Correlation Analysis Of Aristolochic Acid Exposure And Hepatocellular Carcinogenesis Based On Clinical Samples And Animal Experiments

Objective:Liver cancer is a malignant tumor with high incidence in china,among which hepatocellular carcinoma(HCC)is the most important pathological type.The etiology of liver cancer is complex,closely related to hepatitis B virus(HBV)in China.In 2017,Science Translational Medicine reported that the high incidence of liver cancer in Taiwan and other Asian regions is related to aristolochic acid(AA),which has attracted high social attention.AA is a common component of Aristolochiaceae,which is widely distributed in China,and some herbs containing AA are widely used in China.Domestic and foreign studies have identified AA as nephrotoxic and carcinogens in the urinary system,but have never listed the liver as a target organ.Based on the above background,this study aims to explore whether AA exposure is a direct cause of HCC,and to elaborate whether there is a synergistic effect between HBV infection and AA.In addition,it provides scientific data for the prevention and control of HCC and the safety risk assessment of AA related traditional Chinese medicine.Methods:In this study,we used aristolochic acid-DNA adduct to determine the AA exposure in 772 HCC samples.Meanwhile,whole genome sequencing(WGS)of 51 HCC samples were carried out.The single nucleotide variants(SNV)and high frequency mutation gene of HCC samples were obtained by sequencing data,and the A>T mutation rate of HBV-associated and non-HBV-associated HCC samples were also analyzed.In addition,we also used TCGA public database to obtain high-throughput sequencing data for multiple tumors.Subsequently,sequencing data were used for pathway enrichment analysis of tumor prognosis-related genes and the interaction network of prognostic-related genes.We used adult mouse models to observe the effect of AA-I on body weight,total survival rate,and tumor incidence in mice.The effect of AA-I on hepatic fibrosis was observed by Sirius red staining of mouse liver.AA-I combined with CCl₄ were used to observe whether AA-I further aggravate liver fibrosis in mice and to compare the effects of AA-I on HCC in the context of normal liver and liver fibrosis.The proportion of A>T mutations in liver and forestomach of mice were analyzed by WGS.Besides,we also examined the accumulation of AA-DNA adducts in different organs of mice after AA-I administration.Tg HBV-C57BL/6J mice and Tg HBV-BALB/c mice were given AA-I to observe whether HBV affected AA-I causing liver fibrosis,tumorigenesis,overall survival and AA-DNA adducts in mice and to explore the interaction between AA-I and HBV.In this study,C57 mice of 14 days were injected with AA-I to explore their genotoxicity to liver.Then,we will observe whether AA-I causes liver cancer in infant mice model,and sequence the liver of infant mice to analyze the rate of A>T mutation.In addition,we also tested the AA-DNA adducts in the liver and kidney of infant mice model.Moreover,through AA-I combined CCl₄to observe whether the CCl₄ promotes the HCC in infant mice model,further explore the difference between AA-I and common chemical inducers of liver cancer.Results:The positive rate of AA-DNA adduct in clinical multicenter HCC samples was only 4.27%.Besides,the rate of A>T mutation in clinical HCC samples was not high,and there was no significant difference in the proportion of A>T mutation between HBV-associated HCC and non-HBV-associated HCC.In addition,genes such as TP53,CTNNB1,TTN,MUC4 and MUC16 are high-frequency mutations in HCC samples.T cell receptor signaling pathway related genes were found to be associated with favorable outcomes through the TCGA database.We found that genes such as ACTN4,PTK2 and TNFRSF10B were associated with adverse outcomes,whereas NFKBIA,PRKCD and TOLLIP were associated with favorable outcomes.The liver is not the main target organ of AA-I in adult mouse models.AA-I could significantly reduce the weight of mice,and high dose AA-I could cause increase of ALT and AST.But AA-I does not cause liver fibrosis in adult mice,nor does it further aggravate liver fibrosis caused by CCl₄.Besides,forestomach is the main carcinogenic organ of AA-I in adult mouse models,and AA-I combined with CCl₄ does not increase the incidence of HCC.In adult mice,forestomach carcinoma have a large number of A>T mutations and liver have a small amount of AA-DNA adducts.We observed that AA-I had less effect on liver in transgenic mice than wild type,and AA-I did not aggravate liver fibrosis caused by CCl₄ in transgenic mice.There was no significant statistical difference in the incidence of HCC between transgenic mice and wild type.AA-I can induce HCC at 16 months in infant mice model and there are a lot of A>T mutations in HCC.In addition,the kidney is the target organ for formation of AA-DNA adducts.Moreover,AA-I combined with CCl₄ promote HCC in infant mice model.The carcinogenic effect of AA-I in infant mice model is comparable to di-ethylnitrosoamine(DEN),and the carcinogenic effect of AA-I increases with increasing dose.Conclusion:This study found that AA-DNA adducts had a low positive rate in large samples of HCC.The proportion of A>T mutation in HCC samples was not high,and there was no significant difference between HBV and non-associated HCC samples.These results indicate that AA may be not the main cause of HCC in our study.In adult mouse models,AA-I did not induce HCC and HBV have no synergistic effect with AA-I on the development of HCC.However,AA-I have a strong carcinogenic effect on liver of infant mice,so such populations should still be careful to use Chinese medicinal materials containing AA.