Study On The Molecular Pathological Mechanism Of Aristolochic Acid I Promoting Liver Damage (precancerous Lesions

Objective:Aristolochic acid is a mixture of nitrophenanthrene organic acids extracted from Aristolochia genus,Asarum genus and other Aristolochia family plants.Its main components include Aristolochic acid Ⅱ(AAⅠ)and its demethoxy derivative Aristolochic acid Ⅱ(AAⅡ).Traditional Chinese medicine,patent medicine and prescription containing aristolochic acid(AA)are widely used in clinic at present.Aristolochic acid I(AAⅠ)is a well-known nephrotoxic carcinogen and have a have high carcinogenic risk to upper urinary tract epithelium.Reductive metabolism of AA with DNA formed AA-DNA adducts combined with accumulation of AA-DNA adducts induced gene mutations,the key to carcinogenicity,was corroborated by report.Aristolochian acid trigger gene mutations in RAS(mouse)and TP53(human)which progress to tumors furtherly.However,AA was currently reported to be also associated with hepatocellular carcinoma(HCC)which caused the internal and external discussions and speculation on the potential safety risks of traditional Chinese medicine.Whether AAⅠ is a direct hepatocarcinogen or just performing as the indirect promoter remains controversial.In this study we investigated the association between AAⅠ exposure and HCC in adult rats using a sensitive rat liver bioassay with several cofactors.To seek the relationship betwwn dose level and toxicity of AA.To definite the carcinogenicity of AA and its derivatives exposure and to identifiy the toxicitiy and target organs.Providing the reference for the clinic medication to monitior and supervise the carcinogenicity of Aristolochic acid.Method:In this study we investigated the association between AAⅠ exposure and HCC in adult rats using a sensitive rat liver bioassay(Ito module)with several cofactors at different time point(single dose,8 weeks,16-26 weeks and 27-52 weeks).Formation of glutathione S-transferase placental form-positive(GST-P)foci was used as the marker for preneoplastic changes/clonal expansion.The number and area of GST-P positive foci in liver were compared by digital image analysis of whole sections,and the distribution and positive rate of nuclear proliferation antigen(PCNA)in liver transformation foci were analyzed.Hepatocellular apoptosis inside and outside foci was estimate by TUNEL.Content of Aristolochic acid DNA adduct(DA-AL-I)in liver,kidney and stomach were analyzed by LC-MS/MS.The mutation characteristics of TP53 gene and the enrichment of AT-TA transposition inside and peri GST-P positive were collected and analyzed by using combitation of laser microscopy,polymerase chain reaction and Sanger sequencing technology.More than 60 genes,including oncogenic promoter genes,apoptosis and necrosis genes,and chromosome modification were compared and verified by wholegenome sequencing.The total number of mutations,mutation sites and the enrichment of AT-TA transversion in gene mutations were investigated.Results:Formation of glutathione S-transferase placental form-positive(GST-P)foci was used as the marker for preneoplastic lesions/clonal expansion.We first conducted a medium-term(8 weeks)study to investigate whether AAⅠ had any tumor-initiating or-promoting activity.Then a long-term(52 weeks)study was conducted to determine whether AAⅠ can directly induce HCC.Oral administration of single dose of AAⅠ at100 mg/kg did not induce GST-P positive foci after 6 weeks.The content of DNA adduct in kidney is higher than that in liver.Single administration of high dose AA had no hepatocarcinogenesis initiating.Oran administratin of AAⅠ for 6 weeks in combination with PH induced significantly increased number of number and area of GST-P positive foci with dose-dependent relationship.Content of AA-DNA adduct was also high in kidney than in liver.The mutation frequency of TP53 increased significantly in DEN+AAⅠ 10mg/kg GST-P positive transformation focus,and AT-TA inversion was the main mutation form of TP53.The results of whole gene analysis showed that AA could increase the number of mutations in oncogenes IRF2,KMT2 D,PCNA,STATE3,ALB,BAD and CXCR1,and the main types of mutations were C-T andA-G,but there was no significant increase in the number of mutations in genes related to apoptosis and physiological function of necrotic cells.Unscheduled die occurred at 16 th weeks for the rat adimistrated AAⅠ at 10 mg/kg in combination with PH meanwhile at 25 th weeks for the rat whitout combination of PH.AAⅠ-DNA adducts accumulated in the forestomach,kidney and liver with a time-and dose-dependent trendency and highest in the kidney,followed by the liver and the stomach.Conclusion:[1] The accumulation of AAⅠ-DNA adducts and the key gene mutation TP53 are important biomarkers for estimateion the toxicity of AAⅠ.[2] AAⅠ work as preneoplastic lesions promoter to promotes clonal expansion only at10 mg/kg with GST-P positive foci in adult rats.No clonal expansion changes was found in rats at 0.1 mg/kg.[3] AA mediate the liver injury with a dose-dependent trendency and caused the found dead in rat at 10mg/kg for the neplastice lesion in forestomach,intesiten and in kidney.[4] AAⅠ can trigger hepatocelluar necrosis in adult rats without induced any nodules or HCC in the liver microsocpically which indicated that AA can promote the occurrence of precancerous lesions under the premise of liver injury.[5] AAⅠ can increase the number of oncogene mutations,C-T andA-G are the main types of mutations.