Interspecies variation in the hepatic biotransformation of : Evidence for bio-inactivation of mycoestrogen zearalenone in sturgeon fish

Item Type article

Authors Malekinejad, H.; Agh, N.

Download date 26/09/2021 22:55:36

Link to Item http://hdl.handle.net/1834/37624 Iranian Journal of Fisheries Sciences 15(1) 415- 425 2016

Interspecies variation in the hepatic biotransformation of zearalenone: Evidence for bio-inactivation of mycoestrogen zearalenone in sturgeon fish

Malekinejad H.*1; Agh N.2

Received: November 2013 Accepted: August 2014

Abstract Zearalenone (ZEA) as mycoestrogen is found in human foods and animal feeds. Its estrogenic potency depends on its biotransformation fate. The hepatic biotransformation of ZEA in two species of sturgeon fish (Acipenser persicus and Huso huso) was investigated. ZEA was incubated with the hepatic microsomal and post- mitochondrial sub-fractions in the presence of NADPH and the metabolites were determined by means of HPLC. Moreover, the rate of glucuronidation for ZEA and its metabolites were estimated in the presence of uridine diphosphateglucuronic acid. - zearalenol (-ZOL) was found to be the major metabolite of ZEA by both sub-

fractions. Enzymatic kinetics studies revealed that the maximum velocity (Vmax) in microsomal and post-mitochondrial fractions for -ZOL production was found 5- and 7-folds in Huso huso and 8- and 12-folds in A. persicus higher than that for -ZOL production, respectively. The H. huso hepatic post-mitochondrial fraction mainly Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 glucurinated ZEA while in A. persicus, the metabolites and in particular -ZOL were glucuronidated. Data suggest that the hepatic biotransformation of ZEA in studied sturgeons resulted in detoxification of ZEA as the main metabolite tends to be -ZOL with weaker estrogenic property. Moreover, clear differences in glucuronidation profile are indicating interspecies variety in hepatic biotransformation of ZEA.

Keywords: Acipenser persicus; Huso huso; Hepatic biotransformation; Glucuronidation; Subcellular fractions; Zearalenone.

1- Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, P.O. Box: 1177, Urmia University, Urmia, Iran. 2- Department of Aquaculture, Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran. [email protected] * Corresponding author's Email: [email protected] 416 Malekinejad and Agh, Interspecies variation in the hepatic biotransformation of Zearalenon…

Introduction The concentration of ZEA in feed Sturgeons as one of the most valuable materials can vary from a few aquatic resources are specifically found micrograms up to 276 mg/kg in large rivers, lakes, coastal waters and (Vrabcheva et al., 1996). Since ZEA inner seas of the northern hemisphere. binds to receptors (ERs) after In addition of being a major source of ingestion and acts as an estrogenic income and employment, sturgeons’ compound, thus it is referred as a meat and caviar as unfertilized sturgeon mycoestrogen (Malekinejad et al., roe are among the most valuable foods 2005). Animals exposed to ZEA or for human. Like other fish species, ZEA-contaminated feed, show successful sturgeon farming also symptoms of hyperestrogenism. In this depends on the proper management and regards, previous data indicated that high quality water and feed. Several pigs are the most sensitive species to factors influence the feed quality ZEA (Decasto et al., 1995; Yang et al., including having complete nutrients and 1995). The estrogenisity of ZEA in being free of contaminations. rainbow trout has also been previously Mycotoxins are among the main feed demonstrated by using the relative contaminants, the substances which are binding affinity method (Knudsen and produced by various fungi. Previous Pottinger, 1999). studies have shown that plant Previous investigations on effects of ingredients including vegetable oil, ZEA in fish have focused mainly on the soybean meal, corn and wheat gluten reproduction of zebrafish (Johns et al., make up the main parts of fish diet 2009). It is well documented that the (Nizza and Piccolo, 2009). At the same estrogenic potency of ZEA could time it has been reported that largely be affected by hepatic

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 mycoestrogen zearalenone had been biotransformation and the conversion of found at the highest level in the corn ZEA into -zearalenol or -zearalenol. samples used for fish diet preparation. We also have shown that there are Recently ZEA was reported as the more species differences in hepatic prevalent mycotoxin with an average biotransformation of ZEA in terms of level of 67.9 μg kg−1 fish feed (Pietsch et different metabolite production and the al., 2013). rate of glucuronidation (Malekinejad et ZEA, 6-(10-hydroxy-6-oxo-trans-1- al., 2006). Sturgeons have a relatively undecenyl)--, is long life span and depending on the produced by Fusarium species species their sexual maturation starts at including F. culmorum and F. the age of six years onward, thus there graminearum (Hestbjerg et al., 2002). are possibilities of consuming the ZEA These molds contaminate crops such as contaminated diet. Although there is no maize, barley and wheat (Yamashita et direct report about the ZEA- al., 1995; Jimenez and Mateo, 1997). contaminated sturgeon diet, ZEA Iranian Journal of Fisheries Sciences 15(1) 2016 417

contamination has been previously stocked in separate concrete ponds reported in soils, drainage water, rivers containing groundwater freshwater. The and lakes of the United States and fish were cultured in a flow-through several other countries, ranged up to system with a flow rate of 50 L/min. 220 ng/L (Maragos, 2012). In our Dissolved oxygen was maintained previous study, we investigated the above 8 mg/L using constant aeration hepatic biotransformation of ZEA by and fish were exposed to a natural subcellular fractions of rainbow trout photoperiod of approximately 12:12 and demonstrated that the hepatic L:D. The water temperature was 13.5 ± metabolism of ZEA resulted in 1.0˚C, and the pH was maintained detoxification and lessening the between 7.3-7.5. The fish were fed on estrogenic potency of ZEA by commercially standard and mycotoxin converting it to weaker estrogenic free formulated sturgeon diet (Esfahan metabolite of -zearalenol (Malekinejad Mokammel CO., Iran) for a period of et al., 2012). As there is currently a lack three months. of knowledge about the hepatic biotransformation of ZEA in sturgeons, Fish tissue collection and preparation hence in this study the hepatic of the liver subcellular fractions biotransformation of ZEA including The fish were kept in accordance with both phases I and II reactions by sub- the guidelines of the Local Ethical cellular fractions of H. huso and A. Committee roles applying to principles persicus were investigated. of Laboratory Animal Care, NIH publication No. 86-23, revised in 1985 Materials and methods (NIH, 1985). For tissue collection, eight Reagents randomly collected male individuals

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 The test compounds ZEA, α-zearalenol (average wt. 420 ± 20 g) were (α-ZOL), β-zearalenol (β-ZOL), uridine anesthetized by immersion in eugenol diphosphate glucuronic acid (UDPGA), solution (20 mg/L) and immediately and dinucleotide dissected to remove the liver. The phosphate (NADPH) were purchased collected liver samples were rinsed from Sigma Chemical Co. (St. Louis, three times with chilled normal saline to MO, USA). All other chemicals were of get rid of the extra blood. reagent grade. The sub-cellular fractions of the liver were prepared as described previously Animals (Malekinejad et al., 2005). Briefly, Sixty sturgeons fish (average wt. 200 ± immediately after anesthesia, the liver 5 g) including Huso huso (n = 30) and specimens were collected and cut into A. persicus (n= 30) were obtained from small pieces. Two volumes of KCl the Artemia and Aquatic Animals (1.15%) and EDTA (0.1mM) solution Research Institute (Urmia, Iran) and were added to the tissue samples and 418 Malekinejad and Agh, Interspecies variation in the hepatic biotransformation of Zearalenon…

the mixtures were homogenized in a water bath (Memmert, Germany) at 20 Potter-Elvehjem apparatus with a °C for 30 min. The reaction was Teflon pestle (Krackeler Scientific Inc. stopped by transferring the samples to Albany, NY, USA). The homogenates an ice-cold environment followed by were centrifuged (Beckman Coulter extraction with chloroform (1.25 mL). Inc. USA) at 9,000 g for 30 min at 4 °C One milliliter of the organic phase was and an aliquot of the supernatant was collected and evaporated to dryness

collected as the post-mitochondrial under a gentle stream of N2. The fraction. The remaining sample was residue was re-dissolved in the mobile centrifuged at 100, 000 g for 90 min at phase and was subsequently analyzed 4 °C in order to obtain the microsomal by high performance liquid fraction. After centrifugation, the chromatography (Malekinejad et al., supernatant was discarded and the 2005). pellet was carefully dissolved in the same volume of phosphate buffer (0.05 Determination of the extent of M; pH 7.4) with glycerol (20%), and glucuronidation then was homogenized with an ultra- To evaluate the rate of glucuronidation, turrax homogenizer (T 25, IKA, the post-mitochondrial fraction was Germany). The individual fractions dialyzed overnight using cellulose extracted from the fish samples were membranes (width 25 mm, diameter 16 pooled and stored at –70°C until use. mm; retaining proteins with a MW>12 Protein concentrations of the KDa and hence all enzymes) with a microsomal and the post-mitochondrial dialysis buffer containing 1.15% (w/v) fractions were determined according to KCl and 0.1 mM EDTA, which was Lowry method (Lowry et al., 1951). refreshed twice. Subsequently,

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 increasing concentrations of ZEA were Incubation of ZEA with hepatic sub- added to the dialyzed fraction (2.008 cellular fractions (Phase I reactions) mg protein) dissolved in a phosphate To determine the biotransformation buffer (50 mM, pH 7.4) and metabolites, various concentrations of supplemented with 0.5 mM NADPH ZEA (10, 25, 50, 100, 250, 500 M, (pre-dissolved in phosphate buffer) in dissolved in methanol) were added to the absence or presence of 10 mM the reaction mixtures, containing either UDPGA, and incubated in shaking pooled liver microsomes (0.505 mg water bath for 30 min at 20°C. The protein) or the post-mitochondrial reaction was stopped by placing the fraction of the liver (2.008 mg protein) samples on ice and adding 1.25 ml ice- and supplemented with NADPH (0.5 cold chloroform. After vigorous mixing mM) in a final volume of 250 μl and centrifugation at 3000 g for 5 phosphate buffer (50 mM, pH 7.4). The minutes at 4°C, one ml of the organic samples were incubated in a shaking phase was collected and evaporated to Iranian Journal of Fisheries Sciences 15(1) 2016 419

dryness under a stream of nitrogen. The concentration range from zero to 100 residue was subsequently analyzed by M (r2= 0.9992 for ZEA, r2= 0.9975 for HPLC. The rate of glucuronidation (%) -ZOL and r2=0.9985 for -ZOL). was calculated using the following Statistical analysis equation: All results are presented as means ± % of glucuronidation = 100 – (100 × SD. For kinetic studies the Michaelis-

the amount of produced metabolites in Menton constant (Km) and the

the presence of UDPGA / the amount of maximum velocity (Vmax) were produced metabolite in the absence of obtained. Differences between the UDPGA) amounts of the individual products were analyzed with a two-way ANOVA Determination and confirmation of followed by a Bonferroni test, using ZEA metabolites using HPLC method Graph Pad Prism 4.00, Graph Pad ZEA and its metabolites were Software. p<0.05 was considered determined using high-performance significant. liquid chromatography (HPLC), according to previously described Results method (Malekinejad et al., 2005). ZEA was bio-inactivated by both Shortly, the chromatographic system sturgeons’ hepatic biotransformation consisted of an auto sampler Incubation of ZEA with the hepatic (Autosampler Triathlon type 900, subcellular fractions of sturgeons, Germany) and dual pumps (Wellchrom followed by chloroform extraction and HPLC pump, K-1001, KNAUER HPLC analyses, revealed that although Germany). Twenty microlitres of the both known metabolites of ZEA extracted sample was injected into a including - and -ZOL are produced Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 LUNA 5μ C18 (150×4.60 mm, by either studied fractions, however - Phenomenex) column. The mobile ZOL found to be the dominant phase consisted of a mixture of metabolite. Fig. 1 shows both standard methanol- water (70:30, v/v) eluted at a and sample chromatograms with flow-rate of 0.8 mL/min. ZEA and its identical retention time for individual metabolites were detected by means of compounds. The -ZOL production by a fluorescence detector (RF-10AXL Huso huso hepatic microsomes was KNAUER, Germany), set at an found 3 fold higher than that of A. excitation and emission wavelength of persicus. We did not find any 236 and 418 nm, respectively. ZEA and significant (p>0.05) differences its metabolites were quantified by between the level of produced measuring peak areas and comparing metabolites by two post-mitochondrial them to the relevant calibration curves. fractions of the two different species of To obtain calibration curve for each studied sturgeons. single compound, we subjected a 420 Malekinejad and Agh, Interspecies variation in the hepatic biotransformation of Zearalenon…

Comparison of the biotransformation sturgeons was determined. The first capacity of the studied fractions showed finding of the conjugation study that the microsomal fraction produced revealed that there is a clear difference significantly (p<0.05) more quantities between two sturgeons in terms of of both metabolites than those produced glucuronidation profile as H. huso by post-mitochondrial fraction in both hepatic post-mitochondrial fraction examined fishes (Fig. 2). mainly glucurinated ZEA while in A. Enzymatic kinetics studies revealed persicus, the metabolites and in that the maximum velocity (Vmax) in particular -ZOL were glucuronidated. microsomal and post-mitochondrial We found that Huso huso conjugated - fractions of H. huso for -ZOL ZOL significantly (p<0.05) more than production was found approximately 5- -ZOL, while A. persicus post- and 7-fold higher than that for -ZOL mitochondrial sub-fraction mainly production, respectively. The Km glucuronidated -ZOL. Our results value for the -ZOL production by showed a reverse level of hepatic microsomal fraction of H. huso glucuronidation as the high was 1.7-fold higher than that for the - concentration of ZEA, the low ZOL production, while it was found in percentage of glucuronidation, contrary in the post-mitochondrial indicating a saturable condition of fraction as the Km value for -ZOL glucuronidase, which is responsible for production was approximately 3-fold glucuronidation of ZEA and its reduced higher than that for the-ZOL metabolites (Table 2). production (Table 1). At the same time,

the Vmax values for -ZOL production Discussion by the hepatic microsomal and post- The hepatic biotransformation of ZEA Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 mitochondrial subfractions of A. in two types of sturgeon was persicus was estimated 8- and 12-folds investigated. The results showed that, higher than that for -ZOL production. although there are some similarities in The highest Km value was found for - the production of -ZOL as the ZOL production by the post- dominant metabolite by hepatic sub- mitochondrial fraction in A. persicus. fractions of both either sturgeons, but there are also substantial differences in Glucuronidation of ZEA and its the glucuronidation of ZEA and its hydroxylated metabolites metabolites by post-mitochondrial The rate of glucuronidation of ZEA and fractions. its hydroxylated metabolites by the post-mitochondrial fraction of Iranian Journal of Fisheries Sciences 15(1) 2016 421

Table 1: Kinetics of ZEA biotransformation by the sturgeon’s hepatic subcellular fractions. Metabolite Microsomes Post -mitochondrial

Huso huso (α-ZOL) Vmax (pMol/min/mg) 19.5 ± 2.5 2.9 ± 0.09* Km (µMol/L) 91.2 ± 34.6 16.9 ± 2.5* r2 0.68 0.88

β-ZOL Vmax (pMol/min/mg) 98.8 ± 6.5 20.8 ± 1.04* Km (µMol/L) 52.8 ± 11.9 55.25 ± 9.4 r2 0.85 0.91

A. persicus (α-ZOL) Vmax (pMol/min/mg) 3.2 ± 0.08 1.7 ± 0.06* Km (µMol/L) 13.01 ± 1.7 28.5 ± 4.4* r2 0.90 0.89

β-ZOL Vmax (pMol/min/mg) 25.4 ± 1.2 21.3 ± 1.02* Km (µMol/L) 17.3 ± 3.8 72.3 ± 10.9 r2 0.76 0.95

Table 2 : Percentage of glucuronidation of ZEA and its metabolites by the hepatic post- itochondrial fraction of rainbow trout. ZEA (µM) ZEA α-ZOL β - ZOL

Huso huso 10 71.7±9.1 33.1± 2.8 16.3 ± 1.2 25 64.9±8.0 29.0 ± 0.5 9.6 ± 1.8 50 45.7 ± 13.9 27.5 ± 1.3 6.4 ± 0.3 125 40.2 ± 2.7 26.1 ± 2.2 5.5 ± 3.3 250 29.5 ± 12.4 11.9 ± 1.1 4.4 ± 0.6 500 19.7± 4.6 4.7 ± 2.3 3.2 ± 1.1 Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 A. persicus 10 23.6 ± 1.1 100 100 25 24.0 ± 2.3 100 100 50 16.7 ± 2.9 31.2 ± 0.7 100 125 13.2 ± 1.3 28.7 ± 0.4 44.0 ± 0.3 250 7.3 ± 0.5 23.3 ± 3.1 24.5 ± 0.5 500 5.7 ± 0.6 19.2 ± 0.6 12.3 ± 0.9

Very first finding of the current study numerous studies indicating that -ZOL was that the hepatic biotransformation in comparison to -ZOL and the of ZEA resulted in detoxification as - original compound (ZEA) is a weaker ZOL with rather weaker estrogenic mycoestrogen (Le Guevel et al., 2001; activity was found as the main Malekinejad et al., 2005; Filannino et metabolite in both sturgeons. There are al., 2011). 422 Malekinejad and Agh, Interspecies variation in the hepatic biotransformation of Zearalenon…

Figure 1: Standard (A) and sample chromate grams (B) with identical retention time for individual compounds; 1: Zearalenone, 2: -Zearalenol and 3: -Zearalenol.

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 Cosnefroy and co-workers recently the ZEA biotransformation in demonstrated the same order of sturgeons, however our previous studies estrogenic potency for ZEA and its in mammalians showed considerable reduced metabolites by using a interspecies differences between zebrafish specific in vitro stable various ruminants including cattle and reporter gene assay (Cosnefroy et al., sheep (Malekinejad et al., 2006). 2012). Comparing two examined Enzymatic analyses of this study

sturgeons indicates that there are clear clarified that Vmax for production of differences between the amounts of both metabolites in microsomal fraction produced metabolites by the hepatic was higher than that in post- sub-fractions of two sturgeon species, mitochondrial fraction in both tested suggesting interspecies variety in the sturgeons, indicating dense localization ZEA hepatic biotransformation. of enzymes in microsomal fraction. Although there is no direct report about

Iranian Journal of Fisheries Sciences 15(1) 2016 423

A B

125 -ZOL -ZOL 25 a-ZOL b-ZOL 100 20

75 15

50 10

25

5

Metabolites [pM/min/mg] Metabolites [pm/min/mg] 0 0 0 100 200 300 400 500 0 100 200 300 400 500 ZEA M ZEA M

C D

-ZOL -ZOL 30 25 -ZOL -ZOL 25 20

20 15 15

10 10

5

5

Metabolites [pM/min/mg] Metabolites[pM/min/mg]

0 0 0 100 200 300 400 500 0 100 200 300 400 500 ZEA M ZEA M

Figure 2: Biotransformation of ZEA by A) huso huso hepatic microsomes, B) huso huso hepatic post-mitochondrial fraction, C) A. persicus hepatic microsomes and D) A. persicus post-mitochondrial fraction.

This finding is in a good accordance expression and localization of both with previous works (Malekinejad et isoenzymes albeit with differences in

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 al., 2005; Malekinejad et al., 2006; the liver of examined sturgeons. malekinejad et al., 2012). Our data We also investigated the suggest a higher efficiency (Vmax/Km glucuronidation rate of ZEA and its ratio) of the microsomal enzymes than reduced metabolites by the hepatic post-mitochondrial enzymes in post-mitochondrial fraction of examined sturgeon’s liver and this sturgeons. It is known that the rate of finding is not consistent with our glucuronidation mainly depends on the previous finding in the rainbow trout capacity of UDPGTs and availability of (Malekinejad et al., 2012) suggesting UDPGA (Belanger et al., 1990; Tukey various affinity of enzymes toward the and Strassburg, 2000). The estimated ZEA between different species of fish. percentage of the conjugation rate for Additionally, under the assumption that all three compounds indicates that there 3-HSD converts ZEA into -ZOL, the are profound differences between two obtained enzyme kinetics indicate a examined sturgeons in terms of higher efficiency of 3-HSD than 3- glucuronidation profile of ZEA and its HSD in examined sturgeon’s liver. Our metabolites. The main finding in this data also indirectly confirm the regard is indicating that the H. huso 424 Malekinejad and Agh, Interspecies variation in the hepatic biotransformation of Zearalenon…

hepatic post-mitochondrial fraction receptor subtypes by chemicals by mainly glucuronidated ZEA, while using stable reporter gene assay ZEA reduced metabolites were largely developed in a zebrafish liver cell glucuronidated by A. persicus, line. Toxicological Sciences, 125, indicating another difference between 439-449. Decasto, M., Rolando, P., two sturgeons in the biotransformation Nachtmann, C., Ceppa, L. and of ZEA. There is molecular evidence Nebbia, C., 1995. Zearalenone indicating the presence of multiple mycotoxicosis in piglets suckling UGT genes in zebra fish, suggesting the sows fed contaminated grain. fact that the different glucuronidation Veterinary and Human Toxicology, profile of ZEA and its metabolites in 37, 359-361. our study may be related to the Filannino, A., AE-Stout, T., Gadella, presence of distinct UDP- B.M., Sostaric, E., Pizzi, F., glucuronosyltransferases in two Colenbrander, B., Dell’Aquila, examined sturgeons (George and M.E. and Minervini, F., 2011. Dose-response effects of estrogenic Taylor, 2002). Nevertheless, clarifying mycotoxins (zearalenone, alpha- and the presence and function of UGT beta-zearalenol) on motility, enzyme(s) in sturgeons is required. hyperactivation and the acrosome The data described in the present reaction of stallion sperm. study is the first report showing that Reproductive Biology and ZEA is converted mainly into ZOL Endocrinology, 9,134. which could account for detoxification George, S.G. and Taylor, B., 2002. method. Moreover, different Molecular evidence for multiple glucuronidation profile of ZEA and its UDP-glucuronosyl transferase gene metabolites in two examined sturgeons families in fish. Marine confirmed the interspecies variety of Environmental Research, 54, 253- the ZEA hepatic biotransformation. 257. Hestbjerg, H., Nielsen, K.F., Thrane,

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 Acknowledgement U. and Elmholt, S., 2002. This study was financially supported by Production of trichothecenes and the Artemia and Aquatic Animals secondary metabolites by Fusarium Research Institute under the grant culmorum and Fusarium equiseti on number of 004/A/86. common laboratory media and a soil organic matter agar: an ecological References interpretation. Journal of Agriculture Bélanger,A., Couture, J., Caron, S. and Food Chemistry, 50, 7593-7599. and Roy, R., 1990. Determination of Jimenez, M. and Mateo, R., 1997. non-conjugated and conjugated Determination of mycotoxins levels in plasma and prostrate produced by Fusarium isolates from after separation on C-18 columns. banana fruits by capillary gas Annals of New York Academy of chromatography and high- Sciences, 595, 251–259. performance liquid chromatography. Cosnefroy, A., Brion, F., Maillot- Journal of Chromatography A, 778, Mare´chal, E., Porcher, J.M., 363-372. Pakdel, F., Balaguer, P. and Aı¨t- Johns, S.M., Denslow, N.D., Kane, Aı¨ssa, S., 2012. Selective M.D., Watanabe, K.H., Orlando, Activation of zebrafish estrogen Iranian Journal of Fisheries Sciences 15(1) 2016 425

E.F. and Sepulveda, M.S., 2009. central Illinois, USA. Food Additives Effects of and and Contaminants: Part B antiestrogens on gene expression of Surveillance, 5, 55-64. fathead minnow (Pimephales Nizza, A. and Piccolo, G., 2009. promelas) early life stages. Chemical-nutritional characteristics Environmental Toxicology, 26, 195- of diets in aquaculture. Veterinary 206. Research Communications, 33, 25- Knudsen, F.R. and Pottinger, T.G., 30. 1999. Interaction of endocrine Pietsch, C., Kersten, S., Burkhardt- disrupting chemicals, singly and in Holm, P., Valenta, H. and combination with estrogen-, Dänicke, S., 2013. Occurrence of androgen-, and corticosteroid- deoxynivalenol and zearalenone in binding sites in rainbow trout commercial fish feed: An initial (Oncorhynchus mykiss). Aquatic study. Toxins, 5, 184-192. Toxicology, 44, 159–170. Tukey, R.H. and Strassburg, C.P., Le Guevel, R. and Pakdel, F. 2001. 2000. Human UDP- Assessment of oestrogenic potency glucuronosyltransferases: of chemicals used as growth metabolism, expression, and disease. promoter by in-vitro methods. Annual Review of Pharmacology and Human Reproduction, 16, 1030- Toxicology, 40, 581-616. 1036. Vrabcheva, T., Gessler, R., Usleber, Lowry, OH., Rosebrough, NJ., Farr, E., and Martlbauer, E., 1996. First AL., and Randall, RJ., 1951. Protein survey on the natural occurrence of measurement with the folin phenol Fusarium Mycotoxins in Bulgarian reagent. Journal of Biology and wheat. Mycopathologia, 136, 147- Chemistry, 193, 265-275. 152. Malekinejad, H., Maas-Bakker, R.F. Yamashita, A., Yoshizawa, T., Aiura, and Fink-Gremmels, J., 2005. Y., Sanchez, PC., Dizon, E.I. and Bioactivation of zearalenone by Arim, R.H.,1995. Fusarium porcine hepatic biotransformation. mycotoxins (fumonisins,

Downloaded from jifro.ir at 22:04 +0330 on Friday February 23rd 2018 Veterinary Research, 36, 799-810. nivalenol,and zearalenone) and Malekinejad, H., Maas-Bakker, R.F. aflatoxins in corn from Southeast and Fink-Gremmels, J., 2006. Asia. Bioscience, Biotechnology and Species differences in the hepatic Biochemistry, 59, 1804-1807. biotransformation of Zearalenone. Yang, H.H., Aulerich, R.J., Helferich, The Veterinary Journal, 172, 96- W., Yamini, B., Miller, E.R. and 102. Bursian, S.J., 1995. Effect of Malekinejad, H., Agh, zearalenone and/or on N.,Vahabzadeh, Z.,Varasteh, S. swine and mink reproduction. and Alavi, M.H., 2012. In vitro Journal of Applied Toxicology, 15, reduction of zearalenone to β- 223-232. zearalenol by rainbow trout (Oncorhynchus mykiss) hepatic microsomal and post-mitochondrial subfractions. Iranian Journal of Veterinary Research, 3, 28-35. Maragos, C.M., 2012. Zearalenone occurrence in surface waters in