Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe

Journal of Basic Microbiology 2012, 52, 1–11 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe

Eszter Horvath1, Gabor Nagy2, Melinda Turani2, Eniko Balogh2, Gabor Papp1, Edit Pollak3, Istvan Pocsi2, Miklos Pesti1 and Gaspar Banfalvi2

1 Department of General and Environmental Microbiology, University of Pecs, Pecs, Hungary 2 Department of Microbial Biotechnology and Cell Biology, University of Debrecen, Debrecen, Hungary 3 Department of General Zoology, University of Pecs, Pecs, Hungary

The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large-scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 μM patulin concentration were characterized by elongated nuclei containing sticky fibrillary chro-matin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin conden-sation. Short (60 min) incubation of S. pombe cells in the presence of high (500 μM) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 μM) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects.

Keywords: Protoplast / Transmission electronmicroscopy / Scanning electronmicroscopy / Fluorescence microscopy / Long-term scanning / Chromatin structure; cell death

Received: October 12, 2011; accepted: November 03, 2011

DOI 10.1002/jobm.201100515

Introduction*

Patulin is a water soluble broad-spectrum antibiotic against a wide range of disease-causing bacteria. This lactone mycotoxin is produced by several molds, espe-cially by Aspergillus and Penicillium and is commonly found in rotting apples. It exerts antibacterial, carcino- Correspondence: Prof. Gaspar Banfalvi, University of Debrecen, Depart-ment of Microbial Biotechnology and Cell Biology, Life Sciences Building 1.102, 1 Egyetem tér, Debrecen 4010, Hungary E-mail: [email protected] Phone: (36) 52 512 900 ext. 62319 Fax: (36) 52 512 925

genic and mutagenic activities [1, 2]. The cytostatic effect of patulin is related to its potential to reduce the barrier properties of tight junctions [3], inhibiting the potassium uptake [4] and activation of p38 kinase [5]. This notion was confirmed in cells exposed to 50 μM patulin treatment, resulting in lipid peroxidation, abrupt calcium influx, extensive blebbing, and total LDH release suggesting the loss of structural integrity of the plasma membrane. Although, the cytotoxicity of patulin was prevented by indole tetramic acids, it was doubted, that this antioxidant potential was responsible for the preventive effect against patulin. Consequently

2 E. Horvath et al. Journal of Basic Microbiology 2012, 52, 1–11


other mechanisms such as surface charge alterations on the cytoplasmic surface of plasma membranes, al-terations of permeability in the plasma and endoplas-mic reticulum membrane could have been involved [7]. Earlier experiments did not clarify whether the patulin caused alterations in plasma membrane allow-ing the efflux of intracellular K+ and influx of Na+ [8] would lead to cellular shrinkage charateristic to apop-tosis or increased cell size indicating necrosis. That apoptosis may prevail was shown by Comet, annexin V and PI staining assays, by the overexpression of the proapoptotic Bax and tumor suppressor p53 proteins [9]. Other investigations revealed that depleted cytokine secretion was not due to the cytotoxic effects of pa-tulin, rather to the draining off of glutathione pool which could be abolished by adding thiol containing compounds [10]. As far as the nuclear effect of patulin is concerned only a limited number of in vitro and in vivo data are available. At low concentrations of patulin without demonstrable cytotoxicity, the mitotic arrest, the for-mation of micronuclei containing whole chromosomes were observed in cultured Chinese hamster V79 cells [11]. Exposure of human leukemia (HL-60) cells to patulin at 2.5 μM showed the phenomenon of nuclear fragmentation and chromatin condensation to about 24% of the culture [12]. In vivo a patulin dose of 0.1 mg kg–1 was administered orally to growing male rats for a period of 60 or 90 days daily and interdigitating den-dritic cells of the thymus were investigated by trans-mission electronmicroscopy. Sixty days after patulin treatment the loss of cristae, chromatin margination and lysis of the nucleus were found. In the nucleus the long-term (90 days) patulin-treatment of rats increased the number of apoptotic bodies and caused chromatin condensation [13]. The reason why S. pombe cells were chosen as a model organism in this study was: 1) that patulin induced chromatin studies in fungal cells in-cluding the fission yeast S. pombe are scarce and 2) the genome size of 13.8 Mb is distributed to three relatively bulky chromosomes of 5.7, 4.6 and 3.5 Mb [14, 15] and allows a better visibility of chromatin toxicity. Recently we have described the genotoxicity of chromate ion Cr (VI) on S. pombe. These experiments revealed that the intermediates of normal chromatin condensation are similar to mammalian cells involving the fibrillary chromatin veil, the formation of chromatin ribbon and the earliest visible interphase chromosomes referred to as chromatin bodies, elongated linear chromosomes and condensed chromosomes. However, compact chro-mosomes that would correspond to the metaphase chromosomes of mammalian cells were not seen, indi-

cating that the pattern of chromosome condensation differs to some extent from the common pathway of chromosome condensation in mammalian nuclei [16, 17]. In higher eukaryotes chromatin changes are among the most sensitive indicators of genotoxicity and more importantly chromatin changes are specific to the genotoxic agent. In this study we asses the antifungal properties of patulin by electronmicroscopy, fluorescence micro-scopy and long time scanning following the cell growth of S. pombe in the presence and absence of patulin. It was also tested how this broad-spectrum antibiotic affects chromatin condensation and whether these genotoxic changes in S. pombe are characteristic to patu-lin.

Materials and methods

Chemicals and reagents Patulin (IUPAC name 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, molecular mass 154.12 g mol−1, molecular formula C7H6O4) was obtained from Sigma-Aldrich (St. Louis, MO).

Cell culture and patulin exposure The rod-like cells of S. pombe of 15 μm length and 4 μm diameter with a rigid wall divide by binary fission. S. pombe yeast cells were cultured in a 3.33-Hz incubator shaker at 30 °C in synthetic minimal liquid medium (SM). The medium contained 1% glucose, 0.5% (NH4)2SO4, 0.05% KH2PO4, 0.01% MgSO4, 0.001% Wicker-ham vitamin solution [18] and 100 μg ml–1 uracil, pH 4.5. Medium was solidified with 2% agar (SMA) [19]. The cells were precultured overnight in SM medium resulting in exponential-phase culture and washed twice in PBS buffer then centrifuged (600 g, 5 min). Cells were recultured in fresh SM medium. The cell cultures were started at a density sufficient to produce an OD595 of 0.05 and cultivated for 12 h. Mid-log-phase cells were used to determine the survival rates of cells exposed to patulin (10–500 μM).

Transmission electronmicroscopy (TEM) For transmission electronmicroscopy (TEM) examina-tion 108 ml–1 mid-log phase S. pombe cells were treated with 50 and 500 μM patulin for 90 and 180 min at 30 °C. For sample preparation the method of [20] was applied with some modifications. Briefly, after the fixa-tion step in potassium permanganate, cells were stained ‘en block’ with uranyl acetate at RT for 1 h, and were dehydrated in increasing ethanol gradient (50, 70,

Journal of Basic Microbiology 2012, 52, 1–11 Morphological changes of the fungistatic toxin patulin 3


95%), and three changes of 100% ethanol. Samples were then inbedded in epoxy resin (Durcupan ACM, Sigma, Hungary). Blocks were cut by the aid of a Rei-chert ultramicrotome, and every tenth ultra thin sec-tion was collected onto copper grid. Sections were counterstained with uranyl acetate and Reynolds lead citrate and were examined by using JEOL 1200 TEM. In each sample 6–900 cells were investigated to evaluate the results.

Scanning electronmicroscopy (SEM) For scanning electronmicroscopy experiments a culture with 106 cell ml–1 starting cell number was incubated for 35 h at 30 °C, in the presence and absence of 500 μM patulin. Cells were harvested and washed in 0.1 M Na-phosphate buffer (pH 7.2) and fixed in 2.5% glutaraldehyde for 1 h at room temperature, then washed twice in Na-phosphate buffer. Postfixation was performed in 2% osmium-tetroxide for 1 h in the dark. Cells were then washed and the pellet was dehydrated in increasing ethanol gradient (30, 50, 70, 80, 90, 95, 100%). Cells were examined by a JEOL JSM 6300 scan-ning electronmicroscope.

Preparation of protoplasts Mid-log-phase cells were collected by centrifugation and washed twice with 0.6 M KCl solution. For proto-plast formation, the suspensions were incubated for 20 min at 30 °C in protoplasting solution that con-tained 10 mg ml–1 lyophilized enzyme prepared from Trichoderma harzianum (Sigma-Aldrich, Budapest, Hun-gary) in 0.6 M KCl as osmotic stabilizer. After incuba-tion the protoplasts were washed twice in stabilizer solution and were treated with patulin (0, 50, 500 μM) for 60 or 90 min.

Isolation of nuclei For mammalian cells we applied reversibly permeabili-zation to open the nucleus any time during the cell cycle. By using the protoplasts of S. pombe cells perme-abilization was omitted, as nuclei per se are permeable. After isolation protoplasts were washed with phosphate buffered saline (PBS) and incubated at 37 °C for 10 min in Swelling Buffer, followed by centrifugation at 500 g for 5 min. Nuclear stuctures were isolated by the slow addition of 20 volumes of standard Clarks’s Fixative (methanol:glacial acetic acid, 3 :1). Nuclei were then centrifuged at 500 g for 5 min, washed twice in Fixative and resuspended in 1 ml Fixative. The Clark’s fixative keeps cells in a “swollen” state, achieved after hypo-tonic treatment. The fixative solution elutes some of the membrane lipids and proteins and makes the

membrane more fragile and suitable for spreading flat on the slide when subjected to the drying techniques [21]. Cellular and nuclear volumes and nuclear diam-eter were determined with Multisizers.

Spreads of nuclear structures Preparation of nuclei for spreads of chromatin struc-tures applied the method developed for metaphase chromosomes. Nuclei in Fixative stored at 4 °C for 1–7 d were spread over glass slides dropwise from a height of approximately 30 cm. Dropping helps to dis-tribute nuclei evenly on the slide surface [21]. Slides were air dried, stored at room temperature overnight, rinsed with PBS and dehydrated using increasing con-centrations of ethanol (70, 90, 95 and 100%).

Visualization of chromatin structures Experiments employed the DNA fluorochrome DAPI for fluorescent staining. Different shades of the blue colour of DAPI fluorescence indicate the degree of chromatin compactness. DAPI binds specifically to A–T rich se-quences in the minor groove of DNA [22]. Dehydrated slides containing chromatin structures were mounted in 35 μl Antifade Medium under 24 × 50 mm coverslips. Blue fluorescence of DAPI was moni-tored with Olympus AX70 fluorescence microscope or Axioplan Universal microscope (Carl Zeiss, Oberkochen, Germany), equipped with HBO 50 microscope illumina-tor, MC 100 spot camera, Sony 3CCD Video Camera (DXC-930 P), Sony camera adapter and Sony Trinitron Color Video Monitor. Magnificaton of ocular (10×) and immersion object (100×) lenses was 1000×. The refrac-tive indexes of condenser and objective lenses were 1.515. Homogeneous path for light was obtained by filling the air gaps with Cargille immersion oil (n D 25:1.515) (Cargille-Sacher Inc., Cedar Grow, NJ).

Time-lapse photography Two inverse microscopes sitting in CO2 incubator were equipped with high sensitivity video cameras, con-nected to a custom-built dual image acquisition com-puter system. Custom built illumination was developed to minimize heat- and photo-toxicity. Operation of the spectrally warm-white light emitting diodes were syn-chronized with image acquisition periods. Cell cultures in T flasks were placed on inverse microscopes. The screen of the computer was divided in two portions showing side-by-side the morphological changes of the control and patulin treated cells. Photographs of S. pombe cells were taken every minute. The time of exposure was indicated in each frame. Exposures were converted to videofilms by speeding up the projection



Figure 1. Representative transmission electronmicrographs of mid-log-phase cells of S. pombe. Control cell (A), patulin-treated cells: 50 μM, (B); 500 μM, (C) for 180 min. Continuous arrows indicate the nucleus, dashed arrows show vacuoles. to 30 exposures/sec. Individual cells were selected for fur-ther analysis. Photographs were selected as panels shown in the figures. Time-lapse photography of indi-vidual cells allowed us to determine the growth profile of individual cells [23]. We have introduced recently time lapse video photography for the analysis of cell death in mammalian and fungal cells [23] and to follow the movement of individual cells after in vitro treatment with Hg(II) acetate [24]. The homepages of videofilms showing the growth and mobility of S. pombe cells before and after patulin treatment are given under the figure legends.

Results

Effect of patulin on cell survival S. pombe cells were precultured overnight in SM me-dium, and exponentially growing cells were harvested by centrifugation (600 g, 5 min). Cells were recultured in fresh SM medium for 12 h. These mid-log-phase cells were used to estimate the MIC50 survival rates of cells exposed to patulin (10–500 μM). The growth of five S. pombe cell cultures was started at 107 cell/ml, respec-tively. The control sample was not treated. After treat-ment for 60 or 90 min the number of colony forming units of the S. pombe strain were counted in the absence of patulin (control) and in the presence of 10, 30, 50 and 500 μM patulin concentrations resulting in 8, 12, 24 and 67% reduction upon 60 min treatment and in 24, 69, 39 and 95% reduction of CFU after 90 min growth, respectively. The survival data of these short 60 and 90 min incubations in the presence of patulin indi-cated a drastic drop in MIC50 concentrations from ~300 to ~35 μM indicating the high toxicity of my-cotoxin on S. pombe.

Patulin induced ultrastructural alterations Cultures of control and patulin treated cells for 90 and 180 min were collected, fixed, mounted, and sectioned for transmission electronmicroscopy analysis (Fig. 1).

The sections of cells incubated with 50 and 500 μM patu-lin for 90 and 180 min unequivocally showed complex ultrastructural alterations. In comparison with the con-trol, the length of the treatment influenced considerably the dimension of cells. Treatment for 90 and 180 min at higher (500 μM) patulin concentration affected 34 to 50% of the cells, respectively. At lower (50 μM) concen-tration and treatment for 90 and 180 min the percentage of affected cells was 33 and 43%, respectively. Relative to the control (Fig. 1A) lower patulin concentration caused some blebbing and the shrinkage of the nucleus (Fig. 1B) typical features of apoptosis. Higher patulin concentra-tion causing swelling and triggered marginal chromatin condensation (Fig. 1C) may be the result of unbalanced osmotic regulation. Extensive vacuolization and de-creased staining density (Fig. 1B and C), support the notion of loosing intracellular substances [25]. S. pombe cells were visualized by scanning electron microscopy (Fig. 2) after 35 h incubation in the pres-

Figure 2. Representative scanning electronmicrographs of statio-nary-phase cells of S. pombe. Control cells (a, c), and 500 μM pa-tulin-treated cells (b, d). Magnifications: 3000× (a, b); 5000× (c, d).



ence or absence of 500 μM patulin. Control cells in sta-tionary phase terminated the division and occurred solely. In contrast, numerous patulin treated cells showed elongated cell morphology, suggesting an ab-normal or lagging cell division process (Fig. 2b, d). Patulin treated cells became slightly swollen.

Time-lapse photography Two inverse microscopes in the CO2 incubator were equipped with high sensitivity digital cameras, con-nected to a custom-built dual image acquisition system. Operation of spectrally cold-white light custom built illumination was synchronized with image-acquisition

periods to minimize heat and phototoxicity [23]. The growth of two identical cell cultures in T-flasks were started at about 60% confluency corresponding to an average of 100 (±10) cells in the visual field of the mi-croscope. Initially only slightly elongated S. pombe cells were visible in the control that did not contain patulin. A gradual growth of untreated cells was observed for 11 h approaching ~80% confluency and a 2-fold in-crease in cell number (Fig. 3). In the presence of 500 μM patulin an imminent change in cellular shape took place with most of the cells rounded up and becoming oval shaped (first panel, 0 min). The minor cell growth did not exceed 10% dur-

Figure 3. Time-lapse microscopy of cell growth visualized in S. pombe cell culture. Cells were grown in the absence of patulin as described in the Materials and methods from low to high confluency. White numbers at the bottom of each panel show the time passed in minutes from the beginning of the growth of control cell culture. See more details in the videofilm at http://youtu.be/aL4BYYh0fYc.



ing the 11 h incubation (Fig. 4). Patulin treated cells graually became elongated ~2/3 of them seen as hy-phae, while ~1/3 of the cells maintained their oval or round shape (Fig. 4, last panel, 660 min).

Inhibition of cell growth and cellular movement by patulin The time-lapse photography system was also used to quantify the small but visible cell growth in the pres-ence of 500 μM patulin. In these experiments photo-graphes were also taken every minute as in Fig. 4, but cells were incubated for a longer (20 h) period of time to confirm the growth tendency. Fig. 5a shows that dur-

ing this 20 h incubation in the absence of patulin a 4-fold increase in cell number was registered, while in its presence (500 μM) the growth was only 1.6-fold corre-sponding to an 80% inhibition by patulin. The fluctua-tion in cell number, especially at higher densities is a reflection of the cellular movement in the visual field. These data clearly indicate that those cells that sur-vived the short term effect of the mycotoxin underwent a long term adaptation to patulin, pointing to the my-costatic rather than to a mycocidic effect of patulin against S. pombe. The total cell covered area in the presence and ab-sence of 500 μM was expressed as the pixel numbers of

Figure 4. Time-lapse microscopy after patulin treatment. S. pombe cell culture at low confluency was subjected to 500 μM patulin. White numbers at the bottom indicate the time in minutes passed since the beginning of cell growth. In the last panel long black arrows (→) show the elongation and the sticking together of cells, triangles (�) point to rounded up patulin treated cells. See videofilm at http://youtu.be/g8KreF6D3o4.



the computer screen. The increase in the surface area of control cells covering the T-flasks (Fig. 5a) shows a similar tendency to that after patulin treatment (Fig. 5b). The fluctuation of the cell covered area at higher densities is similar to the oscillation of the con-trol cell number and is attributed to the cellular movement within microscopic sight. The surface area of patulin treated cells reflected a 2× increase in 20 h without significant cellular movement. The average cell size of control cells showed a gradual increase (up to 30%) from the initial single-cell yeast form to the fila-mentous growth form (Fig. 5c). There was a 30% in-crease in average particle size of control cells during the 20 h incubation. This is accounted for by the di-morphic switch from the single-cell yeast form to the filamentous invasive growth form [26]. The average particle size of the 500 μM patulin treated S. pombe cells was initially significantly (30%) higher than that of the control cells (Fig. 5c). However, this was followed by only a slight further enlargement (10–15%) of cellular size as registered by the long-term scanning photography. There seems to be a relationship between the moderate cell growth and suppressed tran-sition from the dimorphic to filamentous forms of the patulin treated cells (Fig. 5c).

Figure 5. Inhibition of cell growth by patulin monitored by the time-lapse photography system. Of the two identical cell cultures grown in 25 ml T-flasks at 25 °C one was the control, the other was treated with 500 μM patulin placed on two reverse microscopes in a CO2

incubator. Photographs were taken every minute by custom built cameras attached to microscopes and connected to the computer. A. Changes in cell number (ordinate) versus time of incubation (abscissa) was traced in control (black graphicon), and in patulin treated (grey curve) populations of S. pombe cells. B. Total cell covered area, in control (black) and after patulin (grey) treatment. C. Average particle size: control (black), patulin (grey).

Chromatin structures of normal untreated S. pombe cells For the visualization of interphase chromatin struc-tures in mammalian cells nuclei have been isolated from reversibly permeabilized cells. The reversal of permeabilization served a dual purpose: it maintained the viability of cells, and allowed to open nuclei not only in metaphase but also in interphase, revealing different stages of the complex chromatin condensa-tion process. The chromatin folding pattern was char-acterized by a series of transient geometric forms re-ferred to as decondensed fibrillar, coiled ribbons, chro-matin bodies, thin and thick fibers, elongated and precondensed chromosomal forms (Fig. 6) similarly to mammalian cells [27]. The fact that similar forms were present in different mammalian (Indian Muntjac, CHO, murine preB, human K562 erythroleukemia) nuclei pointed to a common condensation mechanism at least in mammalian cells [16]. In Drosophila nuclei the pres-ence of many small subchromosomal particles indi-cated that chromosomes of the higher arthropod Droso-phila cells consisted of smaller units called rodlets, pro-

Figure 6. Intermediates of chromatin condensation in control S. pombe cells. Protoplasts were prepared from exponentially growing S. pombe cells not subjected to patulin treatment. Nuclei were isolated from protoplasts, spread over glass slides and stained with DAPI as described in the Methods. The images of frequently occuring patterns were selected to illustrate: a, decondensed chro-matin that maintained the oval shape of the nuclei, b, c, rounded up and polarized chromatin, d–k Chromatin ribbon formation. l –o,early elongated chromosomal forms, p, bent forms of the three (1, 2, 3) condensed chromosomes of S. pombe, q, magnification of chromo-some 1 shown in p. Bar, 10 μm each.



viding flexibility to condensing chromosomes [28]. Re-cently, we have isolated chromatin structures from the protoplasts of exponentially growing fission yeast S. pombe cells. Since we have described these intermedi-ates [17], Fig. 6 is presented only to confirm the exist-ence of these forms representing different stages of chromosome condensation, and serves as a control for the patulin treated chromatin changes. These interme-diates of chromatin condensation include elongated nuclei, rounded up and polarized nuclei containing lo-cal condensation of the fibrillary chromatin veil, super-coiling of chromatin veil turning to chromatin ribbon and large coiled individual chromosomes. Highly com-pacted structures similar to metaphase chromosomes were not seen.

Local condensation and fibrillary expansion of chromatin at low (50 μM) concentration of patulin Lower concentrations of gentoxic agents normally cause the apoptotic shrinking of cells and nuclear con-densation. We did not observe significant chromatin changes between 1–10 μM patulin concentrations. The uneven condensation of chromatin pretreated with 50 μM patulin for 60 min is reflected as the polariza-tion of condensing chromosomes accompanied by the rejection of the decondensed fibrillary chromatin (Fig. 7,

Figure 7. Effect of low concentration of patulin on chromatin con-densation in nuclei of S. pombe cells. Nuclei were isolated from protoplasts, spread over glass slides as described in Fig. 5 with the exception that cells were subjected to patulin treatment. Chromatin structures were stained with DAPI and visualized as described in the Methods. Appearance of fibrillar extrusions upon treatment of cells with 50 μM patulin for 60 min (upper panels) or 90 min (lower panels). Bar 10 μm each.

upper panels). However, the enlargement of chromatin structures does not support the idea of apoptosis. The notion that patulin is not an apoptotic agent was con-firmed when longer (90 min) incubation was used in the presence of 50 μM patulin and large fibrous chro-matin structures were observed under the fluorescent microscope with locally condensed chromatin patches seen as white spots (Fig. 7, lower panels).

Local condensation of chromatin and nuclear breakage at high (500 μM) concentration of patulin Higher concentrations of gentoxic agents normally lead to the necrotic explosion of the cells and subcellular particles including their nuclei. When cells were sub-jected to high concentration of patulin (500 μM) for 60 min, the nuclear expansion was accompanied by many small highly condensed chromatin particles sour-ronded by decondensed fibrillar chromatin (Fig. 8, up-per panels a–d). Beside the chromatin that was already partially condensed by the time patulin treatment took place, other distorted chromatin structures of fibrous and ribboned structures became visible with fewer highly condensed chromatin patches. Longer (90 min) incubation in the presence of 500 μM patulin caused nuclear fragmentation (Fig. 8, lower panels). The partial

Figure 8. Effect of high patulin concentration on chromatin conden-sation. Nuclei were isolated from protoplasts, spread over glass slides as described in Fig. 5 with the exception that cells were sub-jected to patulin treatment. Chromatin structures were stained with DAPI and visualized as described in the Methods. Appearance of many highly condensed chromatin and desintegration of nuclei upon treatment of cells with 500 μM patulin for 60 min (upper panels) or 90 min (lower panels). Bar 10 μm each.



disruption of nuclei is not characteristic to necrosis. The deformed fragments did not resemble round sha-ped chromatin bodies excluding the possibility of apop-tosis.

Discussion

The contamination of foods and animal feeds with my-cotoxins produced by the filamentous fungi of Aspergil-lus, Penicillium, Fusarium, and Alternaria species is a worldwide concern. To interphere with fungal growth and minimize the risk of mycotoxin contamination of food and feeds chemical and biological protections have been attempted by using synthetic and natural agents. Patulin is one of the mycotoxins that is produced by a number of fungi involved in food decay, particularly Penicillium expansum, the primary cause of “blue mould rot” disease found on storage rotted apples. Patulin present in juices and isolated from rotten apples [29] is toxic [30], teratogenic [31], and carcinogenic [1] to ani-mals. In fermented apple cider made from the same decayed fruit the patulin concentration was reduced by over 99% [32]. Canadian and British fermentation methods that added sufficient sugar and Saccharomyces spp. supplement cultures resulted in little or no patulin after fermentation [33, 34]. French ciders that relied upon the natural sugars and wild organisms present in the apple juice without sugar or nutrient addition dis-closed patulin in more than 60% of industrial ciders [35]. Recently the awareness of the apple industry to patulin contamination in fruit and importance of cider fermentation lead to the implementation of improved techniques contributing to the quality of the apple products world-wide. Although, it is well documented that patulin disappears during fermentation of apple juice, but so far no explanation was proposed to explain its degradation. As potential natural agents bacteria have been stud-ied to control the mycotoxins production and fungal growth in food. The action of patulin on Saccharomyces cerevisiae was only transient and after the recovery of yeast growth, patulin disappeared from the medium. The growth recovery of the budding yeast S. cerevisiae indicated that the degradation of the mycotoxin might have been the result of an inducible enzymatic system resisting high patulin concentrations [36, 37]. The fact that patulin disappears during fermentation are con-gruent with our recent observation that S. pombe cells show a rapid adaptation to high concentrations of patu-lin [38] and could indicate that fission yeast strains are involved in the degradation of patulin.

Although, the secondary metabolite patulin is known to exhibit cellular toxicity, but the nature of cell death associated with patulin exposure has not yet been elu-cidated and is still a subject of debate. Patulin causes various toxicities including oxidative stress [25, 39], DNA damage, chromosome aberration and micronu-cleus formation have been observed in mammalian cells [40–42]. Membrane blebbing, nuclear fragmenta-tion and DNA ladder formation, accompanied by cyto-chrome c release from mitochondria and Bcl-2 expres-sion decrease without the involvement of p53 indicated apoptosis in human promyelocytic leukemia cells [12]. Others suggested that p53 protein may be involved in patulin induced signaling of pro- and anti-apoptotic Bcl-2 family proteins and p53 may modulate the activation of caspases [43]. That the tumor suppressor gene P53 mediates the activation of programmed cell death, in part by up-regulation of mitochondrial Bax expression has also been reported [44]. Bax in turn as a key com-ponent for apoptosis acts through mitochondrial stress [45]. That patulin has a potential to induce DNA dam-age leading to p53 mediated cell cycle arrest along with intrinsic pathway mediated apoptosis was also sug-gested by others [9]. The in vivo cellular damage of patu-lin was demonstrated by a dose of 0.1 mg kg−1 bw day−1 patulin given to rats for a period of 90 days daily and apoptotic body formation and apoptosis in interdigitat-ing cells of rat thymus was observed [13]. To resolve the conflicting results related to cell growth and cell death generated by patulin we have visualized ultrastructural changes by transmission and electronmicroscopy, cellular movement of individual cells by long-term scanning photography system [23] adapted to S. pombe cells and large scale chromatin changes by fluorescent microscopy. The morphological alterations seen in transmission and scanning elec-tronmicroscopy could be explained by the patulin in-duced unbalance of osmoregulation and by the altered biophysical properties of S. pombe cell membrane [38]. Long-term scanning of S. pombe cells that showed inhi-bition of cell growth at high patulin concentration was followed by a gradual growth recovery of the fission yeast, in accordance with the growth recovery of the budding yeast S. cerevisiae resisting high patulin concen-trations [36, 37]. The growth recovery could be explai-ned by the disappearance of patulin by biodegradation of the adapted S. pombe fission yeast, by the conversion of the mycotoxin patulin to a less toxic compound such as desoxypatulinic acid formation in Saccharomyces cere-visiae [46], resembling the cider processing methods by Saccharomyces spp. supplementation resulting in little or no patulin after fermentation [33, 34].



As far as the morphological cellular and nuclear changes are concerned we could see the shrinkage of cells at lower (50 μM) patulin concentration typical to apoptosis, while the nuclear structure was characterized by elongated nuclei containing sticky fibrillary chroma-tin and enlarged round shaped nuclei retained in the fibrillary stage of chromatin condensation characteristic to necrosis. Although, high (500 μM) patulin concentra-tion generated condensed chromatin patches inside the nucleus, but was associated with nuclear expansion and the rest of chromatin remained in fibrillary form. Longer incubations in 500 μM patulin resulted in fewer condensed chromatin patches and in nuclear fragmen-tation, without typical, round shaped chromatin bodies. The data of computer analysis of long-term scanning photography corresponded to those of microscopic nuclear changes. Beside the moderate cellular shrink-age at lower (50 μM) patulin concentration no apoptotic body formation was registered, which could have been a definitive morphologic evidence of patulin-induced apoptosis. On the other hand the moderate swelling of nuclei without disruption or explosion excluded the possibility of necrotic cell death.

Concluding remarks Based on the observations of others and on our experi-ments we assume that: a) Patulin is more toxic to mammalian cells at relatively low (~μM) concentra-tions, than to the fission yeast S. pombe. b) The growth of S. pombe is only temporarily inhibited by this my-cotoxin. The long-term recovery of cell growth in the presence of high (500 μM) patulin seems to confirm the adaptation of S. pombe cells to patulin. c) There are dif-ferent yeast strains including S. pombe that are involved in the degradation of patulin. d) Patulin induced cell death is neither typical to apoptosis nor to necrosis. This notion is in conformity with the idea that cell death may occur in many transitory forms, depending on the characteristic death pathway of the genotoxic agent. Alternatively, the long-term adaptation may contribute to the reversal of at least some of the intra-cellular processes that would lead to cell death.

Acknowledgement

This work was supported by Hungarian Scientific Re-search Fund (OTKA grant) T 42762 grant to G.B.

Conlict of interest statement Any potential conflict of interest including finantial, personal or other relationships with other people or organizations is disclosed that would bias this work.

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Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe