Metabolism, Distribution, and Elimination of Mequindox in Pigs

The disposition and elimination of MEQ in rats, pigs, and chickens was comprehensively investigated to identify the marker residue and target tissue o...
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Metabolism, Distribution, and Elimination of Mequindox in Pigs, Chickens, and Rats Lingli Huang,† Fujun Yin,‡ Yuanhu Pan,† Dongmei Chen,‡ Juan Li,‡ Dan Wan,‡ Zhenli Liu,‡ and Zonghui Yuan*,†,‡ †

MOA Laboratory of Risk Assessment for Quality and Safety of Livestock and Poultry Products and ‡National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei 430070, China ABSTRACT: Mequindox (MEQ), a quinoxaline-N,N-dioxide antibacterial agent used to control bacterial enteritis in various food-producing animals, is a potential violative residue in food animal-derived products. The disposition and elimination of MEQ in rats, pigs, and chickens was comprehensively investigated to identify the marker residue and target tissue of MEQ in food animals for residue monitoring. Following a single oral administration, 62−71% of MEQ was rapidly excreted via urine and feces in all species within 24 h. Urinary excretion of radioactivity was 84 and 83.5% of the administered dose in rats and pigs, respectively. More than 92% of the administered dose was excreted in all species within 15 days. Radioactivity was found in nearly all tissues at the first 6 h after dosing, with the majority of radioactivity cleared within 4−6 days. The highest radioactivity and longest persisting time were found to be in the liver and kidney. Totals of 11, 12, and 7 metabolites were identified in rats, chickens, and pigs, respectively. No parent drug could be detected in any of the tissues of pigs and chickens. 3-Methyl-2-acetyl quinoxaline (M1), 3-methyl-2-(1-hydroxyethyl) quinoxaline-N4-monoxide (M4), and 3-methyl-2-(1-hydroxyethyl) quinoxaline1,4-dioxide (M6) were the common and major metabolites of MEQ in all three species. Additionally, 3-methyl-2-(1hydroxyethyl) quinoxaline (M5), 3-hydroxymethyl-2-ethanol quinoxaline-1,4-dioxide (M7), and 3-methyl-2-(1-hydroxyethyl) quinoxaline-N1-monoxide (M8) were the major metabolites of MEQ in rats, pigs, and chickens, respectively. M1 was designated to be the marker residue of MEQ in pigs and chickens. These results provide scientific data for the determination of marker residues and withdrawal time of MEQ in food animals and improve the understanding of the toxicity and disposition of MEQ in animals. KEYWORDS: [3H]-mequindox, isotopic tracing, excretion, metabolism, tissue depletion, marker residue, target tissue

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INTRODUCTION Mequindox (MEQ; Figure 1), 3-methyl-2-acetyl-N-1,4-dioxyquinoxaline or C11H10N2O3, is a synthetic quinoxaline antibacterial

indicate that the application of MEQ in food animals could cause adverse health effects on humans through the food chain if MEQ levels in food animal-derived products exceed a safe level. Hence, food safety monitoring of MEQ in animals is essential for the health of the consumers. However, little is known about the food safety of MEQ, and no marker residue and target tissues have been identified up to now, resulting in the withdrawal time (WDT) of MEQ in food animals remaining unavailable. Drug metabolism and disposition determine the safety of animal-derived foods by affecting the composition and content of the drug residues in edible tissues. Previous studies showed that quinoxalines, such as CBX and OLA, could be transformed into several active metabolites that are toxicologically more relevant than their parent compounds.13−15 MEQ was structurally similar to CBX and OLA and could be metabolized via a similar pathway. Moreover, a recent in vitro study reported that >80% of MEQ could be metabolized when it was incubated with liver microsomes of pigs, chickens, or rats, and about 10 metabolites could be detected, suggesting MEQ is metabolized extensively in animals.16 A recent paper reported the metabolism of MEQ in chickens,17 but this study focused mainly on the identification

Figure 1. Chemical structure of mequindox.

agent and has been used in pigs and chickens in China since the 1980s. MEQ is an effective therapeutic drug for piglet diarrhea because of its strong inhibitory activities against both Grampositive and Gram-negative bacteria, especially Treponema hyodysenteriae.1−3 However, accidental poisonings have been frequently reported in pigs and chickens in recent years.4−6 Because carbadox (CBX) and olaquindox (OLA), well-known members of the quinoxaline family, had been banned or limited to use in food animals in many countries due to their toxicity, the food safety of MEQ is of great concern. Recent studies have demonstrated that MEQ can disrupt the endogenous metabolism of mice and rats and cause acute and reproductive toxicity, adrenal toxicity, and genotoxicity in Wistar rats.7−−12 These data © XXXX American Chemical Society

Received: June 4, 2015 Revised: September 10, 2015 Accepted: September 16, 2015

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DOI: 10.1021/acs.jafc.5b02780 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Recovery of Total Radioactivity in the Excreta of Rats, Pigs and Chicken after a Single Oral Administration of [3H]-MEQ at 10 mg/kg bw rats time (days) 0.5 1 3 6 10 15 0−15 total

urine (%) 31.7 28.1 12.1 10.0 2.1

± ± ± ± ±

pigs feces (%)

2.1 3.0 1.7 1.3 1.1

2.9 3.2 3.0 0.8

84.0 ± 5.4

± ± ± ±

urine (%) 29.2 30.7 9.0 7.3 6.2 1.3 83.5

0.5 0.4 0.4 0.3

9.8 ± 2.0 93.8 ± 4.3

1.2 1.2 0.8 0.6 0.8 0.4 1.8

2.9 2.3 1.3 1.0 0.9 8.4 91.9 ± 1.9

of the metabolites, and no quantitative analysis of the metabolites was conducted, resulting in the main metabolites and marker residue remaining unknown. Moreover, no in vivo metabolism data were reported in pigs and rats. Several papers have reported the pharmacokinetics of MEQ and its several metabolites in pigs, chickens, and rats.18−20 In these studies, however, only the plasma concentrations of MEQ and several proposed metabolites were measured; tissue data were not available, which are closely associated with the assignment of marker residue, target tissues, and WDT for food safety monitoring. Thus, the overall metabolism profile, distribution, and elimination of MEQ in both laboratory animals and food animals remain to be investigated, which is essential to further toxicology studies and food safety assessment. Isotopic tracing is a scientific and effective technique to reveal the disposition of a drug in animals, which could locate and quantify the existence of the drug-related substances at any time by detection of the radioactive isotope.21,22 The present study aimed to investigate the metabolism, distribution, and elimination of MEQ in rats, chickens, and pigs using radioisotope tracing coupled with high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). The complete in vivo metabolic profile and quantification analysis of MEQ in body fluids and different organs and tissues of rats, pigs, and chickens were investigated and compared for the first time. The possible toxic metabolites and marker residues of MEQ in animals are proposed. These results would contribute to the determination of marker residue and WDT of MEQ in food animals and improve our understanding of the disposition and toxicity of MEQ in animals.

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± ± ± ± ± ± ±

chickens feces (%) ± ± ± ± ± ±

0.3 0.3 0.3 0.1 0.1 0.7

excreta (%) 39.9 31.3 11.1 6.6 5.9

± ± ± ± ±

1.4 2.4 1.0 0.7 1.1

94.7 ± 3.0 94.7 ± 3.0

Table 2. Structures and Spectra Information of Metabolites of MEQ in Animals

MATERIALS AND METHODS

Chemicals. MEQ (≥98%) was purchased from China Institute of Veterinary Drug Control (Beijing, China). 4-[3H]-O-nitroaniline ([3H]-ONA) was provided by the Shanghai Institute of Applied Physics, Chinese Academy of Sciences. ONA and 10% Pd/C were purchased from Sigma-Aldrich (Shanghai, China). Benzofuroxan (BFO) (≥99.5%) was provided by the Institute of Veterinary Pharmaceuticals (Wuhan, China). Solvable (tissue solubilizing fluid), Ultima Gold and Monophase S were purchased from PerkinElmer Life and Analytical Sciences (Groningen, The Netherlands; Waltham, MA, USA, respectively). Stop Flow AD scintillation liquid was obtained from AIM Research Co. (Hockessin, DE, USA). 3-Methyl-2-acetyl quinoxaline (M1) (≥98.5%), 3-methyl-2-(1-hydroxyethyl) quinoxalineN4-monoxide (M4) (≥99.0%), 3-methyl-2-(1-hydroxyethyl) quinoxaline (M5) (≥99.0%), and 3-methyl-2-(1-hydroxyethyl) quinoxaline-1,4dioxide (M6) (≥99.0%) were synthesized by the Institute of Veterinary Pharmaceuticals (Wuhan, China). [3H]-MEQ was synthesized as previously described.23 In brief, the process involved two steps of chemical reactions. The first step was to prepare [3H]-BFO, which was through deoxidizing [3H]-ONA with

isopropanol and NaClO at 25 °C with constant stirring for 3 h. The second step was the preparation of [3H]-MEQ, which was obtained via reaction of [3H]-BFO with acetylacetone at room temperature for 15 h. The purity of the product was >98%. The radiochemical purity of B

DOI: 10.1021/acs.jafc.5b02780 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 3. Concentrations of MEQ and Its Metabolites in the Tissues of Rats after a Single Administration of [3H]-MEQ at 10 mg/kg bw (n = 4, Mean ± SD) concentrations of the metabolitesa (μg/kg) withdrawal time (days)

M0

liver

0.25 1 2 4

185 ± 63 (13.9)b 152 ± 42 (13.7) ND ND

452 409 394 252

± ± ± ±

71 39 28 23

(34.0) (36.8) (48.5) (70.9)

kidney

0.25 1 2 4

48 ± 17 (5.6) 37 ± 9 (11.1) ND ND

219 139 132 106

± ± ± ±

62 32 14 14

(25.6) (41.8) (63.7) (66.9)

ND ND ND ND

79 ± 27 (9.0) 17 ± 7 (5.2) 12 ± 3 (5.7) ND

muscle

0.25 1 2 4

ND ND ND ND

149 ± 40 (30.8) 59 ± 15 (69.7) 24 ± 6 (77.2) ND

ND ND ND ND

90 ± 20 (18.7) 8 ± 2 (9.8) 6.8 ± 1.7 (21.4) ND

tissue

M1

M2

M4

28 ± 5 (2.1) NDc ND ND

145 133 127 11

± ± ± ±

53 (10.9) 15 (12.0) 12 (15.6) 3 (3.0)

M5 194 177 131 47

± ± ± ±

12 26 23 11

99 43 35 25

± ± ± ±

M6

M10

56 ± 7 (4.2) ND ND ND

89 ± 9 (6.7) 78 ± 16 (7.0) ND ND

28 (11.6) 11 (12.9) 6 (17.1) 7 (15.4)

93 ± 22 (10.9) ND ND ND

68 ± 26 (7.9) 45 ± 13 (13.4) ND ND

76 ± 15 (15.7) ND ND ND

66 ± 14 (13.6) ND ND ND

ND ND ND ND

(14.6) (15.8) (16.1) (13.1)

a M0, mequindox; M1, 3-methyl-2-acetyl quinoxaline; M2, 3-methyl-2-acetyl quinoxaline-N4-monoxide; M4, 3-methyl-2-isopropyl alcohol quinoxaline-N4-monoxide; M5, 3-methyl-2-isopropyl alcohol quinoxaline; M6, 3-methyl-2-isopropyl alcohol quinoxaline-1,4-dioxide; M10, 3-methyl2-acetyl(1-hydroxyl) quinoxaline. bPercentage of individual metabolites compared with the total concentration of MEQ-related. cND, below the LOD (10 μg/kg).

[3H]-MEQ was determined to be >99%. The specific activity of [3H]-MEQ was 16.9 mCi/mg. The radiochemical purity of [3H]-MEQ remained unchanged for 7 months. A stock solution of 100 mg/mL with a specific activity of 169 mCi/g of [3H]-MEQ was prepared 2 weeks before dosing and diluted with 1% aqueous carboxymethyl cellulose to obtain a final dosing suspension of 10 mg/mL with a specific activity of 16.9 mCi/g for animal study. The [3H]-H2O exchange was determined by comparing the radioactivity of the incurred samples before and after freeze-drying. The [3H]-H2O exchange in different tissues was 10% in tissues of rats, pigs, and chickens, respectively, suggesting that M5, M7, and M8 are the main metabolites of MEQ in rats, pigs, and chickens, respectively. The designation of target tissues and marker residue is vital for residue control to guarantee food safety. Given that the target tissue is defined as the tissue in which the residues persist the longest time with the longest elimination half-life, the liver is proposed to be the target tissue for MEQ in animals. The marker residue, defined as the compound that persists the longest in the target tissue, showed similar depletion characteristics with the total residues. The present study demonstrated that bisdeoxymequindox (M1) persisted the longest and showed exactly the same depletion

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AUTHOR INFORMATION

Corresponding Author

*(Z.Y.) Mail: Department of Basic Veterinary College of Animal Science, Huazhong Agricultural University, Shizishan Street, Wuhan, Hubei 430070, China. Phone: 0278728 7186. Fax: 0278767 2232. E-mail: [email protected]. Funding

This work was financially supported by the Special Fund for Agroscientific Research in the Public Interest (No. 201203040), the National Natural Science Foundation of China (Grant 31302143), the National Basic Research Program of China (973 program (No. 2009CB118800), and the Fundamental Research Funds for the Central Universities (2012ZYTS052). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Zhoumeng Lin of the Institute of Computational Comparative Medicine at Kansas State University for critically reviewing the manuscript.

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ABBREVIATIONS USED MEQ, mequindox; CBX, carbadox; OLA, olaquindox; LCMSIT-TOF, high-performance liquid chromatography−mass spectrometry−ion trap−time-of-flight; ONA, onitroaniline; BFO, benzofuroxan; [3H]ONA, 4-[3H]-O-nitroaniline; LSC, liquid scintillation counting

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DOI: 10.1021/acs.jafc.5b02780 J. Agric. Food Chem. XXXX, XXX, XXX−XXX