.00 ± 0.00 0.00 ± 0.00 0.28 ± 0.46 0.29 ± 0.66 0.28 ± 0.64 0.19 ± 0.41
0.10 ± 0.31 0.00 ± 0.00
Data are means ± SD (Standard deviation). NC: Negative Control (Dimethylsulfoxide - DMSO 2% in distilled water). MMS I: 10
µg/mL of Methylmethanesulfonate dissolved in DMSO 2%. MN: Micronucleus. NB: Nuclear Bud. CB: Chromosomal Breaking.
BC: Binucleated Cell. CA: Chromosomal Adherence. Cm: C-metaphase. CL: Chromosomal Loss. CBr: Chromosomal Bridge. MA:
Multipolar Anaphase. NA: Nuclear Alteration. Pre-treatment (piperine or capsaicin + MMS). Simultaneous treatment (piperine or
capsaicin added simultaneously with MMS). Post-treatment (MMS + piperine or capsaicin). + Significant in the Kruskal-Wallis test
with a posteriori Student-Newman-Keuls test (+ p <0.05; ++ p <0.01) when compared to MMS I. The results refer to the analysis of
5,000 cells per treatment. The DMSO 2% was used as a negative control, but how the results were statistical identical to solvent
(distilled water), the data using water were omitted. The MMS I and MMS II (dissolved only in distilled water) also were statistical
identical, the data using MMS II were omitted.
In general, the tested compounds modulated
the genotoxic effect of MMS I. In the pre, the
isolated compound (piperine or capsaicin)
may have interacted directly with MMS I in the
intracellular environment in A. cepa cells. For
the simultaneous, the reduction in damage to
the cell can be a result of both the demutagenic
and bioantimutagenic action (Nantes et al. 2014)
by the tested bioactive agents. In posttreatment,
piperine also promoted a reduction in damage
induced by MMS I by the bioantimutagenic action,
which acts in DNA repair mechanisms, inducing
the reversion of the mutagenic effect and/or
preventing the fixation of mutations (Dametto
et al. 2017). In capsaicin, a similar result for the
posttreatment was observed only in the highest
concentration. These results reinforce the
interaction of piperine or capsaicin, modulating
the genotoxic action of MMS, however there was
no protective effect, since these molecules alone
were genotoxic with a significant production
of MN and NB. Moreover, the %RD was also a
result of a significant reduction of the same
alterations, reinforcing the possible interaction
between isolates with MMS I.
Only the lowest concentration of piperine (25
µM) was not genotoxic and showed a protective
effect in all protocols (pre, simultaneous,
and post), showing demutagenic and
bioantimutagenic action, which increases the
interest in further studies on this concentration
as a chemoprotective. Other studies have also
shown satisfactory results regarding the protective
effect of piperine in the test for chromosomal
changes in mouse bone marrow cells induced
by cyclophosphamide and mitomycin C (Wongpa
et al. 2007, Abo-Zeid & Farghaly 2009). Piperine
also decreased the genotoxic (comet assay) and
mutagenic (test micronucleus) effect induced by
aflatoxins in chickens (Cardoso et al. 2016). The
cytochrome P450 enzyme is responsible for 50%
of the metabolism of therapeutic agents, and
the comparison of the presence of this enzyme
complex leads to the conclusion that plants have
a lower concentration of antioxidant enzymes
compared to mammals and insects (Leme &
Marin-Morales 2009, Rocha et al. 2016).
An Acad Bras Cienc (2021) 93(Suppl. 3)
e20201772
11 | 15
MARCONDES S. DIAS et al.
TOXICOGENETIC EFFECT OF PIPERINE AND CAPSAICIN
MMS was used in the present study as
a DNA damage inducer in the A. cepa assay.
There are two main mechanisms by which
this compound can act. The first is its known
capacity for alkylation and methylation, which
can cause breaks in the double strand of DNA
and inhibition of the replication fork (Chatterjee
& Walker 2017). The second is its induction of
high levels of oxidative stress, which can lead
to apoptosis, cell death and DNA damage
(Lackinger et al. 2001, Jiang et al. 2016). Studies
demonstrate the ability to deplete GlutathioneS-transferase (Liu et al. 1996) and Glutathione
(Siddique et al. 2019) levels by MMS, which
impairs cellular antioxidant defenses and
leads to the accumulation of ROS generated as
byproducts of normal cellular metabolism (Raza
2011).
Piperine or capsaicin probably neutralized
and/or modulated the action of MMS I by the
two mechanisms mentioned, since the direct
genotoxic action of MMS I was reduced in the
protocols used. Moreover, piperine or capsaicin
may also have acted by neutralizing the ROS
resulting from the action of MMS, since the
isolates are alkaloids and have antioxidant
activities, neutralizing the action of free radicals
(Kaur & Arora 2015, Tsoi et al. 2015).
Based on the results obtained in the present
study, piperine and capsaicin showed a cytotoxic
effect, except for the lowest concentration,
associated mainly with the reduction of
prophases in A. cepa, and genotoxic effect
with emphasis on MN and NB, except for the
lowest concentration of piperine. Even with no
cytoprotective effect, the analyzed compounds
reduced chromosomal alterations (MN and
NB) in most protocols and concentrations,
which reinforces the possible interaction with
MMS. However, only the lowest concentration
of piperine (25 µM) was not genotoxic and
showed a protective effect in all protocols,
while the other concentrations of the tested
molecules alone were genotoxic. Thus, the
lowest concentration of piperine demonstrated
important chemopreventive activity, which is
indirectly correlated with the prevention and/
or treatment of genetic diseases, such as cancer.
Nevertheless, further studies are required to
elucidate possible mechanisms of interaction
between biocompounds and MMS.
Acknowledgments
The Universidade Federal do Piauí (UFPI) and the
Coordenação de Aperfeiçoamento Pessoal de Nível
Superior (CAPES). The authors would like to thank the
Universidade Estadual do Piauí for providing some of
the necessary laboratory facilities for this work.
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ERASMO P.V. JUNIOR2
https://orcid.org/0000-0002-7538-3822
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https://orcid.org/0000-0001-5431-6818
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PEDRO M. DE ALMEIDA2
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https://orcid.org/0000-0003-2598-2621
Programa de Pós-Graduação em Genética e Melhoramento,
Universidade Federal do Piauí /UFPI, Laboratório de
Genética, Ininga, 64049-550 Teresina, PI, Brazil
2
Universidade Estadual do Piauí, Centro de Ciências
Naturais (CCN), Laboratório de Genética, Rua João
Cabral, 2231, 64002-150 Teresina, PI, Brazil
3
Universidade Federal de Tecnologia, Paraná /UTFPR,
Departamento de Biodiversidade e Conservação da Natureza,
Campus Campo Mourão, Via Rosalina Maria dos Santos,
1233, Caixa Postal 271, 87301-899 Campo Mourão, PR, Brazil
Correspondence to: Pedro M. de Almeida
E-mail: pedromarcos@ccs.uespi.br
Author contribuitions
Marcondes Soares Dias conducted the entire study, analysis
and interpretation of data and writing of the manuscript.
Erasmo Pereira do Vale Junior and Bianca Cristina dos Santos
participated in the analysis and interpretation of the data.
Francielle Alline Martins contributed to the discussion and
text review and Ana Paula Peron, co-supervised the research,
was responsible for the study design and review. Pedro Marcos
de Almeida participated in all stages from the idealization
and design of the study, as well as analysis, review and
interpretation of data.
How to cite:
DIAS MS, JUNIOR EPV, DOS SANTOS BC, MARTINS FA, DE ALMEIDA PM &
PERON AP. 2021. Cytogenotoxicity and protective effect of piperine and
capsaicin on meristematic cells of Allium cepa L. An Acad Bras Cienc 93:
e20201772. DOI 10.1590/0001-3765202120201772.
Manuscript received on November 12, 2020;
accepted for publication on March 2, 2021
An Acad Bras Cienc (2021) 93(Suppl. 3) e20201772
15 | 15