Article pubs.acs.org/JAFC
Metabolic Disposition and Elimination of Cyadox in Pigs, Chickens, Carp, and Rats Huang Lingli,†,§ Xu Ning,‡,§ Sechenchogt Harnud,†,§ Pan Yuanhu,†,§ Chen Dongmei,‡,§ Tao Yanfei,‡,§ Liu Zhenli,‡,§ and Yuan Zonghui*,†,‡,§ †
MOA Laboratory of Risk Assessment for Quality and Safety of Livestock and Poultry Products, ‡National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, and §Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China ABSTRACT: The metabolism, distribution, and elimination of cyadox (CYA) is investigated in pigs, chickens, carp, and rats to identify the marker residue and target tissue of CYA in food animals for food safety concerns. Following a single oral gavage of [3H]-CYA, the total radioactivity was rapidly excreted, with more than 95% of the dose excreted within 14 days in the four species. Fecal excretion of the total radioactivity was 66.2% and 51.6%, and urinary excretion of the total radioactivity was 28.35% and 44.3% in rats and pigs, respectively. Radioactivity was observed in nearly all of the tissues in the first 6 h after 7 days of consecutive oral dosing. The highest radioactivity and longest persistence were in the livers and kidneys, where the majority of the radioactivity was cleared within 7 days. A total of 15 metabolites were identified in rats, pigs, chickens, and carp, and eight new metabolites were identified for the first time in vivo. No parent drug could be detected in the tissues of rats and pigs. The major metabolites of CYA were Cy1, Cy3, and Cy6 in pigs, Cy1, Cy5, and Cy6 in chickens, Cy1, Cy2, and Cy4 in carp, and Cy1, Cy2, Cy4, and Cy5 in rats. Cy1 was suggested to be the marker residue, and the kidneys were identified as the target tissue of CYA in pigs and chickens. These results provide comprehensive information for the food safety evaluation of CYA in food animals and will improve the understanding of the pharmacology and toxicology of CYA in animals. KEYWORDS: [3H]-cyadox, isotopic tracing, metabolism, disposition, tissue depletion, marker residue, target residue
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microflora of rats, pigs, and chickens.11−13 These studies demonstrate that CYA can be transformed into abundant metabolites when it interacts with different metabolic systems in vitro. However, these studies only focused on the identification of the metabolites of CYA in vitro. Data on the in vivo metabolism and pharmacokinetics of CYA in the literature are quite scarce. Several methods for the quantitative analysis of CYA and its several metabolites in animal tissues have been described,14−16 and recent studies described the pharmacokinetics and depletion of CYA and three of its known metabolites in pigs after oral administration.17,18 However, these studies focused on the quantitative analysis of CYA and three of its known metabolites in the plasma and edible tissues of pigs. The comprehensive in vivo metabolic profile, distribution, and depletion of CYA in both laboratory and food animals were not completely investigated, and the marker residue and target tissues remain unknown. Radiolabeled drugs are excellent investigative tools that are widely used to investigate the disposition of drugs in animals. Because radioactivity offers a unique mode method for quantifying all drug-related molecules, substances related to the parent drug can be identified, located, and quantified at any time by detection of the radioactivity.19,20 Radiolabeled drugs are often employed to identify and elucidate the metabolites
INTRODUCTION Cyadox (CYA, Figure 1), 2-formylquinoxaline-N1,N4-dioxide cyanoacetylhydrazone, is an antimicrobial growth promoting
Figure 1. Structure of [3H]-CYA (T = tritium).
agent of quinoxalines. It has been proven to be effective against most of the pathogenic bacteria in food-producing animals1,2 promote the average daily gain and feed conversion ratio, and prevent Escherichia coli infection in pigs and chickens.3−6 Furthermore, CYA is much safer for animals than carbadox and olaquindox, well-known members of the quinoxaline family that are banned in food animals due to their toxicity.7−10 In addition to optimal safety for animals, food safety is an equally important consideration for medications for food animals. The marker residue and target tissue are the most essential targets for controlling the food safety of veterinary drugs. The metabolism, distribution, and depletion of the drug in animals are the key pieces of information for the designation of the marker residue and target tissue. However, limited information about the disposition of CYA in animals has been reported. Several recent papers have described the metabolism of CYA in the liver microsomes, primary hepatocytes, and intestinal © XXXX American Chemical Society
Received: April 7, 2015 Revised: May 12, 2015 Accepted: May 14, 2015
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DOI: 10.1021/acs.jafc.5b01745 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
500 chickens (weight 1.0−1.5 kg) were purchased from Wuhan Chai Tai Co., Ltd. (Wuhan, People’s Republic of China). Carp (weight 0.4− 0.6 kg) were purchased from Wuhan Fish Breeding Farm (Wuhan, People’s Republic of China). The animals were fed a basal diet without antimicrobial agents/compounds and acclimatized for 1 week before the experiment. The animals and fish were maintained under standard environmental conditions using the routine methods of animal husbandry and aquaculture. Pigs and broilers were housed singly in all-steel metabolism cages specifically designed for the separate collection of urine and feces in a temperature-controlled room (20 ± 2 °C) with a 12 h light/dark cycle. Carp were held separately in a standalone water tank in the same temperature-controlled room (20 ± 2 °C). Throughout the study period, feed was withheld approximately 12 h before through 4 h after drug administration, while water was available ad libitum. The experimental procedures involving the animals in this study were approved by the Animal Care Center, Hubei Academy of Medical Sciences. All of the in vivo experiments complied with the policy on the care and use of laboratory animals. Feed. The feed used in this study was purchased from Wuhan Lvhong Bioscience and Technology Co., Ltd. (Wuhan, People’s Republic of China). The feed formula was strictly in accordance with the recommended formula of the U.S. National Research Council. Premixed (5%; 3.0 g/kg CYA concentration) and complete feed (150 mg/kg CYA concentration) were initially prepared. The blank feed and the medicated feed were analyzed using the HPLC method described by Wu et al.22 to determine the drug content. The results indicated that no quinoxaline compound was detected in the blank feed, and only CYA (150 ± 8 mg/kg feed, n = 9) was found in the medicated feed. Mass Balance Study. Six rats (three male and three female), six chickens, six carp, and four pigs were administered a single oral dose of [3H]-CYA (11.3 mCi/g) by gavage at 20 mg/kg bw (30 mg/kg bw for rats), respectively. Urine and feces samples were collected in preweighed dry ice cooled containers protected from light before dosing at intervals of 0−6, 6−12, and 12−24 h after dose administration and then daily until two consecutive samples from urine and feces had
