Aristolochic acid I promoted clonal expansion but did not induce hepatocellular carcinoma in adult rats

Aristolochic acid I (AAI) is a well-known nephrotoxic carcinogen, which is currently reported to be also associated with hepatocellular carcinoma (HCC). Whether AAI is a direct hepatocarcinogen 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. 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. We showed that oral administration of single dose of AAI (20, 50, or 100 mg/kg) in combination with partial hepatectomy (PH) to stimulate liver proliferation did not induce typical GST-P⁺ foci in liver. In the 8-week study, only high dose of AAI (10 mg · kg⁻¹ · d⁻¹, 5 days a week for 6 weeks) in combination with PH significantly increased the number and area of GST-P⁺ foci initiated by diethylnitrosamine (DEN) in liver. Similarly, only high dose of AAI (10 mg· kg⁻¹· d⁻¹, 5 days a week for 52 weeks) in combination with PH significantly increased the number and area of hepatic GST-P⁺ foci in the 52-week study. No any nodules or HCC were observed in liver of any AAI-treated groups. In contrast, long-term administration of AAI (0.1, 1, 10 mg· kg⁻¹· d⁻¹) time- and dose-dependently caused death due to the occurrence of cancers in the forestomach, intestine, and/or kidney. Besides, AAI-DNA adducts accumulated in the forestomach, kidney, and liver in a time- and dose-dependent manner. Taken together, AAI promotes clonal expansion only in the high-dose group but did not induce any nodules or HCC in liver of adult rats till their deaths caused by cancers developed in the forestomach, intestine, and/or kidney. Findings from our animal studies will pave the way for further large-scale epidemiological investigation of the associations between AA and HCC.