Inadvertent exposure to aristolochic acids (AAs) is causing chronic renal disease worldwide, with aristolochic acid I (AA-I) identified as the primary toxic agent. This study employed chemical methods to investigate the mechanisms underlying the nephrotoxicity and carcinogenicity of AA-I. Aristolochic acid II (AA-II), which has a structure similar to that of AA-I, was investigated with the same methods for comparison. Despite their structural similarities, findings from cultured human cells and gut sac experiments showed that AA-I is absorbed more effectively than AA-II (similar to 3 times greater for AA-I than for AA-II; p < 0.001). This increased absorption, along with the previously observed higher activity of reductive activation enzymes for AA-I, results in greater DNA damage and oxidative stress, both of which are key factors in AA-related toxicity. The similar patterns of cell mortality (34.4 +/- 2.3% vs 9.7 +/- 0.1% for AA-I and AA-II at 80 mu M; p < 0.0001), DNA adduct formation (similar to 3 times greater for AA-I than for AA-II; p < 0.001), and oxidative stress levels in relation to the concentrations of AA-I and AA-II indicate that the higher absorption rate of AA-I is a significant contributor to its greater toxicity. The toxicity of AA-I was also found to be further enhanced by its (natural) coexistence with AA-II. Since AA-I and AA-II differ only by a methoxy group, future research on reducing risks associated with AA exposure should focus on strategies to lower the absorption of these compounds.

Balkan endemic nephropathy (BEN) is a chronic kidney disease that predominantly affects inhabitants of rural farming communities along the Danube River tributaries in the Balkans. Long-standing research has identified dietary exposure to aristolochic acids (AAs) as the principal toxicological cause. This study investigates the pathophysiological role of anemia in BEN, noting its earlier and more severe manifestation in BEN patients compared to those with other chronic kidney diseases. Utilizing a mouse model, our research demonstrates that prolonged exposure to aristolochic acid I (AA-I) (the most prevalent AA variant) leads to significant red blood cell depletion through DNA damage, such as DNA adduct formation in bone marrow, prior to observable kidney function decline. Furthermore, in vitro experiments with kidney cells exposed to lowered oxygen and pH conditions mimicking an anemia environment show enhanced DNA adduct formation, suggesting increased AA-I mutagenicity and carcinogenicity. These findings indicate for the first time a positive feedback mechanism of AA-induced anemia, DNA damage, and kidney impairment in BEN progression. These results not only advance our understanding of the underlying mechanisms of BEN but also highlight anemia as a potential target for early BEN diagnosis and therapy.