Genotoxic effects of aristolochic acid I on functional human-induced hepatocyte-like cells

Aristolochic acid I (AAI) is a carcinogen associated with various human cancers. However, its causal relationship with hepatocellular carcinoma remains controversial, and inconsistent results from rodent studies have suggested species-specific differences. Here we evaluated AAI genotoxicity using functional human-induced hepatocyte-like cells (hiHep cells), a model that closely mimics primary human hepatocytes in gene expression and function, thereby shedding light on its potential hepatocarcinogenic risk in humans. First, we assessed AAI genotoxicity by evaluating AAI-DNA adducts and micronucleus frequency. In hiHep cells, AAI (0.7-2.5 µM) induced up to 105 adducts per 108 nucleotides, indicating high metabolic activation of AAI. A concentration-dependent increase in micronucleus frequency indicated a significant increase in chromosomal aberrations in hiHep cells. Considering the evidence of AAI inducing oxidative stress, we assessed 8-hydroxy-2'-deoxyguanosine and reactive oxygen species levels to evaluate DNA oxidative damage. For both indicators, significantly elevated levels were observed. A mechanism involving oxidative damage was further supported by observations of mitochondrial dysfunction, including changes in mitochondrial membrane potential and mitochondrial complex activity. Ascorbate treatment decreased AAI-induced oxidative DNA damage and DNA adduct formation, providing direct cellular evidence for free radical intermediates in AAI metabolic activation-a mechanism previously hypothesized but not experimentally validated in a human-relevant hepatocyte model. Our study findings revealed the genotoxic effects of AAI on hiHep cells and implicated oxidative stress as the key mechanism. These findings strengthen the association between AAI exposure and liver disease and highlight the potential role of antioxidant therapies in mitigating AAI-associated carcinogenesis.