Component Identification And Toxicity Mechanism Studies Of Thermally Treated Ceftiofur

Ceftiofur(CEF)is the third-generation cephalosporin antibiotic and the first animal-specific cephalosporin antibiotic which exerts an efficient bactericidal effect by inhibition of bacterial cell wall synthesis through penicillin-binding protein.CEF has a broad antibacterial spectrum and has strong antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and some anaerobic bacteria.Moreover,CEF has become one of the most widely used antibiotics in animal husbandry because of its advantages of safety and short drug withdrawal period.However,public safety issues such as increased bacterial resistance and drug residues have gradually increased with the large-scale use of CEF,which has caused potential harm to human health and disease treatment.Studies have proved that CEF is sensitive to light,p H,microorganisms,and temperature,and CEF can degrade under a variety of conditions.CEF is heat-labile,which degrades at high temperature.Animal-derived foods should be heated in industrial processing or cooking,therefore,the residual CEF in food will degrade.It has been reported that the residual CEF in milk and water degraded at high temperature.However,the toxicity studies of CEF degradation products have not been reported.In this study,the toxicity of thermally treated CEF(TTC)was investigated after boiling which was a common cooking method.The relationship between the heating time and the toxicity of CEF was determined through analysing the optical density(OD)value at different heating time point and the result of cytotoxicity.Cytotoxicity test,Hoechst 33342/PI fluorescence staining,flow cytometry were used to study the cytotoxicity of TTC.Subsequently,the composition of TTC was investigated and characterized by high performance liquid chromatography tandem mass spectrometry(LC-MS).At the same time,the composition of thermally treated desfuroylceftiofur(DFC),metabolite of CEF in vivo,was also characterized.CEF-1,a thermal degradation product of CEF,was synthesized by chemical synthesis.The effects of CEF-1 on cytotoxicity,ROS level and mitochondrial membrane potential were studied.Finally,the toxicity of CEF in vivo was studied by acute oral toxicity study,acute intraperitoneal toxicity study and repeated dose 28-day oral toxicity study.Our results showed that the color and OD value of CEF changed after thermal treatment,suggesting that CEF had degraded.The inhibitory effect of TTC on the proliferation of human normal liver cells(LO2),human embryonic kidney cells(293),and human embryonic diploid lung cells(MRC-5)was significantly greater than that of CEF.The result of Hoechst 33342/PI fluorescence staining showed that TTC could induce cell apoptosis and necrosis.The results of flow cytometry analysis showed the similar results to fluorescence staining tests.By LC-MS,we found that ceftiofur-aldehyde(CEF-1)was the only one thermal degradation byproduct of TTC and CEF-1 also presented in thermally treated DFC.By studying the cytotoxicity of CEF-1,we found that the cytotoxicity of TTC to LO2 was caused by the addition of CEF-1 and incompletely decomposed CEF.Thus,CEF-1 was the only toxic byproduct of TTC.In addition,the results of ROS level measurement and mitochondrial membrane potential test showed that CEF-1 could increase ROS release,decrease mitochondrial membrane potential and induce apoptosis.The results of acute oral toxicity study showed that TTC had no acute oral toxicity,but it could lead to weight gain,hepatocyte steatosis,hypercholesterolemia,mitochondrial damage of liver cells and mitochondrial ? oxidation disorder.The results of acute intraperitoneal toxicity study showed that the median lethality dose(LD50)of thermally treated CEF for 30 min(TTC30)was 1.2 times of that of CEF,while the LD50 of thermally treated CEF for 60 min(TTC60)was 1.4 times of that of CEF.At 1500 mg/kg dose,the CEF group had a 100 % survival rate within 336 h,while the TTC30 group had a 25 % survival rate within 3 h and the TTC60 group had a 0 % survival rate within 3 h.In addition,TTC induced pulmonary congestion whichcould further develop into pulmonary edema,prolonged coagulation time,increased level of ROS in the alveolar cavityand serum that prompted oxidative stress damage.TTC showed liver damage and reduced the number of lymphocytes in mice of the repeated dose 28-day oral toxicity study.Additionally,the decreasing relative organ weights of the lungs and the increasing of the relative organ weights of the spleen of the male mice were observed,while the relative organ weights of the reproductive organs of female mice was reduced.In summary,we found that CEF-1,the thermal degradation byproduct of CEF,could induce cell apoptosis and necrosis through oxidative stress,causing significant cytotoxicity.In vivo tests,acute administration of TTC could also lead to increased ROS level and trigger oxidative stress damage,and TTC had repeated-dose oral toxicity,which could cause changes in the relative organ weights,lymphocytes count and liver function in mice.CEF or DFC remaining in animal-derived foods has the conditions to produce CEF-1 after heating.Therefore,the residual CEF may cause potential public health and food safety issues.