Regulation of patulin-induced oxidative stress processes in the fission…

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Regulation of patulin-induced oxidative processes in the fission yeast pombe.

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Regulation of oxidative stress processes in the yeast

Schizosaccharomyces pombe

Pappa, Eszter Horváthb, Mikea, Zoltán Gazdaga, Belágyic, Zoltán Gyöngyid,

Bánfalvie, László Hornokf, Pestia,⇑

aDepartment of General and Microbiology, Faculty of Sciences, of Pécs, H-7602 Pécs, POB Hungary

bDepartment of Limnology, of Pannonia, Veszprém, Hungary

of Biophysics, Faculty of Medicine, of Pécs, Pécs, Hungary

of Public Health Medicine, of Medicine, University of Pécs, Hungary

eDepartment of Microbial and Cell Biology, University of Debrecen, Hungary

fFaculty of and Environmental Sciences, Department of Szent István University, o, Hungary

a r t i c l ei n f o

Article history:

26 March 2012

Accepted 1 2012

Antioxidant enzyme






a b s t r a c t

Patulin is one of the most widely disseminated found in agricultural products. In

study the PAT-induced accumulation of oxygen species (ROS) and the of the specific

activities of antioxidant were investigated in the single eukaryotic organism Schizosaccha-

pombe. In comparison with the cells, 500 lM PAT treatment caused a 43% in

the concentration of the main intracellular glutathione (GSH); this of GSH initiated

a 2.44- and a 2.6-fold of superoxide anion and hydrogen respectively, but did not

increase the concentration of radicals; the reduction of ROS-induced processes via

the activation of Pap1 factor resulted in significantly specific activities of Cu/Zn

dismutase, catalase and glutathione to protect the cells against the

unbalanced redox state. no change was measured in the activities of reductase, glu-

tathione and glucose-6-phosphate dehydrogenase. It seems to assume that the

temporary ROS accumulation plays a crucial in adaptation processes. The adverse

of PAT may be exerted mainly through the of cellular membranes and protein/enzyme


2012 Elsevier All rights reserved.

1. Introduction

water-soluble, heat-stable mycotoxin by 60 species of

moulds encompassing 30 genera, has frequently been

in apples, apple-based products and other foods, including

vegetables, pears and peaches. PAT a number of

health risks to and animals, comprising both (ede-

ma, ulceration, gastrointestinal and damage, etc.) and

chronic immunotoxic and neurotoxic, etc.) This

explains why the content of PAT in has been limited to 50 lM


in many countries (for see Moake et al. 2005; Sant’Ana

et al.

In order to attain an understanding of the effects of PAT

and to characterize its time- and biolog-

ical activity, were earlier carried out at a le-

vel, using both and eukaryotic microorganisms and various

of tissue cultures of higher (Moake et al. 2005).

PAT is considered to cells and cellular processes via

ple electrophilic reactivity, leading to the of adducts

with nucleofiles as sulfhydryl (-SH) and amino groups.

The adducts formed glutathione (GSH), free or cys-

teine-, lysine-, and a-amino acid-containing proteins

are cross-linked compounds (Fliege and

1999; Lee and Röschenthaler, 1986). of the cyto- and geno-

toxic of PAT infection, such as the inhibition of

enzymes, protein prenylation, the of transcription,

translation and DNA synthesis et al. 2012a; Pfeiffer

et al. 2005; et al. 1993; Moake et al. 2005), mem-

brane disruption et al. 2002; Riley et al. 1990),

0278-6915/$ — see front 2012 Elsevier Ltd. All reserved.

Abbreviations: catalase; G6PD, glucose-6-phosphate GPx,

glutathione peroxidase; glutathione S-transferase; GR, glutathione

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in the phase-transition temperature of the plasma

caused by modification of the 3D protein and the loss

of cellular compounds light at 260 nm, e.g. nucleotides,

and free bases (Horváth et al. are regarded as

consequences of this activity of the toxin.

GSH, a component of all living cells, is of

scavenging reactive oxygen (ROS). It is a co-factor of vari-

ous enzymes, such as glutathione (GR), glu-

tathione (GPx), glutathione S-transferase and

also glutaredoxin and thioredoxin, maintain an optimal re-

dox state in the through the reduction of protein Under

normal conditions, the milieu is a reducing environ-

thanks to high concentrations (up to 10 mM) of GSH

2001; Pócsi et al. 2004). In rat slices PAT treatment causes

GSH in a concentration-dependent manner as a result

of adduct formation (Pfeiffer et al.

ROS injury was originally detected in epithelial (LLC-PK1)

cells to 50 lM PAT, which resulted in lipid per-

oxidation and Showker, 1991). Significantly levels

of peroxides and hydroperoxides measured in rat hepato-

cytes 1000 lM PAT treatment for 20 min (Barhoumi and

1996). PAT causes oxidative DNA (Liu et al.,

2003), DNA breaks and DNA-DNA cross-links

et al. 2006). PAT-induced apoptosis through the mithoc-

hondrial was observed in human promyelocytic

(HL-60) cells as a consequence of ROS H2O2) accumulation (Liu

et al. Wu et al. 2008). Saxena et al. (2009) that

ROS generation is involved in the of PAT-mediated

cytotoxicity. Although are great differences in reactivity,

and decomposition of superoxide (O2??),

(H2O2) and hydroxyl radical (Gille and Sigler, 1995), ‘‘to-

tal’’ ROS concentrations measured in most studies at

identification of the effects of PAT on ROS production via use of

the dye 20,70-dichlorodihydrofluorescein diacetate.

The increased of ROS resulted in the activation of mito-

protein kinases (MAPKs), p38 and c-Jun N-

terminal kinase in human embryonic kidney 293) cells

(Liu et al. A single topical application of PAT p38,

ERK and JNK MAPKs and the downstream proteins c-fos, c-

Jun and NF-(j)B factors. PAT treatment led to signif-

reductions of superoxide dismutase GPx and GR activ-

ities in those (Saxena et al. 2011). The role of

in attenuating apoptotic phenotypes by abiotic stressors,

including has been demonstrated in Fusarium a

mycotoxin-producing plant pathogenic Indicators of pro-

grammed …, such as the levels of mitochondrial mem-


underwent significant increases stress conditions in a

DFphog1 of the fungus obtained by targeted disrup-

tion of Fphog1, a (high osmolarity glycerol) gene,

as compared with its parental strain (Ádám et al.

The complexity of the action of PAT on cells was by

a transcriptome analysis of the budding Saccharomyces cere-

visiae. Of the ORFs identified in the yeast 490 and

477 annotated genes were to be up- or downregulated,

respectively, after of the cells with a subinhibitory

centration of the toxin (50 lM). PAT activated or stimu-

lated a of genes involved in the defense oxidative

stress, the metabolism of sulfur-containing acids, and pro-

tein (Iwahashi et al. 2006).

To acquire knowledge on this field, the aim of the

ent study was the separate measurement of the in levels of

O2. H2O2, and?OH in PAT-treated cells. The levels of the

reducing factor GSH and the specific of

the most important antioxidant (SODs, catalase (CAT),

GR, glucose-6-phosphate dehydrogenase (G6PD) and

andnuclear disintegration,

were determined to follow the whole of cellular reg-

ulation in to the oxidative stress caused by PAT

The single-cell eukaryotic model Schizosaccharomyces

pombe (S. pombe) was in these experiments so as to obtain re-

comparable with those in our studies (for reviews,

Giga-Hama and Kumagai, 1997; and Kumagai,

2003; Horváth et al. 2012a,b).

2. Materials and methods

Strains, culture conditions and of minimal inhibitory concentrations

of PAT in signal transduction mutants

The S. uracil auxotroph (ura4-D18) (h?) strain was used in

all and earlier (Horváth et al. 2010, 2012b) experiments, except in

the of MICs of S. pombe MAPK mutants (see below). The

liquid SM medium containing 1% 0.5% (NH4)2SO4, 0.05%

0.01% MgSO4, and 0.001% vitamin solution (Spencer and

1996) with 100 mg l?1uracil was for cultivation at pH 5.6. The cells pre-

cultured in the SM medium at 30 ?C. This culture was washed by

fugation (1017 g, 5 min) and to prepare mid-log phase (15 h) in a

3.33-Hz incubator shaker at 30 ?C, a starting number of 106cells

density, OD595nm= 0.05). The were washed twice in NaCl solution before

experiment, according to Iwahashi et al. The stock solution of PAT was

prepared to Murillo et al. (2008).

The MICs of PAT on S. MAPK deletion mutants determined by the

standard microdilution of NCCLS M27-A (NCCLS, S. pombe strains

Dwis1 leu 1–32 ura4-D18, h?), (sty1::ura4 leu 1–32 ura4-D18

h+), Datf1 (atf1::ura4 leu ura4-D18, h+), and Dpap1

leu 1–32 ura 4-D18, h+) were all from the parental strain 1–32 ura4-

D18 his 7–366 h+). The deletion mutants gifts from Dr. J. Millar

of Yeast Genetics, National for Medical Research, The Ridgeway,

Hill, London). Cells maintained on the surface of YES medium

0.5% yeast extract, 3% 225 mg l?1amino acids necessary for

and 2% agar at pH 4.5 ( The experiments

performed in agar-free liquid YEL at pH 5.6. MIC100(the minimal

tration required for 100% inhibition) values were on 96-well

flat-bottomed microtiter (Costar, Corning Incorporated, NY, USA)

by measuring the OD595nmvalues of the cultures. A series of 2-fold

was prepared from the PAT stock in the solvent acetonitrile to yield a

tion that contained 250 the final concentration required for the in the

broth microdilution assays. intermediate solution was further in YEL

to twice the final required S. pombe cell inocula prepared from

1-day-old grown on YES slants. The cell was diluted with YEL

medium. (100 ll) containing various of PAT diluted in

YEL were mixed 100 ll of cell suspension on microtiter The final con-

centrations of PAT and the under investigation in the wells

from 0 to 600 lM. The initial cell was 5. 103cells ml?1. The micro-

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were incubated for 48 h at 30 ?C, and the OD595nmwas with a

microtiter plate (Multiskan EX, Thermo). Uninoculated was used

as background for the spectrophotometric the growth control wells

cell suspension in drug-free For calculation of the extent of

inhibition, the the drug-free control cultures was set at growth

in each case. All were repeated three

2.2. Measurements of ROS, enzyme activities and other procedures

Cell-free extracts prepared by using X-press 10, S-412 70

Göteborg, Sweden). The content of the cell-free extract was by

a modified Lowry method 1983). To estimate the intracellular

levels, the dye dihydrorhodamine 123 (10 M) and dihydroethidium

(10 M) used, respectively (Carter et al. Henderson and Chappell,

1993). ml?1were treated for 1 h in the presence of an amount

of PAT, then with 0.9% NaCl Rhodamine and ethidium formation

measured and quantified simultaneously a BD FACSCalibur flow cytom-

(Henderson and Chappell, 1993; et al. 1994). The excitation wavelength

was 488 nm for dyes, while the emission were 585 and 530 nm

for ethidium and rhodamine, The SD values were less 5% in each


The in vitro of?OH and the reduction of chromate [Cr(VI)] to

Cr(V) in S. pombe monitored by electron paramagnetic (EPR) spec-

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concentration (10 M). cells were treated (i) 2 mM K2Cr2O7[Cr(VI)];

(ii) 2 mM Cr(VI) + 2 mM (iii) 2 mM Cr(VI) + 3 mM H2O2; 2 mM

Cr(VI) + 2 mM NADPH + 3 mM H2O2for 5 min mixing with 0.1 M PBN (which

forms an adduct with?OH)-containing buffer solution at pH 7.6. The

values of the concentrations were from measurements on three

preparations. Their values not higher than 10–15% of the integrals

(Belágyi et al. 1999; et al. 2000).

For measurement of the specific of enzymes and GSH concentration,

mid-log-phase were collected by centrifugation and in fresh SM

medium. Suspensions 108cells ml?1were treated 500 lM PAT for

60 min in a 3.33-Hz incubator shaker at 30 ?C, collected and washed in Sören-

sen buffer (pH 7.0) at 0 ?C. 7.5. in 4 ml were stored at ?80 ?C

until and cell-free extracts were obtained by X-pressing at ?20 ?C. The

specific of Cu/Zn-SOD and Mn-SOD (Oberley and 1984), GST (War-

holm et al. GR (Pinto et al. 1984.), GPx (Chiu et al. G6PD (Emri et al.,

and CAT (Roggenkamp et al. 1974) and the intracellular of

GSH and oxidized GSH (glutathione disulfide, (Anderson, 1985) were

mined by means of well-established assays. Four independent

ments were used to the SD.

DNA fragmentation was analyzed by pulsed gel electrophoresis (Birren and

Lai, and single cell gel electrophoresis 1995). Exposed phosphatidylser-

a marker of apoptosis, was investigated by Annexin V Alexa Fluor?488

and propidium iodide staining as by the manufacturer (Invitro-

gen). In experiments cells were with 500 lM PAT for 180 min.


All of the chemicals used in this were of analytical grade and ob-

tained from Sigma–Aldrich Budapest, Hungary, except dihydroethidium

was purchased from Buchs, Switzerland.

3. Results and

3.1. PAT-induced toxicity and of oxidative stress sensitive

factor Pap1

The ura4-D18, heterothallic strain of S. pombe

was because of its suitability for transformation

ments with the non-integrative pUR18 N vector (Koósz

et al. In all experiments (except for the determination of

of MAPK mutants), mid-log-phase cultures multiplied

in SM medium used to obtain cells in the physiological

status and to eliminate the effects of complete yeast

media (Gazdag et al. 2011). 500 lM the

optimal concentration, was applied in and earlier experi-

ments to the multiplicatory effects of PAT (Horváth et

2010, 2012a,b). The uptake of PAT by S. cells could be di-

vided two phases: 14% of the toxin was taken up

20 min by biosorption, and 20% within 120 min by bioaccumula-

The growth-inhibitory effect of PAT was determined in

kencultures,starting with

500 lM PAT caused a 32% in cell proliferation, with a

delay in entering the log phase et al. 2010). Abnor-

mally yeast cells were found in cultures grown

for 35 h in medium (Fig. 1), indicating the

tence of an adaptation mechanism to the effect of PAT

in S. pombe, which has confirmed by Horváth et al. (2010).

treatment with 10 lM PAT induced a

mented adaptation process: the colony-forming ability of a

cell treated with 500 lM PAT was increased to

when the cells were with 10 lM PAT and then chal-

with 500 lM of the toxin (Horváth et al. Scanning

electron microscopy that a high number of

(OD595nm= 0.05):

concentrations of PAT in the 0–600 lM for the parental

strain, for its MAPK deletion mutants and Dsty1,

and for two other mutants, Datf1, deprived of the abil-

ity to the transcription factors Pap1, and respec-

tively. Pap1 target genes in response to low

(0.2 mM) of H2O2, whereas controls primarily the transcrip-

response to stronger oxidative induced by 25 mM

H2O2, (Quinn et al. PAT added at 150 lM did not cause a sig-

decrease in the growth yield of the mutants Dwis1,

Dsty1 and or of the parental strain. 600 lM PAT caused

a 10% decrease in the growth yield of the strain, but re-

duced the growth of the Dwis1, Dsty1, and Datf by

95%, and 65%, respectively 2). Deletion of the Pap1 transcrip-

factor-encoding gene, however, in extreme sensitivity

to PAT in S. pombe: the of growth of the Dpap1 mutant was

by 98% in the presence of 50 lM PAT, and 75 lM of the

toxin complete … in the mutant population

(Fig. 2). Under the conditions, the growth yield of the

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