Toxic Effect And Mechanism Of Diclofenac Sodium On Broiler Chicken

Diclofenac sodium(DFS)is a derivative of phenylacetic acid,which belongs to the third generation of powerful non-steroidal anti-inflammatory drugs(NSAIDs)with potent analgesic,anti-inflammatory and antipyretic properties.Clinically,DFS is extensively applied to rheumatoid arthritis(RA),ankylosing spondylitis(AS),osteoarthritis(OA),postoperative pain,and fever of various origins.Compared with traditional NSAIDs(such as aspirin,metamizole,etc.),DFS is considered to be the first choice for the treatment of acute or chronic pain and inflammation with quick action,strong efficacy and fewer side effects.Given its outstanding advantages over similar drugs,DFS has also been used in veterinary clinics in recent years,mainly for the treatment of arthritis,neuritis,fever caused by various inflammation and postoperative pain in mammals,etc.,with safe and significant effects.However,birds are extremely sensitive to DFS,which could cause significant toxicity and even death to birds,and visceral urate deposition is considered the primary manifestation.At present,there are relatively few studies related to the toxicity of DFS on poultry.This study aims to investigate the toxic effect of DFS on broiler chicken from the aspects of clinical manifestation,pathological anatomy,histopathology,serum biochemical indexes and proteomics,which could provide a theoretical basis for elucidating the toxic mechanism of DFS on broiler chicken.The primary research contents and results are as follows:1.The acute toxicity study of DFS in broiler chickenTo determine the toxic effects of DFS on broiler chicken,an acute oral toxicity test(Horn method)was performed to obtain the oral median lethal dose(LD50)values while aspirin and metamizole were selected as control drugs.The results showed that the LD50 of DFS to broiler chicken was 23.3 mg/kg(95%confidence interval:16.0-39.9 mg/kg),which was 1260 mg/kg(95%confidence interval:926-1710 mg/kg)and 2710 mg/kg(95%confidence interval:2000-3690 mg/kg)for aspirin and metamizole,respectively.The present study not only demonstrated that broiler chicken was far more sensitive to DFS than similar drugs such as aspirin and metamizole but also provided a reference for the selection of drug dosage in subsequent research.2.Study on the tissue distribution of DFS in broiler chickenTo determine the distribution of DFS in broiler chicken,a high-performance liquid chromatography(HPLC)method was developed for the quantification of DFS in plasma and tissue samples.DFS concentrations in plasma and tissue samples were determined at 2 h,4 h,8 h,and 12 h after oral administration at a dose of 10 mg/kg,and the results indicated that DFS exists in a variety of tissues.DFS could be absorbed through the gastrointestinal tract and then transported into different tissues of broiler chicken,which were the basis and theoretical foundation for its toxic effects.3.Evaluation of the toxic effect of DFS on broiler chickenAccording to the LD50 obtained from the acute oral toxicity test,DFS was administered by oral gavage at appropriate dosages(10 and 20 mg/kg),and the toxic effect of DFS on broiler chicken was evaluated systematically in terms of clinical symptoms,anatomical changes,histopathology and serum biochemical indexes.Subsequently,the lower single doses of DFS(2.5 and 5.0 mg/kg)were administered to broilers via oral gavage,and blood samples were obtained at multiple time points before and within 72 h after DFS administration to explore the association between the toxicity of DFS and the uric acid content in serum.After DFS poisoning via oral administration,the main clinical manifestations of broiler chicken were depression and inability to stand,and gross anatomy showed that the surface of visceral organs was covered with diffuse urate.In addition,histopathologic observations indicated that the visceral organs of broiler chicken were damaged to varying degrees after the exposure to DFS,especially the kidney,liver,and duodenum.Serum biochemical analysis suggested that the levels of AST,Crea,UA,LDH and K⁺were significantly elevated,while significantly decreased levels of GLU,Ca²⁺and TG were observed.Compared with control group,the greatest magnitude of changes was observed in Crea and UA,with Crea up-regulated by about5-fold and UA up-regulated by about 7-fold.The UA testing results revealed that the UA content in serum was rapidly and strongly increased after DFS administration.The peak time of UA content in the 2.5 mg/kg group was 2 h and the peak concentration was expressed as around 2.5-fold of controls,while that of the 5.0 mg/kg group was 8 h and the peak concentration was expressed as around 7.5-fold.Overall,the visceral organs of broiler chicken were damaged after the exposure to DFS,especially the kidney and liver.Broiler chicken subjected to DFS exhibited significantly elevated serum UA levels in a dose-dependent manner and the toxic effect of DFS in broiler chicken was associated with the metabolism of UA.4.Nephrotoxic mechanism of DFS on broiler chicken revealed by i TRAQ-based proteomics analysisThe previous studies suggested that the kidney was one of the primary impaired organs for the toxic effect of DFS on broiler chicken.To investigate the nephrotoxic mechanism of DFS,proteome changes in kidney samples of broiler chicken were revealed after oral administration of DFS(10 mg/kg)via i TRAQ-based proteomic analysis.The m RNA expression levels were measured by quantitative real-time polymerase chain reaction(q RT-PCR)to validate the results obtained by proteomics.Proteins with fold change?1.2 and p-value?0.05 were filtered as the differentially expressed proteins(DEPs).434 DEPs were screened in kidney samples between the administration group and control group,of which 277were up-regulated and 157 were down-regulated.GO enrichment analysis revealed that DEPs were widely involved in metabolic processes,such as“organic substance metabolic process”,“primary metabolic process”,“cell metabolic process”,and“nitrogen compound metabolic process”.KEGG pathway analysis indicated that these DEPs were mainly enriched in metabolism-related pathways,such as“protein processing in endoplasmic reticulum”,“carbon metabolism”,“alanine,aspartate and glutamate metabolism”,“biosynthesis of amino acids”,“glyoxylate and dicarboxylate metabolism”,“propanoate metabolism”,“glycine,serine and threonine metabolism”,and“nitrogen metabolism”.DFS could induce apoptosis via endoplasmic stress in chicken renal cells;DFS could affect the biosynthesis of purine ring by upregulating GSL,GPT2,PIPOX and ASNS while downregulating ABAT,GLDC,AGXT2,GLUD1 and GATM,which in turn affect the production of uric acid;DFS could also prevent renal uric acid excretion by inhibiting the urate transporter OAT2(gene name:SLC22A7).Moreover,chicken embryo kidney(CEK)cells were prepared and the cell viability of CEK cells was determined by a CCK-8 assay after 24 h of exposure to different concentrations of DFS.The half maximal inhibitory concentration(IC50)value of DFS in CEK cells was determined as 1.355 m M(95%confidence interval:1.280-1.434 m M).5.Hepatotoxic mechanism of DFS on broiler chicken revealed by i TRAQ-based proteomics analysisTo investigate the hepatotoxic mechanism of DFS,proteome changes in liver samples of broiler chicken were revealed after oral administration of DFS(10 mg/kg)via i TRAQ-based proteomic analysis.The m RNA expression levels were measured by q RT-PCR to validate the results obtained by proteomics.Proteins with fold change?1.2 and p-value?0.05 were filtered as the DEPs.201 DEPs were obtained in liver samples,among them 154 were down-regulated and 47 were up-regulated.GO enrichment analysis demonstrated that DEPs were mainly involved in metabolic processes including“organic substance metabolic process”,“nitrogen compound metabolic process”,“primary metabolic process”,“cellular metabolic process”,and“catabolic process”.KEGG pathway analysis indicated that these DEPs were mainly enriched in metabolism-related pathways,such as“protein processing in endoplasmic reticulum”,“retinol metabolism”,“steroid hormone biosynthesis”,“linoleic acid metabolism”,“metabolism of xenobiotics by cytochrome P450”,“glycine,serine and threonine metabolism”,etc.metabolic signaling pathways.DFS could induce apoptosis via endoplasmic stress in chicken hepatocytes;DFS could affect the retinol metabolism by upregulating CYP3A5 while downregulating CYP3A4,CYP2C45,BCO1 and UGT1A1,which in turn induce oxidative stress injury in hepatocytes;DFS could also affect the biosynthesis of purine ring by downregulating TDH,LOC101747660 and GAT,which in turn affect the production of uric acid.In conclusion,DFS exhibited extremely strong toxicity to broiler chicken and the LD50was 23.3 mg/kg.After the established method for the quantification of DFS in plasma and tissue samples based on HPLC,the assay results suggested that DFS could be absorbed through the gastrointestinal tract and then transported into different tissues of broiler chicken,which were the basis and theoretical foundation for its toxic effects.DFS could lead to multiorgan or tissue damage,significant increases in serum UA content and hyperkalemia in broiler chicken.The proteomic analysis indicated that the nephrotoxicity of DFS on broiler chicken was achieved via inducing apoptosis in chicken renal cells,interfering with purine metabolism and inhibiting the urate transporter OAT2 while the hepatotoxicity was achieved via inducing apoptosis in chicken hepatocytes,interfering with retinol and purine metabolism.The present study not only provided a rationale for explaining the toxicity mechanism of DFS on broiler chicken but also provided a reference for the risk assessment of DFS to poultry,which has a certain ecological significance as well.