Advances in Metabolism and Metabolic Toxicology of Quinoxaline 1,4-Di-N-oxides.

Quinoxaline 1,4-di-N-Oxides (QdNOs) have been used as synthetic antimicrobial agents in animal husbandry and aquaculture. The metabolism and potential toxicity have been also concerns in recently years. The metabolism investigations showed that there were 8 metabolites of Carbadox (CBX), 34 metabolites of Cyadox (CYA), 33 metabolites of Mequindox (MEQ), 35 metabolites of Olaquindox (OLA), and 56 metabolites of Quinocetone (QCT) in different animals. Among them, Cb3 and Cb8, M6, and O9 are metabolic residual markers of CBX, MEQ and OLA, which are associated with N → O reduction. Toxicity studies revealed that QdNOs exhibited severe tumorigenicity, cytotoxicity, and adrenal toxicity. Metabolic toxicology showed that toxicity of QdNOs metabolites might be related to the N → O group reduction, and some metabolites exhibited higher toxic effects than the precursor, which could provide guidance for further research on the metabolic toxicology of QdNOs and provide a wealth of information for food safety evaluation.

Determination of marker residues of quinoxaline-1,4-di-N-oxides and its prototype identification by liquid chromatography tandem mass spectrometry.

Quinoxaline-1,4-di-N-oxides (QdNOs), such as carbadox, olaquindox, mequindox, quinocetone, etc. are a class of antibacterial drugs. Prototype drugs residues can not be detected due to their rapid metabolism in animals. Quinoxaline-2-carboxylic acid (QCA) and 3-methyl-QCA (MQCA) are their common marker residues, so it has been always a challenge to trace the specific QdNOs drug used in food animal production. Herein, a liquid chromatography tandem mass spectrometry method was developed to determine QCA and MQCA, and meanwhile, the prototype drugs were identified by analyzing bis-desoxy QdNOs metabolites in single ion-pair monitoring mode. The method indicated that the average recoveries for QCA and MQCA were from 90 % to 105 % with relative standard deviations below 10 %, and the limits of quantification were 1.0 µg/kg. The limits of detection of five bis-desoxy QdNOs (qualitative markers) reached 0.5 µg/kg. This new analytical strategy can effectively solve the identification problem of QdNOs drugs in animal-derived food.

Hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion extraction of carbadox and olaquindox in feeds.

BACKGROUND: Carbadox and olaquindox have been banned from feeds since 1998 by the EU because of their mutagenic, photoallergic, and carcinogenic effects. Unfortunately, owing to their outstanding effect, they are frequently abused or misused in animal husbandry. There is an urgent need to develop a sensitive and reliable method for monitoring these drugs in animal feeds. RESULTS: This work reported a new method of hydrophilic-interaction-based magnetically assisted matrix solid-phase dispersion (MMSPD) extraction coupled with reversed-phase liquid chromatography-mass spectrometry for simultaneous determination of carbadox and olaquindox in animal feeds. 3-Trimethoxysilylpropyl methacrylate (γ-MAPS)-modified attapulgite (ATP) was crosslinked with γ-MAPS-modified iron(II,III) oxide (Fe3 O4 ), 1-vinyl-3-(butyl-4-sulfonate) imidazolium (VBSIm), acrylamide (AM), and N,N'-methylene-bis(acrylamide) (MBA) to synthesize ATP@Fe3 O4 @poly(VBSIm-AM-MBA) particles. The resultant particles were characterized by scanning electron microscopy, energy dispersive spectrometer, transmission electron microscopy, vibrating sample magnetometer, and Fourier transform infrared spectroscopy. Crosslinking of ATP into the magnetic particles has significantly increased the adsorption capacity of the particles. Under optimum conditions, the limits of detection (S/N = 3) were 0.3 µg kg-1 and 0.9 µg kg-1 for carbadox and olaquindox respectively. The intra-day and inter-day recoveries of the spiked targets in feed samples were in the range 83.5-98.3% with relative standard deviations of 1.0-8.3%. CONCLUSION: With a simplified procedure and a low amount of sample, the proposed hydrophilic-interaction-based MMSPD method is not only useful for the determination of carbadox and olaquindox in feeds but also holds great promise for the analysis of other polar targets in solid or semisolid matrices. © 2021 Society of Chemical Industry.

Swine growth promotion with antibiotics or alternatives can increase antibiotic resistance gene mobility potential.

Even though the use of antibiotics for food-producing animals may contribute to the emergence of antimicrobial resistance, antibiotics are still used as growth promoters. Due to consumer and regulatory pressures, the use of alternatives to antibiotics as growth promoters is increasing, thus more information is needed on their capability to disseminate antimicrobial resistance compared to antibiotics. We investigated the impacts of carbadox (antibiotic), copper sulfate and zinc oxide (metals) and mushroom powder (natural product) on the pig fecal resistome and microbiome. Antibiotic resistance gene (ARG) and mobile genetic element (MGE) abundances were measured using a high-throughput qPCR array with 382 primer pairs. Bacterial community composition was determined by 16S rRNA gene sequencing. More ARGs co-occurred with MGEs in the growth promoter group samples than in the control group samples. Community composition could not be linked to resistome in the growth promoter group samples, indicating a potential decoupling of ARGs and phylogeny. Additionally, machine-learning methods aided in defining the community and resistome differences in response to treatments. Since increased ARG mobility potential was the primary response to the dietary additives used in this study, we suggest that ARG mobility should be considered when designing antimicrobial use policies and antimicrobial resistance surveillances.

Bacillus subtilis: a potential growth promoter in weaned pigs in comparison to carbadox.

The study was conducted to investigate the efficacy of a probiotic Bacillus subtilis strain on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli and to compare the efficacy of B. subtilis with that of carbadox. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of four dietary treatments: negative control (NC, control diet without E. coli challenge), positive control (PC, control diet with E. coli challenge), and supplementation of 50 mg/kg of carbadox (antibiotic growth promotor [AGP]) or 2.56 × 109 CFU/kg of B. subtilis probiotics (PRO). The experiment lasted for 28 d with 7 d before and 21 d after the first E. coli inoculation. Fecal and blood samples were collected on days 0, 3, 7, 14, and 21 post inoculation (PI) to analyze ß-hemolytic coliforms and complete blood cell count, respectively. Diarrhea score was recorded daily for each pig to calculate the frequency of diarrhea. All pigs were euthanized at day 21 PI to collect jejunal and ileal mucosa for gene expression analysis. Pigs in AGP had greater (P < 0.05) BW on days 7, 14, and 21 PI than pigs in PC and PRO groups. Supplementation of PRO enhanced pigs' BW on day 21 PI compared with the PC. Escherichia coli F18 challenge reduced (P < 0.05) average daily gain (ADG) and feed efficiency from day 0 to 21 PI, while supplementation of carbadox or PRO enhanced ADG and feed efficiency in E. coli F18-challenged pigs from day 0 to 21 PI. Pigs in AGP and PRO groups had reduced (P < 0.05) frequency of diarrhea throughout the experiment and fecal ß-hemolytic coliforms on day 7 PI than pigs in the PC. Pigs in PRO had greater (P < 0.05) gene expression of CLDN1 in jejunal mucosa than pigs in the PC. Supplementation of carbadox or PRO reduced (P < 0.05) the gene expression of IL6 and PTGS2 in ileal mucosa of E. coli-infected pigs compared with pigs in the PC. Pigs in the PRO group had lower (P < 0.05) white blood cell number and neutrophil count, and serum haptoglobin concentration on day 7 PI, and less (P < 0.05) monocyte count on day 14 PI, compared with PC. In conclusion, supplementation of probiotic B. subtilis could enhance disease resistance and promote the growth performance of weaned pigs under disease challenge conditions. The potential mechanisms include but not limited to enhanced gut barrier integrity and local and systemic immune responses of weaned pigs.

Novel Electrochemical Sensor Fabricated for Individual and Simultaneous Ultrasensitive Determination of Olaquindox and Carbadox Based on MWCNT-OH/CMK-8 Hybrid Nanocomposite Film.

A hybrid nanocomposite consisting of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) and cube mesoporous carbon (CMK-8) was applied in this study to construct an MWCNT-OH/CMK-8/gold electrode (GE) electrochemical sensor and simultaneously perform the electro-reduction of olaquindox (OLA) and carbadox (CBX). The respective peak currents of CBX and OLA on the modified electrode increased by 720- and 595-fold relative to the peak current of GE. The performances of the modified electrode were investigated with electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry. Then, the modified electrodes were used for the individual and simultaneous determination of OLA and CBX. The fabricated sensor demonstrated a linear response at 0.2-500 nmol/L in optimum experimental conditions, and the detection limits were 104.1 and 62.9 pmol/L for the simultaneous determination of OLA and CBX, respectively. As for individual determination, wide linear relationships were obtained for the detected OLA with levels of 0.05-500 nmol/L with LOD of 20.7 pmol/L and the detected CBX with levels of 0.10-500 nmol/L with LOD of 50.2 pmol/L. The fabricated sensor was successfully used in the independent and simultaneous determination of OLA and CBX in spiked pork samples.

Determination of olaquindox, carbadox and cyadox in animal feeds by ultra-performance liquid chromatography tandem mass spectrometry.

Olaquindox, carbadox, and cyadox are chemically synthesised antibacterial and growth-promoting agents for animals. At high doses they may exert mutagenicity and hepatic and adrenal toxicities in animals. Regrettably, these substances are frequently abused or misused when added into animal feeds. Thus, developing a sensitive and reliable method for simultaneous determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds is crucially important for food safety monitoring. In this paper we optimised instrumental conditions, extraction solvents, solid phase extraction cartridges, and pH of the loading solvents on the Oasis HLB cartridge. Under the optimal conditions, mean recoveries ranged from 74.1 to 111%, and intra-day and inter-day variations were lower than 14.6% and 10.8%, respectively. The limits of quantification for olaquindox, carbadox, and cyadox were 0.05 mg kg-1, 0.10 mg kg-1, and 0.025 mg kg-1, respectively. The proposed method uses ultra-performance liquid chromatography tandem mass spectrometry and is sensitive and reliable for the simultaneous determination of olaquindox, carbadox, and cyadox in three kinds of animal feeds (specifically, mixed feed, concentrated feed, and additive premixed feed). This method has good precision, high sensitivity, and good reproducibility, and thus it can be used for convenient and accurate determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds.

Bacterial Whack-a-Mole: Reconsidering the Public Health Relevance of Using Carbadox in Food Animals.

Carbadox is an antibiotic used to control dysentery and promote growth in swine in the United States; however, the drug also causes tumors and birth defects in laboratory animals. Despite this and because the drug has no analogs in human medicine, it is not considered "medically important" and can be used in livestock without veterinarian oversight. In their recent study, T. A. Johnson et al. (mBio 8:e00709-17, 2017, https://doi.org/10.1128/mBio.00709-17) demonstrated that carbadox has profound effects on the swine gut microbiome, including the induction of transducing phage carrying tetracycline, aminoglycoside, and beta-lactam resistance genes. In swine production, carbadox can be used in conjunction with other antibiotics (e.g., oxytetracycline) that could fuel the emergence of strains carrying phage-encoded resistance determinants. Johnson et al.'s findings underscore the potential unforeseen consequences of using antibiotics in livestock production and call into question our current methods for classifying whether or not a veterinary drug has relevance to human health.

The In-Feed Antibiotic Carbadox Induces Phage Gene Transcription in the Swine Gut Microbiome.

Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo longitudinal effects of carbadox on swine gut microbial gene expression (fecal metatranscriptome) and phage population dynamics (fecal dsDNA viromes). Microbial metagenome, transcriptome, and virome sequences were annotated for taxonomic inference and gene function by using FIGfam (isofunctional homolog sequences) and SEED subsystems databases. When the beta diversities of microbial FIGfam annotations were compared, the control and carbadox communities were distinct 2 days after carbadox introduction. This effect was driven by carbadox-associated lower expression of FIGfams (n = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism (q < 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FIGfams related to all stages of the phage lytic cycle 2 days following the introduction of carbadox (q ≤0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. These effects were diminished by 7 days of continuous carbadox in the feed, suggesting an acute impact. Additionally, the viromes included a few genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics but these did not change in frequency over time or with treatment. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes in swine gut bacterial communities as a result of carbadox administration.IMPORTANCE FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Interestingly, the present study shows greater prophage gene expression in feces from carbadox-fed animals than in feces from nonmedicated animals 2 days after the initiation of in-feed carbadox treatment. Importantly, the phage genetic material isolated in this study contained genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered.

Aberrant cell cycle regulation in rat liver cells induced by post-initiation treatment with hepatocarcinogens/hepatocarcinogenic tumor promoters.

The present study aimed to determine the onset time of hepatocarcinogen/hepatocarcinogenic tumor promoter-specific cell proliferation, apoptosis and aberrant cell cycle regulation after post-initiation treatment. Six-week-old rats were treated with the genotoxic hepatocarcinogen, carbadox (CRB), the marginally hepatocarcinogenic leucomalachite green (LMG), the tumor promoter, ß-naphthoflavone (BNF) or the non-carcinogenic hepatotoxicant, acetaminophen, for 2, 4 or 6 weeks during the post-initiation phase using a medium-term liver bioassay. Cell proliferation activity, expression of G2 to M phase- and spindle checkpoint-related molecules, and apoptosis were immunohistochemically analyzed at week 2 and 4, and tumor promotion activity was assessed at week 6. At week 2, hepatocarcinogen/tumor promoter-specific aberrant cell cycle regulation was not observed. At week 4, BNF and LMG increased cell proliferation together with hepatotoxicity, while CRB did not. Additionally, BNF and CRB reduced the number of cells expressing phosphorylated-histone H3 in both ubiquitin D (UBD)(+) cells and Ki-67(+) proliferating cells, suggesting development of spindle checkpoint dysfunction, regardless of cell proliferation activity. At week 6, examined hepatocarcinogens/tumor promoters increased preneoplastic hepatic foci expressing glutathione S-transferase placental form. These results suggest that some hepatocarcinogens/tumor promoters increase their toxicity after post-initiation treatment, causing regenerative cell proliferation. In contrast, some genotoxic hepatocarcinogens may disrupt the spindle checkpoint without facilitating cell proliferation at the early stage of tumor promotion. This suggests that facilitation of cell proliferation and disruption of spindle checkpoint function are induced by different mechanisms during hepatocarcinogenesis. Four weeks of post-initiation treatment may be sufficient to induce hepatocarcinogen/tumor promoter-specific cellular responses.

Dual-label quantum dot-based immunoassay for simultaneous determination of Carbadox and Olaquindox metabolites in animal tissues.

A novel and reliable dual-label direct competitive fluorescence-linked immunosorbent assay (dc-FLISA) based on quantum dots (QDs) was developed for the simultaneous determination of the major metabolites of Carbadox and Olaquindox residues in animal tissues, using anti-QCA monoclonal antibodies and anti-MQCA polyclonal antibodies labeled with QD520 and QD635, respectively. The limits of detection for QCA and MQCA were 0.05 and 0.07ng/ml, respectively. The method was used to analyze fortified samples and analyte recoveries ranged from 81.5% to 98.2% (QCA) and 84.2% to 95.7% (MQCA). Good correlations between the dc-FLISA method and HPLC were demonstrated for the determination of QCA and MQCA residues in swine tissue samples, confirming the reliability of the proposed method.

Disruption of spindle checkpoint function ahead of facilitation of cell proliferation by repeated administration of hepatocarcinogens in rats.

We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and ß-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

HPLC-MS/MS method validation for the detection of carbadox and olaquindox in poultry and swine feedingstuffs.

Carbadox (CBX) and olaquindox (OLA) were used in poultry and swine feed for growth promotion, to improve feed efficiency and increase the rate of weight gain. However, the use of these agents in feedingstuffs was prohibited because of concerns about their toxicity. Regulatory laboratories are required to have suitably validated analytical methods to ensure compliance with the ban. A quantitative and confirmatory method for determining the presence of CBX and OLA in poultry and swine feed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed, optimized, and validated. The analytes extraction was performed with a mixture of water and acetonitrile (1:1v/v) and cleanup with hexane and C18 (dispersive phase). The method was evaluated by the following parameters: specificity, linearity, matrix effect, decision limits (CCα), detection capability (CCß), accuracy, precision, limits of detection (LoD), limits of quantification (LoQ) and measurement uncertainty. The validated method presented a broad linear study range and no significant matrix effect. The limit of detection (LoD) was defined at 9 µg kg(-1) for CBX and 80 µg kg(-1) for OLA, and the limit of quantification (LoQ) was defined at 12 µg kg(-1) and 110 µg kg(-1) for CBX and OLA, respectively. The accuracy of the method was adequate for CBX and OLA. The recovery values found in the repeatability conditions were 99.41% for CBX and 104.62% for OLA. Under intralaboratory reproducibility conditions, the values were 98.63% for CBX and 95.07% for OLA. It was concluded that the performance parameters demonstrated total method adequacy for the detection and quantification of CBX and OLA in poultry and swine feedingstuffs.

Pharmaceuticals and other anthropogenic tracers in surface water: a randomized survey of 50 Minnesota lakes.

Water from 50 randomly selected lakes across Minnesota, USA, was analyzed for pharmaceuticals, personal care products, hormones, and other commercial or industrial chemicals in conjunction with the US Environmental Protection Agency's 2012 National Lakes Assessment. Thirty-eight of the 125 chemicals analyzed were detected at least once, all at parts per trillion concentrations. The most widely detected was N,N-diethyl-m-toluamide, present in 48% of the lakes sampled. Amitriptyline, a widely used antidepressant, was found in 28% of the lakes. The endocrine active chemicals bisphenol A, androstenedione, and nonylphenol were found in 42%, 30%, and 10% of the lakes, respectively. Cocaine was found in 32% of the lakes, and its degradation product, benzoylecgonine, was detected at 28% of the locations. Carbadox, an antibiotic used solely in the production of swine, was also present in 28% of the lakes sampled. The means by which these and other chemicals were transported to several of the remote lakes is unclear but may involve atmospheric transport.

In vivo studies to highlight possible illegal treatments of rabbits with carbadox and olaquindox.

For the treatment of rabbit dysentery and bacterial enteritis, veterinary practitioners often adopt veterinary medicinal products authorised for other food-producing species, but in some cases non-authorised drugs frequently used in the past, such as carbadox and olaquindox, might be illegally adopted. To verify the carbadox and olaquindox distribution and persistence in rabbit tissues, two independent in vivo studies were carried out. In the first study, 24 healthy rabbits received water medicated with carbadox at 100 mg l(-1) over a period 28 days, whereas in the second one, 24 healthy rabbits were administered water containing olaquindox at 100 mg l(-1). In each study rabbits were randomly assigned to four groups to be sacrificed respectively at 0, 5, 10 and 20 days from treatment withdrawal, for depletion studies. A control group of six animals was adopted for control and as a reservoir of blank tissues. Muscle and liver samples collected from each treated animal were stored at -20°C pending the analysis. Sensitive and robust liquid chromatography-tandem mass spectrometry analytical methods were set up for the parent compounds and their main metabolites quinoxaline-2-carboxylic acid, desoxycarbadox and 3-methylquinoxaline-2-carboxylic acid to verify their residual. Data collected demonstrate that the combination of liver as target matrix, quinoxaline-2-carboxylic acid and 3-methylquinoxaline-2-carboxylic acid as marker residue and enzymatic digestion is strategic to evidence carbadox and/or olaquindox illegal treatments in rabbits, even 20 days after treatment withdrawal at concentration levels higher than 0.5 µg kg(-1). This findings suggests that liver should be proposed as target matrix for official control in national monitoring plan.

Discovery of novel antigiardiasis drug candidates.

Giardiasis is a severe intestinal parasitic disease caused by Giardia lamblia, which inflicts many people in poor regions and is the most common parasitic infection in the United States. Current standard care drugs are associated with undesirable side effects, treatment failures, and an increasing incidence of drug resistance. As follow-up to a high-throughput screening of an approved drug library, which identified compounds lethal to G. lamblia trophozoites, we have determined the minimum lethal concentrations of 28 drugs and advanced 10 of them to in vivo studies in mice. The results were compared to treatment with the standard care drug, metronidazole, in order to identify drugs with equal or better anti-Giardia activities. Three drugs, fumagillin, carbadox, and tioxidazole, were identified. These compounds were also potent against metronidazole-resistant human G. lamblia isolates (assemblages A and B), as determined in in vitro assays. Of these three compounds, fumagillin is currently an orphan drug used within the European Union to treat microsporidiosis in immunocompromised individuals, whereas carbadox and tioxidazole are used in veterinary medicine. A dose-dependent study of fumagillin in a giardiasis mouse model revealed that the effective dose of fumagillin was ∼ 100-fold lower than the metronidazole dose. Therefore, fumagillin may be advanced to further studies as an alternative treatment for giardiasis when metronidazole fails.

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry.

This paper presents LC-MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3-methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0µgkg(-1). Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45°C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexION™ technology to reduce matrix effect was used. The decision limit (CCα) ranged from 1.04µgkg(-1) to 2.11µgkg(-1) and the detection capability (CCß) ranged from 1.46µgkg(-1) to 2.89µgkg(-1). The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC.

Complexation facilitated reduction of aromatic N-oxides by aqueous Fe(II)-tiron complex: reaction kinetics and mechanisms.

Rapid reduction of carbadox (CDX), olaquindox and several other aromatic N-oxides were investigated in aqueous solution containing Fe(II) and tiron. Consistent with previous work, the 1:2 Fe(II)-tiron complex, FeL2(6-), is the dominant reactive species as its concentration linearly correlates with the observed rate constant kobs under various conditions. The N-oxides without any side chains were much less reactive, suggesting direct reduction of the N-oxides is slow. UV-vis spectra suggest FeL2(6-) likely forms 5- or 7-membered rings with CDX and olaquindox through the N and O atoms on the side chain. The formed inner-sphere complexes significantly facilitated electron transfer from FeL2(6-) to the N-oxides. Reduction products of the N-oxides were identified by HPLC/QToF-MS to be the deoxygenated analogs. QSAR analysis indicated neither the first electron transfer nor N-O bond cleavage is the rate-limiting step. Calculations of the atomic spin densities of the anionic N-oxides confirmed the extensive delocalization between the aromatic ring and the side chain, suggesting complex formation can significantly affect the reduction kinetics. Our results suggest the complexation facilitated N-oxide reduction by Fe(II)-tiron involves a free radical mechanism, and the subsequent deoxygenation might also benefit from the weak complexation of Fe(II) with the N-oxide O atom.

Hydroxylation of quinocetone and carbadox is mediated by CYP1As in the chicken (Gallus gallus).

Quinoxaline derivatives (quinoxalines) comprise a class of drugs that have been widely used as animal antimicrobial agents and feed additives. Although the metabolism of quinoxaline drugs has been mostly studied using chicken liver microsomes, the biochemical mechanism of biotransformation of these chemicals in the chicken has yet to be characterized. In this study, using bacteria produced enzymes, we demonstrated that both CYP1A4 and CYP1A5 participate in the oxidative metabolism of quinoxalines. For CYP1A5, three hydroxylated metabolites of quinocetone were generated. In addition, CYP1A5 is able to hydroxylate carbadox. For CYP1A4, only one hydroxylated product of quinocetone on the phenyl ring was identified. Neither CYP1A5 nor CYP1A4 showed hydroxylation activity towards mequindox and cyadox. Our results suggest that CYP1A4 and CYP1A5 have different and somewhat overlapping substrate specificity in quinoxaline metabolism, and CYP1A5 represents a crucial enzyme in hydroxylation of both quinocetone and carbadox.

Lysozyme as an alternative to antibiotics improves growth performance and small intestinal morphology in nursery pigs.

Lysozyme is a 1,4-ß-N-acetylmuramidase that has antimicrobial properties. The objective of this experiment was to determine if lysozyme in nursery diets improved growth performance and gastrointestinal health of pigs weaned from the sow at 24 d of age. Two replicates of 96 pigs (192 total; 96 males, 96 females) were weaned from the sow at 24 d of age, blocked by BW and gender, and then assigned to 1 of 24 pens (4 pigs/pen). Each block was randomly assigned 1 of 3 dietary treatments for 28 d: control (two 14-d phases), control + antibiotics (carbadox/copper sulfate), or control + lysozyme (100 mg/kg diet). Pigs were weighed and blood sampled on d 0, 14, and 28 of treatment. Blood was analyzed for plasma urea nitrogen (PUN) and IgA. At 28 d, pigs were killed, and samples of jejunum and ileum were collected and fixed for intestinal morphology measurements. An additional jejunum sample was taken from the 12 pigs with the median BW per treatment to determine transepithelial electrical resistance (TER). Pigs consuming antibiotics or lysozyme grew at a faster rate than control pigs (0.433 ± 0.009 and 0.421 ± 0.008 vs. 0.398 ± 0.008 kg/d, respectively; P < 0.03), which resulted in heavier ending BW (20.00 ± 0.31, 19.8 ± 0.29, and 18.83 ± 0.32 kg, respectively; P < 0.03). Feed intake was not different (P > 0.48), but G:F was improved in pigs consuming antibiotics or lysozyme (0.756 ± 0.014, 0.750 ± 0.021, and 0.695 ± 0.019 kg/kg; P < 0.05). Immunoglobulin A (P < 0.03) and PUN (P < 0.01) increased during the experiment, regardless of dietary treatment (P > 0.48). Dietary treatment did not affect TER (P > 0.37), but gilts had lower TER compared with barrows (P < 0.04). No differences in villi height or crypt depth were observed in the ileum (P > 0.53). However, jejunum villi height was increased and crypt depth was decreased in pigs consuming antibiotics or lysozyme (P < 0.001), resulting in an increased villi height:crypt depth of 72% (P < 0.001). Thus, we concluded that lysozyme is a suitable alternative to carbadox/copper sulfate diets fed to pigs weaned from the sow at 24 d of age.