Application Of High-Resolution Mass Spectrometry Combined With Omics In Early Detection Of 3-MCPD Esters-Induced Nephrotoxicity

Fatty acid esters of 3-monochloropropane 1,2-diol?3-MCPD?are a group of processing-induced toxicants,which have become a real food safety concern due to their nephrotoxicity,whereas the molecular mechanism?s?and early detection of their nephrotoxicity remains unclear.Considering the fact that more than 85%of 3-MCPD esters were diesters in human foods,and high content of 3-MCPD dipalmitate in food,this study choosed 3-MCPD dipalmitate as a candidate compound to investigated the biomarker for early detection of 3-MCPD dipalmitate-induced nephrotoxicity in vivo,which based on the study of the nephrotoxic effect,toxicokinetics and metabonomics used high-resolution mass spectrometry and nontargeted metabonomics.1.This study investigated whether and how necroptosis may play a role in the nephrotoxic effect of 3-MCPD dipalmitate?2.5 g/kg BW?in C57BL/6 mice.The results showed that 3-MCPD dipalmitate induced nephrotoxicity by necroptosis and inflammation via activated the receptor-interacting protein 1?RIPK1?/RIPK3/mixed lineage kinase domain-like protein?MLKL?signaling pathway.In addition,miRNA analysis revealed that 38 and 40 differentially expressed miRNA?DE-miRNA?of known miRNAs and novel miRNAs were detected in 3-MCPD dipalmitate-induced nephrotoxicity.Of these DE-miRNAs,miR-223-3p was significantly up-regulated in kidney during 3-MCPD dipalmitate exposed.The luciferase reporter assay further confirmed that miR-223-3p was able to inhibit RIPK3expression by directly targeting the 3?un-translated region?3?UTR?of RIPK3,suggestting that the nephrotoxic effect induced by 3-MCPD dipalmitate may be attenuated by miR-223-3p.2.The present study was conducted to investigate the toxicokinetics of3-MCPD dipalmitate at a dose of 1.6 g/kg BW in Sprague Dawley?SD?.As the results,the calculated mean of peak plasma concentration(C_max)was135.00 ng/mL,which occured at 2.5 hours(T_max:time to reach C_max)after an oral gavage of 1.6 g/kg BW 3-MCPD dipalmitate.The elimination half-life(T_1/2),mean residence time?MRT?,clearance rate?CL?,and volume of distribution?V_d?were 3.87 h,5.08 h,3.50 L/h/g and 21.34 L/g,respectively.The total area under the plasma concentration-time curve(AUC_0-?)for 3-MCPD dipalmitate in rat plasma is 458.47 h ng/mL on average.The distribution of 3-MCPD dipalmitate in tissues,such as brain and testicle,might explain its suggesting their potential to pass through the blood-brain barrier and blood-testis barrier.A total of 17 metabolites were identified,and16 of that were reported for the first time,these metabolites were metabolites of phase I reactions?hydrolysis and dichlorination etc.?and phase II reactions?endogenous substance conjugation etc.?.The phase I metabolites could further undergo possible conjugation reactions to form more polar metabolites for urine excretion?phase II metabolites?.In addition,3-MCPD diesters and the other metabolites might excrete with faeces.The prototype and metabolites of 3-MCPD dipalmitate cannot exist in the body circulation for a long time,and their concentrations in blood are at ng/mL level or lower,so that they are not suitable biomarkers for early detection of3-MCPD dipalmitate-induced nephrotoxicity.3.For early detection of 3-MCPD dipalmitate-induced nephrotoxicity,the high-resolution mass spectrometry combined with nontargeted metabonomics analysis was using for identification of potential biomarkers in SD rat.Twelve significant differential metabolites were detected and identified in plasma,including indoxyl sulfate,phenol sulphate,p-cresol sulfate,2-phenylethanol glucuronide,p-cresol glucuronide,p-cresol,allantoin,phenylacetylglycine,pyrocatechol sulfate,phenyllactic acid,5-hydroxyindoleacetic acid and creatinine.Combination with the different metabolites in the four important tissues?kidney,liver,spleen and testis?,3-MCPD dipalmitate was supposed to disturb the amino acid?phenylalanine,tryptophan,tyrosine and glycine?,fatty acid,purine and gut microbiota metabolism.In addition,the biomarkers found in plasma were high stability,sensitivity and significant change metabolites,suggesting that the non-invasive measurement of these biomarkers in plasma might be a surrogate for early detection of 3-MCPD esters-induced nephrotoxicity.