Menghong Dai

Antibiotic alternatives: the substitution of antibiotics in animal husbandry?

It is a common practice for decades to use of sub-therapeutic dose of antibiotics in food-animal feeds to prevent animals from diseases and to improve production performance in modern animal husbandry. In the meantime, concerns over the increasing emergence of antibiotic-resistant bacteria due to the unreasonable use of antibiotics and an appearance of less novelty antibiotics have prompted efforts to develop so-called alternatives to antibiotics. Whether or not the alternatives could really replace antibiotics remains a controversial issue. This review summarizes recent development and perspectives of alternatives to antibiotics. The mechanism of actions, applications, and prospectives of the alternatives such as immunity modulating agents, bacteriophages and their lysins, antimicrobial peptides, pro-, pre-, and synbiotics, plant extracts, inhibitors targeting pathogenicity (bacterial quorum sensing, biofilm, and virulence), and feeding enzymes are thoroughly discussed. Lastly, the feasibility of alternatives to antibiotics is deeply analyzed. It is hard to conclude that the alternatives might substitute antibiotics in veterinary medicine in the foreseeable future. At the present time, prudent use of antibiotics and the establishment of scientific monitoring systems are the best and fastest way to limit the adverse effects of the abuse of antibiotics and to ensure the safety of animal-derived food and environment.

Benefits and risks of antimicrobial use in food-producing animals.

Benefits and risks of antimicrobial drugs, used in food-producing animals, continue to be complex and controversial issues. This review comprehensively presents the benefits of antimicrobials drugs regarding control of animal diseases, protection of public health, enhancement of animal production, improvement of environment, and effects of the drugs on biogas production and public health associated with antimicrobial resistance. The positive and negative impacts, due to ban issue of antimicrobial agents used in food-producing animals, are also included in the discussion. As a double-edged sword, use of these drugs in food-animals persists as a great challenge.

Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats.

Mequindox (MEQ) is a synthetic quinoxaline 1,4-dioxides (QdNOs) derivative which can effectively improve growth and feed efficiency in animals. This study was to investigate the dose-dependent long-term toxicity in the adrenal of male rats exposed to 180 days of MEQ feed. Our data demonstrated that high doses of MEQ in the diet for 180 days led to adrenal damage and steroid hormone decrease, combined with sodium decrease and potassium increase in rat plasma. Significant changes of GSH and SOD in plasma were observed in the high doses (110, 275 mg/kg) groups. At the same doses, MEQ treatment down-regulated the mRNA levels of CYP11A1, CYP11B1 and CYP11B2 which located in mitochondria, but up-regulated mRNA levels of CYP21 and 3beta-HSD which located in endoplasmic reticulum. In conclusion, we reported the dose-dependent long-term toxicity of MEQ on adrenal gland in male rats, which raise awareness of its toxic effects to animals and consumers, and its mechanism may involve in oxidative stress and steroid hormone biosynthesis pathway.

JAK/STAT Pathway Plays a Critical Role in the Proinflammatory Gene Expression and Apoptosis of RAW264.7 Cells Induced by Trichothecenes as DON and T-2 Toxin

Deoxynivalenol (DON) and T-2 toxin commonly affect cells of the immune system and cause inflammation and apoptosis. Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is highly associated with inflammatory process and apoptosis and is worth investigating its role when cells were exposed to trichothecenes. The results showed that DON and T-2 upregulated the messenger RNA (mRNA) expressions of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, JAK1-2, STAT1-3, and suppressors of cytokine signaling members and activated the tyrosine phosphorylation of STAT1 and STAT3 with a dose-dependent manner in RAW264.7 cells. AG490 and Stattic, the specific inhibitors of JAK/STAT pathway, blocked the STAT1 and STAT3 tyrosine phosphorylation and decreased the gene expressions of proinflammatory cytokines induced by trichothecenes. Interestingly, the time when the mRNA levels of STAT1 and STAT3 were significantly upregulated was at 12 h, which was much later than the time when mitogen-activated protein kinase was activated, indicating that STATs might be the downstream targets of the trichothecenes. With the intervention of AG490 and Stattic, DON and T-2 toxin induced apoptosis in a strengthened way, with the loss of mitochondrial membrane potential and the decrease ratios of the B-cell leukemia/lymphoma 2 (Bcl-2)/bcl-2-associated X (Bax) and B-cell lymphoma-extra large (Bcl-xL)/Bax. After exposing to DON and T-2 toxin, cells exhibited G2/M and G0/G1 phase arrest, respectively. The increased mRNA expressions of STAT target genes p21 and cyclin D1 for DON and the increases in p21 mRNA and the decreases in cyclin D1 for T-2 toxin were observed. These results demonstrated for the first time that the activation of JAK/STAT might be a critical mediator to induce the inflammatory response and apoptosis in macrophage in response to trichothecenes.

Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high‐performance liquid chromatography combined with ion trap/time‐of‐flight mass spectrometry

Olaquindox (N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1-M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N --> O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3-M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals.

Acute and sub-chronic oral toxicological evaluations of quinocetone in Wistar rats

To provide a detailed toxicity with wide spectrum of doses for quinocetone, a new antimicrobial growth promoting agent, acute and sub-chronic toxicological studies were conducted. For acute study, quinocetone was administered singly by oral gavage to Wistar rats and Kunming mice. Calculated LD50 was 8687.31 mg/kg b.w./day in rats and 15848.93 mg/kg b.w./day in mice. In sub-chronic study, quinocetone was fed to Wistar rats at dietary levels of 0, 50, 300 and 1800 mg/kg or olaquindox (300 mg/kg), approximately equivalent to quinocetone 5, 30, 180 or olaquindox 30 mg/kg b.w./day. There was significant decrease in body weight in both genders, total protein and creatinine in females and alkaline phosphatase in males fed with 1800 mg/kg diet, while alkaline aminotransferase values decreased in all treated groups. Significant increase in relative weights of liver and kidneys in both genders and testis in male rats were noted at 1800 mg/kg diet. Histopathological observations revealed that 1800 mg/kg quinocetone diet and 300 mg/kg olaquindox diet could induce proliferation of bile canaliculi in the portal area. In conclusion, quinocetone can induce hepatic histological changes as well as leaking of different serum enzymes. The no-observed-adverse-effect level of quinocetone was considered to be 300 mg/kg diet.

Metabolism of cyadox in rat, chicken and pig liver microsomes and identification of metabolites by accurate mass measurements using electrospray ionization hybrid ion trap/time‐of‐flight mass spectrometry

Cyadox (CYX), (2-formylquinoxaline)-N(1),N(4)-dioxide cyanoacetylhydrazone, is a growth promoter, which is more efficient and less toxic to animals. Few studies have been performed to reveal the metabolism of CYX in animals till now. In this study, the metabolic fate of CYX in the liver microsomes of animal was investigated firstly using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. CYX was incubated with rat, chicken and pig liver microsomes in the presence of a NADPH-generating system. Multiple scans of metabolites in MS and MS(2) modes and accurate mass measurements were performed simultaneously through data-dependent acquisition. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The structures of metabolites and their fragment ions were easily and reliably characterized based on the accurate MS(2) spectra and known structure of CYX. The relative biotransformation of CYX into characterized metabolites was estimated based on the UV absorption and the assumption that all metabolites had the same extinction coefficient as the parent compound at 305 nm. Totally, seven metabolites were identified as three reduced metabolites (cyadox 1-monoxide (Cy1), cyadox 4-monoxide (Cy2) and bisdesoxycyadox (Cy4)), three hydrolysis metabolites of the amide bond (N-decyanoacetyl cyadox (Cy5), N-decyanoacetyl cyadox 1-monoxide (Cy6) and N-decyanoacetyl bisdesoxycyadox (Cy7)) and a hydroxylation metabolite of Cy1 (Cy3). Cy1-Cy6 could be detected in rat, chicken and pig liver microsomes while metabolite Cy7 could only be observed in pig. The amounts of the metabolites in three species are different. For the formations of Cy1 and Cy3, the rank order was rat approximately chicken > pig. For Cy4 and Cy5, the order was pig > rat > chicken. Cy1 and Cy4 have been previously reported, whereas the other five metabolites were novel. The N-->O group reduction and hydroxylation were the main metabolic pathways for CYX in the three species.

Development of a high performance liquid chromatography method and a liquid chromatography–tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumers health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70°C) and pressure (1400 psi). After clean-up with hydrophilic-lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC-MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC-MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC-MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues.

Development of a high performance liquid chromatography method and a liquid chromatography―tandem mass spectrometry method with pressurized liquid extraction for simultaneous quantification and confirmation of cyromazine, melamine and its metabolites in foods of animal origin

Simple and sensitive methods have been developed for simultaneous detection of cyromazine, melamine and their metabolites (ammeline, ammelide and cyanuric acid) in samples of animal origins. These include a high performance liquid chromatography (HPLC) method and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and are useful in regular monitoring and in toxicity studies of these molecules. Representative samples used in this study include muscles and livers of swine, bovine, sheep and chicken, kidneys of swine, bovine and sheep, and milk powder. A new sample preparation procedure with pressurized liquid extraction (PLE) at 1400psi and 70°C was investigated. Quantification of these five compounds by HPLC was achieved using an APS-2 column with UV detection at 230 nm. Limit of detection (LOD) was at 10 μgkg(-1), and limit of quantification (LOQ) was at 40 μgkg(-1). Recoveries of the five analytes in spiked samples ranged from 72.2% to 115.4% with RSD less than 12%. Confirmatory analysis of the analytes was performed using LC-MS/MS in selected reaction monitoring (SRM) mode. The LOD and LOQ were 5 μgkg(-1) and 15 μgkg(-1), respectively. This is the first simultaneous analysis of cyromazine, melamine, ammeline, ammelide and cyanuric acid residues in complex tissue samples using PLE and HPLC. It is expected that these methods will find many practical applications in evaluating the safety of cyromazine, melamine and their metabolites.

Permethrin-induced oxidative stress and toxicity and metabolism. A review

Permethrin (PER), the most frequently used synthetic Type I pyrethroid insecticide, is widely used in the world because of its high activity as an insecticide and its low mammalian toxicity. It was originally believed that PER exhibited low toxicity on untargeted animals. However, as its use became more extensive worldwide, increasing evidence suggested that PER might have a variety of toxic effects on animals and humans alike, such as neurotoxicity, immunotoxicity, cardiotoxicity, hepatotoxicity, reproductive, genotoxic, and haematotoxic effects, digestive system toxicity, and cytotoxicity. A growing number of studies indicate that oxidative stress played critical roles in the various toxicities associated with PER. To date, almost no review has addressed the toxicity of PER correlated with oxidative stress. The focus of this article is primarily to summarise advances in the research associated with oxidative stress as a potential mechanism for PER-induced toxicity as well as its metabolism. This review summarises the research conducted over the past decade into the reactive oxygen species (ROS) generation and oxidative stress as a consequence of PER treatments, and ultimately their correlation with the toxicity and the metabolism of PER. The metabolism of PER involves various CYP450 enzymes, alcohol or aldehyde dehydrogenases for oxidation and the carboxylesterases for hydrolysis, through which oxidative stress might occur, and such metabolic factors are also reviewed. The protection of a variety of antioxidants against PER-induced toxicity is also discussed, in order to further understand the role of oxidative stress in PER-induced toxicity. This review will throw new light on the critical roles of oxidative stress in PER-induced toxicity, as well as on the blind spots that still exist in the understanding of PER metabolism, the cellular effects in terms of apoptosis and cell signaling pathways, and finally strategies to help to protect against its oxidative damage.