Toxicity Studies Of Cyadox Using Metabonomics

As an antimicrobial growth promoter, cyadox has great potential to be used as additive in the feedstuff for livestock and poultry. However, a comprehensive food safety assessment should be performed before becoming feed additives. So far, there is no a report on how cyadox impacts on endogenous metabolites of animals, though many traditional toxicological studies of cyadox have been conducted. Therefore, in this paper, NMR-based metabonomic techniques were used to study the impact of cyadox on the endogenous metabolisms of Kunming mice. To further study metabolic responses of different species, endogenous metabolome changes induced by cyadox in Wistar rats were also investigated. On the basis of these two experiments, we also have designed a sub-chronic toxicological experiment on Wistar rats, in order to explore how endogenous metabolome changes under long-term cyadox exposure. In addition, transcriptomics profile was also used to investigate the variance of liver on transcriptome level.In the study of metabolic influence of acute cyadox exposure on Kunming mice, three groups of mice were respectively given a single dose of cyadox at three different concentrations (100,650and4000mg/kg body weight) via gavage. We present here the metabolic alterations of urine, plasma, liver and renal medulla extracts induced by cyadox exposure. The metabolic alterations induced by cyadox exposure are dose dependent and metabolic recovery is only achieved for low and moderate levels of cyadox exposure during experimental period. Cyadox exposure resulted in a disturbance of gut microbiota, which is manifested in the depleted levels of urinary hippurate, trimethylamine-N-oxide, dimethylamine and trimethylamine. In addition, cyadox exposure on high levels caused accumulations of amino acids and depletions of nucleotides in the liver. Furthermore, marked elevations of nucleotides and a range of organic osmolytes, such as myo-inositol, choline and glycerophosphocholine and decreased levels of amino acids are observed in the renal medulla of cyadox exposed mice. These results suggest that cyadox exposure causes inhibition of amino acids metabolism in the liver and disturbance of gut microbiota community, influences osmolytic homeostasis and nucleic acids synthesis in both the liver and the kidney.In the study of metabolic influence of acute cyadox exposure on Wistar rats, three groups of Wistar rats were respectively given a single dose of cyadox at three different concentrations (50,325and2000mg/kg body weight) via gavage. Compared to the mice, rats were more sensitive to cyadox mainly represented in the changes on the plasma profiles, an indication of inbalanced organism homeostasis. A series of significant changes including increased leucine, valine, methionine, asparagine, and creatine in moderate dose group and the elevated level of alanine, succinic acid, methionine, glucose and lactic acid and decreased lipid in the high-dose group were observed. In addition, no significant inhibition of amino acids metabolism in rat liver and no disturbance in the rat kidney were observed. However, disturbances of nucleic acid metabolism in the liver and gut microbiota were also observed in the rats.In the study of metabolic and transcriptome influences of sub-chronic cyadox exposure on Wistar rats, three groups of Wistar rats were respectively fed with foods containing three different concentrations of cyadox (50,150,2500mg/kg feed). Two weeks recovery were conducted before experiment ended. Results of metabonomic analysis showed that metabolites in urine of rats in low-and moderate-dose changed freely and were not in some rule. Kidney showed variance on the metabolism of amino acids and nucleic acids but no change was observed after two weeks recovery. Variations in the profiles of plasma were only observed in the high dose group during the entire experiment time. Cyadox seemed to have a cumulative effect on the profiles liver, The metabolic displacement increased as the dosage becomes high level. No obvious recovery was observed for rats exposed to moderate and high levels of cyadox. Moreover, disturbance even appeared in the rats of the low dose group, after two weeks recovery time, though no abnormal changes were observed during the dosed time. We further investigated gene expressions of the liver of high-dose group after12weeks cyadox administration. Genes involved drug metabolism such as Cyplal, Hsdllb1and Cyp2a2, involved lipid metabolism such as Scd, Acotl/2/4, Acsm2and Pexlla, involved glycolytic pathway and TCA cycle pathway such as Slc34a2and Pfkfbl, involved amino acid metabolic pathway such as Daao, involved transcriptional regulatory pathways such as Nfe2, Pir and Scyllbpl, involved inflammatory reaction pathway such Cxcll3, Tmem55a, Rtl-N and Icaml and involved nervous system pathway such as Fam134b, Scyllbpl, Colq, Gabbprl, Trim2, Epha4, Rtn4rll and Hspbl all changed significantly, which were mostly consistent with the metabolic changes.Mechanisms of cyadox action on organism were investigated in this thesis from transcriptome and metabolome aspects. Liver is the major metabolic organ for cyadox. A certain amount of reactive oxygen species (ROS) will be released during the process of cyadox metabolism and long-term stimulation of reactive oxygen species might cause lipid peroxidation, resulting disturbances in a series of metabolic pathways, such as in the lipid metabolism, amino acid metabolism, TCA cycle, nucleotide metabolism and so on. In addition, hydropic degeneration, punctate necrosis and fatty degeneration of hepatic cells were also observed in liver pathology. Moreover, as a broad-spectrum antibacterial agent, cyadox also suppressed the activity of the gut microbiota significantly. Overall, dose-dependent effect and different actions on different species were observed. Wistar rats were more sensitive to cyadox than Kunming mice. The dose of cyadox with50mg/kg feed is the recommended dose used as feed additives. In the sub-chronic toxicological experiments, rats’responses on this dosage on either urine or kidneys are very slight and can be restored and no abnormalities were observed on the plasma. However, long-term feeding of cyadox on this dose may cause a certain degree of influence on the liver. In short, it’s practicable on the metabolic aspect for cyadox’s used in livestock farming when the dosage was strictly controlled and some recovery time was given.In summary, this thesis provides a comprehensive view of the toxicological effects of cyadox with the help of metabolomics and transcriptomics profile, which is important in animal and human food safety.