Effects Of Maduramicin On Hepatopancreas Drug-metabolizing Enzymes Of Procambarus Clarkia And Toxicological Studies On Rhabdomyolysis In Mice

Maduramcin is a polyether ion carrier antibiotic,which is mainly used to control coccidiosis in broiler chicken industry.Due to its rapid metabolism in the target animal chickens,prototype drugs are often exceeded in feces,polluting the ecological environment including aquatic environment.To date,there are hundreds cases of Haff disease caused by crayfish(Procambarus clarkia)consumption,and the symptoms are very similar to those of maduramicin poisoning.In the present study,we aimed to investigate the effects of maduramicin on hepatopancreas drug enzymes of Procambarus clarkia and the toxicity and toxicokinetic characteristics of two tissues of Procambarus clarkia containing maduramicin on mice,and to further explore the association between maduramicin and Haff disease and then to assess the risk.The findings are as follows:1 Effects of maduramicin on drug metabolizing enzymes in the hepatopancreas of Procambarus clarkiaThe 192 male Procambarus clarkia were randomly divided into four groups,one of which was a blank control group,and the other three groups were exposed to 0.7 mg·L-1,3.5 mg-L-1 and 7 mg· L-1 maduramicin for 28 d,respectively.At the 7th,14th,21th and 28th day after treatment,12 Procambarus clarkia hepatopancreas samples of all tested groups were collected for the determination of cytochromes P450 and cytochromes b5,and the activities of phase I drug metabolizing enzymes(ERND,APND,AH)and phase ? drug metabolizing enzymes(GSTs,UGTs,NATs).Meanwhile,the mRNA expression levels of CYP450,gst and CYP b5 genes were detected by real-time fluorescence quantitative PCR.The results showed that the content of CYP450 in the 7 mg·L-1 treatment group was significantly higher than that in the control group at all time periods.while the content of CYP b5 was not significantly changed by maduramicin treatment.Only the activity of ERND was significantly increased in the 0.7 mg·L-1 treatment group.while the activities of phase I drug metabolizing enzymes ERND,APND,AH and phase ? drug metabolizing enzymes GSTs,UGTs and NATs were significantly increased in the 7 mg·L-1 treatment group.The expression of gst and CYP450 genes related to liver drug enzyme were significantly up-regulated by maduramicin,and there was no significant change in CYP b5 gene except on 7th day.These findings suggested that maduramicin could induce drug metabolizing enzymes in hepatopancreas of Procambarus clarkia.2 Toxicity of maduramicin derived from Procambarus clarkia tissues on miceTwo pathways were used to expose maduramicin to mice through the tissue of Procambarus clarkia.Drug bath administration group:Procambarus clarkia were exposed to 0.7 mg·L-1,3.5 mg·L-1 and 7 mg·L-1 maduramicin for 72 h in a drug bath.After Procambarus clarkia were sacrificed,muscle and hepatopancreas of each group were stripped to prepare homogenate for mice administration.Artificial administration:three groups of prepared maduramicin solution with different concentrations were mixed with blank muscle and hepatopancreas homogenate and make the doses of gavage mice were 0.7 mg·kg-1,3.5 mg·kg-1 and 7 mg·kg-1,respectively.Mice were continuous administrated maduramicin for 7 d,clinical status,weight change and feed intake of mice were observed and recorded,the determination of blood biochemical parameters,and the preparation of four groups(heart,liver,kidney,skeletal muscle)pathological section.The results showed that the mice in the drug bath group were not significantly different in mental state,body weight,blood biochemical indexes and pathological tissues.The mice in the artificial drug group were depressed and sleepy,and their body weight and food intake decreased significantly with the increase of maduramicin.Serum biochemical indexes of ALT,AST,BUN,LDH and CK were significantly increased in the 3.5 mg·kg-1 and 7 mg·kg-1 treatment groups.Histopathological lesions were observed in the 7 mg·kg-1 treatment group in a number of organs,including vesicular degeneration of liver cells,exfoliation of renal epithelial cells,disorders of myocardial and skeletal muscle filaments,and partial dissolution.The results showed that 7 mg·kg-1 maduramicin had a more serious toxic effect on mice,but maduramicin was not the primary factor of Haff disease in Procambarus clarkia.3 Toxicokinetics of maduramicin with two different exposure models in miceThe blank muscle and hepatopancreas of Procambarus clarkia were homogenized with maduramicin solution to prepare muscle and hepatopancreas homogenization buffer.234 male ICR mice were randomly divided into three groups:muscle homogenate group,hepatopancreas homogenate group and maduramicin drug solution group,and the concentrations of maduramicin to gavage mice in the three groups were the same(7 mg-kg1).Plasma in mice was collected from 6 mice in each group at 0.083,0.167,0.25,0.5,0.75,1,2,4,8,24,48,72,96 h after administration of maduramicin.Mice plasma was extracted with acetonitrile,and after nitrogen blowing concentration and mobile phase redissolution,the multi-reaction monitoring mode(MRM)was selected for analysis under electrospray positive ion mode(ESI+)by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Nigericin was selected as internal standard substance for quantitative analysis.The toxicokinetic parameters of maduramicin in mice was analyzed by using WinNonLin software.The validation of the methodology showed that,within the linear range of 1-400 ng mL-1,,the linearity was good(R2>0.99),the recovery rate in the mouse plasma was:y=1.69737x+1.62269,and the intraday and daytime precision was:82.87%?117.74%and 0.99?12.18%,indicating that the method had strong specificity,high sensitivity and good repeatability,and was suitable for the determination of the concentration of mardomethium in the mouse plasma.The first group parameters were as followed:the mean elimination half-life(Ti/2x)was(23.60±7.38)h,the peak time(Tmax)was(0.83±0.13)h,the peak concentration was(Cmax)was(1489.36±366.08)ng·mL-1,the mean area under the concentration time curve(AUCo?t)was(20431.47±2604.85)h·ng·mL-1.The second group parameters were as followed:T1/2? was(21.62±3.92)h,Tmax was(0.79±0.19)h,Cmax was(1613.46±309.81)ng·mL-1,AUCo?t was(19575.29±1893.97)h.ng·mL-1.The third group parameters were as followed:T1/2? was(20.30±3.54)h,Tmax was(0.79±0.19)h,Cmax was(1536.78 ±352.56)ng·mL-1,AUC0?t was(24221.00±3054.99)h·ng·mL-1.