Screening And Validation Of Novel Biomarkers For Cadmium-induced Liver Injury Based On Targeted Bile Acid Metabolomics

Objective: This study aims to explore the role of BA in Cd-induced liver injury,and identify reliable and sensitive biochemical indicators for the diagnosis of Cd-induced liver injury.In order to provide clues for further research on the mechanism of Cd-induced liver injury,and provide an effective reference for the prevention and treatment of Cd-induced liver injury in the future.Methods: 1.There had 48 male Sprague-Dawley rats are provided,and divided into 6 groups,which were treated with normal saline or Cd Cl2（2.5,5,10,20,40mg/kg/d）for 12 weeks,respectively.The blood of abdominal aorta,liver,caecal contents,and faeces were collected.The obtained samples were analyzed and determined: liver biochemical and fibrosis indicators,liver pathological degeneration,liver Cd level,BA profiles in liver,serum,caecal contents,and faeces.2.A total of 403 subjects from the DDGXL cohort living in Cd polluted area（n =268）and control area（n = 135）were selected,and their blood were collected.The obtained samples were analyzed and determined: liver injury indices;BCd level;BA profiles in serum.Results: 1.Cd-induced liver injury and its relationship with BA in rats（1）Cadmium caused liver injury and affected the liver BA compositions in ratsAfter Cd exposure at different doses（0,2.5,5,10,20,and 40 mg/kg）,compared with the control group,liver biochemical parameters（AST,ALT and CHE）and liver fibrosis indices（HA and LN）increased significantly in Cd exposure groups（P < 0.05 or 0.01）.Histopathological examination of liver showed that fibrous hyperplasia in portal vein region was observed in the liver tissues of Cd exposure groups,which increased with the increase of Cd exposure dose.In addition,the rats’ liver BA spectrum was significantly changed,and compared with the control group,GCA,GCDCA,G/TUDCA,G/TDCA and G/TLCA increased significantly with the increase of Cd exposure dose（P < 0.05 or 0.01）.These toxic BAs（GCA,GCDCA,G/TUDCA,G/TDCA and G/TLCA）were positively correlated with liver injury indices（AST,ALT,CHE,HA and LN）（P < 0.05 or 0.01）,and negatively correlated with TC（P <0.05 or 0.01）.（2）Screening of targeted BAs for Cd-induced liver injury in ratsAccording to PCA and PLS-DA,it was found that the liver BA spectrum of rats was significantly different in control group,mild Cd-induced liver injury group（2.5,5and 10 mg/kg）and severe Cd-induced liver injury group（20 and 40 mg/kg）.The differences of GUDCA,GLCA,TDCA,and TLCA among the three groups were statistically significant,and their levels increased significantly with the increase of the severity of Cd-induced liver injury（P < 0.05 or 0.01）.In addition,GUDCA,GLCA,TDCA,and TLCA were significantly positively correlated with liver Cd levels（P <0.05 or 0.01）,and ROC curve analysis of these biochemical parameters showed the AUC was 0.899,0.910,0.979,0.954,the sensitivity was 1.000,0.857,1.000,1.000,and the specificity was 0.742,0.871,0.871,0.903,respectively.（3）Cadmium affected BA compositions in rat’ serum,caecal contents,and faecesTotal bile acid and hepatotoxic BAs（GCA,GCDCA,G/TUDCA,G/TDCA,and G/TLCA）increased with the increase of Cd exposure dose in liver and serum（P <0.05 or 0.01）.There was a significant positive correlation between the same BAs in the rat’ serum and liver,between BAs and liver injury indices（P < 0.05 or 0.01）.However,the changes in the caecal contents and faecal BAs were not the same as those in the liver.2.Validation of targeted BAs for Cd-induced liver injury in humansSerum BAs（GUDCA,GLCA,TDCA,and TLCA）were significantly positively correlated with BCd level and liver biochemical indicators（AST,ALT,TBIL,DBIL,IBIL,ALP,GGT,and CHE）（P < 0.05 or 0.01）,indicating that these four BAs can reflect the BCd level and liver function abnormalities in humans.Specifically,the levels of GUDCA,GLCA,TDCA and TLCA increased significantly with the increase of BCd level（P < 0.05 or 0.01）,and ROC curve analysis of these biochemical parameters showed the AUC was 0.868,0.776,0.826,0.731,the sensitivity was 0.875,0.788,0.733,0.945,and the specificity was 0.761,0.663,0.750,0.489,respectively.Conclusion: Cd exposure caused cholestasis and increased toxic BAs,leading to liver injury.Among them,GUDCA,GLCA,TLCA,and TDCA were expected to be potential biomarkers for early detection of Cd-induced liver injury in rats.Furthermore,serum GUDCA,GLCA,TDCA,and TLCA were verified to be of value to evaluate Cd-induced liver injury and Cd exposure level in humans.These findings provided evidence for screening and validation of additional biomarkers for Cd-induced liver injury based on targeted BA metabolomics.