??Background
Aristolochic acids (AAs) are a potent class of environmental mutagens found in plants of the genus Aristolochia. Human exposure occurs through the intentional use of traditional herbal medicines or through inadvertent dietary intake from contaminated crops. Although AA exposure has long been linked to renal and other cancers, the scale, sources, and geographic boundaries of exposure remain elusive. Recent findings from the Mutographs Grand Challenge¹ indicate that both the prevalence and mutational burden of AA-associated signatures in Romania are substantially higher than previously estimated, suggesting widespread exposure of largely unknown origin with important public health implications. Scalable approaches to systematically detect and monitor AA exposure across populations are therefore needed.
Objective
To establish a focused mutational epidemiology framework integrating tumor whole-genome sequencing (WGS) and high-resolution epidemiological data to map the spatial, temporal, and environmental footprint of AA exposure and inform cancer prevention.
Methods
We assembled a three-part series to capture AA exposure across contemporary and historical timeframes and diverse geographies. First, we established a newly recruited prospective collection of 240 renal cell carcinoma (RCC) cases collected between 2020 and 2024 from high-risk and neighboring regions in Romania, Serbia, Ukraine, Hungary, Greece, Bulgaria, and Croatia. This contemporary series was complemented by a well-curated biorepository of 536 RCC European cases collected between 2006 and 2016, previously reported by Mutographs¹. To assess potential environmental exposure, we additionally collected normal kidney and liver tissues from environmentally exposed animals (cows and sheep; n=50) in Eastern Romania, the region with the highest observed prevalence of AA-associated signatures.
All human cases underwent whole-genome sequencing and were accompanied by harmonized epidemiological data, including residential history, lifestyle factors such as herbal remedy use, and external exposure variables, including historical distribution of Aristolochia plants and regional soil characteristics.
Results
AA-associated mutational signatures were highly prevalent in RCC from Central and Eastern Europe, with approximately 90% (84/93) of renal cancers from Romania showing clear evidence of AA exposure. Signatures exhibited strong geographic clustering, with the highest burden in Eastern Romania and a decreasing gradient into Bulgaria (40%; 2/5), Serbia (57%; 44/77), and Croatia (10%; 2/20). In contrast, AA-associated signatures were largely absent in cases from Hungary, Ukraine, and Greece, delineating clear boundaries of the exposure zone. Signature burden varied by birth cohort and residential history, consistent with long-term and potentially ongoing exposure. Distinct sub-patterns within SBS22 (SBS22a, SBS22b, and the newly identified SBS22c) suggest contributions from multiple AA analogues or metabolic derivatives, pointing to a more complex exposure landscape. Ongoing integration of geospatial data and animal tissue analyses may clarify further a potential environmental route of exposure beyond individual herbal use.
Conclusions
AA exposure in parts of Central and Eastern Europe is more extensive, persistent, and heterogeneous than previously recognized. This study establishes a scalable mutational surveillance framework that integrates genomic, environmental, and spatial data to reconstruct carcinogenic exposures at the population level, providing a strong foundation for targeted cancer prevention in high-risk regions and a model for monitoring environmental mutagens.
References
?1. Nature 629, 910 (2024) (PMID: 38693263)