Interspecies Variation in the Hepatic Biotransformation of Zearalenone: Evidence for Bio-Inactivation of Mycoestrogen Zearalenone in Sturgeon Fish
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Interspecies variation in the hepatic biotransformation of zearalenone: 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 estrogen 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)--resorcylic acid lactone, 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 nicotinamide 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