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Food Chemistry 141 (2013) 3967–3976

Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Modulation of the antioxidant/pro-oxidant balance, cytotoxicity

and antiviral actions of grape seed extracts
Codrutßa Ignea a,⇑, Cristina Mihaela Dorobantßu b, Christopher Paul Mintoff c, Norica Branza-Nichita b,
Michael R. Ladomery d, Panagiotis Kefalas a, Veronica Sanda Chedea a,d,e,⇑
a
Department of Food Quality and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania, Centre International de Hautes Etudes Agronomiques
Méditerranéennes, Chania, PO Box 85, 73100 Chania, Crete, Greece
b
Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, Splaiul Independentei 296, Sector 6, Bucuresti 77700, Romania
c
Peter MacCallum Cancer Centre, St. Andrews Pl, East Melbourne, VIC 3002, Australia
d
Centre for Research in Bioscience, Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Frenchay, Bristol BS16 1QY, UK
e
Laboratory of Animal Biology, National Research Development Institute for Animal Biology and Nutrition Balotesßti (IBNA), Calea Bucuresti 1, Balotesßti, Ilfov 077015, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system
Received 4 March 2013 (regarding the cellular growth and growth recovery from H2O2 insult). GSEs antioxidant activity was
Received in revised form 10 May 2013 detected in wild-type and mutant strains Dcta1, Dgsh1 and Doye2glr1, while pro-oxidant activity in
Accepted 20 June 2013
Dsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2
Available online 29 June 2013
2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white
grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected
Keywords:
by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual
Antioxidant/prooxidant activity
Antiviral action
antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deficiencies and exhi-
Cytotoxicity bit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV.
Grape seed extracts Ó 2013 Elsevier Ltd. All rights reserved.
Yeast

1. Introduction
Winemaking by-products are of particular interest because grape
is the world’s largest fruit crop, with more than 68 million tons pro-
duced per year, of which the European Union produce approximately
24.5 million tons per year in 2010 (FAO STAT Database in http://fao-
stat.fao.org (March. 27th, 2012). Grape seeds are waste products of
Abbreviations: FRAP, ferric reducing antioxidant power; FBS, fetal bovine serum;
GAE, gallic acid equivalents; GSE, grape seed extract; HBeAg, hepatitis B ‘‘e’’
antigen; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; HBV,
human hepatitis B; MAPK, mitogen-activated protein kinase; OYE, old yellow
enzymes; GSSG, oxidised glutathione; PI3K, phosphatidylinositide 3-kinase; PCD,
programmed cell death; ROS, reactive oxigen species; RW, red grape seed aqueous
extract; GSH, reduced glutathione; TP, total polyphenolic content; WW, white grape
seed aqueous extract; Dcta1, Dsod1, and Dgsh1, yeast mutants derived from the
wild-type (wt) strain BY474, harbouring deletion of the genes CTA1, SOD1, or GSH1
respectively.
⇑ Corresponding authors. Address: Laboratory of Animal Biology, National
Research Development Institute for Animal Biology and Nutrition Balotesßti (IBNA),
Calea Bucuresti nr. 1, Balotesßti, Ilfov 077015, Romania. Tel.: +40 21 351 20 81 (V.S.
Chedea). Department of Food Quality and Chemistry of Natural Products, Mediter-
ranean Agronomic Institute of Chania, Centre International de Hautes Etudes
Agronomiques Méditerranéennes, Chania, PO Box 85, 73100 Chania, Crete, Greece.
Tel.: +30 28210 35051 (C. Ignea).
E-mail addresses: igneacodruta@yahoo.com (C. Ignea), chedea.veronica@ibna.ro
(V.S. Chedea).
the winery and grape juice industry, containing lipid, protein, carbo-
hydrates, and 5–8% polyphenols, depending on the variety. Grape
seed polyphenols contain, aside from phenolic acid precursors (gallic
acid), the monomeric flavan-3-ols (catechin, epicatechin, gallocate-
chin, epigallocatechin and epicatechin 3-O-gallate) and procyanidin
dimers, trimers, and highly polymerised procyanidins (Chedea et al.,
2011). The pharmacological and health benefits of grape seed ex-
tracts (GSEs) include antioxidant (Chedea, Braicu, & Socaciu, 2010),
cardioprotective (Schewe, Sadik, Klotz, Yoshimoto, Kühn, & Sies,
2001), hepatoprotective, neuroprotective, anti-inflammatory, anti-
diabetic, anti-carcinogenic, and anti-ageing effects (Nassiri-Asl &
Hosseinzadeh, 2009; Xia, Deng, Guo, & Li, 2010).
Oxidative stress, as a gap between production of reactive oxy-
gen species (ROS) and cellular antioxidant defence, is a key phe-
nomenon in chronic disorders: diabetes mellilitus, cardiovascular
diseases, and cancer (Schewe et al., 2001). The harmful effects of
oxidative processes in living organisms can be diminished by the
dietary intake of polyphenols like flavan-3-ols and procyanidins,
present in GSEs (Chedea et al., 2010). Redox cell signalling and
the antioxidant potential of various natural compounds have been
widely evaluated in vivo using yeast, as an eukaryotic model sys-
tem (Dani, Bonatto, Salvador, Pereira, Henriques, &, Eleutherio,
2008; Zyracka, Zadrag, Koziol, Krzepilko, Bartosz, &, Bilinski,
2005). This takes advantage of the existing elaborate enzymatic

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.06.094
 Author's personal copy
3968 C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976
redox machinery, consisting of catalases, superoxide dismutases,
reductases, peroxiredoxins, glutaredoxins, and glutathione trans-
ferases (Herrero, Ros, Belli, & Cabiscol, 2007). Saccharomyces cerevi-
siae strains lacking the antioxidant machinery mimic the altered
intracellular redox circumstances that are frequently encountered
in human pathological conditions, and can be used to assess the
ability of antioxidant compounds to protect against oxidative dam-
age (Zyracka et al., 2005). The consumption of polyphenol-rich
foods exerts valuable outcomes in oxidative stress-related chronic
diseases, including some forms of cancer (Surh, 2003). The antican-
cer effects of dietary polyphenols occur via a variety of mecha-
nisms, such as the inhibition of cell growth proliferation, the
modulation of cancer cell signalling and antioxidant enzymatic
machinery, the induction of apoptosis and cell cycle arrest or the
elimination of carcinogenic agents (Ramos, 2008). Paradoxically,
dietary polyphenols may also perform anti-cancer effects as a re-
sult of their pro-oxidant activity (Halliwell, 2008; Surh, 2008).
Polyphenols are among the 400 compounds that have been listed
as potential chemopreventive agents, 40 of these being currently
under clinical evaluation (Cimino et al., 2012).
To address the antioxidant/pro-oxidant capacity of aqueous
GSEs to promote/prevent cell survival from oxidative damage we
focused on yeast model systems, to simulate common cellular dys-
functions that occur in oxidative stress using strains compromised
in important components of their antioxidant machinery. The cyto-
toxic effect on tumour cells, the antiviral potential of GSEs and the
possible link between these two effects was also investigated. This
study is ultimately aimed at increasing the use of wine industry
waste through inexpensive processing with a view to exploit the
material in nutrition to prevent disease, and even to develop novel
pharmacological uses.
2. Materials and methods
2.1. Plant material
Varieties of red and white grapes cultivated in Greece were
used for all seed samples studied (Chedea, Moussouni, Socaciu, &,
Kefalas, 2012), red grape seed aqueous extract (RW) and white
grape seed aqueous extract (WW). The grapes were harvested at
optimum technological maturity. Grape berries were manually
deseeded and seeds were mixed, frozen in liquid nitrogen, and
stored in the freezer (20 °C) until analysed.
2.2. Polyphenol extraction
5 g of seeds were ground with liquid nitrogen, and extracted for
1 h by adding 40 ml of boiled water to the seed powder. After sed-
imentation of the large solid particles, the supernatant was centri-
fuged for 10 min at 1500g, filtered, and then aliquoted and stored
at 20 °C until further analysis. The total polyphenolic (TP) content
was measured by the Folin–Ciocalteu method and expressed as mg
of gallic acid equivalents (GAE)/g seeds (Chedea et al., 2012).
2.3. Yeast strains
The wild-type (wt) strain BY4741 and the derived mutants
Dcta1, Dsod1, and Dgsh1, harbouring deletion of the genes CTA1,
SOD1, or GSH1, respectively, were obtained from Research Genet-
ics. The double-knockout strain Doye2glr1 was as previously de-
scribed (Odat et al., 2007). The yeast strains employed show
defects in free radical-scavenging enzymatic system and glutathi-
one metabolism.
2.4. Cell lines
The PC3 cell line derived from prostate metastatic androgen
independent tumour was purchased from the Health Protection
Agency Culture Collections (http://www.hpacultures.org.uk/collec-
tions/ecacc.jsp). PC3 cells were seeded at 25% confluence (i.e.
3  105 in a 6-well plate), fed with RPMI medium supplemented
with L-glutamine, containing 10% foetal bovine serum (FBS),
50 units/ml penicillin, and 50 lg/ml streptomycin.
The hepatocarcinoma–derived HepG2 2.2.15 cells (kind gift
from Dr. David Durantel, INSERM U871, Lyon, France), stably trans-
fected with two head-to-tail dimers of the Human Hepatitis B
(HBV) genome, were grown in RPMI 1640 medium (Euroclone)
containing 10% foetal bovine serum (FBS), 50 units/ml penicillin,
50 lg/ml streptomycin and 2 mM Glutamax (Invitrogen), supple-
mented with 200 lg/ml of G418 (Gibco).
2.5. Growth recovery curves
Yeast strains were routinely cultivated at 30 °C in YEPD med-
ium containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 2%
(w/v) glucose. Single yeast colonies from fresh YEPD plates were
used to inoculate starter cultures in 50 ml YEPD broth at
A600 = 0.1, which were incubated at 30 °C with shaking (160 rpm)
for 14–18 h. Aliquots were taken at regular intervals and the absor-
bance was measured. When A600 > 1, aliquots were serially diluted
and new measurements were taken. The assay was conducted as
previously described (Amari, Fettouche, Samra, Kefalas, Kampranis,
&, Makris, 2008).
2.6. Viability colony assay
BY4741 and the mutant Dsod1 strains were freshly grown over-
night in rich YEPD medium. Viability colony assays were per-
formed as previously described (Amari et al., 2008). All
experiments were performed independently in triplicate. The re-
sults were statistically analysed by the GraphPad statistical
software.
2.7. Cytotoxicity (MTS) assay
PC3 and HepG2.2.2.15 cells were seeded at confluence in 96
wells plates (Greiner) in a final volume of 100 ll/well culture med-
ium. Wells loaded with 0.1 ll milliQ water were used as negative
control. GSEs to the final concentration of 1 lM GAE, 5 lM GAE,
10 lM GAE, 25 lM GAE, 50 lM GAE and 100 lM GAE were added
to each well. Supplemented cells were left to incubate at 37 °C for
24 and 48 h. The cell proliferation was assayed using WST-1 re-
agent (Roche Diagnostics Gmbh, Manheim, Germany). Cells were
incubated with 10 ll of WST-1 for 30 min. The measurements were
done in triplicate for each sample. Colour change was assayed by
measuring the absorbance at 420 nm with a microplate reader (op-
tima BMG labtech). The assay quantifies enzymatic activities in
metabolically active cells; therefore, dead cells do not contribute
to the colour change.
2.8. Quantification of HBV subviral particles secretion by ELISA
Aliquots of supernatants from GSE-treated HepG2 2.2.15 cells
were analysed for the secreted HBsAg, using the Monolisa HBsAg
Ultra Kit (Bio-Rad). The results were registered as ratios of signal
to cutoff and were converted to percentages of HBsAg secretion
form control, untreated samples.
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C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976
2.9. Quantification of HBV viral DNA secretion by real-time PCR
Secretion of HBV virions by GSE-treated HepG2 2.2.15 cells was
monitored by purification of encapsidated viral DNAs from super-
natants using phenol–chloroform extraction, as previously de-
scribed (Lazar et al., 2007). The DNA was quantified using the
Corbett Rotor Gene 6000 real-time PCR system and the Maxima
SYBR Green qPCR Master Mix (Fermentas). Primers were designed
to amplify a 279-bp HBV-specific fragment: HBV3575-3854_for, 50 -
TCCAGGATCCTCAACAACCAGCACG-30 and HBV3575-3854_rev, 50 -
TGGCCCCCAATACCACATCATCC-30 .
2.10. Statistical analysis
Statistical analysis was performed by using GraphPad Prism six
Software. Experimental data was analysed by RM one-way ANOVA.
Equal variability of differences was assumed with no correction.
Fisher’s LSD test was used for multiple comparisons of treated
samples with untreated-control samples.
3. Results
The chemical composition of the GSEs assessed in the present
work for their in vivo antioxidant/pro-oxidant activity, cytotoxicity
and antiviral actions was characterised in a previous study (Chedea
et al., 2012). The polyphenol composition of RW and WW extracts
was determined by LC–DAD–MS in ESI+ mode (Chedea et al., 2012).
Gallic acid, catechin, epicatechin, procyanidine dimers, trimers,
tetramers, pentamers and hexamers were identified. Procyanidin
oligomers with a low degree of galloylation were extracted using
hot water (Chedea et al., 2012). TP for RW was 40.11 ± 0.90 and
for WW 38.05 ± 0.86 mg GAE/g seeds (Chedea et al., 2012). The
antioxidant activity of the two extracts was assessed by the ferric
reducing antioxidant power (FRAP), as well as by the DPPH radical
and luminol chemiluminescence methods. The behaviour in bulk
oil medium was evaluated by the Rancimat test (Chedea et al.,
2012).
3.1. Effect of GSEs on yeast cell growth and H2O2-induced oxidative
stress
Antioxidant and pro-oxidant activities of natural (dietary)
agents are important health-promoting properties, ensuring pro-
tective mechanisms or inducing important detoxifying enzymes
(Azam, Hadi, Khan, & Hadi, 2004). Our previous study showed that
the oxidation products of polyphenols, either as pure molecules or
in GSEs, are formed within the cellular matrix and the extracellular
medium; the tested GSEs, considered as an antioxidant nutritive
supplement, might have pro-oxidant activity as well, depending
on dose, duration of administration and other dietary components
(Chedea et al., 2010).
To evaluate the capacity of the two GSEs to assist yeast cells
growth and protect cells compromised in their enzymatic antioxi-
dant machinery, we used four deletion strains and their isogenic
wild-type counterpart in cell growth and growth recovery assays.
Cells deleted in superoxide dismutase (Dsod1), catalase A (Dcta1),
glutathione synthase (Dgsh1) and double knockout in old yellow
enzyme 2 and glutathione reductase 1 (Doye2glr1) were subjected
to 1.25 mM H2O2 pro-oxidant insult, which induces programmed
cell death (PCD) in a percentage of the population. The cell growth
and the growth recovery of the surviving cells in complete minimal
media supplemented with low (5 lM GAE) or high (50 lM GAE)
antioxidants was monitored for a period of 12–18 h. All cell growth
and recovery assays were performed independently in triplicate.
Statistically significant differences in cell growth could be seen
3969
starting at 4 h for wild-type and Doye2glr1 cells, at 6 h for the
slower growing Dsod1 and Dcta1 cells and 8 h for the very slow
growing Dgsh1. The H2O2 stress applied to the yeast cultures ap-
pears to last for up to 10 h, as judged by the lag in cell division time
compared to untreated cells.
Unlike the mutant strains, treatment of the wt strain with GSEs
exhibited only a minor influence on cellular growth (Figs. 1A and
2A). An improved growth rate of wt cells, statistically significant
when compared with untreated cells, was acquired in the presence
of 50 lM GAE WW, exhibiting a 12% enhancement over the un-
treated cells (Fig. 1A). Addition of WW extract determined an in-
creased recovery from the oxidative insult in a dose-dependent
fashion, ranging from 6 to 15.5% at 5 and 50 lM GAE, respectively
(Fig. 3A). Under H2O2 toxic stress, the wt strain cells attained the
same tolerance to the induced injury after administration of
50 lM GAE RW. By contrast, a pro-oxidant effect was observed at
low dose (5 lM GAE) RW, statistically significant, which increased
stress by 17% compared to control cells (Fig. 4A).
The first candidate strain carries a deletion in the catalase A
gene (Dcta1). This enzyme is responsible for neutralizing H2O2
and is primarily found in the peroxisomal matrix and mitochondria
(Petrova, Drescher, Kujumdzieva, & Schmitt, 2004). The Dcta1 mu-
tant strain showed evidence of healthier growth in the presence of
both extracts. The overall benefit gained from GSE supplementa-
tion was found to be dose-independent, around 15% (Figs. 1B and
2B), but only 50 lM GAE WW treatment appeared statistically sig-
nificant when compared with untreated cells. Both extracts were
highly protective in the compromised deletion strain Dcta1 after
H2O2 treatment. A low dose of provided GSEs improved recovery
by around 35%, while addition of 50 lM GAE WW and RW rescued
Dcta1 cells from oxidative injury by 25% and 38%, respectively as
compared to control cells (Figs. 3B and 4B). The antioxidant activity
of GSEs in Dcta1 cell was found to be statistically significant in all
treatments.
The free radical-scavenging enzyme, superoxide dismutase
Sod1 is one of the first line of cell defence against oxidative injury,
catalysing the dismutation of superoxide O2 to H2O2. Deletion of
sod1 sensitises cells to pro-oxidant insult, increases endogenous
ROS, and causes methionine auxotrophy (Amari et al., 2008). Sup-
plementation of Dsod1 cells with WW did not contributed to cell
growth (Fig. 1C), with a statistically significant inhibition of 10%
at 5 lM GAE WW being observed. RW rendered cells more sensi-
tive to endogenous ROS and a dose-independent growth inhibition
between 13% and 16% was measured (Fig. 2C). A significant pro-
oxidant effect, reducing cell recovery in a range of 22–26%, was
shown in H2O2-treated Dsod1 cells incubated with WW (Fig. 3C).
The same effect was monitored for RW, which inhibited cell
growth recovery in a dose-depended manner by 11% at 5 lM
GAE RW and 28% at 50 lM GAE RW (Fig. 4C). Overall the observed
growth inhibition and pro-oxidant effect generated by GSEs in
Dsod1 cells were statistically significant by comparison with un-
treated Dsod1 cells. According to these findings, the protection pro-
vided by GSEs against H2O2 insult requires the cytosolic superoxide
dismutase, since the Dsod1 strain was not able to acquire tolerance
when treated with GSEs.
Glutathione reductase is an enzyme responsible for recycling
oxidised glutathione back to the reduced form. Old yellow en-
zymes (OYE) have been implicated in modulating oxidative stress
responses by altering actin polymerisation (Odat et al., 2007). Dele-
tion of glr1 and oye2 causes cellular sensitivity to oxidative stress
and H2O2-induced programmed cell death (PCD), respectively
(Odat et al., 2007). The double-knockout strain Doye2glr1, exhibits
a radically altered ratio of reduced glutathione to oxidised glutathi-
one (GSH/GSSG) in favour of the latter, higher than single mutant
Dglr1 cells (Odat et al., 2007). Growth enhancement, varying from
15% to 18%, was monitored for the Doye2glr1 cells in the presence
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3970 C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976

Figure 1. Effect of white grape seeds extract (WW) on yeast cell growth. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells (E)
were assessed untreated (N). Cells were supplemented with WW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h. Statistically
significant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt – 5 lM P = 0.0673
(ns), 50 lM P = 0.0005 (⁄⁄⁄); Dcta1 – 5 lM P = 0.0269 (⁄), 50 lM P = 0.1230 (ns); Dsod1 – 5 lM P = 0.0116 (⁄), 50 lM P = 0.4929 (ns); Doye2glr1 – 5 lM P = 0.0945 (ns), 50 lM
P = 0.0348 (⁄); Dgsh1 – 5 lM P = 0.1071 (ns), 50 lM P = 0.1513 (ns).

of WW (Fig. 1D) and a favourable, dose-independent, effect of 26%
was seen after RW supplementation (Fig. 2D), being statistically
significant for both RW treatments and 50 lM GAE WW treatment.
The generated damage of Doye2glr1 cell exposure to H2O2 oxida-
tive stress was more efficiently counteracted by addition of 5 lM
GAE WW and 50 lM GAE RW, by 14% and 20% respectively
(Figs. 3D and 4D), only the last being statistically significant when
compared with untreated cells. Other tested doses did not affect
cell recovery.
The gene GSH1 encondes a c-glutamyl cisteine synthetase cata-
lysing the first step in glutathione biosynthesis. Yeast cells har-
bouring a gsh1 deletion are unable to grow in CM media unless
extensively supplemented with GSH. Medium supplementation
with 100 nM GSH enables Dgsh1 cells to grow at a lower rate
(Amari et al., 2008). The capacity of GSEs to improve the growth
properties and recover Dgsh1 cells from oxidative injury was sub-
sequently assayed. Supplementation with WW brought a minor
improvement in growth properties of Dgsh1 cells ranging from 7
to 9% (Fig. 1E), while RW addition shown a constant benefit of
8% in cell growth (Fig. 2E) of which only the treatment with
5 lM GAE RW was statistically significant when compared with
untreated cells. The antioxidant ability of WW was accomplished
by increasing the tolerance of Dgsh1 cells to H2O2 insult by 11%
at 5 lM GAE and 29% at 50 lM GAE (Fig. 3E). Peroxide stress was
more aggressive in Dgsh1 cells supplemented with a low concen-
tration of RW, a minor cell recovery of 5% being monitored, but a
significant protective effect of 22% was evident at 50 lM GAE
RW (Fig. 4E). Antioxidant activity in Dgsh1 was found statistically
significant only after treatment with high concentration of GSEs.
To evaluate the comparability of the growth recovery assay data
with the frequently employed viability colony assay, wild-type and
Dsod1 strains were selected as reference and highly responsive,
respectively. Cells were grown to midlog phase at OD600 = 0.5 in
glucose CM media. Fresh media containing 50 lM GSEs was inoc-
ulated with aliquots of cells. After addition of 1.5 mM H2O2, cells
were incubated at 30 °C for 2 h. The numbers of viable cells were
counted prior and subsequent to H2O2 treatment. As seen in
Fig. 5A and B the percentage of surviving colonies after H2O2 treat-
ment was reduced by more than half to an average of 41% for wild-
type cells and 38% for Dsod1 cells. Supplementation with GSEs was
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C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976 3971

A wt B Δcta1
200 200
180 ns **
ns 175
160 **
150
millions of cells 140
120 125

millions of cells
100 100
80 75
60
50
40
20 25
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14
me (h) time (h)

C Δsod1 D Δoye2glr1
100 210
** ***
80 *** 180
***
150
million of cells

millions of cells
60
120
40 90
60
20
30
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14
me (h) me (h)

E Δgsh1
160
140
*
ns
120
100
millions of cells

80
60
40
20
0
0 2 4 6 8 10 12 14 16
me (h)

Figure 2. Effect of red grape seeds extract (RW) on yeast cell growth. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells (E) were
assessed untreated (N). Cells were supplemented with RW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h. Statistically significant
changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt – 5 lM P = 0.9728 (ns), 50 lM
P = 0.1316 (ns); Dcta1 – 5 lM P = 0.0018 (⁄⁄), 50 lM P = 0.0049 (⁄⁄); Dsod1 – 5 lM P = 0.0013 (⁄⁄), 50 lM P = 0.0004 (⁄⁄⁄); Doye2glr1 – 5 lM P = 0.0002 (⁄⁄⁄), 50 lM P = 0.0004
(⁄⁄⁄); Dgsh1 – 5 lM P = 0.0307 (⁄), 50 lM P = 0.1802 (ns).

beneficial to cell survival for the wild type strain which increased for the evaluation of chemopreventive properties of natural
to 67% for WW and 58% for RW. Treatment with both extracts re- products, due to high incidence, long latency and identifiable pre-
sulted in a significant reduction in viability of H2O2 treated Dsod1 neoplastic lesions (Cimino et al., 2012). Chronic infection with hep-
cells, between 15% and 20%. The effects of supplementation were atitis HBV causes hepatocellular carcinoma (HCC), one of the most
statistically significant in all cases and the results of colony viabil- common cancers worldwide. Phenolic antioxidants have been
ity assays are highly comparable to the corresponding growth identified as HCC chemopreventive agents, which induce cytopro-
recovery assays (Fig 5). tective genes (Nair et al., 2006).
In this context, the present study aimed to also determine the
3.2. Cytotoxicity assessment of PC3 and HepG2.2.2.15 cells treated cytotoxic activity of GSEs against two different cell lines, PC3
with GSEs which is an in vitro model for prostate cancer and HepG2.2.2.15,
which is a liver tumour-derived cell line, replicating high amounts
There is a growing body of evidence suggesting a correlation of of HBV (Sells, Chen, & Acs 1987). To this end, we employed the MTS
oxidative stress with pathological conditions including various assay as a measure of cell viability, this assay being routinely used
forms of cancer, such as those from the prostate, liver, gut, skin, to assess the chemosensitivity of tumour cells by measuring the
and so forth (Kumar, Koul, Khandrika, Meacham, & Koul, 2008; inhibition of proliferation.
Stehbens, 2004). Cell proliferation plays a central role in the devel- The human prostate carcinoma PC3 cell line is widely investi-
opment of severe diseases, and it has been shown that the use of gated in drug resistance studies (Kumar et al., 2008). Untreated
specific natural (dietary) or synthetic agents may prevent, delay, cells were considered to exhibit 100% viability. Fig 6A and B
or slow down this process. Prostate cancer is a model target disease presents the percentage of inhibition or stimulation of cell
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3972 C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976

A wt B Δcta1
200 180
ns **
175 160
* *
140
150
120
125
millions of cells

millions of cells
100
100
80
75 60
50 40
25 20
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14
me (h) time (h)

C Δsod1 D Δoye2glr1
60 180
* ns
160
50 ** ns
140
40 120

millions of cells
millions of cells

100
30
80
20 60
40
10
20
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14
me (h) me (h)

E Δgsh1
160
ns
140 *
120
100
millions of cells

80
60
40
20
0
0 2 4 6 8 10 12 14 16
me (h)

Figure 3. Effect of white grape seed extract (WW) on growth in H2O2-treated cells. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1
cells (E) were treated with 1.25 mM H2O2 (N). Cells were supplemented with WW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h.
Statistically significant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt – 5 lM
P = 0.2088 (ns), 50 lM P = 0.0156 (⁄); Dcta1 – 5 lM P = 0.0054 (⁄⁄), 50 lM P = 0.0405 (⁄); Dsod1 – 5 lM P = 0.0260 (⁄), 50 lM P = 0.0037 (⁄⁄); Doye2glr1 – 5 lM P = 0.2708 (ns),
50 lM P = 0.9674 (ns); Dgsh1 – 5 lM P = 0.3186 (ns), 50 lM P = 0.0154 (⁄).

proliferation, measured by the MTS reduction to formazan, for cells cells, but very strong cytotoxicity was observed in cells treated
co-incubated with GSEs. The proliferation rate of PC3 cells treated with 100 lM GAE RW especially after 24 h of incubation, when
with 1 lM GAE WW was about 70% after 24 h incubation and 80% only 10% of cell proliferation occurred (Fig. 6B).
after 48 h incubation, compared to control. The cytotoxic effect in- The cytotoxicity of GSEs was also assessed on the HepG2 2.2.15
creased after treatment with 5 lM GAE WW, to only 60% cell pro- cell line supporting replication, but also assembly and secretion of
liferation after 24 h incubation and 50% after 48 h. No response to both subviral and infectious HBV particles. The results, shown in
treatment was observed for PC3 cells incubated with 10 lM GAE Fig. 6C and D, indicate a similar cytotoxicity pattern following
WW for 24 h, but significant cytotoxic effect was monitored after treatment of HepG2 2.2.15 cells with both extracts, wth cell prolif-
48 h (more than 60% inhibition of cell proliferation). Addition of in- eration being slightly stimulated at 10 lM GAE and decreasing pro-
creased concentrations of 25 and 50 lM GAE WW resulted in 80% portionally with increased concentrations. The highest cytotoxicity
cell proliferation after 24 h incubation, while at 100 lM GAE WW, was observed at 100 lM GAE GSEs, independent of the incubation
60% cell proliferation was monitored. The cytotoxic effect of WW at period, the treatment resulting in reduced proliferation rate, to
higher doses was constantly stronger after 48 h incubation, result- around 40% to 60% from that of control.
ing in 50% cell proliferation. Together, the toxicity profile of WW
exhibited the highest activity on PC3 tumour cells at doses be- 3.3. Anti HBV activity of GSEs
tween 10 and 25 lM GAE, after a longer incubation time (48 h)
(Fig 6A). In the case of RW treatment of PC3 tumour cells the dura- The HepG2 2.2.15 cell line is a useful system to assess the anti-
tion of incubation influences the cytotoxic effect at concentrations viral activity of GSEs due to the capacity to produced and secrete
of 1 lM GAE, 25 lM GAE and slightly at 100 lM GAE. Generally, HBV DNA, hepatitis B surface antigen (HBsAg) and hepatitis B ‘‘e’’
the cell proliferation occurs at half rate compared with untreated antigen (HBeAg) in supernatants. The HBsAg secretion was
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C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976 3973

A wt B Δcta1
180 180
* **
160 160
ns
***
140 140
120 120

millions of cells
million of cells

100 100
80 80
60 60
40 40
20 20
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14
me (hr) me (h)

C Δsod1 D Δoye2glr1
60
ns 210 ns
50 *** 180 *
40 150

millions of cells
millions of cells

30 120
90
20
60
10 30
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14
me (h) me (h)

E Δgsh1
140
ns
120 *
100
millions of cells

80
60
40
20
0
0 2 4 6 8 10 12 14 16
me(h)

Figure 4. Effect of red grape seed extract (RW) on growth in H2O2-treated cells. BY4741 wild type-cells (A), Dcta1 cells (B), Dsod1 cells (C), Doye2glr1 cells (D), and Dgsh1 cells
(E) were treated with 1.25 mM H2O2 (N). Cells were supplemented with RW at 5 lM GAE (h) and 50 lM GAE (j). Growth was monitored by measuring OD600 every 2 h.
Statistically significant changes in growth assays conducted in triplicate are observed for multiple comparisons of treated samples with untreated-control samples: wt – 5 lM
P = 0.0457 (⁄), 50 lM P = 0.8239 (ns); Dcta1 – 5 lM P = 0.0045 (⁄⁄), 50 lM P = 0.0007 (⁄⁄⁄); Dsod1 – 5 lM P = 0.6696 (ns), 50 lM P = 0.0008 (⁄⁄⁄); Doye2glr1 – 5 lM P = 0.5871
(ns), 50 lM P = 0.0189 (⁄); Dgsh1 – 5 lM P = 0.7882 (ns), 50 lM P = 0.0414 (⁄).

A BY4741 B Δsod1
120% 120%

100% 100%
% of viable cells
% of viable cells

80% 80%

60% 60%

40% 40%

20% 20%

0% 1 2 3 4 5
0% 1 2 3 4 5

** *** *** ***

- --- WW RW - --- WW RW
H2O2 - --+ S -+ -+ H2O2 - --+ ---- + -+

Figure 5. Viability colony assay in H2O2-treated cells suplemented with GSEs. Wild type (A) and Dsod1 (B) cells grown at midlog phase were used to inoculate fresh media
supplemented with 50 lM GAE GSEs. Cells were immediately challenged with 1.5 mM H2O2 and incubated for 2 h. Viable cells were counted prior and subsequent to
treatment by plating dilutions on YPD plates. Statistically significant changes in viability colony assays conducted in triplicate are observed WW: wt – P = 0.001 (⁄⁄), Dsod1 –
P = 0.0007 (⁄⁄⁄); RW: wt – P = 0.0005 (⁄⁄⁄), Dsod1 – P = 0.0004 (⁄⁄⁄).
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3974 C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976

A WW B RW
120 120
24hrs 24 hrs
100 48 hrs 100 48 hrs

% of proliferation
% of proliferation

80 80

60 60

40 40

20 20

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
µM GAE of WW µM GAE of RW

C WW D RW
140 140
24 hrs 24 hrs
120 48 hrs 120 48 hrs
% of proliferationl
100 100
% of proliferation

80 80

60 60

40 40

20 20

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
µM GAE of WW µM GAE of RW

E 120 F 120

100 100
% of HBV virions secreted
% of HBsAg secretion

80 80

60 60

40 40

20 20

0 0
Control 15µM WW 25µM WW 15µM RW 20µM RW Control 15µM WW 25µM WW 15µM RW 20µM RW

Figure 6. Assessment of cell proliferation by the MTS assay on tumour PC3 and HepG2 2.2.15 cells and the antiviral activity of GSEs against HBV. PC3 cells were treated with
WW (A) and RW (B) and on HepG2 2.2.15 infected cells treated with WW (C) and RW (D) incubated for 24 h (j) and 48 h (N). Statistically significant changes in MTS assays
conducted in triplicate are observed WW: PC3 – P = 0.006 (⁄⁄), HepG2 2.2.15 – P = 0.007 (⁄⁄); RW: PC3 – P = 0.0001 (⁄⁄⁄), HepG2 2.2.15 – P = 0.001 (⁄⁄). ELISA assay of HBsAg
secretion (E) and HBV viral DNA quantification by RT-PCR (F) in GSE-adapted HepG2 2.2.15 cells after 72 h of incubation. All experiments were performed independently in
triplicate.

determined by ELISA (Fig. 6E) and quantification of viral DNA studies have shown that polyphenols are extensively metabolized
secretion was performed by real-time PCR (Fig. 6F). As shown in in vivo, resulting in significant alteration of their redox potential
Fig. 6E, HBsAg secretion was not affected in cells treated with (Amari et al., 2008). Therefore, it is essential to screen the efficacy
WW, and only a minor inhibition was detected in cells treated with of these compounds in vivo. The physiological role of dietary anti-
15 lM GAE RW. The amount of viral DNA measured by real-time oxidants depends on their absorption and biotransformation
PCR in the cell supernatant did not change between the control mechanisms. In this study, the action of GSEs was assessed both
and supplemented samples, indicating that neither replication, in vivo, using yeast as a model organism and in vitro in two
nor assembly or trafficking of the virions was affected by the treat- different biological systems: prostate cancer cells and HBV-
ment (Fig. 6F). replicating hepatoma cells. The active role of GSEs in oxidative
stress or cell proliferation is thus supported by in vivo and
4. Discussion in vitro studies.
Despite the limitations inherent to the use of such a simple
The antioxidant potential of GSEs has been investigated single cell experimental model, S. cerevisiae is very useful as a
through in vitro analysis (Chedea. et al., 2012), although previous first screening tool to investigate oxidative stress at the cellular
 Author's personal copy
C. Ignea et al. / Food Chemistry 141 (2013) 3967–3976
level. This microorganism conserves not only most enzymatic
equipment and cellular structures, but also the redox balance
and the response to oxidative stress specific to higher
eukaryotes. The defects in the antioxidant yeast system studied
here are linked to several human diseases, such as familial
amyotrophic lateral sclerosis associated with mutations of Sod1
(Rosen et al., 1993), ageing and Parkinson’s disease, connected
with glutathione homeostases (Patsoukis et al., 2005), or
glutathionylation of actin mediated by Oye2 protein (Odat
et al., 2007).
The yeast cell growth and growth recovery were the assays of
choice, as populations of yeast cells can be maintained in chemi-
cally controlled media supplemented with various doses of GSEs
during the whole assay period, which closely resembles a real life
setting. This enables testing concentrations of GSEs at physiologi-
cally relevant levels that the organisms encounter (5 lM GAE
low dose and 50 lM GAE high dose), especially when the yeast ef-
flux system is taken into consideration. Our results clearly empha-
sise the role of the GSEs supplementation in yeast cell growth and
growth recovery assays, which can vary from antioxidant to pro-
oxidant depending on the cellular antioxidant machinery defi-
ciency. Incubation period and GSE concentration have a limited
contribution to their antioxidant/pro-oxidant activity. Growth aug-
mentation upon GSE supplementation in cells exhibiting deficiency
of catalase activity or glutathione level could indicate a potential
role in controlling intracellular peroxide concentration and avoid-
ing oxidative damage to membranes (Haliwell, 2006) or a possible
involvement in the GSH metabolic pathway. GSEs operated as pro-
oxidant agents in cells lacking superoxide dismutase activity which
could comply to the common mechanism for anti-cancer and che-
moprotective properties of plant polyphenols. The mutant defi-
cient strain Dcta1 treated with GSEs exhibited the highest
capacity of recovery from H2O2 damage, indicating that GSEs can
either succesfully replace Cta1 activity or trigger the super expres-
sion of other antioxidant systems as a form of compensation (Zyra-
cka et al., 2005). In the absence of cytoplasmatic superoxide
dismutase, the level of ROS produced was elevated, toxicity rising
up by 2.5-fold in a dose-dependent fashion. Sod1 contributes
essentially to the elimination of ROS achieved by GSEs under
H2O2 stress.
Chemoprevention is a promising approach to reducing the inci-
dence of cancer. The two GSEs investigated in the present study,
exhibited statistically significant cytotoxic activity against two hu-
man tumour-derived cell lines, prostate cancer PC3 and hepatoma-
derived HepG2 2.2.15 cells. Generally, higher concentration of ex-
tracts increased cytotoxicity, with the most convincing effect
appearing in PC3 cells treated with RW. The period of administra-
tion clearly influences the inhibition of cell proliferation in HepG2
2.2.15 infected cells, the cytotoxicity increasing after longer
incubation.
No antiviral effect was observed by measuring the secreted vir-
ion particles in HepG2 2.2.15 cells treated with GSEs. Nevertheless,
it would be very interesting to further investigate whether the
HBeAg secretion in HepG2 2.2.15 cells is affected by GSEs treat-
ment and to which extent the secreted HBV virions are still
infectious.
While there has been a major focus on the antioxidant proper-
ties of natural products, there is an emerging view that these com-
pounds, and their metabolites, may affect signalling pathways that
modulate several cell responses (Cimino et al., 2012), like mitogen-
activated protein kinase (MAPK) and phosphatidylinositide 3-ki-
nase (PI3K) pathways involved in cancer cell proliferation, or cas-
pase pathway that leads to apoptosis. Dietary agents, being
extractive and not synthetic products, may elicit a great variability
of therapeutic consequences.
3975
5. Conclusions
The action of GSEs was explored in the present study. Taken to-
gether, the antioxidant yeast system deficiencies affect the capac-
ity of GSEs in vivo to sustain cellular growth and to protect cell
viability from oxidative damage, while insights of the cytotoxic
properties of GSEs in PC3 and HepG2 2.2.15 cell lines, but no anti-
viral action against HBV, were revealed. However, future work is
necessary to fully understand these effects and the mechanisms
by which they protect the organisms against various injuries.
Acknowledgements
We thank Dr. Antonios Makris for providing Doye2glr1 yeast
strain. Dr Veronica S. Chedea was supported by a Royal Society
International Travel Grant (reference TG092176).
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