105 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 1: 105–114, 2016, ISSN 2543-8832 Agnieszka Chrustek, Magdalena Twarużek*, Ewa Zastempowska, Jan Grajewski Department of Physiology and Toxicology, Institute of Experimental Biology, Faculty of Natural Sciences, Kazimierz Wielki University, Bydgoszcz, Chodkiewicza 30, 85-064 Bydgoszcz, Poland, *twarmag@ukw.edu.pl Mycotoxin – induced apoptosis in swine kidney epithelial cells Introduction Apoptosis is a  programmed cell death that requires energy input and the activation of many genes controlled by a variety of proteins primarily of Bcl-2 family. Promot- ing proteins include, among others: Bax, Bad, Bid, and Bcl-xs, while the inhibiting ones are Bcl-2, Bcl-x1, Bcl-w (Haake, Polakowska, 1993; Ockner, 2001; Elmore, 2007; Stępień et al., 2007). Programmed cell death can be caused by a number of factors such as UV and gamma radiation, cytostatics, bacteria, toxins, reactive oxygen species, ox- idative stress or DNA damage (Stępień et al., 2007). Process of apoptosis occurs in three stages. At �rst, the process induction signal is generated. In the second stage, caspases activation occurs. �en, during the third stage, phagocytosis of apoptotic bodies by macrophages can be observed (Kopacze- wska, Kopaczewski, 2004). A given cell death can be distinguished on a basis of mo- lecular, biochemical and morphological changes. �e latter include: condensation of cytoplasm and chromatin, shrinkage and nucleus fragmentation, as well as formation of blisters and apoptotic bodies. �ere is also the release of cytochrome c from mito- chondria, decrease in mitochondrial potential, cell dehydration, increase in calcium ion concentration, activation of serine proteases and caspases, loss of asymmetry in cell membrane phospholipids distribution, as well as degradation of actin �laments and DNA. Apoptosis can occur in di�erent ways, depending on the pathway. �ere are ex- ternal, internal, pseudo-receptor, sphingomyelin-ceramide and stress-induced types distinguished. In the �rst type, the pathway is induced by combining the ligand with a  membrane receptor, resulting in death signal transmission and caspase cascade activation. Contradictory to that, the internal pathway, also known as mitochondri- al, is invoked by, among others, the in�uence of reactive oxygen species, oxidative stress or DNA damage. �is leads to the opening of mitochondrial channels and 106 A gn ie sz ka C hr us te k, M ag da le na T w ar uż ek , E w a Za st em po w sk a, J an G ra je w sk i cytochrome c release. Presence of pseudo-receptor pathway was observed in the nat- ural killer (NK) cells and cytotoxic T lymphocytes, while sphingomyelin-ceramide pathway may be activated by viral infections or ionizing radiation. �e last apoptosis pathway (stress-induced) was discovered in 2000 (Nakagawa et al., 2000). It arises from the accumulation of incorrectly folded proteins in the endoplasmic reticulum (Kerr et al., 1972; Haake, Polakowska, 1993; Ockner, 2001; Fadeel, Orrenius, 2005; Elmore, 2007). Apoptosis is an important physiological process occurring in the postnatal period and during embryogenesis. Disturbance of this process can lead to the occurrence of such diseases as cancer, Parkinson’s, Huntington’s or Alzheimer’s diseases, heart attack or brain stroke (Elmore, 2007). Process of apoptosis can be tested in many various ways (Darzynkiewicz et al., 1997; Smolewski, Darzynkiewicz, 2003; Elmore, 2007). �ere are cytometric and non-cytometric methods. �e latter include the cell morphology analysis using dyes such as hematoxylin and eosin or �uorochromes, e.g. DAPI. Changes are assessed by means of light, �uorescence and electron microscope. One of the apoptosis symp- toms is DNA fragmentation that can be analyzed using agarose gel electrophoresis and by comet assay. �e cytometric methods include: analysis of laser light scattering by apoptotic cells, study of cell membrane permeability disturbances using dyes such as: trypan blue, ethidium bromide, 7-AAD (7-aminoactinomycin), as well as study of conformational changes in cell membrane using annexin V. �e study frequently analyses the phenomena associated with the participation of mitochondria in pro- grammed cell death, e.g. mitochondrial potential drop using dyes (rhodamine 123), translocation of Bax to the mitochondria or the release of cytochrome c from mito- chondria into cytoplasm by means of Western blotting using antibodies conjugated with a �uorochrome (Darzynkiewicz et al., 1997; Smolewski, Darzynkiewicz, 2003). Mycotoxins are secondary metabolites produced by a  wide range of di�erent molds including Aspergillus, Penicillium, Fusarium and Stachybotrys spp. Biosynthesis of these compounds can take place during storage and processing of raw materials and during plant growth as well. Mycotoxins are a  potential threat to the health of animals and humans. �ey enter the organism not only by ingestion, but also through inhalation of contaminated air. Secondary metabolites of molds can cause mycotoxi- cosis, which causes damage to internal organs and skin tissues (Grajewski, 2006). �e number of adverse e�ects of mycotoxins on an animal organism have been shown, e.g. dermotoxic, estrogenic, hepatotoxic, immunotoxic, carcinogenic, mutagenic, terato- genic and toxic actions to the hematopoietic system (Wróbel, 2014). �e most common mycotoxins include: a�atoxins, fumonisins, ochratoxin A, trichothecenes (T2, HT2, deoxynivalenol) and zearalenone. Trichothecenes are sec- ondary metabolites of Fusarium that occur in cereals and cereal products. �ey have 107 M ycotoxin – induced apoptosis in sw ine kidney epithelial cells dermatoxic and hemorrhagic action (Chen et al., 2008; Liu et al., 2014). A�atoxins are primarily synthesized by Aspergillus and are being found in peanuts, spices, dried fruits, cotton pulp and maize grain (Twarużek et al., 2013). A�atoxin B1 is the most toxic metabolite. �e milk can contain a�atoxin M1 (Zastempowska et al., 2016). A�a- toxins are potent poisons having teratogenic, mutagenic and carcinogenic properties (Wróbel, 2014; Liu et al., 2015a; Peng et al., 2016). �ere is also ochratoxin A synthe- sized by Penicillium and Aspergillus, and occurs in cereal grain, dried fruits, co�ee, spices, animal-origin food and wine (Kosicki et al., 2016). It causes irreversible dam- age to the nephrons and reveals carcinogenic and immunotoxic action (Grajewski et al., 2007; Zhang et al., 2009; Chopra et al., 2010; Liu et al., 2015b). �e aim of this study was to analyze the impact of mycotoxins: a�atoxin B1, ochra- toxin A  and toxin T2 on the cells of swine kidney (SK) epithelial cell line and their apoptosis. Materials and methods Cell culture and treatment conditions �e SK (swine kidney epithelial) cell line derived from the Department of Physiol- ogy and Toxicology of the Kazimierz University of Bydgoszcz were the material for research. �e cells were cultured in MEM (Minimum Essential Medium Eagle) sup- plemented with antibiotics (penicillin and streptomycin) and 5% fetal calf serum. Cultures (adjusted to 4×105 cells/ml) were grown for 24 h at 37°C, at the access for 5% CO2 and at 95% humidity. �en cells were incubated for 24 h with mycotoxins in concentrations chosen experimentally and based on literature: T2 toxin at 2.5 µM and 25 µM dose, a�atoxin B1 at concentrations of 10 µM and 30 µM and ochratoxin A of 50 µM and 80 µM. Cell line SK cultured in MEM supplemented with antibiotics and fetal calf serum constituted the control. Analysis of apoptosis Flow cytometer Muse Cell Analyzer, as well as Muse Annexin V & Dead Cell Kit (Merck) were used for evaluation of apoptosis. �e cytometer uses laser-based �uo- rescent detection and microcapillary technology to deliver quantitative cell analysis. Cells used in the culture were separated from the base using 0.25% trypsin and then incubated for 20 min with Muse Annexin V & Dead Cell Kit in accordance with the attached protocol. Muse Annexin V & Dead Cell Kit contains �uorescently labelled annexin V, which, as the coagulant, binds with negatively charged phospholipids such as phosphatidylserine moving outside the plasma membrane upon the onset of ap- optosis. �is procedure allows to detect cells in various stages of apoptosis: live cells, dead cells, early and late apoptotic cells. 108 A gn ie sz ka C hr us te k, M ag da le na T w ar uż ek , E w a Za st em po w sk a, J an G ra je w sk i Results and discussion Studies have shown sensitivity of SK cell line on mycotoxins, namely T2 toxin, a�a- toxin B1 and ochratoxin A (Tab. 1, Fig. 1–2). �e data indicates a relationship between the dose of mycotoxins and the occurrence of apoptosis. �e highest percentage of apoptotic cells was observed in those treated with a�atoxin B1 at a dose of 30 µM, and ochratoxin A at both concentrations. �ere were no clear di�erences in the e�ect of ochratoxin A at both concentrations, i.e. 50 µM and 80 µM (Tab. 1, Fig. 1–2). Myco- toxin the least interacting on SK cell line turned out to be T2 toxin. Tab. 1. In�uence of mycotoxins on the presence of apoptosis in the swine kidney epithelial cell line Toxin (dose) Live [%] Early apoptotic [%] Late apoptotic [%] Total apoptotic [%] Dead [%] Control 93.90 5.35 0.15 5.50 0.60 T2 (2,5 µM) 93.04 3.36 3.53 6.90 0.06 T2 (25 µM) 73.55 15.10 11.25 26.35 0.10 a�atoxin B1 (10 µM) 85.90 12.80 0.70 13.50 0.60 a�atoxin B1 (30 µM) 55.05 26.05 14.55 40.60 4.35 ochratoxin A (50 µM) 31.49 64.77 3.74 68.51 0 ochratoxin A (80 µM) 39.59 52.47 7.94 60.41 0 Mycotoxins such as trichothecenes (T2 toxin, HT2, deoxynivalenol), a�atoxins, ochratoxin A, fumonisins and zearalenone can lead to the cell death. Most o�en, apop- tosis and necrosis can be observed. Apoptosis is a programmed cell death and, in con- trast to necrosis, is the active process that needs some energy input and gene activation (Majno, Joris, 1995; Trump et al., 1997; Stępień et al., 2007). During this process, chro- matin condensation, DNA fragmentation, formation of apoptotic bodies and phago- cytosis take place. Programmed cell death is a physiological process occurring in the postnatal period and during embryogenesis. Disorders during its course can lead to the occurrence of many diseases (Elmore, 2007). In contrast to apoptosis, necrosis is a pathological process a�ecting the cell groups. It brings cells to swell, causes disintegra- tion of organelles and induces in�ammation (Majno, Joris, 1995; Trump et al., 1997). �ese studies have con�rmed the ability of selected mycotoxins to induce apoptosis. Our study showed that T2 toxin proved to be the least toxic mycotoxin (at least at the concentrations used in the experiment). �e T2 toxin is derived from tricho- thecenes group. �e exact mechanism of this substance action is not known. It is be- lieved that T2 toxin is an inhibitor of protein synthesis, induces lipid peroxidation and 109 M ycotoxin – induced apoptosis in sw ine kidney epithelial cells inhibits synthesis of DNA and RNA (Doi et al., 2008). It is well known that T2 toxin induces apoptosis (Shinozuka et al., 1997; Li et al., 1997; Wang et al., 2012), but the research of molecular mechanism of this process is still unknown. Scientists suspect that apoptosis can probably occur through the extrinsic pathway with the use of membrane receptors and their ligand (Chen et al., 2008). �e death receptors include TNF (tumor necrosis factor), while TNF-alpha or FasL as its ligand (Haake, Polakowska, 1993; Ockner, 2001; Fadeel, Orrenius, 2005; Elmore, 2007). Com- bination of the relevant molecules activates the caspase cascade, which in consequence leads to death. Chen et al. (2008) studied the e�ects of T2 on human chondrocytes. �eir results showed the increment of Fas, p53, Bax, caspase-3 and a decrease of Bcl- 2 and, therefore, indicated the presence of apoptosis. It is believed that it might have occurred with the use of an external pathway. �e researchers suggest that the T2 toxin can cause Kashin-Beck disease, which causes damage to the joints (Chen et al., 2008). When administered orally, parenterally and in contact with the skin, T2 induces apop- tosis in thymocytes, in the thymus lymphocytes, hematopoietic cells, epithelial cells of the intestinal crypts, hepatocytes and keratinocytes in animals. �is process was also noted in the red and white pulp of the spleen, and in Peyer’s patches as well (Chen et al., 2008; Doi et al., 2008; Liu et al., 2014). It is worth mentioning that the toxin pene- trates the placenta and a�ects the fetus, among others, by damaging its brain (Doi et al., 2008). In addition to T2, ochratoxin A at concentrations of 50 µM and 80 µM was also tested in the present study. It has been shown that both doses caused apoptosis in about 60% of the cells. Ochratoxin A is classi�ed as a human carcinogen. Liu et al. (2015b) studied the e�ect of the substance on Het-1A cells (cancer of the esophagus). Fig. 1. In�uence of mycotoxins on the presence of apoptosis in the swine kidney epithelial cell line 0 20 40 60 80 100 Control T2 2,5µM T2 25µM a�atoxin B1 10µM a�atoxin B1 30µM ochratoxin A 50µM ochratoxin A 80µM [%] Toxin Live Total apoptotic 110 A gn ie sz ka C hr us te k, M ag da le na T w ar uż ek , E w a Za st em po w sk a, J an G ra je w sk i Fig. 2. �e occurrence of apoptosis in the swine kidney (SK) epithelial cells a�er treatment with mycotox- ins at di�erent concentrations: a – control, B – 2.5 µM T2, C – 25 µM T2, D – 10 µM a�atoxin B1, E – 30 µM a�atoxin B1, F – 50 µM ochratoxin A, G – 80 µM ochratoxin A. �e cells have been divided into four distinct populations: live (the lower le� square), early apoptotic (right lower square), late apoptotic (right upper square) and dead (le� upper square). Results have been expressed as percentages 111 M ycotoxin – induced apoptosis in sw ine kidney epithelial cells �e DNA strand breaks were observed along with chromosomal aberrations, and arrest of the cell cycle in the G2 phase. Moreover, there was an increase of caspase-3, which suggests the occurrence of apoptosis in Het-1A cells. Elevated activation of caspase-3 was also observed in a study upon the immunotoxicity of ochratoxin a in cells of H9 line (human T lymphocytes). In addition, changes were seen at the cell morphology level (condensed cytoplasm, chromatin condensation, apoptotic bodies, swollen mitochondria), the increase in TNF-alpha, and a decrease of IL-2. It is suggested that necrosis has the priority over ap- optosis at higher concentrations of ochratoxin A. �e researchers turned their attention to the possibility of the apoptosis occurrence through the mitochondrial pathway, other- wise known as internal pathway (Darif et al., 2016). Mitochondrial channels are opened under the in�uence of stress-inducing factors and cytochrome c is released into the cyto- plasm. �is compound binds to cytoplasmic factor Apaf 1 and inactive caspase-9. �en, activation of caspase-9 and executive caspases takes place, which leads to cell death (El- more, 2007; Stępień et al., 2007; Darif et al., 2016). Ochratoxin A has nephrotoxic, hepa- totoxic and immunotoxic action. Other studies have shown the dose-dependent manner increase in cytotoxicity on neural/nerve cells (Zhang et al., 2009). In the present experiment, we demonstrated a  dose-dependent manner e�ect of a�atoxin B1 on the SK line cells. a dose of 10 µM resulted in an increase of apoptotic cells by 8%, while 30μM dose by 35% in comparison to the control version. A�atoxin B1 interferes with the porcine oocyte maturation by inducing the oxidative stress, and also causes the occurrence of excessive autophagy and apoptosis (Liu et al., 2015a). Both apoptosis and autophagy are a  type of programmed cell death (Stępień et al., 2007). Autophagy is present in all eukaryotic cells and is activated in a  response to a shortage of development-inducing nutrients, as well as damage due to toxins. It is characterized by a lack of the nucleus fragmentation, degradation of the Golgi appa- ratus or the endoplasmic reticulum, and partial chromatin condensation. Peng et al. (2016) studied the role of mitochondria, death receptors and endoplasmic reticulum in the toxin-invoked pathways of thymocytes apoptosis induced by a�atoxin B1. �e study revealed the importance of external and internal pathways in the occurrence of apoptosis. It was also noted that the mycotoxin induces apoptosis, but also necrosis in human lymphocytes (Al-Hammadi et al., 2014). 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(1997). �e pathways of cell death: oncosis, apop- tosis, and necrosis. Toxicologic Pathology, 25(1), 82–88. Twarużek, M., Błajet-Kosicka, A., Grajewski, J. (2013). Occurrence of a�atoxins in selected spices in Po- land. Journal of Consumer Protection and Food Safety, 8(1), 57–60. DOI: 10.1007/s00003-013-0813-4 Wang, X., Liu, Q., Ihsan, A., Huang, L., Dai, M., Hao, H., Cheng, G., Liu, Z., Wang, Y., Yuan, Z. (2012). JAK/STAT pathway plays a  critical role in the proin�ammatory gene expression and apoptosis of RAW264.7 cells induced by trichothecenes as DON and T-2 toxin. Toxicological Sciences, 127(2), 412–424. DOI: 10.1093/toxsci/kfs106 Wróbel, B. (2014). Zagrożenia zwierząt i ludzi toksynami grzybów pleśniowych zawartych w paszach i żywności. Water-Environment-Rural Areas, 3(47), 159–176. [In Polish] Zastempowska, E., Grajewski, J., Twarużek, M. (2016). Food-borne pathogens and contaminants in raw milk – a review. Annals of Animal Science, 16(3), 623–639. DOI: 10.1515/aoas-2015-0089 Zhang, X., Boesch-Saadatmandi, C., Lou, L., Wol�ram, S., Huebbe, P., Rimbach, G. (2009). Ochratoxin A  induces apoptosis in neuronal cells. Genes & Nutrition, 4(1), 41–48. DOI: 10.1007/s12263-008- 0109-y Abstract Apoptosis, as the programmed cell death, plays a signi�cant role in proper functioning of an organism, both in the postnatal period and during embryogenesis. Disturbances in this process can lead to the occurrence of several dysfunctions, e.g. cancer, stroke, Alzheimer’s disease and others. Apoptosis can be triggered by factors such as oxidative stress, free radicals, UV radiation and cytotoxic drugs, but also mycotoxins, e.g. a�atoxins, ochratoxin A  and trichothecenes. �ese toxins are produced primarily by fungi of Aspergillus, Penicillium, Fusarium and Stachybotrys genera. �e aim of the study was to inves- tigate the e�ect of mycotoxins on the occurrence of apoptosis in the swine kidney (SK) epithelial cells. For this purpose, trichothecene T2 toxin was used at a concentration of 2.5 µM and 25 µM, a�atoxin B1 at a dose of 10 µM and 30 µM, and ochratoxin A concentrations of 50 µM and 80 µM. �e results were assessed using �ow cytometer Muse Cell Analyzer (Merck). Studies have shown high sensitivity of the cell line SK on mycotoxins. Apoptosis was caused by all kinds of toxins and depended on the dose of ex- 114 A gn ie sz ka C hr us te k, M ag da le na T w ar uż ek , E w a Za st em po w sk a, J an G ra je w sk i amined substance. T2 toxin at a concentration of 2.5 µM caused apoptosis in 6.9% of the cells, whereas at a concentration of 25 µM in 26.35% of the cells. A�atoxin B1 used at concentrations of 10 µM and 30 µM caused apoptosis in 13.5% and 40.6% of the cells, respectively. �e use of ochratoxin A in concentrations of 50 µM and 80 µM caused the occurrence of apoptosis respectively in 68.51% and 60.41% of the cells. Key words: a�atoxins, apoptosis, mycotoxins, ochratoxin A, swine kidney cell line, T2 toxin Received: [2016.08.16] Accepted: [2016.10.26] Apoptoza w komórkach nabłonkowych nerki świni indukowana mykotoksynami Streszczenie Istotną rolę w prawidłowym funkcjonowaniu organizmu odgrywa apoptoza jako programowa śmierć ko- mórki, zarówno w  okresie poporodowym, jak i  podczas embriogenezy. Zaburzenia tego procesu mogą prowadzić do wystąpienia różnych schorzeń, np. raka, udaru mózgu, choroby Alzheimera i innych. Apo- ptoza może być efektem takich czynników jak: stres oksydacyjny, wolne rodniki, promieniowanie UV, leki cytotoksyczne, w tym również mykotoksyny. Do najczęstszych mykotoksyn należą: a�atoksyny, ochratok- syna A  i  trichoteceny. Są one produkowane głównie przez grzyby Aspergillus, Penicillium, Fusarium oraz Stachybotrys. Celem pracy było zbadanie wpływu mykotoksyn na występowanie apoptozy w  komórkach nabłonkowych nerki świni (SK). W  eksperymencie zastosowano toksynę T2 w  stężeniu 2,5 μM i  25 μM, a�atoksynę B1 w dawce 10 μM i 30 μM oraz ochratoksynę A w stężeniu 50 μM i 80 μM. Wyniki oceniano za pomocą cytometru przepływowego Muse Cell Analyzer (�rmy Merck). Badania wykazały wysoką wraż- liwość linii komórkowej SK na mykotoksyny. Apoptoza była wywoływana przez wszystkie rodzaje toksyn i była zależna od dawki badanej substancji. Toksyna T2 w stężeniu 2,5 μM powodowały apoptozę u 6,9% komórek, podczas gdy w stężeniu 25 μM stwierdzono jej obecność u 26,35% komórek. A�atoksyna B1 stoso- wane w stężeniach 10 μM i 30 μM powodowały apoptozę komórek odpowiednio u 13,5% i 40,6% komórek. Zastosowanie ochratoksyny A w stężeniu 50 μM i 80 μM powodowało wystąpienie apoptozy u 68,51% oraz 60,41% komórek. Słowa kluczowe: a�atoksyny, apoptoza, mykotoksyny, ochratoksyna A, linia komórkowa SK, T2 toksyna Information on the authors Agnieszka Chrustek She is interested in the identi�cation and analysis of pathways leading to the occurrence of cell death (apoptosis, necrosis, mitotic catastrophe). �e research are carried out using light, �uorescence and elec- tron microscopy, �ow cytometry and Western blot method. Magdalena Twarużek Her main research interests revolve around the following thematic groups: the occurrence of mold and mycotoxins in food, feed and environment of humans and animals, the e�ect of molds and mycotoxins on human and animal health and the use of in vitro culture in the evaluation of contamination. Ewa Zastempowska �e subject of her scienti�c interests are microorganisms that are causative agents of mammary gland in�ammation (mastitis) in cows. She is also interested in problems related to virulence, drug resistance and cytotoxicity of the pathogenic microorganisms isolated from the animal milk and the presence of mycotoxins in milk produced by molds as well. Jan Grajewski Professor of agricultural sciences. His research interests include food and feed safety in aspects of molds and their secondary metabolites – mycotoxins.