Studies On Antibacterial Activity And Cytotoxocity Of Chicken Primary Liver Cells Of Mequindox And Its Metabolites

People constantly pay attention to the residues of veterinary drug in animal food, olaquindox and carbadox were limited to use as animal feed additives because of the mutagenicity, developmental and reproductive toxicities and carcinogenicity of the metabolites 3-methyl-quinoline-2-carboxylic acid and quinoline-2-carboxylic acid, respectively. Mequindox (MEQ), a new synthetic veterinary medicine in quinoline 1,4-dioxides, owing to its antibiotic activity against gram-negative bacterial including salmonella and Escherichia coli, has been used as an animal feed additive and veterinary medicine for diseases. Currently, many papers reported the antibiotic activity and toxicity of MEQ. However, we discovered that MEQ was metabolized to other compounds quickly after administration to animals. There were few papers about the antibiotic activity or cytotoxicity of the metabolites. Here we researched the antibiotic activity of five synthesized metabolites of MEQ in vivo and intro. And the material basis of antibiotic action and cytotoxicity action of MEQ were investigated. It would take a basis for safety evaluation of MEQ residues in animal products.Testl Synthesis and characterization of five major mequindox metabolitesBased on our previous study, we successfully synthesized MEQ’s five main metabolites (Ml, M2, M3, M4 and M5) by using Na2S2O4 or NaBH4 as a reducing agent, and purified them by recrystallization. In the synthesis process, we monitored the process of synthesis of metabolites at any time to detect the sample by HPLC-MS. The compounds synthesized were confirmed by mass spectrometry and nuclear magnetic resonance. Then we determined the content of each metabolite with area normalization method. The results showed that several mequindox metabolites were successfully obtained. The purity of these metabolites was greater than 96%, and the resulting compound content meet toxicological requirements.Test 2 Antibacterial activity of mequindox and its metabolites in vivo and in vitro in chickenMinimum inhibitory concentration of mequindox and its metabolites on chicken Escherichia coli, Staphylococcus aureus, and Salmonella pullorum was detected by using 96-well plates.100 13-day-old chicken were randomly divided into A (E. coli group), B (Salmonella group), C (S.aureus group), D (Control group) to research therapeutic effects of mequindox and Ml on E. coli, Salmonella, Staphylococcus aureus, respectively. Each group of chicken was made artificial bacterial infection onset, then we gave the chicken oral gavage MEQ, M1, once every five days, to observe treatment effects. We weighed each group of chicken and recorded the results before inoculation of bacteria and at the end of the test. When the test was finished, the mortality rate and the relative growth rate were calculated for each group. And the ratio of the average weight gain of each infected group and of the healthy control group was also calculated. The results showed that mequindox minimum inhibitory concentration for Escherichia coli, Salmonella and Staphylococcus aureus were respectively 2μg/mL,8μg/mL and 16μg/mL. The minimum inhibitory concentration of E. coli of metabolite Ml was 32μg/mL. The minimum inhibitory concentration of Salmonella and Staphylococcus aureus were 128μg/mL. In addition, there was a therapeutic effect on chicken E. coli and salmonella of metabolite Ml, and it could promote the growth of chickens, but the treatment effect and the promoting growth of M1 are less than the group by MEQ.Test 3 Cytotoxocity of mequindox and its metabolites in chicken primary liver cellsThe cell cycle and apoptosis of chicken primary hepatocytes induced by MEQ, Ml, M2, M3, M4 and M5 were detected by flow cytometry. The results showed that after treated respectively by 200μg/mL MEQ, M1, M2, M3, M4 and M5, the early apoptosis ratio in chicken primary hepatocytes were presented by 68.2%,26.7%,20.3%,31.4%,24.5% and 19.5%. There was a greater difference compared with the control group with 10%. Meanwhile, the cycle of chicken primary hepatocytes was also changed after treated with compounds. The cell proliferation was blocked in S phase by MEQ, M1, M5, while it has no significant cell cycle arrest in cells induced by M2, M3 and M4.