Subchronic Toxicity Study Of 3-chloropropane-1,2-diol Esters In Rats

3-chloro-1,2-propanediol esters,also known as 3-MCPD esters,are the fatty acid ester compounds formed by free 3-chloropropanol and long chain fatty acids.3-MCPD esters are potential toxic substances formed during food thermal processing.Therefore,the safety of 3-MCPD esters has attracted extensive attention in the food science community.However,up to now,the subchronic toxicity mechanisms of 3-MCPD esters are not clear.The researches on the changes of endogenous substances caused by long-term exposure of model animal to 3-MCPD esters are also scarce.In this study,two representative 3-MCPD monoesters,3-MCPD1-stearate and 3-MCPD 1-oleate were selected as the research objects to simulate the possible dosage conditions of 3-MCPD ester in daily diet.First,high purity monomer 3-MCPD 1-stearate and 3-MCPD 1-oleate were synthesized,and their structures and purities were confirmed by liquid chromatography-mass spectrometry(LC-MS)and nuclear magnetic resonance spectroscopy(NMR).Then,Sprague-Dawley(SD)rat was used as the model animal to investigate their subchronic toxicity.Pathological results showed that 3-MCPD 1-stearate and 3-MCPD1-oleate had similar toxic effects under low and high dose conditions,among which the long-term toxic target organs were mainly kidney and testis.3-MCPD esters also had specific damage to thymus and lung,and caused a small amount of inflammation in liver and intestinal tissue,and the toxic effects were dose-dependent.In addition,this study also showed that compared with those of the control group,the kidney injury related indicators such as serum uric acid,urea nitrogen and creatinine and the inflammatory indicators such as IFN-? and TGF-? were significantly up-regulated in 3-MCPD esters treated groups,the testis injury related indicator such as testosterone was significantly down-regulated in3-MCPD esters treated groups.These changes were in a dose-dependent manner.To further investigate the subchronic toxic mechanisms of 3-MCPD esters in vivo,proteomics approaches were employed to examine the changes of related proteins in kidney and testis tissues of rats after oral administration of 3-MCPD esters.GO and KEGG pathway analysis were used to evaluate the related protein changes between the control and3-MCPD esters treated groups.Proteomics results showed that 3-MCPD1-stearate and 3-MCPD 1-oleate had similar subchronic nephrotoxicity and testicular toxicity.Proteins deregulated in kidney by 3-MCPD esters were mainly related to ion transport,which leaded to the accumulation of calcium ions,thus triggering cell apoptosis and causing the activation of II enzymes and transport enzymes.Various endogenous biological metabolism pathways also showed significant changes,including carbohydrate,amino acid,nitrogen,lipid and fatty acid metabolism,as well as the tricarboxylic acid cycle.In addition,it was also found that the toxicity of 3-MCPD esters was mainly attribuited to cell necrosis induced by inflammation in testes.Furthermore,after reducing the expression of related genes by si RNA interference technology,rat renal tubular duct epithelial cell NRK-52 E model was used to verify the molecular mechanisms of renal toxicity caused by endogenous apoptosis mediated by Caspase-9,and mice Leydig cell TM3 model was used to verify the molecular mechanisms of testicular toxicity induced by cell necrosis mediated by RIPK1 and MLKL.In addition,the endogenous metabolisms of 3-MCPD esters in rats was preliminarily investigated in this study.Through high resolution mass spectrometry combined with non-targeted metabonomics analysis,the biological toxic markers induced by 3-MCPD esters were screened,and11 biomarkers were identified in rats' plasma,urine and/or feces,including L-homocysteic acid,4-heptanone,N-acetyl-L-phenylalanine,L-gamma-glutamyl-L-isoleucine,3-keto-2-methylbutyrate,methylmalonylcarnitine,Lyso PC,elaidic carnitine,15-deoxy-d-12,14-PGJ2,13,14-dihydro PGF-1? and tetrahydrodeoxycortisol.These biological markers provide scientific basis for subchronic exposure diagnosis and safety evaluation of 3-MCPD esters.