Mechanisms of Aristolochic Acid Resistance in Specialist Butterflies and Evolutionary Insights for Potential Protective Pathways

Aristolochic acids (AAs) are natural compounds found in Aristolochiaceae plants, to which humans are frequently exposed through environmental and medicinal sources. AAs are highly nephrotoxic and carcinogenic, mediated by oxidative stress and bioactivation-induced DNA damage and mutagenicity. Nevertheless, some Lepidoptera, including Pachliopta aristolochiae, feed exclusively on Aristolochiaceae and sequester AAs as a chemical defense. This is uncommon in nature and it is not yet fully understood how these insects avoid the lethal effects of AAs. To address this question, we investigate Pac. aristolochiae's AA-resistance mechanisms by employing metabolic analyses, multiomics analyses, in situ imaging and more. Our findings indicate that AAs may be detoxified through biotransformation and a robust antioxidant system, involving candidate genes such as 15-oxo-prostaglandin 13-reductases (PGRs), cytochrome P450s, and catalases. Unexpectedly, DNA adducts, the covalent binding products from activated AA intermediates, are detected across most life stages of Pac. aristolochiae, revealing that Pac. aristolochiae can maintain genomic integrity despite a substantial burden (reaching over 1800 AA-DNA adducts per 10⁸ nucleotides in adults, approximately 1 adduct per 55 000 nucleotides). Interestingly, however, no detectable DNA adducts are observed in wing discs, a representative organ undergoing metamorphosis, and AAs in testes are confined to somatic but not germ cells. Therefore, the strategies to protect against AA-induced mutagenicity likely include restricted AA distribution in critical tissues and enhanced DNA repair. Using butterflies as an evolutionary model, we identify PTGR1, the human PGR homolog, as a potential target of AA resistance, which is associated with human acute kidney injury. Validation in human cells further demonstrates its role in reducing AA-induced cytotoxicity and lipid peroxidation. Our study highlights insect AA tolerance as a means to discover human protective mechanisms, thereby suggesting new avenues for preventing AA-related diseases.