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DOSE-RESPONSE OF PORCINE OVARIAN GRANULOSA CELLS TO AMYGDALIN TREATMENT COMBINED
WITH DEOXYNIVALENOL

Marek Halenár*, Marína Medveďová, Nora Maruniaková, Dagmara Packová, Adriana Kolesárová

Address(es): Ing. Marek Haklenár,
Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak
Republic.
*Corresponding author: [email protected]
ABSTRACT
Amygdalin is one of many nitrilosides, which are natural cyanide-containing substances abundant in the seeds of apricots, almond, Received 24. 10. 2013 peaches, apples, and other rosaceous plants. It is a controversial anti-tumor natural product that has been used as an alternative cancer Revised 26. 11. 2013 drug for many years. On the other hand, one of the most widely distributed mycotoxin contaminating food and animal feed is Accepted 16. 12. 2013 deoxynivalenol (DON). Deoxynivalenol has adverse effects on humans, animals, and crops that result in illnesses. The aim of the in Published 1. 2. 2014 vitro study was to investigated the effect of natural substance amygdalin at the selected doses (1, 10, 100, 1000, 10 000 µg/mL) in combination with deoxynivalenol (1000 ng/mL) on secretion of steroid hormones (progesterone and estradiol) by ovarian granulosa cells (GCs) from cyclic pigs. Our results showed that the releasing of progesterone and estradiol by ovarian granulosa cells was affected by amygdalin plus DON addition. The secretion of progesterone by ovarian GCs was significantly (P≤0.05) affected by administration of both compounds in all experimental groups. Similarly, estradiol releasing by GCs was significantly (P≤0.05) increased in experimental groups with amygdalin (10, 100 and 10 000 µg/mL) plus DON (1000 ng/mL) addition. Amygdalin treatment combined with DON caused increase of steroid hormones release by ovarian granulosa cells. Our findings suggest possible involvement of these natural substances (amygdalin and deoxynivalenol) in the regulation process of steroidogenesis. In conclusion, results from this experiment contribute to knowledge about interaction between two different natural compounds and their positive or negative interferences with ovarian functions. Keywords: Amygdalin, deoxynivalenol, steroid hormones, ovarian granulosa cells

INTRODUCTION
adipose tissue and reproductive tissues. Thus, mycotoxin exposure that alters granulosa cells steroid hormone production may also alter oocyte development, Natural plant compound as amygdalin is still a major part of traditional medicine. ovulation, reproductive tract function and pregnancy outcome (Medveďová et
Amygdalin (D-mandelonitrile-β-D-gentiobioside, Fig. 1), sometimes termed to as al., 2011).
a Vitamin B17, is found in the seeds of apricots, almonds, peaches and other Steroid hormones, such as progesterone and estradiol are produced by ovarian plants (Fukuda et al., 2003; Chang et al., 2006; Nabavizadeh et al., 2011). This
cells and both are substantial for normal ovarian cycles (Hagan et al., 2008;
controversial natural substance is composed of two molecules of glucose, one of Arnhold et al., 2009), contribute to regulation of ovarian follicular development
benzaldehyde, which induces an analgesic action, and one of hydrocyanic acid, and remodelling (Mahajan, 2008).
which is an anti-neoplastic compound. It has been reported that amygdalin canbe In the present study, we observed the potential effects of both natural compounds used in medicine for effectively prevent and treat cancers, chronic inflammation, amygdalin and deoxynivalenol on secretion of steroid hormones (progesterone migraine, relieve fever and pain (Yan et al., 2006, Fukuda et al., 2003, Zhou et
and estradiol) by porcine granulosa cells from cyclic ovaries. al., 2012). However, the Food and Drug Administration (FDA) has not approved
amygdalin as a cancer treatment owing to insufficient clinical evidence of its MATERIAL AND METHODS
efficacy and potential toxicity. Despite the failure of clinical tests to demonstrate the anticancer effects of amygdalin in the U.S.A. and in Europe, amygdalin Preparation, culture and processing of granulosa cells from ovaries
continues to be manufactured and administered as an anticancer therapy in northern Europe and Mexico (Chang et al., 2006; Kwon et al., 2010).
Ovaries from cyclic pigswere obtained from healthy Slovakian White gilts without obvious reproductive abnormalities. Isolated ovaries were transported to the laboratory in containers at 4 ºC and washed in sterile physiological solution. Follicular fluid was aspirated from 3-5 mm follicles. Granulosa cells were isolated by centrifugation for 10 min at 200xg followed by washing in sterile DMEM/F12 1:1 medium (BioWhittakerTM, Verviers, Belgium) and resuspended in the same medium supplemented with 10% fetal calf serum (BioWhittakerTM, Verviers, Belgium) and 1% antibiotic-antimycotic solution (Sigma, St. Louis, Mo, USA) at the final concentration of 106 cells/mL (as detected by haemocytometer). Portions of the cell suspension were dispensed to 24-welled culture plates (NuncTM, Roskilde, Denmark, 1ml/well; for Enzyme Linked ImmunoSorbent Assay,ELISA). The well plates were incubated at 37 °C and 5% Figure 1 Chemical structure of amygdalin
CO2 in humidified air until a 75% confluent monolayer was formed (4-5 days), at this point, the medium was renewed and ovarian granulosa cells were incubated Deoxynivalenol (DON) is one of the most important and occurring Fussariu with the similar supplements (DMEM/F12 1:1 medium, 10% fetal calf serum, mmycotoxin (Łazicka and Orzechowski, 2010; Klem et al., 2007). Occurrence
without 1% antibiotic-antimycotic solution) and without (control) or with of thismycotoxin is mainly in grains such as wheat, barley and maize (Creppy,
amygdalin (1, 10, 100, 1000, 10 000 µg/mL) (99 % purity, Sigma-Aldrich, St. 2002). DON could be rapidly absorbed after oral administration passively
Louis, Mo, USA) combined with deoxynivalenol (1000 ng/mL) (Romer Labs throughout the gastrointestinal tract and actively in the kidneys, liver, muscle, J Microbiol Biotech Food Sci / Halenár et al. 2014 : 3 (special issue 2) 77-79
Division Holding GmbH, Tulln, Austria) for 24h. Experimental group P1/E1 represents culture medium with amygdalin (1 µg/mL) plus deoxynivalenol (1000 ng/mL), other groups represent culture medium with amygdalin P2/E2 (10 µg/mL), P3/E3 (100 µg/mL), P4/E4 (1000 µg/mL) and P5/E5 (10 000 µg/mL) plus in each group deoxynivalenol (1000 ng/mL). After 24h of incubation the culture media from well plates were aspirated and kept at –80°C for subsequent assay. The concentrations of steroid hormones progesterone and estradiol were assayed using ELISA (Dialab, Wiener Neudorf, Austria) according to the manufacturer´s instructions. Statistical Analysis
Each experimental group was represented by four culture wells of granulosa cells. Assay of hormone level in the incubation media was performed in duplicate.Significance of differences between the control and experimental groups were evaluated by one-way ANOVA and t-testusing statistical software Sigma Plot 11.0 (Jandel, Corte Madera, USA). The data are expressed as means ± SD. Differences were compared for statistical significance atthe P – level less than 0.05 (P≤0.05). Figure 3 The effect of amygdalin in combination with deoxynivalenol on
progesterone release by porcine ovarian granulosa cells Control represents culture medium without amygdalin and deoxynivalenol addition. Experimental group E1 represents culture medium with amygdalin (1 Release of progesterone by porcine ovarian granulosa cells
µg/mL) plus deoxynivalenol (1000 ng/mL), other groups represent culture medium with amygdalin E2 (10 µg/mL), E3 (100 µg/mL), E4 (1000 µg/mL) and The secretion activity of granulosa cells from cyclic porcine ovaries after E5 (10 000 µg/mL) plus in each group deoxynivalenol (1000 ng/mL). Signs a,b addition of natural compounds amygdalin (1, 10, 100, 1000, 10 000 µg/mL) denote values significantly different from control group (P≤0.05) evaluated by combined with DON (1000 ng/mL) was determined (Figs. 2, 3). The release of one-way ANOVA and t-test. ELISA. steroid hormone progesterone by ovarian granulosa cells was significantly (P≤0.05) stimulated after amygdalin administration in combination with DISCUSION
deoxynivalenol in all experimental groups (P1, P2, P3, P4, P5) compared to control group without addition of both natural compounds (Fig. 2). In the present in vitro study, possible response of porcine ovarian granulosa cells to amygdalin addition in combination with deoxynivalenol (DON) was examined. Our isolated ovarian granulosa cells were able to survive, growth in culture and release hormonal substances (progesterone and estradiol) after experimental addition of natural substances amygdalin and DON. The results from our investigation demonstrate that the release of both steroid hormones (progesterone, estradiol) was influenced by natural product amygdalin in combination with mycotoxin deoxynivalenol. In the late 1970s and early 1980s, amygdalin was reported to selectively kill cancer cells at the tumor site without systemic toxicity and to effectively relieve pain in cancer patients (Zhou et al., 2012).Recent study showed preventive and
therapeutic effects of amygdalin on absolute alcohol-induced gastric ulcer in rats. The results of this study showed that amygdalin protected gastric mucosa from alcohol-induced gastric ulcer. This gastroprotection was mediated via gastric mucosal nitric oxide production and TNF-α suppression (Nabavizadeh et al.,
2011).
Previous studies examined the effects of natural compounds on different parts of
animal reproductive system (Kolesárová et al., 2012a; 2012b; 2011; Tanyildizy
and Bozkurt, 2004; Yasui et al., 2003; Randel et al., 1992). Our recent in vitro
investigation showed that the release of steroid hormone progesterone by granulosa cells from cyclic and non-cyclic porcine ovaries was not affected by Figure 2 The effect of amygdalin in combination with deoxynivalenol on
the amygdalin addition (1, 10, 100, 1000, 10 000 µg/mL) (Halenár et al.,
progesterone release by porcine ovarian granulosa cells 2013a). But on the other hand, amygdalin (at 10 000 but not at 1, 10, 100, 1000
Control represents culture medium without amygdalin and deoxynivalenol µg/mL) combined with DON (1000 ng/mL) significantly (P≤0.05) stimulated the addition. Experimental group P1 represents culture medium with amygdalin (1 release of steroid hormones progesterone and estradiol by granulosa cells from µg/mL) plus deoxynivalenol (1000 ng/mL), other groups represent culture non-cyclic porcine ovaries (Halenár et al., 2013b).The results from this study
medium with amygdalin P2 (10 µg/mL), P3 (100 µg/mL), P4 (1000 µg/mL) and showed that amygdalin combined with deoxynivalenol caused significant P5 (10 000 µg/mL) plus in each group deoxynivalenol (1000 ng/mL). Signs a,b (P≤0.05) stimulation of progesterone release by granulosa cells from cyclic denote values significantly different from control group (P≤0.05) evaluated by porcine ovaries in all experimental groups. Similarly, release of estradiol by GCs one-way ANOVA and t-test.ELISA. from cyclic porcine ovaries were affected by addition of amygdalin (10, 100, 1000, 10 000 µg/mL) in combination with DON (1000 ng/mL), but not in Release of estradiol by porcine ovarian granulosa cells
experimental group with the lowest dose (1 µg/mL) of amygdalin. Steroid hormones, such as progesterone and estradiol are produced by ovarian The release of estradiol by ovarian granulosa cells after addition of amygdalin (1, cells and both play irreplaceable role in ovarian cycles (Hagan et al., 2008;
10, 100, 1000, 10 000 µg/mL) in combination with DON (1000 ng/mL) was Arnhold et al., 2009), contribute to regulation of ovarian follicular development
detected. Significant (P≤0.05) stimulation of the estradiol release by ovarian and remodeling (Mahajan, 2008).The possible impact of different naturally
granulosa cells was detected in experimental group E2, E3, E4 and E5 compared cyanide-containing substances on the male reproductive system, focused on to control group without addition of both natural substances. On the other hand, sperm motility and morphological abnormality in bull sperm, was observed no significant (P≤0.05) differences in estradiol release by porcine ovarian previously(Tanyildizy and Bozkurt, 2004). Many studies have described the
granulosa cell were found out between control group without administration of dose-dependent effects of different mycotoxins on the secretion activity of these compounds and experimental group E1 with the lowest dose (1 µg/mL) of porcine (Medveďová et al., 2011, Maruniaková et al., 2013, Ranzenigo et al.,
amygdalin treatment combined with DON (1000 ng/mL). 2008) and rats ovarian cells (Kolesárová et al., 2011). Steroid secretion by
porcine ovarian granulosa cells after deoxynivalenol addition was examined by
Medveďová et al. (2011). The release of progesterone by porcine ovarian
granulosa cells was stimulated by DON treatment at the doses 1000 ng/mL but
not at 10 and 100 ng/mL. Previous data also indicated dose-depended effects of
DON on ovarian granulosa cells (Ranzenigo et al., 2008). Exposure to toxic
concentrations of deoxynivalenol, resveratrol and their combination on the
release of progesterone by porcine ovarian granulosa cells was studied by
Kolesárová et al. (2012a). Results from this in vitro study suggested that
J Microbiol Biotech Food Sci / Halenár et al. 2014 : 3 (special issue 2) 77-79
reproductive toxicity of animals induced by a mycotoxin – deoxynivalenol can be addition of T-2 toxin and its combination withgrowth factor IGF-I. Journal of inhibited by a protective natural substance - resveratrol. Microbiology, Biotechnology and Food Sciences, 2, 1864-1874. There is now considerable evidence suggesting that production of steroid MEDVEĎOVÁ, M., KOLESÁROVÁ, A., CAPCAROVÁ, M., LABUDA, R., hormones by ovarian granulosa cells in vitro is affected by addition of some SIROTKIN, A.V., KOVÁČIK, J., BULLA, J. 2011. The effect of deoxynivalenol on the secretory activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro. Journal of Environmental Science and Health Part B, 46(3), 213- CONCLUSION
NABAVIZADEH, F., ALIZADEH, A.M., SADROLESLAMI, Z., ADELI, S. 2011. Gastroprotective effects of amygdalin on experimental gastric ulcer: Role Potential effects of reportedly anti-tumor natural substances amygdalin combined of NO and TNF-α. Journal of Medical Plants Research, 5(14), 3122-3127. with mycotoxin deoxynivalenol on secretion of steroid hormones by granulosa RANDEL, R.D., CHASE, C.C.JR., WYSE, S.J. 1992. Effects of gossypol and cells from porcine cyclic ovaries was demonstrated in this study. Amygdalin cottonseed products on reproduction of mammals. Journal of Animal Science, 70, treatment combined with DON caused increase of steroid hormones release by ovarian granulosa cells. In conclusion, our findings suggest possible involvement RANZENIGO, G., CALONI, F., CREMONESI, F., AAD, P.Y., SPICER, L.J. of these natural substances (amygdalin and deoxynivalenol) in the regulation 2008. Effects of Fusariummycotoxins on steroid production by porcine granulosa process of steroidogenesis. cells. Animal Reproduction Science, 107(1-2), 115-130. TANYILDIZI, S., BOZKURT, T. 2004. In Vitro Effects of Linamarin, Acknowledgments: For provide mycotoxin, we would like to thank Romer Labs
Amygdalin and Gossypol Acetic Acid on Hyaluronidase Activity, Sperm Motility Division Holding GmbH from Tulln, Austria. This work was financially and Morphological Abnormality in Bull Sperm. Turkish Journal of Veterinary supported by the Ministry of Education, Science, Research and Sport of the and Animal Sciences, 28, 819-824. Slovak Republic projects no. 1/0790/11, 1/0022/13, APVV-0304-12, and YAN, J., TONG, S., LI, J., LOU, J. 2006. Preparative Isolation and Purification European Community under project no 26220220180: Building Research Centre of Amygdalin from Prunusarmeniaca L. with High Recovery by High-Speed „AgroBioTech". Countercurrent Chromatography. Journal of Liquid Chromatography & Related Technologies, 29, 1271-1279. REFERENCES
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