Sclerotinia Sclerotiorum (White Mold): Cytotoxic, Mutagenic, and Antimalarial Effects In Vivo and In Vitro

Abstract

This work aimed includes performing the sclerotia chemical profile and evaluates their biological effects on mutagenesis, oxidative stress, cancer, and malaria. A chemical profile was determined by ultraperformance liquid chromatography mass spectrometry (UHPLC–HRMS) analysis dereplicating norditerpenoid dilactone, sclerolide, and other compounds. The GI50 values to cancer cells (19.8 to 277.6 µg/mL) were higher than normal (16.05 µg/mL), meaning high cytotoxicity. Regarding the oxidative stress, the results showed that the all AcOET fraction concentrations tested on IMR90 noncancer cell increased reactive oxygen species (ROS) production in more intense way (by fivefold) than in tested cancer cells. The in vivo study showed an increase of the following biomarkers (by 296.00%): % DNA in comet tail in peripheral blood and liver cells; micronucleated erythrocytes and colon cells and lipid serum peroxidation. These results indicate the sclerotia as genotoxic and mutagenic agent and its contamination may lead to fungal toxic effects with a risk to human health.

Full text

Toxicology & Chemical
Food Safety
Sclerotinia Sclerotiorum (White Mold): Cytotoxic,
Mutagenic, and Antimalarial Effects In Vivo and
In Vitro
Carolina Girotto Pressete, Laila Santos Vieira Giannini, Daniela Aparecida Chagas de Paula, Mariana Ara´
ujo Vieira do Carmo,
Diego Magno Assis, M´
ario Ferreira Conceic¸˜
ao Santos, Jos´
e da Cruz Machado, Marcos Jos´
e Marques, Marisi Gomes Soares,
and Luciana Azevedo
Abstract: This work aimed includes performing the sclerotia chemical profile and evaluates their biological effects
on mutagenesis, oxidative stress, cancer, and malaria. A chemical profile was determined by ultraperformance liquid
chromatography mass spectrometry (UHPLC–HRMS) analysis dereplicating norditerpenoid dilactone, sclerolide, and
other compounds. The GI50 values to cancer cells (19.8 to 277.6 µg/mL) were higher than normal (16.05 µg/mL),
meaning high cytotoxicity. Regarding the oxidative stress, the results showed that the all AcOET fraction concentrations
tested on IMR90 noncancer cell increased reactive oxygen species (ROS) production in more intense way (by fivefold)
than in tested cancer cells. The in vivo study showed an increase of the following biomarkers (by 296.00%): % DNA in
comet tail in peripheral blood and liver cells; micronucleated erythrocytes and colon cells and lipid serum peroxidation.
These results indicate the sclerotia as genotoxic and mutagenic agent and its contamination may lead to fungal toxic
effects with a risk to human health.
Keywords: apoptosis, cytotoxicity, fungus, malaria, mutagenicity, oxidative stress
Introduction
It is known that natural compounds may behave as beneficial
or toxic to health and we confronted with a variety of those ones
within our everyday life (Wang, Ouyang, & Lin, 2018). Toxins
are hazardous substances, causing illness or damage to an exposed
organism if inhaled, swallowed, or absorbed through the skin
(Schilter, Constable, & Perrin, 2014). Fungi (yeasts and molds) and
bacteria are capable of producing toxic secondary metabolites that
can contaminate food and cause adverse effects on human health
(Aichinger et al., 2018), which effects include acute or chronic
toxicity, genotoxicity, mutagenicity, carcinogenicity, neurotoxicity
(Tournas, 2005), pulmonary infection, allergies, osteomyelitis,
endocarditis, keratitis (Fr ˛
ac, Jezierska-Tys, & Yaguchi, 2015),
hepatotoxicity, and teratogenicity upon consumption (Kong et al.,
2014; Shin, Bae, Choi, & Woo, 2014). Conversely, many of them
cause a broad variety of beneficial biological activities including
anticancer, anti-inflammatory, antimicrobial (Chow & Ting, 2015;
Liu, Zhao, Sun, Li, & Liu, 2018; Shin et al., 2014), antibacterial,
antiviral, and antiprotozoal therapies (Bhadury, Mohammad, &
Wright, 2006). Thus, a large number of secondary metabolites
from fungi occupy a significant position in the pharmaceutical in-
dustry and have been integrated into drug development (Bhadury
JFDS-2019-0849 Submitted 5/31/2019, Accepted 10/5/2019. Authors Pressete,
Giannini, Vieira do Carmo, and Azevedo are from Nutrition Faculty, Federal Univ.
of Alfenas, Alfenas, Minas Gerais, Brazil. Authors de Paula, Santos, and Soares
are from Federal Univ. of Alfenas, Chemistry Inst., Alfenas, Minas Gerais, Brazil.
Author Assis is from Bruker do Brasil, Atibaia, S˜
ao Paulo, Brazil. Author Machado
is from Federal Univ. of Lavras, Seed Pathology Laboratory, Lavras, Minas Gerais,
Brazil Author Marcos Marques is from Federal Univ. of Alfenas, Inst. of Biomedical
Sciences, Alfenas, Minas Gerais, Brazil Direct inquiries to author Azevedo (Email:
lucianaazevedo2010@gmail.com).
et al., 2006). Compounds that are ultimately selected for devel-
opment of new drugs must meet the requirements of safe drugs
and that do not show any overt toxicity to human health (Fidock,
Rosenthal, Croft, Brun, & Nwaka, 2004), however, this require-
ment is not always met. Sadorn et al. (2018) showed that crude ex-
tracts from fungus Cytospora eugeniae had cytotoxic activity against
human breast adenocarcinoma MCF-7, papilloma carcinoma KB,
and lung NCI-H187 cancer cells, but also demonstrate toxic
effect in noncancerous cells (Vero, African green monkey kidney
fibroblasts).
The fungus Sclerotinia sclerotiorum is remarkable for its extremely
broad host range and for its aggressive host tissue colonization
(Liang & Rollins, 2018). This nonspecific plant pathogen may
infect economically important crops and vegetables such as sun-
flower, bean, soybean, canola, cotton, potatoes, peas, and tomatoes
(Duan et al., 2018; Heard, Brown, & Hammond-Kosack, 2015;
Xu, Liang, Hou, & Zhou, 2015). Its infection causes stem rot or
white mold and represents one of the major challenges for agri-
cultural production (Bolton, Thomma, & Nelson, 2006; Malenˇ
ci´
c
et al., 2010). The food contamination with S. sclerotiorum may be
derived from their sclerotia. These structures are characterized by
hard melanized rind enclosing compact dark bodies and they are
not always removed dur ing harvest and postharvest procedures,
which may lead to their human consumption (Azevedo et al.,
2016). Unfortunately, little is still known about the effects of hu-
man consumption of contaminated food with S. sclerotiorum since
their compounds can be a potential health hazard to individuals.
Azevedo et al. (2016) pointed out that sclerotia aqueous extract
presented mutagenic and cytotoxic effects against colon adeno-
carcinoma (HT29). Herein, we aimed at performing the chemical
profile of sclerotia and to assay their in vivo and in vitro toxicological
activities, furthermore determined their possible antimalarial and
anticancer potential.
C2019 Institute of Food Technologists R
3866 Journal of Food Science rVol. 84, Iss. 12, 2019 doi: 10.1111/1750-3841.14910
Further reproduction without permission is prohibited

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Materials and Methods
Sample preparation and chemical profile analyses
Sclerotinia sclerotiorum was obtained in sclerotia form from con-
taminated soybean seed lots from Seed Pathology Laboratory of
the Federal Univ. of Lavras (UFLA/MG). The sclerotia of S. scle-
rotiorum were then separated from the soybean seeds by manual
separation process and storage at −80 °C. Only one batch of fungi
was used, therefore, there are no differences among extractions.
After manual separation, 1.69 Kg of sclerotia was ground once
with 2 L of ethanol, which was evaporated and concentrated to
500 mL. The resulting ethanol extract was diluted with 50 mL of
H2O and partitioned with ethyl acetate (3 ×500 mL), which pro-
vided the ethyl acetate (AcOET) fraction acquisition. This fraction
was subjected to a C18 reversed-phase column chromatography
with a H2O/MeOH gradient as the eluent and three AcOET frac-
tions were obtained: 100% H2O(F1),1:1H
2O/MeOH (F2), and
100% MeOH (F3). Concerning the in vivo experiment, we ap-
plied the AcOET fraction by representing the whole ethyl acetate
matrix. Differently, we perfor med in vitro biological evaluation
with F3 fraction, once the two other fractions possessed mainly
sugar-containing components. The UHPLC–HRMS analyses of
AcOET fraction and its F3 fraction were performed using a Shi-
madzu UHPLC instrument coupled with a photodiode array spec-
trophotometer and mass spectrometer QTOF Bruker Compact.
The UHPLC–HRMS analyses were performed using a C18 col-
umn (2.1 ×100 mm, 2.2 µm particle size, AcclaimTM RSLC 120,
Dionex, USA) with a flow rate of 0.4 mL/min and the following
gradient elution: 5 to 99% of acetonitrile in water containing 0.1%
of formic acid through 15 min; 99% of acetonitrile until 17.5 min,
99 to 99.5% of acetonitrile until 17.6 min, and 5% of acetoni-
trile until 20 min. The main MS parameters were related to the
standard of the equipment QTOF compact, for negative/positive
mode, such as collision gas supply flow rate 35.0%, dry gas source
10.0 L/min, dry heater source 200 °C, and extraction trigger time
100 µs.
The molecular formula (MF) was determined using the seven
golden rules for MF determination from data obtained by accurate
mass spectrometry (Kind & Fiehn, 2007; Theodoridis et al., 2011).
In addition, the dereplicated compounds were only those whose
MF matched with the in-house database built with compounds
from Sclerotinia genus (Chagas-paula, Zhang, Costa, & Edrada-
ebel, 2015; Kind & Fiehn, 2007; Wolfender, Litaudon, Touboul,
& Queiroz, 2019). The data were treated to decovolution, align-
ment and putative identification of the chemical features (m/z
and retention time) on the software Target Analysis (Bruker) and
MZmine 2.33. The Venn diagram was elaborated on the software
Ve n n y 2 . 1 .
Cytotoxicity and cell proliferation in vitro
The following cancer cell lines were obtained from the Rio
de Janeiro Cell Bank (BCRJ): human lung adenocarcinoma ep-
ithelial (A549), ileocecal colorectal adenocarcinoma (HCT8), and
normal lung cell (IMR90). These cells were cultured as detailed
in Migliorini et al. (2019).
Cell viability was assessed as described by De Menezes
et al. (2016), using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. Briefly, the cells were seeded
in 96-well plates at density of 5 ×103cells/well (A549 and HCT8)
and 2 ×103cells/well (IMR90). After 24 hr, cells were treated
with serial concentrations of AcOET fraction and F3 fraction (10
to 500 µg/mL) of S. sclerotiorum.TheIC
50, GI50,andLC
50 param-
eters were performed in accordance with the method described
by Carmo et al. (2018).
Reactive oxygen species (ROS) generation
The effects of S. sclerotiorum fractions on scavenging of in-
tracellular ROS in IMR90 nor mal cells (2 ×104cells/well),
HCT8, and A549 cancerous cells (6 ×104cells/well) were inves-
tigated by DCFH-DA (2,7-Dichlorofluorescin diacetate). Cells
were treated for 1 hr with different concentrations of S. sclerotiorum
AcOET fraction (5 to 100 µg/mL), diluted in DCFH-DA solution
(25 mmol/L), whose AcOET concentrations were determined by
GI20 from cell viability . Following the treatment, it was added
post-treatment with H2O2at 15 µmol/L for all the wells (Escher
et al., 2018). The fluorescence intensity was measured at an ex-
citation wavelength of 485 nm and at an emission wavelength of
538 nm. The data were expressed as percentage of fluorescence
intensity relative to the untreated group (negative control).
Antimalarial activity, selectivity index (SI), and chemical
injury to erythrocytes
Plasmodium falciparum resistant (W2) and chloroquine-sensitive
(3D7) strains used for antimalarial study were obtained from the
Oswaldo Cruz Foundation, Ren´
e Rachou Research Center. The
parasites were maintained using erythrocytes type O+from hu-
man healthy local donor. The culture media consisted of standard
RPMI 1640 (Sigma-Aldrich, Germany) supplemented with 10%
Albumax II (Gibco, Grand Island, NY, USA), 100 µMhypox-
anthine (Sigma-Aldrich, St. Louis, MO, USA), 25 mM HEPES
(Sigma-Aldrich, St. Louis, MO, USA), 12.5 µg/mL gentamicine
(Sigma-Aldrich, Dorset, UK), and 25 mM NaHCO3(Sigma-
Aldrich, St. Louis, MO, USA) and it was kept according to Radfar
et al. (2009).
A SYBRGreen I Rmicrofluorimetric DNA-based assay was
used to monitor parasite growth inhibition. Synchronized rings
from stock cultures were used to test different amounts 50 to
1200 µg/mL of AcOET fraction and F3 fraction of S. sclerotiorum,
besides 0.01 to 3.0 µg/mL serial dilutions of chloroquine (Sigma-
Aldrich, St. Louis, MO, USA) in 96-well culture microplates.
Thus, 150 µL of parasites at 4% hematocrit, 0.15% parasitemia
chloroquine resistant strain (W2), and 0.25% parasitemia chloro-
quine sensitive strain (3D7) were allowed to grow for 48 hr with
the above treatments. The microplates with parasites were then
centrifuged at 600 gfor 10 min and resuspended in 150 µLof
saponin (0.15%, w/v in phosphatebuffered saline) to lyse the ery-
throcytes. The pellet was washed by the addition of 150 µLofPBS,
centrifuged again and resuspended in 100 µL of PBS, and 100 µL
of SYBRGreen I Rdiluted in TE buffer. Plates were incubated for
30 min in the dark and the fluorescence intensity was measured at
485 nm excitation and 538 nm emission. Growth inhibition was
calculated in accordance with the method described by Moneriz,
Mar´
ın-Garc´
ıa, Bautista, Diez, and Puyet (2009).
The SI value was calculated as the ratio between cytotoxic IC50
values of normal lung cell (IMR90) and 3D7 or W2 parasitic IC50
values (Jansen et al., 2012).
Regarding the chemical injury, 200 µL of erythrocytes were in-
cubated with the treatments equal to the highest (1200 µg/µL)
used dose in the antiplasmodial assay with same assay condi-
tion. After 24 hr of incubation, smears stained with Giemsa
3% (v/v) were microscopically observed for any morphological
changes, which were compared with those in erythrocytes that
were uninfected and not exposed to AcOET and F3 (Dutta et al.,
2017).
Vol. 84, Iss. 12, 2019 rJournal of Food Science 3867

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
In vivo toxicological analyses of AcOET fraction
The toxicological analyses of ethyl acetate fraction (AcOEt)
were ver ified in 66 Swiss mice (weaned mice 15 ±5g)ob-
tained from Federal Univ. of Alfenas (UNIFAL-MG), Brazil. All
the experiments and procedures were approved by the Ethical
Committee for Animal Research (Protocol 408/2012). The ex-
periment was carried out with the following groups (n=11):
groups 1, 2, and 3 received commercial pelleted diet, group 4
received pellet crushed diet with addition of 6 mg of AcOET
fraction/100 g diet (corresponding to 0.06 g AcOET/kg diet
and 4.50 g sclerotia/kg diet), group 5 received crushed diet +
60 mg of AcOET fraction/100 g diet (corresponding to 0.60 g
AcOET/kg diet and 45.00 g sclerotia/kg diet), and group 6 re-
ceived crushed diet +600 mg of AcOET fraction/100 g diet
(corresponding to 6.00 g AcOET/kg diet and 450.00 g sclero-
tia/kg diet). On the 14th day of the experiment, the animals
were treated with: group 1 (negative control), physiological so-
lution (NaCl 0.9% w/v); group 2 (positive control), doxorubicin
chloridate (DXR 30 mg/kg b.w., ip); group 3 (positive control)
N’-dimethylhydrazine via oral (DMH, 30 mg/kg b.w., via gavage);
and groups 4, 5, and 6 with AcOET fraction plus physiological
solution. At the end of the study (15th day), all animals were
anesthetized with ketamine 10% (0.1 mg/100 g b.w.) and xylazine
(0.05 mg/100 g b.w.). At necropsy, blood, bone marrow cells,
colon, and liver were collected from all animals.
Bone marrow and colon micronucleus analysis and
apoptosis analysis
The mutagenicity of the AcOET fraction was evaluated using
the bone marrow micronucleus test as described in Azevedo et al.
(2019). The in vivo gut micronucleus test was performed according
to Silva et al. (2014). For the identification of apoptotic cells, a
total of 20 perpendicular well-oriented crypts were examined in
each animal, counting the total number of epithelial cells in each
crypt (Chang, Chapkin, & Lupton, 1997). The apoptotic cells
were identified as previously described by Risio et al. (1996). The
apoptosis was estimated as the percentage of apoptotic cells in
relation to the total number of cells.
Comet assay and oxidative stress
The peripheral blood and liver comet assay were executed, as
described in (Azevedo et al., 2019). The fluorescent labeled DNA
was visualized using a Nikon Eclipse 80i fluorescence microscope
at 200 ×magnification, with a green filter. The parameters for
DNA damage analyses included tail moment (TM), tail length, and
% DNA in comet tail. Fifty cells were counted from each slide for a
total of 100 cells per animal. Resulting images were captured and
processed with image analysis software (TriTek CometScoreTM
v1.5, Sumerduck, VA).
The oxidative stress assay was carried out in accordance with
the protocol by Uchiyama and Mihara (1978). Briefly, the liver
samples (0.5 g) were homogenized with 1.15% (w/v) potassium
chloride (KCl) and centrifuged, and the supernatant was used to
determine lipid peroxidation by malondialdehyde (MDA). The
protein content was quantified by the Bradford method (Brad-
ford, 1976) and the results were expressed in nmol/mg protein
(Uchiyama & Mihara, 1978).
Statistical analysis
One-way analysis of var iance (ANOVA) was used, followed by
Tukey’s test for ROS, antimalarial, lipid peroxidation analysis, and
comet assay. Cytotoxicity analysis of the dose–response was deter-
mined by nonlinear regression (curve fit) and t-test was performed
for all in vitro analysis, and χ2 for the micronucleus and apopto-
sis assays. The experiments were analyzed by GraphPad Prism R
(GraphPad Software, Inc., San Diego, CA, USA) software. All tests
were considered statistically significant with P-values of Pࣘ0.05.
Results and Discussion
Chemical composition of sclerotia fractions
The results of UHPLC–HRMS chemical profile analyses of
AcOET fraction and F3 fraction reveal that the main differences
between the chemical composition of the F3 fraction and AcOET
fraction was most pronounced in positive mode (Table 1). The
literature lists some volatile compounds emitted by S. sclerotio-
rum sclerotia such as: 2-methyl-2-bornene, 1-methylcamphene,
2-methylisoborneol, 2-methylenebornane, and a diterpene with
a molecular weight of 272 (Fravel, Connick, Grimm, & Lloyd,
2002). The characteristic musty earthy odor that fungus S. sclero-
tiorum exhales is from the compound 2-methylisoborneol (Fravel
et al., 2002). Another compound identified as part of the chemi-
cal composition of this fungus was 5-O-(α-d-galactopyranosyl)-d-
glycero-pent-2-enono-1,4-lactone that aids in the production of
oxalic acid, an acid that has a toxic effect, attacking and degrading
the complex structure of the host plant cell wall (Keats, Loewus,
Helms, & Zink, 1998; Komatsu, Tsuda, Omura, Oikawa, & Ikeda,
2008). In addition to these compounds, steroids, such as ergosterol,
ergosterol peroxide, and fatty acids, were identified by Dembit-
sky (2015) from S. sclerotiorum, who highlighted their possible
antimalarial, antibacterial, cytotoxic, and other pharmacological
activities as an important source of leads for drug discovery.
We dereplicated on AcOET fraction and F3 fraction (Figure 1A
and B, Table 1) and the samples showed 122 substances in common
to both samples and 211 only on the AcOET fraction and 770 only
on the F3 fraction (Figure 2C). The β-d-glucan, previously de-
scribed by Ohno and Yamode (1987), was detected exclusively to
the AcOET fraction samples (Table 1). This extracellular polysac-
charide was previously observed in Sclerotinia libertiana and it was
associated to antitumor properties (Ohno & Yadomae, 1987).
In vitro studies in noncancer and cancer cell lines
Regarding the biological activity of samples from sclerotia (IC50,
GI50,andLC
50, Table 2), AcOET fraction showed low GI50 val-
ues against HCT8 cancer cell (19.8 µg/mL) and IMR90 non-
cancer cells (16.05 µg/mL), indicating high cytotoxicity. In con-
trast, lower cytotoxicity was observed for both AcOET and F3
fractions against A549 cancer cell line (101.8 µg/mL to AcOET
and 277.6 µg/mL to F3). Besides, AcOET fraction presented
better antiproliferative effects against the HCT8 cells (IC50 48.0;
GI50 19.8 µg/mL) compared to F3 fraction (IC50 250.5; GI50
123.1 µg/mL), the below results highlighted that AcOET frac-
tion exhibited potential cytotoxic effects against both normal
and malignant cell lines. In contrast, Azevedo et al. (2016) us-
ing sclerotia aqueous extract pointed out that HT-29 cancer cells
(0.01598 mg/L) had almost three times lower GI50 values than
noncancer cell CCD-18Co (0.04712 mg/L), which turn evident
the importance of chemical profile and their interactions obtained
from each extract and fraction.
Furthermore, it is clear that the fungus AcOET fraction in-
duced ROS generation in noncancer cell (Figure 3) in more in-
tense way (by fivefold) than in studied cancer cells (by twofold
for highest concentration, which may explain its cytotoxic and
3868 Journal of Food Science rVol. 84, Iss. 12, 2019

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Table 1–Putative identification of compounds from sclerotia (Sclerotinia sclerotiorum) using in-house data base.
Retention time Ion Molecular formula Error (mDa) mSigma Compound Area in AcOET Area in F3
0.1 [M+H]+C18H32 O16 2.5 28.9 β-D-glucan 75,776 -
1.3 [M+H]+C11H19 N3O3S14.6 −Sclerothionine 19,156 −
4.2 [M+H]+C13H14 O4−1.5 −Sclerin 17,348 −
5.4 [M−H]−C14H20 O60.1 15 Colletodiol 1,420 3,674
[M+H]+0.4 1.4 3,027 13,157
6.3 [M+H]+C18H16 O54.7 −Sclerodione −11,187
6.9 [M−H]−C21H23 Cl1O51.9 −Sclerotiorin 517 1,097
7.3 [M-H]−C13H16 O5−1.9 3.4 Antibiotic LL−253 α11,91,531 149,251
7.6 [M+H]+C18H14 N2O30.4 25.0 Sclerominol 63,989 23,975
7.6 [M−H]−C12H14 O44.0 37.9 Sclerotin C −13,675
7.0 [M+H]+1.8 47.8 11,681
7.9 [M−H]−C18H16 O63.7 34.1 Scleroderolide 922 8,633
8.4 [M+H]+C15H22 O31.5 37.1 11-hydroxy-sclerosporin 6,651 −
8.8 [M+H]+C15H22 O23.6 −Sclerosporin 24,743 14,753
9.9 [M+H]+0.4 36.9 10,650 −
10.3 [M+H]+C16H16 O66.0 15.7 Norditerpenoid dilactone 2,025 2,321
12 [M+H]+C23H36 N4O43.2 26.6 Sclerotiotide E 1,90,158 −
12.5 [M+H]+C21H38 O40.2 3.5 Glycerol monolinolate 14,62,260 −
13.4 [M+H]+C12H14 O4−0.8 10.7 Sclerolide 99,576 −
15.6 [M+H]+C21H32 N4O50.9 −Sclertotiotide F −41,646
Figure 1–Base peak chromatogram of AcOET fraction (blue line) and F3 (red line) detected on the UHPLC–HRMS in negative mode (A) and positive
mode (B). (C) Number the compounds detected in UHPLC–HRMS. Some compounds are in both samples and others exclusively of AcOET or F3 fractions.
antiproliferative effects). Healthy cells have developed specific
adaptations to overcome the damaging effects of ROS, through
the balanced generation of these species and sufficient antioxidant
activity (Moloney & Cotter, 2018). However, in the present study,
the increase of ROS production in IMR90 noncancer cells showed
that cell antioxidant repair systems, such as superoxide dismutase,
glutathione reductase, and glutathione peroxidase, have not neu-
tralized the overproduction of free radicals leading these normal
cells to death. In contrast to our study, Azevedo et al. (2016) ob-
served that sclerotia aqueous extract increased ROS production
in more intensity in HT-29 cancer cells than in noncancer cell
CCD-18Co, leading this cancer cell to triggering the intrinsic
and extrinsic apoptotic pathways by ROS overproduction.
Thus, these results clearly pointing out the AcOET chemical
profile as cell inductor of ROS generation and cytotoxicity, whose
main differences from F3 fractions are the presence of compounds
such as β-d-glucan, sclerothionine, sclerin, sclerolide and scle-
rotiotide. Although the lack of data about biological effects of
majority of these fungi compounds in the literature, the purified
compounds sclerotiorin, scleroderolide, and sclerodione have al-
ready been associated with antiproliferative properties against can-
cer and normal cell lines (Giridharan, Verekar, Khanna, Mishra, &
Vol. 84, Iss. 12, 2019 rJournal of Food Science 3869

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Figure 2–Compounds from Sclerotinia sclerotiorum detected on the samples described in the Table 1.
Table 2–Cytotoxicity and inhibition of proliferation of human
lung adenocarcinoma epithelial (A549), ileocecal colorectal ade-
nocarcinoma (HCT8), and normal lung cell (IMR90) after 48 hr
exposure to AcOET and F3 fractions.
AcOET fraction F3 fraction
Cell lines (µg/mL) (µg/mL)
IMR90 IC50 103.64 ±0.59 127.63 ±0.38
GI50 16.05 ±2.94 12.73 ±4.22
LC50 205.9 ±2.67 177.3 ±1.73
HCT8 IC50 48.03 ±0.85 250.50 ±3.96
GI50 19.80 ±1.12 123.10 ±4.68
LC50 282.2 ±9.77 384.1 ±6.42
A549 IC50 107.80 ±7.98 259.40 ±3.65
GI50 101.80 ±2.11 277.60 ±3.17
LC50 180.08 ±0.5 >500
IC50: the concentration of the agent that inhibits growth by 50%, is the concentration at
which (T/C)×100 =50, where T=number of cells, at time t of treatment; C=
control cells at time t of treatment.
GI50: the concentration of the agent that inhibits growth by 50%, relative to untreated
cells, is the concentration at which ([T−T0]/[C−T0]) ×100 =50, where Tand C
are the number of treated and control cells, respectively, at time t of treatment and T>
T0; T0 is the number of cells at time zero.
LC50: the concentration of the agent that results in a net loss of 50% cells, relative to the
number at the start of treatment, is the concentration at which ([T−T0]/T0) ×100 =
−50.
Deshmukh, 2012; Li et al., 2018). Herein, it is important to stress
that AcOET fraction represents the whole ethyl acetate matrix,
which comprises an extremely rich source of individual bioactive
compounds. This explains why in many cases whole matrices give
a better specific bioactivity than isolated/single nutrients (Martins,
Barros, & Ferreira, 2016). In this sense and taking the results to-
gether, the toxicological effects of sclerotia constituents should be
more investigated, since these fungi present cytotoxic effects and
may lead to their human consumption.
In vitro studies in Plasmodium falciparum strains
There is continuing need for new and improved drugs to tackle
malaria, which remains a major public health problem, especially
in tropical and subtropical regions of the world (Ateba et al., 2018).
However, compounds that are ultimately selected for development
of new antimalarial drugs must meet the requirements of rapid ef-
ficacy to counter the spread of malaria parasites, safe drugs, and
that do not show any overt toxicity to human health (Fidock et al.,
2004). Herein, the AcOET fraction and F3 fraction do not show
effective activity against chloroquine resistant (W2) and chloro-
quine sensitive (3D7) P. falciparum strains, once it is necessary to
use high concentrations of the treatments to inhibit the growth
of the parasite (IC50 values: 330.4 to 465.1 µg/mL). In line with
WHO guidelines and basic criteria for antiparasitic drug discovery
(Fidock et al., 2004; Pink, Hudson, Mouri`
es, & Bendig, 2005),
the AcOET fraction is classified as low cytotoxicity to this strain
or weak activity (Table 3), based on four classes according to their
IC50 values: high activity (IC50 ࣘ5µg/mL); promising activity
(5 µg/mL <IC50 ࣘ15 µg/mL); moderate activity (15 µg/mL <
IC50 ࣘ50 µg/mL); weak activity (IC50 >50 µg/mL), and a pure
compound is defined as highly active when its IC50 ࣘ1µg/mL
(Jansen et al., 2012). Furthermore, microscopic observation of un-
infected erythrocytes added with AcOET and F3 and uninfected
erythrocytes showed no morphological differences after 24 hr of
incubation. Herein, the antimalarial behavior was different from
other fungi extracts. Ateba et al. (2018) observed that extracts from
Paecilomyces lilacinus,Penicillium,andPaecilomyces sp. exerted highly
potent activities against chloroquine-resistant P. falciparum (IC50
<1µg/mL); extracts from Mucor falcatus and Aspergillus aculea-
tus showed moderate potency (IC50 between 10 and 25 µg/mL),
while the extract from Aspergillus tamarri was considered inactive
3870 Journal of Food Science rVol. 84, Iss. 12, 2019

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Figure 3–Results of intracellular ROS generation in human lung adenocarcinoma (A549), ileocecal colorectal adenocarcinoma (HCT8), and normal lung
cell (IMR90) after treatment with AcOET fraction or 15 µmol/L H2O2(positive control) or medium (negative control) for 1 hr. Quantitative data are
the mean ±standard deviation. Different letters within same parameter indicate a significant difference (P<0.05, Tukey test).
Table 3–Antiplasmodial activity and cytotoxicity of Sclerotinia sclerotiorum, against chloroquine resistant strain (W2) and chloroquine
sensitive strain (3D7) after 48 hr exposure to AcOET and F3 fractions.
SI IC50
IMR90/IC50W2-3D7 Remark
Samples
Activity against P.
falciparum (W2
strain) IC50 (µg/mL)
Activity against P.
falciparum (3D7
strain) IC50 (µg/mL)
Cytotoxicity against
IMR90 cell line
IC50 (µg/mL) SI W2 SI 3D7 W2 3D7
AcOET 330.4 ±0.84 449.9 ±1.93 103.6 ±1.12 0.31 0.23 Weak
activity
We a k
activity
F3 362.3 ±1.64 465.1 ±4.22 127.6 ±4.68 0.35 0.27 Weak
activity
We a k
activity
Chloroquine 0.471 ±5.2 0.0265 ±2.7 16.24 ±5.19 34.47 612.83 High
activity
High
activity
SI: selectivity index =IC50 (IMR90)/IC50 (W2 or 3D7, respectively). The inhibition of parasite growth was expressed as IC50 (µg/mL): the concentration of the agent that inhibits
Plasmodium falciparum growth by 50% Antiparasitic activities of AcOET and F3 were classified into four classes according to their IC50 values: high activity (IC50 ࣘ5µg/mL);
promising activity (5 µg/mL <IC50 ࣘ15 µg/mL); moderate activity (15 µg/mL <IC50 ࣘ50 µg/mL); and weak activity (IC50 >50 µg/mL).
(IC50 >100 µg/mL). These disagreements may be explained by
the affinity among the bioactive compounds present in each fungi
extracts and the extracting medium polarity.
Moreover, the side effect of S. sclerotiorum samples by selectivity
index (IC50 (IMR90)/IC50 ( P. f a l c i p a r u m )) lies between 0.23 and 0.35,
depending on the sample and the plasmodium strain (Table 3),
which confirms they presented cytotoxic activities more so to
the noncancer and cancer cells than to malaria parasite (Tables 2
and 3). The norditerpenoid dilactone detected in our samples,
although previously described with antiplasmodial activity, also
had cytotoxic activity against mammalian kidney fibroblasts (Vero
cells) (Herath et al., 2010). The same behavior was found by
Sadorn et al. (2018) applying C. euge niae extracts that exhibited
excellent antimalarial activity and cytotoxicity against cancerous
(MCF-7, KB, NCI-H187) and noncancerous (Vero, African green
monkey kidney fibroblasts) cells. On the other hand, Ateba et al.
(2018) pointed out three fungi extracts with great activity against
chloroquine-resistant P. falciparum and little cytotoxic against the
human embryonic kidney HEK293T cells.
Although the weak activity observed in this study, it is, how-
ever, the first report of the S. sclerotiorum antiplasmodial properties
against chloroquine resistant strain (W2) and chloroquine sensitive
strain (3D7) of P. falciparum. Furthermore, this fungus should be
more studied once it is known by producing compounds, such as
ergosterol, ergosterol peroxide, which are recognized as source of
antimalarial and pharmacological metabolites for drug discovery
(Dembitsky, 2015).
In vivo tests: comet, micronucleus, apoptosis, and oxidative
stress
The in vivo analyses were carried out to explore the muta-
genic and genotoxic effects of AcOET fraction by micronucleus
assay and comet, respectively. In relation to feed consumption,
the results showed that the animals were not affected by their diet
Vol. 84, Iss. 12, 2019 rJournal of Food Science 3871

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Figure 4–Monitored variables for DNA damage analysis in peripheral blood and liver cells, where G1: 10 mL/Kg b.w. of 0.9% NaCl (negative control);
G2: DXO: doxorubicin 30 mg/kg b.w. (positive control); G4: diet added to AcOET 6 mg/100 g; G5: diet added to AcOET 60 mg/100 g; G6: diet added to
AcOET 600 mg/100 g. Quantitative data are the mean ±standard deviation. Different letters within same parameter indicate a significant difference
(P<0.05, Tukey test).
consumption, which was similar among all experimental groups
and indicates an animal well-being. Taking into account the diet
consumption, the group 4 (6 mg/100 g diet) consumed 25 mg
AcOET/kg b.w. corresponding to 2.1 g sclerotia/kg b.w.; the
group 5 (60 mg/100 g diet) was 240 mg AcOET/kg b.w. corre-
sponding to 20.9 g sclerotia/kg b.w. and group 6 (600 mg/100 g
diet) was 2600 mg AcOET/kg b.w. corresponding to 135.0 g
sclerotia/kg b.w.
Regarding the fungus genotoxic effects, the results demonstrate
that the AcOET fraction increased % DNA in comet tail at lev-
els significantly above the negative control by 129.0, 212.0, and
160% for peripheral blood and 284.0, 296.0, and 260.0% for
liver cells, in the three tested doses 6, 60, and 600 mg/100 g
diet b.w., respectively. The same results were obtained for TM,
where there was an increase of 166.0, 380.0, and 271.0% for pe-
ripheral blood and 660.0, 639.0, and 429.0% for liver cells, in
respective treated groups (Figure 4). Similar damages were ob-
served to micronuclei assay, whose damages were significantly
above the negative control by 147.82 to 239.13% for erythro-
cytes and 173.68 to 223.68% for colon (Figure 5). Thus, we
stress that injury levels observed by comet assay and colon mi-
cronuclei reached comparable levels of damage effects as same
as caused by doxorubicin and N’-dimethylhydrazine, the geno-
toxics and antioneoplastics agent used as positive control. Taking
into account that micronuclei explore the mutagenic effects from
chromosomal damage that are displaced to the cytoplasm of the
daughter cells (Hayashi et al., 2000), and the comet assay explore
genotoxic effects presents a measure for DNA damage assessed
before repair and cell division (Møller, 2006), it is possible to
conclude that AcOET fraction presented both genotoxic and mu-
tagenic activity. These events may be linked in many different
ways with spindle fiber changing and chromosome rearrange-
ments (Schmid, 1975); inhibition of the enzyme topoisomerase
II, ligases and helicases; formation of adducts with DNA (Desai
et al., 2013), and inducing chromosome segregation (Fenech &
Ferguson, 2001). Controversially, this AcOET fraction presented
different behaviors compared to aqueous fungi extract used by
Azevedo et al. (2016), which compounds have caused mutation
by micronucleus test but not induced DNA breakage by comet
parameters.
When dealing with citotoxicity and ROS generation, one of
the most observed pathway triggered is the apoptosis event. Thus,
the apoptotic assay in the intestinal epithelium was performed to
further explore these mechanisms. Results from this study showed
that positive control (G3) increased 3.01% the frequency of apop-
totic cell in enterocytes, while the negative control (G1) identified
a rate of 0.62% the frequency of apoptosis (Figure 5). Furthermore,
the tested doses of AcOET fraction increased apoptosis at levels
above the negative control by 500.0% and increased apoptotic cells
at levels similar the DMH. Indeed, Azevedo et al. (2016) also ob-
served this effect, which was triggered by extrinsic and intrinsic
pathway, leading to the activation of Caspase-8, p53, Bax, and
the production of ROS, that induce the release of Cytochrome
C. Apoptosis is considered a major mechanisms in cancer therapy
and plays a crucial role in the cellular progress of proliferation,
differentiation, senescence, and death (Ma et al., 2014). Thus,
herein, this increase in the number of apoptotic cells indicates
that S. sclerotiorum activated mechanisms of cell death. Since that
apoptosis is an event of eradication of cells that have suffered
DNA damage due to the presence of mutagenic and/or genotoxic
compounds (Badawi et al., 2018), these results agree with damage
observed by comet assay. The β-d-glucan, which was detected ex-
clusively to the AcOET fraction, was descr ibed by Queiroz et al.
(2015) as exerting an antiproliferative effect in breast cancer MCF-
7 cells, that was associated with apoptosis, necrosis, and oxidative
stress. Furthermore, another compound detected in our sample,
the sclerotiorin, was also found to induce apoptosis in colon cancer
(HCT-116) cells through the activation of BAX, and downregu-
lation of BLC-2, those further activated cleaved caspase-3 causing
apoptosis of cancer cells (Giridharan et al., 2012).
In the present study, the MDA analysis was performed, evaluat-
ing one of the most studied consequences of oxidative stress: lipid
peroxidation, which is among the processes implicated in oxidative
stress-induced cellular damage and is associated with cytotoxicity.
The treated groups with two higher doses (60 and 600 mg/100 g
diet b.w.) of AcOET fraction presented a similar increase in ox-
idative stress when compared to agent doxorubicin at levels signif-
icantly above the negative control by 149 and 200%, respectively
(Figure 4). Queiroz et al. (2015) also observed that mechanisms
underlying to β-d-glucan antiproliferative effect in breast cancer
3872 Journal of Food Science rVol. 84, Iss. 12, 2019

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Figure 5–Results of in vivo experiments, where G1: 10 mL/Kg b.w. of 0.9% NaCl (negative control); G2: DXO: doxorubicin 30 mg/kg b.w. (positive
control); G3: DMH: N’-dimethylhydrazine 30 mg/kg b.w. (positive control); G4: diet added to AcOET 6 mg/100 g; G5: diet added to AcOET 60 mg/100 g;
G6: diet added to AcOET 600 mg/100 g. (A) Micronucleated cell as percentage of total polychromatic erythrocyte within all animals of each same
group (2.000 cells/animal). ∗means that they are different from negative control (only NaCl) Pࣘ0.05 (χ2). (B) Micronucleated cell as percentage
of total colon cells within all animals of each same group (1.000 cells/animal). ∗means that they are different from negative control (only NaCl) Pࣘ
0.05 (χ2). (C) Lipid peroxidation by malondialdehyde (MDA) analysis in mice´s liver. Quantitative data are the mean ±standard deviation. Different
letters within same parameter indicate a significant difference (P<0.05, Tukey test). (D) Frequencies of colon enterocytes apoptosis as percentage of
total epithelial cells within all animals of each same group (total of twenty perpendicular well-oriented crypts were examined in each animal). ∗means
that they are different from negative control (only NaCl) Pࣘ0.05 (χ2)
MCF-7 cells may include the increase of oxidative stress in this
cell. It is known that increased basal oxidative stress could pro-
mote tumor growth, invasion, and metastasis, and furthermore,
lipid peroxidation may lead to the in vivo oxidation of polyunsatu-
rated fatty acids, which are responsible for the maintenance of cell
membranes (Balu, Sangeetha, Haripriya, & Panneerselvam, 2005).
Thus, as same as in vitro study, the animal experiment also evi-
dence that this fungus induces stress oxidative, which may tr iggered
the mutagenesis by possible three ways: (1) promoting redox state
alteration with consequent increase in the free radicals leading to
the inactivation or exhaustion of the antioxidant enzymes (Lazz´
e
et al., 2003), (2) by covalent binding between DNA bases with the
product of lipid peroxidation, such as hydroxyl radicals, and the
protein attack with alteration in membrane properties, structures,
and enzyme functions (Ferguson, 2001; Lazz´
e et al., 2003); (3) by
attacking nucleic acids, especially some spots in purine and pyri-
dine, which results in base substitution and DNA breakage (Reist,
Jenner, & Halliwell, 1998).
Conclusions
A chemical profile analysis of AcOET fraction and F3 fraction
identified β-d-glucan, norditerpenoid dilactone, sclerolide, and
others compounds, which highlighted toxic activities. The GI50
in noncancer cells was lower compared to cancer cells and the in
vivo results pointed out that AcOET fraction also caused geno-
toxic DNA damage, mutations in polychromatic erythrocytes and
colon cells, as well as stimulating the process of apoptosis in en-
terocytes. The oxidative stress assay by ROS generation and lipid
peroxidation tests suggests that AcOET caused overproduction of
free radicals and may lead these cells to apoptosis. According with
results obtained in antimalarial activity, we may conclude that the
AcOET fraction and F3 fraction from S. sclerotiorum presented
cytotoxic activities more so to the human cells than to malaria
parasite. These cytotoxic activities by the fungus needs to be more
investigated, since that food contamination with sclerotia may lead
to consume of fungal toxins with a risk to human health.
Acknowledgments
This work was supported by Fundac¸˜
ao de Amparo `
a Pesquisa do
Estado de Minas Gerais (FAPEMIG) [APQ 0285515] and granted
fellowship to author (Carolina Pressete). We also gratefully ac-
knowledge to Conselho Nacional de Desenvolvimento Cient´
ıfico
e Tecnol´
ogico (CNPq) and Coordenac¸˜
ao de Aperfeic¸oamento de
Pessoal de N´
ıvel Superior (CAPES) for financial support and fel-
lowships to authors.
Conflict of Interest
The authors declare no conflict of interest.
Authors’ Contributions
Pressete CG, Marques MJ, and Carmo MAV performed in vitro
experimental analyses; Giannini LSV performed in vivo experi-
mental analyses, De Paula DAC, Assis DM, Santos MFC, and
Soares MG carried out all chemical analyses, Machado JC con-
tributed with phytopathology experience and acquisition of scle-
rotia, Azevedo, L designed this study and discussed all data. All
authors reviewed and approved the article.
Abbreviations
AcOET ethyl acetate fraction
F3 100% MeOH fraction
Vol. 84, Iss. 12, 2019 rJournal of Food Science 3873

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
MeOH methanol
NaHCO3sodium bicarbonate
PBS phosphate buffered saline
UHPLC-HRMS ultra-performance liquid chromatography
mass spectrometry
HCT rat hepatoma
V79 Chinese hamster lung fibroblasts
HCT-116 colon cancer cells
MCF-7 human breast adenocarcinoma
KB papilloma carcinoma
NCI-H187 lung cancer cells
HT29 colon adenocarcinoma
HCT8 ileocecal colorectal adenocarcinoma
A549 human lung adenocarcinoma epithelial
IMR90 normal lung cell
W2 P. falciparum chloroquine-resistant
3D7 P. falciparum chloroquine-sensitive
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide)
ROS reactive oxygen species
DCFH-DA 2,7-dichlorofluorescin diacetate
H2O2hydrogen peroxide
MDA malondialdehyde
DXO doxorubicin chloridate
DMH N’-dimethylhydrazine
MNPCE micronucleated polychromatic erythrocytes
NaCl sodium chloride
TM tail moment
KCl potassium chloride
References
Aichinger, G., Puntscher, H., Beisl, J., K¨
utt, M. L., Warth, B., & Marko, D. (2018). Delphinidin
protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II.
Toxicology Letters,284, 136–142. https://doi.org/10.1016/j.toxlet.2017.12.002
Ateba, J., Toghueo, R., Awantu, A., Mba’ning, B., Gohlke, S., Sahal, D., . . . Lenta, B. (2018).
Antiplasmodial properties and cytotoxicity of endophytic fungi from Symphonia globulifera
(Clusiaceae). Journal of Fungi,4(2), 70. https://doi.org/10.3390/jof4020070
Azevedo,L.,Chagas-Paula,D.A.,Kim,H.,Roque,A.C.M.,Machado,J.C.,Dias,K.S.
T., . . . Mer tens-Talcott, S. U. (2016). White mold (Sclerotinia sclerotiorum), friend or foe:
Cytotoxic and mutagenic activities in vitro and in vivo. Food Research International,80, 27–35.
https://doi.org/10.1016/j.foodres.2015.11.029
Azevedo, L., de Araujo Ribeiro, P. F., de Carvalho Oliveira, J. A., Correia, M. G., Ramos, F. M.,
de Oliveira, E. B., . . . Stringheta, P.C. (2019). Camu-camu (Myrciaria dubia) from commercial
cultivation has higher levels of bioactive compounds than native cultivation (Amazon Forest)
and presents antimutagenic effects in vivo. JournaloftheScienceofFoodandAgriculture,99(2),
624–631. https://doi.org/10.1002/jsfa.9224
Badawi, A., Biyanee, A., Nasrullah, U., Winslow, S., Schmid, T., Pfeilschifter, J., & Eber-
hardt, W. (2018). Inhibition of IRES-dependent translation of caspase-2 by HuR confers
chemotherapeutic drug resistance in colon carcinoma cells. Oncotarget,9(26), 18367–18385.
https://doi.org/10.18632/oncotarget.24840
Balu, M., Sangeetha, P., Har ipriya, D., & Panneerselvam, C. (2005). Rejuvenation of antioxidant
system in central nervous system of aged rats by grape seed extract. Neuroscience Letters,383(3),
295–300. https://doi.org/10.1016/j.neulet.2005.04.042
Herath, T. H. M., Bandara, Herath, W. H. M. W., Carvalho, P., Khan, S. I., Tekwani, B. L.,
. . . Nanayakkara, N. P. D. (2010). Biologically active tetranorditer penoids from the fungus
Sclerotinia homoeocarpa, causal agent of dollar spot in turfgrass. Journal of Natural Products,72(12),
2091–2097. https://doi.org/10.1021/np900334k.Biologically
Bhadury, P., Mohammad, B. T., & Wright, P. C. (2006). The current status of natural products
from marine fungi and their potential as anti-infective agents. Journal of Industrial Microbiology
and Biotechnology,33(5), 325–337. https://doi.org/10.1007/s10295-005-0070-3
Bolton, M. D., Thomma, B. P. H. J., & Nelson, B. D. (2006). Sclerotinia sc lerotiorum (Lib.) de
Bary: Biology and molecular traits of a cosmopolitan pathogen. Molecular Plant Pathology,7(1),
1–16. https://doi.org/10.1111/j.1364-3703.2005.00316.x
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry,72,
248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Chagas-paula, D. A., Zhang, T., Costa, F. B. Da, & Edrada-ebel, R. (2015). A metabolomic
approach to target compounds from the asteraceae family for dual cox and lox inhibition.
Metabolites,5, 404–430. https://doi.org/10.3390/metabo5030404
Chang, W. C., Chapkin, R. S., & Lupton, J. R. (1997). Predictive value of proliferation,
differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis,
18(4), 721–730. https://doi.org/10.1016/B978-0-12-801732-6.00002-1
Chow, Y., & Ting, A. S. (2015). Endophytic l-asparaginase-producing fungi from plants
associated with anticancer properties. Journal of Advanced Research,6(6), 869–876.
https://doi.org/10.1016/j.jare.2014.07.005
De Menezes, R. R. P. P. B., Mello, C. P., Lima, D. B., Tessarolo, L. D., Sampaio, T. L.,
Paes, L. C. F., . . . Martins, A. M. C. (2016). Involvement of nitric oxide on bothropoides
insularis venom biological effects on murine macrophages in vitro. PLoS ONE,11(3), 1–15.
https://doi.org/10.1371/journal.pone.0151029
Dembitsky, V. M. (2015). Bioactive fungal endoperoxides abstract sterols and its derivatives.
IMedPub Journals,1(1:5), 1–7.
Desai, V. G., Herman, E. H., Moland, C. L., Branham, W. S., Lewis, S. M., Davis, K., . . .
Fuscoe, J. C. (2013). Development of doxorubicin-induced chronic cardiotoxicity in the
B6C3F1 mouse model. Toxicology and Applied Pharmacology,266(1), 109–121.
Carmo, do, V. M. A., Pressete, C. G., Marques, M. J., Granato, D., & Azevedo, L. (2018).
Polyphenols as potential antiproliferative agents: Scientific trends. Cur rent Opinion in Food
Science,24, 26–35. https://doi.org/10.1016/J.COFS.2018.10.013
Duan, Y., Li, T., Xiao, X., Wu, J., Li, S., Wang, J., & Zhou, M. (2018). Pharmacological charac-
teristics of the novel fungicide pyrisoxazole against Sclerotinia sclerotiorum.Pesticide Biochemistry
and Physiology,149, 61–66. https://doi.org/10.1016/j.pestbp.2018.05.010
Dutta, P. P., Bordoloi, M., Gogoi, K., Roy, S., Narzary, B., Bhattacharyya, D. R.,
. . . Mazumder, B. (2017). Antimalarial silver and gold nanoparticles: Green synthe-
sis, characterization and in vitro study. Biomedicine and Pharmacotherapy,91, 567–580.
https://doi.org/10.1016/j.biopha.2017.04.032
Escher, G. B., Santos, J. S., Rosso, N. D., Marques, M. B., Azevedo, L., do Carmo, M.
A. V., . . . Granato, D. (2018). Chemical study, antioxidant, anti-hypertensive, and cyto-
toxic/cytoprotective activities of Centaurea cyanus L. petals aqueous extract. Food and Chemical
Tox i c o l o g y ,118, 439–453. https://doi.org/10.1016/j.fct.2018.05.046
Fenech, M., & Ferguson, L. R. (2001). Vitamins/minerals and genomic stability in hu-
mans. Mutation Research—Fundamental and Molecular Mechanisms of Mutagenesis,475(1–2), 1–6.
https://doi.org/10.1016/S0027-5107(01)00069-0
Ferguson, L. R. (2001). Role of plant polyphenols in genomic stability. Muta-
tion Research—Fundamental and Molecular Mechanisms of Mutagenesis,475(1–2), 89–111.
https://doi.org/10.1016/S0027-5107(01)00073-2
Fidock, D. A., Rosenthal, P. J., Croft, S. L., Br un, R., & Nwaka, S. (2004). Antimalarial drug
discovery: Efficacy models for compound screening. Nature Reviews Drug Discovery,3(6),
509–520. https://doi.org/10.1038/nrd1416
Fr ˛
ac, M., Jezierska-Tys, S., & Yaguchi, T. (2015). Occurrence, detection, and molecular and
metabolic characterization of heat-resistant fungi in soils and plants and their r isk to human
health. Advances in Agronomy,132, 161–204. https://doi.org/10.1016/bs.agron.2015.02.003
Fravel, D. R., Connick, W. J., Gr imm, C. C., & Lloyd, S. W. (2002). Volatile compounds
emitted by sclerotia of Sclerotinia minor, Sclerotinia sclerotiorum,andSclerotium rolfsii.Journal of
Agricultural and Food Chemistry,50(13), 3761–3764. https://doi.org/10.1021/jf0117098
Giridharan, P., Verekar, S. A., Khanna, A., Mishra, P. D., & Deshmukh, S. K. (2012). Anticancer
activity of sclerotiorin, isolated from an endophytic fungus. Indian Journal of Experimental
Biology,50, 464–468.
Hayashi, M., MacGregor, J. T., Gatehouse, D. G., Adler, I. D., Blakey, D. H., Dertinger,
S. D., . .. Sutou, S. (2000). In vivo rodent er ythrocyte micronucleus assay. II. Some
aspects of protocol design including repeated treatments, integration with toxicity test-
ing, and automated scoring. Environmental and Molecular Mutagenesis,35(3), 234–252.
https://doi.org/10.1002/(SICI)1098-2280(2000)35:3<234::AID-EM10>3.0.CO;2-L
Heard, S., Brown, N. A., & Hammond-Kosack, K. (2015). An interspecies comparative analysis
of the predicted secretomes of the necrotrophic plant pathogens sclerotinia sclerotiorum and
botrytis Cinerea. PLoS ONE,10(6), 1–27. https://doi.org/10.1371/journal.pone.0130534
Jansen, O., Tits, M., Angenot, L., Nicolas, J. P., De Mol, P., Nikiema, J. B., & Fr´
ed´
erich,
B. (2012). Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of
urospermal A-15-O-acetate as the main active compound. Malaria Journal,11(289), 1475–
2875. https://doi.org/10.1186/1475-2875-11-289
Keats, R. E., Loewus, F. A., Helms, G. L., & Zink, D. L. (1998). 5-O-(α-d-Galactopyranosyl)-
d-glycero-pent-2-enono-1,4-lactone: Characterization in the oxalate-producing fungus, Scle-
rotinia sclerotiorum.Phytochemistry,49(8), 2397–2401.
Kind, T., & Fiehn, O. (2007). Seven golden rules for heuristic filtering of molecu-
lar formulas obtained by accurate mass spectrometry. BMC Bioinformatics,20, 1–20.
https://doi.org/10.1186/1471-2105-8-105
Komatsu, M., Tsuda, M., Omura, S., Oikawa, H., & Ikeda, H. (2008). Identification and
functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proceedings of the
National Academy of Sciences,105(21), 7422–7427. https://doi.org/10.1073/pnas.0802312105
Kong, W., Wei, R., Logrieco, A. F., Wei, J., Wen, J., Xiao, X., & Yang, M.
(2014). Occurrence of toxigenic fungi and determination of mycotoxins by HPLC-
FLD in functional foods and spices in China markets. Food Chemistry,146, 320–326.
https://doi.org/10.1016/j.foodchem.2013.09.005
Lazz´
e, M. C., Pizzala, R., Savio, M., Stivala, L. A., Prosperi, E., & Bianchi, L. (2003). An-
thocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth
muscle and hepatoma cells. Mutation Research—Genetic Toxicology and Environmental Mutagene-
sis,535(1), 103–115. https://doi.org/10.1016/S1383-5718(02)00285-1
Li, Q., Zhu, R., Yi, W., Chai, W., Zhang, Z., & Lian, X. (2018). Peniciphenalenins A Ffrom
the culture of a marine-associated fungus Penicillium sp. ZZ901. Phytochemistry,152, 4–11.
Liang, X., & Rollins, J. A. (2018). Mechanisms of broad host range necrotrophic pathogenesis
in Sclerotinia sclerotiorum.Fitopatologia,108(10), 191–198.
Liu, Z., Zhao, J.-Y., Sun, S.-F., Li, Y., & Liu, Y.-B. (2018). Fungi: Outstanding source
of novel chemical scaffolds. Journal of Asian Natural Products Research,6020, 1–22.
https://doi.org/10.1080/10286020.2018.1488833
Ma, W. D., Zou, Y. P., Wang, P., Yao, X. H., Sun, Y., Duan, M. H., . . .
Yu, B. (2014). Chimaphilin induces apoptosis in human breast cancer MCF-7 cells
through a ROS-mediated mitochondrial pathway. Food and Chemical Toxicology,70,1–8.
https://doi.org/10.1016/j.fct.2014.04.014
Malenˇ
ci´
c,D.,Kiprovski,B.,Popovi
´
c, M., Prvulovi´
c, D., Miladinovi ´
c, J., & Djord-
jevi´
c, V. (2010). Changes in antioxidant systems in soybean as affected by Sclero-
tinia sclerotiorum (Lib.) de Bary. Plant Physiology and Biochemistry,48(10–11), 903–908.
https://doi.org/10.1016/j.plaphy.2010.08.003
Martins, N., Barros, L., & Ferreira, I. C. F. R. (2016). In vivo antioxidant activity of
phenolic compounds: Facts and gaps. Trends in Food Science and Technology,48, 1–12.
https://doi.org/10.1016/j.tifs.2015.11.008
Migliorini, A. A., Piroski, C. S., Daniel, T. G., Cruz, T. M., Escher, G. B., Vieira do Carmo, M.
A., . . . Rosso, N. D. (2019). Red chicor y (Cichor ium intybus) extract rich in anthocyanins:
3874 Journal of Food Science rVol. 84, Iss. 12, 2019

Toxicology & Chemical
Food Safety
Sclerotinia sclerotiorum (white mold) . . .
Chemical stability, antioxidant activity, and antiproliferative activity in vitro. Journal of Food
Science,85. https://doi.org/10.1111/1750-3841.14506
Møller, P. (2006). The alkaline comet assay: Towards validation in biomonitoring of DNA
damaging exposures. Basic & Clinical Pharmacology & Toxicology,98, 336–345.
Moloney, J. N., & Cotter, T. G. (2018). ROS signalling in the biology of cancer. Semi-
nars in Cell and Developmental Biology,80, 50–64. https://doi.org/10.1016/j.semcdb.2017.05.
023
Moneriz, C., Mar´
ın-Garc´
ıa, P., Bautista, J. M., Diez, A., & Puyet, A. (2009).
Haemoglobin interference and increased sensitivity of fluorimetric assays for quantifica-
tion of low-parasitaemia Plasmodium infected erythrocytes. Malaria Journal,8(1), 1–10.
https://doi.org/10.1186/1475-2875-8-279
Ohno, N., & Yadomae, T. (1987). Two different conformations of the antitumour/3-
d- glucan produced by Sclerotiniu sclerotiorum.Carbohydrate Research,159, 293–302.
https://doi.org/https://doi.org/10.1016/0008-6215(88)85044-4
Pink, R., Hudson, A., Mouri`
es, M. A., & Bendig, M. (2005). Opportunities and chal-
lenges in antiparasitic drug discovery. Nature Reviews Drug Discover y,4(9), 727–740.
https://doi.org/10.1038/nrd1824
Queiroz, E. A. I. F., Fortes, Z. B., Da Cunha, M. A. A., Barbosa, A. M., Khaper, N., & Dekker,R.
F. H. (2015). Antiproliferative and pro-apoptotic effects of three fungal exocellular β-glucans
in MCF-7 breast cancer cells is mediated by oxidative stress, AMP-activated protein kinase
(AMPK) and the Forkhead transcription factor, FOXO3a. International Journal of Biochemistry
and Cell Biology,67, 14–24.https://doi.org/10.1016/j.biocel.2015.08.003
Radfar, A., M´
endez, D., Moneriz, C., Linares, M., Mar´
ın-Garc´
ıa, P., Puyet, A., . . . Bautista,
J. M. (2009). Synchronous culture of Plasmodium falciparum at high parasitemia levels. Nature
Protocols,4(12), 1899–1915. https://doi.org/10.1038/nprot.2009.198
Reist, M., Jenner, P., & Halliwell, B. (1998). Sulphite enhances peroxynitrite-dependent α1-
antiproteinase inactivation. A mechanism of lung injury by sulphur dioxide? FEBS Letters,
423(2), 231–234. https://doi.org/10.1016/S0014-5793(98)00099-4
Risio, M., Lipkin, M., Newmark, H., Yang, K., Rossini, F. P., Steele, V. E., . .. Kelloff, G. J.
(1996). Apoptosis, cell replication, and Wester n-style diet-induced tumorigenesis in mouse
colon. Cancer Research,56(21), 4910–4916.
Sadorn, K., Saepua, S., Boonyuen, N., Boonruangprapa, T., Rachtawee, P., & Pit-
tayakhajonwut, P. (2018). Antimicrobial activity and cytotoxicity of xanthoquinodin
analogs from the fungus Cytospora eugeniae BCC42696. Phytochemistry,151, 99–109.
https://doi.org/10.1016/j.phytochem.2018.04.001
Schilter, B., Constable, A., & Perrin, I. (2014). Naturally occurring toxicants of plant origin.
Food Safety Management: A Practical Guide for the Food Industry,3, 45–57. https://doi.org/
10.1016/B978-0-12-381504-0.00003-2
Schmid, W. (1975). The micronucleus test. Mutation Research,31, 9–15.
Shin, T. Y., Bae, S. M., Choi, J. B., & Woo, S. D. (2014). Simple and rapid screening of
entomopathogenic fungi having radical-scavenging and anticancer activities. Journal of Asia-
Paci fic Ent omo log y,17(3), 543–547. https://doi.org/10.1016/j.aspen.2014.05.002
Silva,J.P.L.,Ferreira,E.B.,Barbisan,L.F.,Brigagao,M.R.P.L.,Paula,F.B.A.,Moraes,G.O.
I., . . . Azevedo, L. (2014). Organically produced coffee exerts protective effects against the
micronuclei induction by mutagensin mouse gut and bone mar row.Food Research Inter national,
61, 272–278. https://doi.org/10.1016/j.foodres.2013.07.006
Theodoridis, G., Gika, H. G., Wilson, I. D., Park, A., Spec, M., Introduction, I.,
& Metabolomics, A. (2011). Mass spectrometry-based holistic analytical approaches
for metabolite profiling in systems biology studies. Mass Spectrometry Reviews,30.
https://doi.org/10.1002/mas.20306
Tour nas, V. H. (2005). Spoilage of vegetable crops bybacter ia and fungi and related health hazards.
Critical Reviews in Microbiology,31(1), 33–44. https://doi.org/10.1080/10408410590886024
Uchiyama, L., & Mihara, M. (1978). Determination of malondialdehyde precursor in tis-
sues by thiobarbituric acid test. Analytical Biochemistry,86, 271–278. https://doi.org/https://
doi.org/10.1016/0003-2697(78)90342-1
Wang, T. L., Ouyang, C. S., & Lin, L. Z. (2018). β-Asarone suppresses Wnt/β-catenin sig-
naling to reduce viability, inhibit mig ration/invasion/adhesion and induce mitochondria-
related apoptosis in lung cancer cells. Biomedicine and Pharmacotherapy,106(1), 821–830.
https://doi.org/10.1016/j.biopha.2018.07.009
Wolfender, J.-L., Litaudon, M., Touboul, D., & Queiroz, E. F. (2019). Innovative omics-based
approaches for prioritisation and targeted isolation of natural products— New strategies for
drug discovery. Natural Product Reports,36, 855–868. https://doi.org/10.1039/c9np00004f
Xu, C., Liang, X., Hou, Y., & Zhou, M. (2015). Effects of the novel fungicide benzothiostrobin
on Sclerotinia sclerotiorum in the laboratory and on sclerotinia stem rot in rape fields. Plant
Disease,99(7), 969–975. https://doi.org/10.1094/PDIS-09-14-0983-RE
Vol. 84, Iss. 12, 2019 rJournal of Food Science 3875