Hindawi Publishing Corporation

Oxidative Medicine and Cellular Longevity

Volume 2015, Article ID 602360, 7 pages
http://dx.doi.org/10.1155/2015/602360

Research Article
Protective Effects of the Flavonoid Chrysin against
Methylmercury-Induced Genotoxicity and Alterations
of Antioxidant Status, In Vivo

Eduardo Scandinari Manzolli,1 Juliana Mara Serpeloni,1 Denise Grotto,1

Jairo Kennup Bastos,2 Lusânia Maria Greggi Antunes,1
Fernando Barbosa Jr.,1 and Gustavo Rafael Mazzaron Barcelos1
1
Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto,
University of São Paulo, Avenida do Café s/n∘ , 14040-903 Ribeirão Preto, SP, Brazil
2
Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo,
Avenida do Café s/n∘ , 14040-903 Ribeirão Preto, SP, Brazil

Correspondence should be addressed to Gustavo Rafael Mazzaron Barcelos; [email protected]

Received 10 December 2014; Revised 10 February 2015; Accepted 11 February 2015

Academic Editor: Silvana Hrelia

Copyright © 2015 Eduardo Scandinari Manzolli et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The use of phytochemicals has been widely used as inexpensive approach for prevention of diseases related to oxidative damage
due to its antioxidant properties. One of dietary flavonoids is chrysin (CR), found mainly in passion fruit, honey, and propolis.
Methylmercury (MeHg) is a toxic metal whose main toxic mechanism is oxidative damage. Thus, the study aimed to evaluate the
antioxidant effects of CR against oxidative damage induced by MeHg in Wistar rats. Animals were treated with MeHg (30 𝜇g/kg/bw)
in presence and absence of CR (0.10, 1.0, and 10 mg/kg/bw) by gavage for 45 days. Glutathione (GSH) in blood was quantified
spectrophotometrically and for monitoring of DNA damage, comet assay was used in leukocytes and hepatocytes. MeHg led to
a significant increase in the formation of comets; when the animals were exposed to the metal in the presence of CR, higher
concentrations of CR showed protective effects. Moreover, exposure to MeHg decreased the levels of GSH and GSH levels were
restored in the animals that received CR plus MeHg. Taken together the findings of the present work indicate that consumption of
flavonoids such as CR may protect humans against the adverse health effects caused by MeHg.

1. Introduction The flavonoid chrysin (CR; 5,7-dihydroxy-2-phenyl-4H-

chromen-4-one) is a natural and biologically active com-
The use of phytochemicals (PhC) has been proposed as pound; it is found mainly in honey, passion fruit (Passiflora
inexpensive approach for prevention of diseases associated sp.), and propolis [8–10]. Previous studies were already
with oxidative stress, such as cardiovascular disorders, degen- performed aiming to determine the concentrations of CR in
erative diseases, and cancer, due to its antioxidant properties honey and propolis; for example, Lachman et al. [11] assessed
[1–3]. PhC are bioactive compounds, nonnutrients found in the CR content in several types of honeys and found values
fruits, vegetables, grains, and other plants that are associated ranging from 0.10 mg/kgg (honeydew honeys) to 5.3 mg/kg
with a decreased risk to develop several diseases [1]. In this (forest honeys); in another study, Siess et al. [12] found levels
context, it is notable that several studies have been carried of CR in propolis as high as 28 g/L, demonstrating that honey
out aiming to assess the potential protective effects of PhC and propolis are a rich source of this flavonoid.
in many laboratory models, showing very promising results Compared with other flavonoids, few studies have been
[3–7]. carried out in order to determine the beneficial effects of CR.
2 Oxidative Medicine and Cellular Longevity

Some of these investigations yielded promising results. For Use of Experimental Animal Resources, University of São
example, Uhl et al. [13] demonstrated antimutagenic prop- Paulo, Brazil (approved protocol number 09.1.457.53.1).
erties against benzo(a)pyrene B(a)P induced DNA-damage
in HepG2 cells and Salmonella typhimurium; Anand et al. 2.3. Experimental Design. The dose of MeHg (30 𝜇g/kg of
[14] showed that CR reduces the disturbances of redox body weight (bw)) was chosen on the basis of previous studies
status, named superoxide dismutase (SOD), catalase (CAT), of our group which showed consistently that this concentra-
glutathione peroxidase (GPx), and glutathione (GSH) in liver, tion is able to induce DNA damage and disturbances of redox
kidney, and brain tissues of rats treated with D-galactose. status and also reflects the exposure levels of individuals from
Fish is considered a healthy food since it is a good source Amazonian region [30–32] and from other regions which also
of proteins, is poor in saturated fats, and has high amounts have high levels of MeHg exposure [33]. Treatments with CR
of polyunsaturated fat acids, which may protect against were chosen according to previous articles [14, 34].
cardiovascular disorders; and populations that traditionally The animals were divided in eight groups (six
consume large amounts of fish generally show low mortality animals/group): (I) control (mineral oil); (II) MeHg
rates from coronary diseases [15, 16]. On the other hand, fish (30 𝜇g/kg bw); (III) CR I (0.10 mg/kg bw); (IV) CR II
consumption is also an important source of human exposure (1.0 mg/kg bw); (V) CR III (10 mg/kg bw); (VI) CR I + MeHg;
to a variety of bioactive toxicants such as methylmercury (VII) CR II + MeHg; and (VIII) CR III + MeHg; the animals
(MeHg) and arsenic (As) [17–19] which may interact with the were treated daily by gavage for a period of 45 days. After the
health effects of fish intake [20–22]. It is well established that treatment, the rats were killed by an overdose of ketamine and
chronic exposure to MeHg is associated with neurological xylazine (300 and 30.0 mg/kg bw, resp.). Subsequently, blood
disorders [23–25] and with adverse effects on the cardiovas- was collected by decapitation and used for comet analyses
cular system [26, 27]. One of the main mechanisms respon- and to determine the GSH levels. The livers were rinsed with
sible for MeHg toxicity is the induction of oxidative stress. phosphate buffered saline (PBS, pH 7.4) before removal.
Earlier data have consistently shown that MeHg exposure can
lead to disturbances in the redox status, causing oxidative 2.4. Comet Assays with Peripheral Leukocytes and Hepato-
damage in macromolecules, such as lipids and DNA [28, 29]. cytes. Whole blood was used for the determination of DNA
Despite the low number of studies concerned with eval- damage in leukocytes [35]. Samples of liver were collected
uating the protective effects of PhC against MeHg-induced after euthanasia, and 0.20 g of each organ was placed in 1.0 mL
adverse effects, the aim of present study was to investigate of chilled Hank’s balanced salt solution in a Petri dish, sliced
potential protective effects of CR against the toxic effects into fragments with a pair of scissors, and filtered through
caused by MeHg, through monitoring of DNA-damage by two layers of gauze [36].
comet assays in peripheral blood cells and hepatocytes and The SCGE assays were carried out according to the
by determination of GSH levels in blood of Wistar rats. protocol of Singh et al. [37]. Briefly, 20 𝜇L of blood or
nuclei from liver suspensions was transferred to agarose-
2. Materials and Methods coated slides which were coverslipped and cooled at 4∘ C
for 20 min. After removal of the coverslips, the slides were
2.1. Chemicals. Methylmercury chloride (CAS 115-09-3), immersed in fresh lysis solution for 1 h at 4∘ C. Thereafter,
reduced glutathione (GSH, CAS 70-18-8), glutathione reduc- they were transferred to an electrophoresis chamber with
tase (GR, CAS 9001-48-3), sodium azide (CAS 26628-22-8), buffer (300 mM NaOH and 1.0 mM EDTA pH > 13) and
trypan blue (CAS 72-57-1), ethidium bromide (CAS 1239- electrophoresis was conducted under standard conditions
45-8), and 5-5󸀠 -dithiobis-(2-nitrobenzoic acid) (DTNB; CAS (25 V; 300 mA; 1.25 V/cm) for 20 min. Subsequently, the slides
69-78-3) came from Sigma-Aldrich (St. Louis, MO, USA). were neutralized, air-dried, and fixed in absolute ethanol for
CR (CAS 480-40-8) was kindly provided by Professor Dr. 10 minutes; then they were stained with ethidium bromide
Jairo K. Bastos from School of Pharmaceutical Sciences of and evaluated with a fluorescence microscope (Nikon, Japan)
Ribeirão Preto, São Paulo, Brazil. The purity of CR used in under 40x magnification. From each sample, two slides were
the experiments was ≥95%. Ketamine and xylazine were from made and from each, 50 cells were evaluated per animal.
Bayer (São Paulo, Brazil). Low melting point agarose (LMP) Comets were scored using the Comet Score software from
and normal melting point (NMP) agarose were obtained Tritek (Sumerduck, VA, USA); the percentage of DNA in
from Invitrogen (California, CA, USA). All other chemicals, tail was determined as a parameter of DNA damage. All
reagents, and buffers were analytical grade products from experiments were carried out according to the guidelines for
Sigma (St. Louis, MO, USA). SCGE assays [36]. The trypan dye exclusion method [38]
was used to determine cell viability immediately before the
2.2. Animals. The experiments were carried out with 2- comet assays and 300 cells were evaluated per group. In all
month-old male Wistar rats, weighing on average 200 ± 20 g, treatments, the viability was higher than 80%.
which were obtained from Central Animal House (University
of São Paulo, Ribeirão Preto, Brazil). The animals were kept 2.5. Total Thiols (GSH) Levels. Total thiols (taken here as
under a 12 h light/dark cycle in an acclimatized room at GSH) were determined in erythrocytes by addition of DTNB
22–25∘ C and had free access to food (standard ration from as described by Ellman [39]. DTNB, a symmetric aryl
Guabi, Campinas, Brazil) and water. The animals were used disulfide, reacts with free thiols to form disulfide plus 2-nitro-
according to the guidelines of the Committee on Care and 5-thiobenzoic acid. The reaction product was quantified by
Oxidative Medicine and Cellular Longevity 3

measurement of absorbance at 412 nm with a spectropho- concentrations between 14.3 and 174.7 nmol/plate were not
tometer (Micronal B380 UV–Vis, São Paulo, Brazil). Results able to induce mutagenic effects in Salmonella typhimurium
are expressed as micromoles per milliliter (𝜇mol/mL) in TA 98 and TA 100 with or without S9 fraction. On the
blood. other hand, Uhl et al. [13] demonstrated that CR induces
MN formation in HepG2 cells in doses ranging from 15
2.6. Statistical Analysis. All data analyses were performed to 35 𝜇g/mL; also, Oliveira et al. [43] reported increase of
with the GraphPad Prism version 6.01 for Windows (La MN induction in HepG2 cells exposed to the flavonoid
Jolla, CA, USA). Results are reported as means ± standard (1.0–15 𝜇M). In the same work, the later authors found that
deviations (SD). The results of different experiments were high doses of CR are able to induce mutagenic effects in S.
analyzed using one-way ANOVA and Dunnett’s test. 𝑃 values typhimurium TA 98 and TA 100 (with or without S9 fraction).
≤ 0.050 were considered as statistically significant. Resende et al. [42] reported that higher concentrations of
CR (116.4 and 174.7 nmol/plate) induce significant number of
3. Results revertants per plate in S. typhimurium TA 102. In this context,
it is notable that flavonoids and other phytochemicals may act
The results of the experiments concerning the impact of as prooxidants at high concentrations [44, 45].
CR on MeHg-induced comet formation are summarized in Despite the large number of in vitro studies which aimed
Figure 1. Exposure of the animals to the metal compound to assess the genotoxicity of CR, to our knowledge, there are
increased the extent of DNA-migration in leukocytes and no in vivo studies and there are no works that evaluated the
liver cells 9.3 and 4.9-fold over the background values while potential genotoxic effects of CR on animals in subchronic
all treatments with CR did not cause significant DNA-damage treatments as the present study. Here, we observed that
under our experimental conditions. Comet formation was CR was not able to induce comet formation in all doses
significantly reduced when the flavonoid was administered tested. This difference between the observations in mammal
in combination with the metal. In leukocytes, the decrease cells culture, such as HepG2, and the present data may be
of comet formation at the highest doses (1.0 and 10 mg/mL) explained, at least partly, to the metabolism system of them.
was 33 and 35%, respectively, while in hepatocytes only the HepG2 cells possess mainly high expression of enzymes of
highest dose was able to reduce MeHg induced DNA-damage. phase I of metabolism [46]; the same occurs in systems that
The results of the measurements of the GSH levels in used the S9 fraction (which also have high levels of phase I
blood are summarized in Figure 2. Treatment with the metal enzymes) [47], while in animals, the metabolism comprises a
reduced the levels of GSH when compared to negative balanced expression of phase I and phase II enzymes [48].
controls. Furthermore, it can be seen that treatment with It is well documented in in vitro and in vivo experiments
the higher doses of CR had a clear impact on the GSH that MeHg exposure leads to formation of reactive species
concentrations and reduced the GSH concentrations by 15 that may cause oxidative damage of macromolecules [28, 29].
and 16%, respectively. Furthermore, it was also shown that the MeHg binds to
Figure 2 also shows the levels of the tripeptide GSH which endogenous biomolecules with –SH groups; this observation
were measured after combined treatment of the animals with explains the decrease of the GSH levels which was seen in
MeHg and different doses of the flavonoid. When the animals the present experiments and is in accordance with previous
were exposed simultaneously to the metal compound and to studies [49, 50].
CR, the GSH concentrations were restored to those found in One of the most important mechanisms to explain
untreated control animals. the oxidative damage induced by Hg exposure is its high
reactivity with sulhydryl groups (−SH) of macromolecules,
4. Discussion which may inactivate them [51]. GSH is the main intracellular
nonprotein free thiol, and it is also one of the most important
Taken together, the findings of the present work indicate that antioxidants in the body [52]. It is conceived that GSH play
consumption of flavonoids such as CR may protect humans a role as a first cellular defense against Hg compounds. The
against the adverse health effects caused by exposure to metal compounds bind to GSH covalently, through cysteine
MeHg. residues and thus, its deleterious effects are minimized. This
The observation of comet formation in white blood protective effect mediated by GSH, however, decreases its
cells and hepatocytes of the animals after treatment with concentrations, and then the cells may be more susceptible
MeHg is in agreement with results of earlier studies [31, to oxidative damage through the accumulation of reactive
32, 40]. In addition, we demonstrated previously that MeHg oxygen species (ROS) normally neutralized by GSH [53, 54].
increases the formation of 8-hydroxy-2󸀠 -deoxyguanosine in The reactive species then attack proteins, DNA and lipids
HepG2 cells [5]. Jin et al. [41] also observed increase of this [55], inducing oxidative damage. Hg interferes with the
parameter in rats that were exposed to the metal compound. activity of several antioxidant enzymes; for example, the
These findings give pieces of evidence that oxidative damage activity of superoxide dismutase (SOD), catalase (CAT), and
accounts for the comet formation which we observed in the glutathione peroxidase (GPx). However, so far, there is no
present study. general consensus among the data; that is, some studies
In vitro results concerning the genotoxic properties of reported increase of activity of certain antioxidant enzymes
CR are, so far, contradictory and are related to the models while others showed a reduction in activity of these enzymes.
that were used. For example, Resende et al. [42] showed that Ariza et al. [40] demonstrated that HgCl2 , inducing the
4 Oxidative Medicine and Cellular Longevity

40 40
35 35 ∗
∗

%DNA in tail (hepatocytes)

%DNA in tail (leukocytes)

30 30 ∗
25 25 ∗#
∗
∗
20 20
∗# ∗#
15 15
10 10 # #
# # #
5 # # # 5
0 0
Ctrl

MeHg

CR I

CR II

CR III

MeHg + CR I

MeHg + CR II

MeHg + CR III

Ctrl

MeHg

CR I

CR II

CR III

MeHg + CR I

MeHg + CR II

MeHg + CR III
(a) (b)

Figure 1: Impact of oral treatment of rats with CR on induction of DNA-damage by MeHg in (a) lymphocytes and (b) hepatocytes. The animals
were treated by gavage with different doses of the flavonoid (CR I: 0.1 mg/kg/bw/day; CR II: 1.0 mg/kg/bw/day; and CR III 10 mg/kg/bw/day)
in combination with the metal (30 𝜇g/kg/bw/day) over a period of 45 days. Bars indicate means ± SD of results obtained with six animals
per group. Stars indicate significant difference from negative control group; hashes indicate significant difference in comparison to the MeHg
group (𝑃 ≤ 0.050; one-way ANOVA and Dunnett’s test).

1.5 an indirect compensatory response of the cells to increased

oxidative stress, such as self-protection mechanism [49].
#
1.2 # We also showed that the higher doses of CR (1.0 and
#
GSH (𝜇mol/mL blood)

# 10 mg/kg bw, resp.) reduce the levels of GSH when compared

∗ ∗
∗ to negative controls. A possible explanation for this obser-
0.9
vation is that antioxidant properties of CR may modulate
a feedback mechanism on the antioxidant system triggered
0.6
by administration of the flavonoid to the animals, which
was already seen in a previous study where female rats were
0.3 exposed to several flavonoids, including CR [57].
The expectation that CR prevents the adverse effects of
0.0 MeHg is based on results of previous studies which indicate
Ctrl

MeHg

CR I

CR II

CR III

MeHg + CR I

MeHg + CR II

MeHg + CR III

that the flavonoid shows DNA-protective effects against radi-

ation and chemically induced DNA-instability. For example,
Benković et al. [58] showed that CR at dose of 100 mg/kg bw
was able to decrease the comet formation in leukocytes
of gamma-irradiated mice; in a further study of the same
Figure 2: Impact of oral treatment of rats with CR on the authors [59] it was observed that pretreatment of CR protects
levels of GSH in blood. The animals were treated by gavage with against DNA-damage induced by gamma radiation in human
different doses of the flavonoid (CR I: 0.1 mg/kg/bw/day; CR II: lymphocytes cell cultures. Uhl and coworkers [13] observed
1.0 mg/kg/bw/day; and CR III 10 mg/kg/bw/day) in combination that CR decreases MN formation induced by B(a)P and 2-
with the metal (30 𝜇g/kg/bw/day) over a period of 45 days. Bars amino-1-methyl-6-phenylimidazo[4,5-b]pyridine exposure.
indicate means ± SD of results obtained with six animals per group. In the same study, the authors also observed protective
Stars indicate significant difference from negative control group;
effects in S. typhimurium strains TA 98 and TA 100 against
hashes indicate significant difference in comparison to the MeHg
group (𝑃 ≤ 0.050; one-way ANOVA and Dunnett’s test). B(a)P. A recent study of Resende et al. [60] gives further
pieces of evidence concerning the antigenotoxic effects of
CR against several indirect and direct mutagens (4-nitro-
o-phenylenediamine, sodium azide, mitomycin C, B(a)P,
formation of H2 O2 , stimulates SOD activity and does not aflatoxin B1 and 2-anthramine) in S. typhimurium TA 98,
affect the activity of CAT and GPx, whereas Hussain et al. 100, and 102, with or without activation mix (S9). Another
[56] showed an increase in the activity of GPx and CAT. In hypothesis is that CR can bind directly to MeHg, chelating the
general, acute exposure to Hg leads to decrease in enzymatic metal, and prevent the direct oxidative damage induced by
activity; on the other hand, exposure to prolonged periods exposure to the metal, which was already reported by several
seems to not change the enzyme patterns, possibly due to studies (for a comprehensive review, see Flora 2009; 2010)
Oxidative Medicine and Cellular Longevity 5

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