Study On The Mutagenicity And Carcinogenicity Of Aristololactam Ⅰ And On The Mechanism Of PIG-A Gene Mutation

Aristolochic acids(AAs)and their derivatives have been used as compounds in many traditional Chinese medicine products for thousands of years.It is widely recognized that AAs induce renal toxicity and tumors of the urinary tract,and have been classified as a Group 1 human carcinogen,by the International Agency for Research on Cancer(IARC).The mechanism of action of AAs-induced urinary tract tumors is not fully understood.Current studies have found that AAs are metabolized to aristolactams nitrogen ions,which react with DNA to form DNA adducts(AAs-DNA adducts).The AAs-DNA adducts are difficult to be metabolized and accumulated in the body chronically,resulting in mutations of the DNA fingerprint of AAs:A:T→T:A transversion mutations.AAs consist of two major compounds,aristolochic acid I(AA-I)and aristolochic acid II(AAⅡ).Studies have shown that both AA-I and AA-II are genetic toxicants,while AA-I is the main cause of nephrotoxicity.The Pharmacopoeia of the People’s Republic of China 2015 and 2020 editions only provide limit standards of AA-I.Aristololactam I(AL-I),the main metabolite of AA-I,presents in plants such as Aristolochiaceae,Annonaceae,Northern Leguminosae,Pepperaceae,Rosaceae,and Perillaceae.In vitro and in vivo animal tests have preliminarily confirmed that,at the same concentration or dose level,AL-I is more toxic to the kidney cells than AA-I,and the toxic effects of AL-I are more serious.However,so far,very few relevant research is reported on the genotoxicity or carcinogenicity of AL-I.ALs was found to cause chromosome aberration in vitro in our previous study,and to form DNA adducts in mice.Based on the findings,this study aims to evaluate the mutagenicity of AL-I with the in vitro PIG-A gene mutation assay on the basis of a standard battery of genotoxicity tests.Moreover,multiple genetic endpoints such as mutagenicity,clastogenicity,and DNA damage were included in a 26-week carcinogenicity test with transgenic animals,to evaluate the genetic toxicity and carcinogenicity of AL-I,and further clarify the basis and the potential mechanism of the carcinogenicity of AAs,and provide a certain theoretical basis for the use and supervision of AL-I-containing botanicals.This study was divided into two parts:First part:The mutagenicity of AA-I and AL-I was evaluated using our early established in vitro PIG-A gene mutation assay with TK6 human lymphoblastoid cells assay.Then the potential carcinogenic effects of AL-I were evaluated in the CB6F1-Tg.rasH2 transgenic mice.The mice were dosed orally with AL-I once daily for 26 weeks.At the same time,CB6F1-Non-Tg.rasH2 wild-type mice were included in a concurrent toxicokinetic study,in which the toxicokinetic profiles of AL-I were studied.In addition,a Pig-a gene mutation and micronucleus analysis in peripheral blood,and comet analysis with multiple organs were integrated into the carcinogenicity study in CB6F1-Tg.rasH2 transgenic mice.To support the target organ and interpretation of toxicokinetic results of AL-I,a mouse tissue distribution test was performed.Second part:On the basis of the first part,the mechanism of action of PIG-A gene mutation and the relationship between phenotypic results and genotype mutations in the PIG-A gene mutation assay with TK6 cells were explored using Next Generation Sequencing(NGS)technology,providing a theoretical basis to assess the effectiveness of in vitro PIG-A gene mutation assay.The main results were as follows:AA-I induced a positive response for PIG-A mutation in TK6 cells in vitro under 4h+S9 condition and a negative response under 4h+S9 and 24h-S9 conditions.In the 26-week transgenic carcinogenicity test of AL-I via oral administration in Tg.rasH2 transgenic mice,no AL-I related changes in clinical chemistry,organ weight,or tumor lesions were found.The toxicity following AL-I treatment included emaciation,decreases of body weight or weight gains,and vacuolation of renal tubules that were non-tumor lesions.Negative results were obtained in the accompanied blood micronucleus,blood Pig-a,or comet(blood,liver,and kidney)assays.Validated analysis method confirmed that there was no AL-I exposure in plasma in the concurrent toxicokinetic study and the tissue distribution assay also indicated that the small amout of AL-I into the blood was distributed in the tissues rapidly.In the whole genome sequencing(WGS)analysis,among the 12 sorted PIG-A positive clones,4/12 cases were PIG-L mutations,2/12 cases were PIG-A mutations,and 2/12 cases were PIG-V mutations.Thus,X-linked PIG-A gene mutation,autosome-linked PIG-L and PIG-V gene point or deletion mutations contribute to the glycosylphosphatidylinositol(GPI)anchor defect.These results suggested that in PIG-A mutagenicity studies in TK6 cells,more types of detectable mutations could be analyzed.The conclusions were drwan as follows:Under the conditions of this study,AL-I induced a negative response in the in vitro PIGA mutation assay in TK6 cells and showed no carcinogenicity on Tg.rasH2 transgenic mice.Detection of concurrent genotoxicity endpoints also demonstrated no mutagenicity,clastogenicity or DNA damage.Furthermore,the effectiveness of the test system of in vitro PIG-A mutation assay in TK6 cells was validated from the mutation mechanism.