CO-ADMINISTRATION OF AMYGDALIN AND DEOXYNIVALENOL DISRUPTED REGULATORY PROTEINS LINKED TO PROLIFERATION OF PORCINE OVARIAN CELLS IN VITRO

Deoxynivalenol (DON) represents one of the most prevalent trichothecene mycotoxin produced by Fusarium species, causing economic and health impacts. On the other hand, amygdalin has been demonstrated to possess both prophylactic and curative properties, thus it has been used as a traditional drug because of its wide range of medicinal benefits, including curing or preventing cancer, relieving fever, suppressing cough, and quenching thirst. The aim of this in vitro study was to evaluate potential effects of natural product amygdalin combined with mycotoxin deoxynivalenol (DON) on the key regulators of cell proliferation and apoptosis in porcine ovarian granulosa cells. Ovarian granulosa cells were incubated for 24h with amygdalin (1, 10, 100, 1000, 10 000 μg.mL) combined with deoxynivalenol (1 μg.mL), while the control group remained untreated. The presence of proliferative (cyclin B1, PCNA) and apoptotic markers (caspase-3) in porcine ovarian granulosa cells after amygdalin treatment (1, 10, 100, 1000, 10 000 μg.mL) combined with deoxynivalenol (1 μg.mL) was detected by immunocytochemistry. The presence of proliferative (cyclin B1, PCNA) and apoptotic markers (caspase3) in porcine ovarian granulosa cells was detected by immunocytochemistry. Co-administration of amygdalin plus DON significantly (p <0.05) increased the number of granulosa cells containing cyclin B1 and PCNA at all tested concentrations, when compared to control. However, percentage of granulosa cells containing major apoptotic marker caspase-3 did not differ after co-administration of amygdalin and DON. In summary, results form this in vitro study indicate that co-exposure of amygdalin and deoxynivalenol may act to stimulate proliferation-associated peptides in porcine ovarian granulosa cells, and thus alter cell proliferation and normal follicular development.


INTRODUCTION
Mycotoxins are a group of toxic secondary metabolites produced by molds and frequently enter the food chain as food contaminants (Vejdovszky et al., 2016).Deoxynivalenol (DON) represents one of the most prevalent trichothecene mycotoxin produced by Fusarium species responsible of Fusarium head blight.Due to a high prevalence of this disease, type B trichothecenes are the most common contaminants of cereal grains in temperate regions of the world (Alassane- Kpembi et al., 2015).They are commonly found on cereals grown in the temperate regions of Europe, America and Asia.The extent of infection is dependent on weather conditions, Good Agricultural Practice and storage conditions of cereal crops (Larsen et al., 2004).A large scale data survey indicates that DON and its metabolites 15-ADON and 3-ADON are present in 57%, 20% and 8%, respectively of food samples collected in the European Union (Alassane- Kpembi et al., 2015).They represent a unique class of mycotoxins that do not only exert toxicity in animal but also are virulence factors in plant disease, which make them one of the major groups causing significant economic and health impacts (Desjardins, 2009).Intoxications following consumption of foodstuffs contaminated with trichothecenes have occurred in both humans and animals with large numbers of people and livestock being affected.Some countries have already established legislative limits in cereals for DON, the most abundant trichothecene, and the European Commission has proposed EU regulatory limits for DON in various raw cereals and their refined products (Larsen et al. 2004).
At the molecular level, trichothecenes display multiple inhibitory effects on primary metabolism of eukaryotic cells including inhibition of protein as well as DNA and RNA synthesis, and their activity may eventually produce harmful levels of oxidative stress due to generation of free radicals (Arunachalam and Doohan 2013).Thus organs and biological functions involving actively dividing cells appear more sensitive to this class of mycotoxins (Parent- Massin, 2004;Pestka et al., 2004).Mycotoxins as contaminants of animal feed can impair growth and/or reproductive effeciency.This is especially prominent in prepubertal gilts (Dänicke, 2002).The adverse health effects of trichothecenes include emesis, nausea, anorexia, growth retardation, hemorrhagic lesions, neuroendocrine changes and immunosuppression (Larsen et al., 2004;Pestka et al., 2004;Alassane-Kpembi et al., 2015).On the other hand, amygdalin as a natural plant compound belongs to the cyanogenic glycosides abundantly present in diverse plants, especially in the rosaceous plant seeds such as bitter almonds, apricots and peaches etc. (Chang et al., 2006, Lee andMoon 2016).This bioactive substance is composed of glucose, benzaldehyde, which induces an analgesic action, and hydrocyanic acid, which is an anti-neoplastic compound (Fukuda et al., 2003;Chang et al., 2006).Amygdalin has been demonstrated to possess both prophylactic and curative anticancer properties, thus it has been used as a traditional drug because of its wide range of medicinal benefits, including curing or preventing cancer, relieving fever, suppressing cough, and quenching thirst (Moertel et al., 1982;Oyewole and Olayinka 2009;Zhou et al., 2012).In addition, the pharmacological activity of amygdalin also include anti-inflammatory, antiatherogenic and anti-asthmatic effects (Song and Xu, 2014).However, the use of the drug was discouraged when it was demonstrated that amygdalin is metabolized in the body to release significant amount of cyanide thus leading to cyanide poisoning (Chandler et al., 1984; Bromley et al., 2005).β-glucosidase, one of the enzymes that catalyzes the release of cyanide from amygdalin, is present in the human small intestine and is also found in a variety of common foods (Strugala et al., 1995;Deng et al., 2002).Side effects of amygdalin ingestion in humans mirror symptoms of cyanide poisoning which includes nausea, vomiting, headache, dizziness, bluish colouration of the skin, liver damage, hypotension, nerve damage, fever, mental confusion, coma and death (Howard-Reuben and Miller 1984).Considering that amygdalin has been frequently used as alternative therapy, modulatory and chemopreventive potential of amygdalin has not been sufficiently studied yet.
Pig is considered an illustrative case of dual-purpose target and model species that benefits agricultural and biomedical research.In addition, because of its similarity, the pig can be regarded as a good model of extrapolation to human (Alassane-Kpembi et al., 2015).
The aim of this in vitro study was to evaluate potential effects of natural product amygdalin combined with mycotoxin deoxynivalenol on the key regulators of cell proliferation and apoptosis in porcine ovarian granulosa cells.

MATERIAL AND METHODOLOGY Preparation, culture and processing of granulosa cells from ovaries
Ovaries of pre-pubertal gilts (n = 12 per experiment) were obtained after slaughter at a local abattoir.Porcine ovaries were obtained from healthy Slovakian White gilts without visible reproductive abnormalities.The ovaries were transported to the laboratory in containers at 4 • C and washed with sterile physiological solution.The follicular fluid was aspirated from 3 -5 mm follicles.The granulosa cells (GCs) were isolated by centrifugation for 10 min at 200 x g followed by washing in sterile DMEM/F12 1 : 1 medium (BioWhittaker TM , Verviers, Belgium) and resuspended in the same medium supplemented with 10% fetal calf serum (BioWhittaker TM , Verviers, Belgium) and 1% antibiotic-antimycotic solution (Sigma, St.Louis, Mo, USA) at a final concentration of 10 6 cells.mL - (as quantified by a haemocytometer).Portions of the cell suspension were dispensed to Lab-Tek 16welled chamber slides (Nunc Inc., International, Naperville, USA, 100 µL per well).The well plates were incubated at 37 °C and 5% CO 2 in humidified air until a 75% confluent monolayer was formed, at this point, the medium was renewed and ovarian GCs were incubated with the same supplements (DMEM/F12 1:1 medium, 10% fetal calf serum, with 1% antibiotic-antimycotic solution), without (control) or with amygdalin (1, 10, 100, 1000, 10 000 µg.mL -1 ) (≥99% purity, from apricot kernels, Sigma-Aldrich, St. Louis, Mo, USA) in combination with DON (1 µg.mL -1 ) (Romer Labs Division Holding GmbH, Tulln, Austria) for 24 h.After 24 h of culture the media from wells were removed and the wells from chamber slides were washed in ice-cold phosphate-buffered saline (PBS) (pH 7.5).Cells were fixed for 1h at room temperature in 4% paraformaldehyde.

Immunocytochemistry
Signaling substances within GCs plated on chamber slides were detected using immunocytochemistry method.The ImmunoCruz Staining System and primary mouse monoclonal antibodies against cyclin B1, Proliferating cell nuclear antigen (PCNA) and caspase-3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used as directed by the manufacturer.Visualisation of the primary antibody binding sites were achieved with a secondary rabbit polyclonal antibody against mouse IGs, labelled with horseradish peroxidase (Sevac, Prague, Czech Republic; dilution 1 : 1000) and diaminobenzidine (DAB) reagent.Chamber slides stained with peroxidase/DAB reagent were mounted with Glycergel (DAKO, Carpinteria, CA, USA) mounting medium.

Statistical analysis
The proportions of cells containing specific immunoreactivity were calculated from inspection of at least 1000 cells per chamber.The data are presented as means of values obtained from three separate experiments performed on separate days using separate pools of ovaries from 10 -12 animals.The significance of differences between the control and experimental groups was evaluated by One-Way ANOVA (Dunnett´s multiple comparison test) using the statistical software GraphPad Prism 3.01 (GraphPad Software Inc., San Diego, CA, USA).The data are expressed as means ±SEM.Differences were compared for statistical significance at the p-level less than 0.05 (p <0.05).

DISCUSSION
Numerous studies demonstrated that granulosa cells play a fundamental role in the process of normal folliculogenesis, oocyte growth and development To assess cell proliferation in porcine ovarian granulosa cells after co-exposure to amygdalin and deoxynivalenol, selected proliferationassociated peptides (cyclin B1 and PCNA) were evaluated in our experiments.Co-exposure of amygdalin and deoxynivalenol at all doses tested significantly increased number of granulosa cells containing both proliferative markers, cyclin B1 and PCNA.Proliferating cell nuclear antigen and cyclin B1 represent the fundamental peptides related to cell cycle, which are involved in the process of ovarian cell proliferation, growth and development (Naryzhny and Lee 2001;Tomanek and Chronowska 2006;Kolesarova et al., 2008Kolesarova et al., , 2015)).
Our previous study revealed dose-dependent response of porcine ovarian GCs to amygdalin alone or in combination with deoxynivalenol (Halenar et al., 2015).The presence of amygdalin alone induced a significant stimulation of 17β-estradiol release, but not progesterone, by porcine GCs at the highest used dose (10 000 µg.mL -1 ).However, combination of amygdalin with DON increased progesterone and 17β-estradiol release in dose-dependent manner.Therefore, these data suggest that amygdalin is shown to be potential regulator of 17-β-estradiol but not progesterone in porcine ovarian GCs.Stimulatory effect of amygdalin combined with DON on progesterone secretion was clearly due to DON addition, not by amygdalin.Taken all together, till obtained results suggest dose and species dependent effects of several natural substances on the cellular processes in ovaries, such as proliferation and programmed cell death -apoptosis.

CONCLUSION
In conclusion, results form this in vitro study indicate that co-exposure of amygdalin and deoxynivalenol may act to stimulate proliferation-associated peptides in porcine ovarian granulosa cells, and thus alter cell proliferation and normal follicular development.Considering the lack of information about the potential effects of natural products on the reproductive function in animals further research should be addressed.
Kolesarova et al. (2012) has suggested protective actions of the natural substance resveratrol in mycotoxin induced reproductive toxicity in vitro.Their results showed stimulatory effects of resveratrol, DON and their combination on the release of progesterone by ovarian granulosa cells in dose-dependent manner.Moreover, resveratrol effectively reduced the stimulatory action of DON on steroid hormone production.Recent studies demonstrated the effect of various natural substances with protective (Kolesárova et al., 2012; Halenár et al., 2015) or toxic (Ranzenigo et al., 2008; Maruniakova et al., 2014) potential on the cellular processes in ovarian cells.Confirming previous results (Medvedova et al., 2011), porcine GCs exposure to DON had stimulatory effect on proliferative markers depending on concentrations of DON.Higher concentrations of DON increased significantly percentage of cyclin B1 as well PCNA positive cells when compared to control.On the other hand, Ranzenigo et al. (2008) demonstrated an inhibitory effect of DON on the proliferation of porcine granulosa cell in vitro.Examination of Maruniakova et al. (2014) indicates modulatory impacts of trichothecene mycotoxins, T-2 and HT-2 toxin, combined with insulinlike growth factor I (IGF-I) on the steroid hormone secretion in porcine ovarian granulosa cells in vitro.Previous studies related to amygdalin have primarily focused on its purification, anti-tumor mechanism, determination in plants, as well as on toxicity caused by the release of cyanide (Rauws et al., 1982; Yildirim and Askin 2010; Zhou et al., 2012).An in vitro study of Makarevic et al. (2014) demostrated that amygdalin was able to block the bladder cancer cell growth by downmodulating cell cycle regulating proteins cyclin A and cyclin dependent kinase 2. Additionaly, amygdalin acted on the cdk1-cyclin B axis in PC3 cells after 2 weeks but not after 24 h, implying a time dependent mode of action (Makarevic et al., 2016).Histological analysis of rabbit femoral bones after intramuscular application of amygdalin was observed by Kovacova et al. (2016).Their results showed a significantly lower value of primary osteons' vascular canals and secondary osteons in bone microstructure of experimental rabbits.However, Tusimova et al. (2016) demonstrated no obvious impact of amygdalin application on the energy profile of rabbits in vivo.As a central substance in death receptor pathway of the apoptosis, caspase-3 plays a key role in mediating nuclear apoptosis and is required for some typical nuclear and other morphological changes (Porter and Janicke 1999; Denault and Salvesen 2008).The present study revealed that the presence of caspase-3 in granulosa cells was not influenced by co-administration of amygdalin and deoxynivalenol, compared to untreated control cells.These findings are in accordance with results of Medvedova et al. (2011), who observed no substantial effect of DON on the expression of caspase-3 in porcine ovarian GCs.Conversely, previous report of Zhu et al. (2012) demonstrated the increase of caspase-3 accumulation in porcine granulosa cells after addition of other mycotoxinzeralanenone, and triggered a caspase-3-depended apoptotic process.Combinatory effects of isoflavone genistein with mycotoxin zearalenone on endometrial adenocarcinoma cell line were studied by Vejdovszky et al. (2016).Authors observed significant increase in alcaline phosphatase activity and cellular protein amounts after addition of both tested substances, suggesting slight proliferative effects and biphasic dose-response.