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Aristolochic acids (AAs) are nephrotoxic and carcinogenic nitrophenanthrene carboxylic acids produced naturally by plants from the Aristolochia and Asarum genera, which have been used extensively as herbal medicines. In addition to consuming AA-containing herbal medicinal products, there is emerging evidence that humans are also exposed to AA through the environment. In 2022, the World Health Organization (WHO) called for global action to remove AA exposure sources and to implement preventative measures against the development of AA-associated cancers. Herein, we report the development of a simple and efficient iron powder-packed reduction column that allows online post-column conversion of the nonfluorescing AA to its corresponding strongly fluorescing aristolactam (AL), facilitating the sensitive and selective detection of AA in herbal medicinal products, food grain, arable soil, or groundwater samples by high-performance liquid chromatography with fluorescence detection. Moreover, AL, a group of naturally occurring derivatives of AA that have demonstrated toxicity to cultured bacteria, human cells, and rats, is monitored and quantified simultaneously with AA in one single run without sacrificing sensitivity. In comparison with existing analytical methods for AA measurement, the newly developed method is not only inexpensive and less laborious, but it also offers improved sensitivity. We believe this novel method will find wide application in identifying the presence of AA in food, herbal medicines, and environmental samples, thus assisting in the identification and removal of AA exposure sources.

Prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicine or AA-contaminated food is associated with the development of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), both public health risks to which the World Health Organization is calling for global action to remove exposure sources. The AA exposure-induced DNA damage is believed to be related to both the nephrotoxicity and carcinogenicity of AA observed in patients suffering from BEN. While the chemical toxicology of AA is well- studied, we investigated in this study the understated effect of different nutrients, food additives, or health supplements on DNA adduct formation by aristolochic acid I (AA-I). By culturing human embryonic kidney cells in an AAI-containing medium enriched with different nutrients, results showed that cells cultured in fatty acid-, acetic acid-, and amino acid-enriched media produced ALI- dA adducts at significantly higher frequencies than that cultured in the normal medium. ALI-dA adduct formation was most sensitive to amino acids, indicating that amino acid- or protein-rich diets might lead to a higher risk of mutation and even cancer. On the other hand, cells cultured in media supplemented with sodium bicarbonate, GSH, and NAC reduced ALI-dA adduct formation rates, which sheds light on their potential use as risk-mitigating strategies for people at risk of AA exposure. It is anticipated that the results of this study will help to better understand the effect of dietary habits on cancer and BEN development.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy affecting residents of rural farming areas in many Balkan countries. Although it is generally believed that BEN is an environmental disease caused by multiple geochemical factors with much attention on aristolochic acids (AAs), its etiology remains controversial. In this study, we tested the hypothesis that environmental contamination and subsequent food contamination by polycyclic aromatic hydrocarbons (PAHs) and phthalate esters are AA toxicity factors and important to BEN development. We identified significantly higher concentrations of phenanthrene, anthracene, diethyl phthalate (DEP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) in both maize and wheat grain samples collected from endemic villages than from nonendemic villages. Other PAHs and phthalate esters were also detected at higher concentrations in the soil samples from endemic villages. Subsequent genotoxicity testing of cultured human kidney cells showed an alarming phenomenon that phenanthrene, DEP, BBP, and DBP can interact synergistically with AAs to form elevated levels of AA-DNA adducts, which are associated with both the nephrotoxicity and carcinogenicity of AAs, further increasing their disease risks. This study provides direct evidence that prolonged coexposure to these environmental contaminants via dietary intake may lead to greater toxicity and accelerated development of BEN. © 2021 American Chemical Society.

Aristolochic acids (AAs) are persistent soil pollutants in the agricultural fields of the Balkan Peninsula that are endemic for Aristolochia clematitis L. This class of carcinogenic and nephrotoxic phytotoxins is taken up by crops through root absorption and contaminates staple foods across the peninsula. Human exposure to AAs via dietary intake has recently been recognized as a cause of Balkan endemic nephropathy. For the sake of public health, human exposure to AAs from all sources should be minimized in a timely manner. However, currently, there is no available remediation method to remove AAs from soil. In this study, we developed the first soil remediation method for AAs using Fenton's reagent (FR), a combination of ferrous ion and hydrogen peroxide, and optimized factors, including pH, temperature, time, and dose of FR, to achieve the best degradation performance. The maximum AA degradation efficiency was found to be >97% in soil with 500 μg kg-1 of AAs. We anticipate that this developed method, mediated via Fenton reaction, will be useful to effectively eliminate AAs from the Balkan farmlands. © 2020 Elsevier B.V.

Balkan endemic nephropathy (BEN) is a slowly progressive interstitial fibrotic disease affecting numerous people living along the Danube River in the Balkan Peninsula, of which aristolochic acids (AAs) produced naturally in Aristolochia plants are key etiological agents. However, the exposure biology of the disease remains poorly understood. Initially, the high incidence of BEN in the Balkan Peninsula was thought to occur through ingestion of bread prepared from flour made with wheat grains comingled with the seeds of Aristolochia clematitis L., an AA-containing weed that grows abundantly in the wheat fields of the affected areas. In this study, by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, we show for the first time that vegetables, in particular root vegetables of endemic areas, are extensively contaminated with AAs taken up through root absorption from the AA-tainted soil. Furthermore, we found a pH dependence of the n-octanol/water partition coefficient (Kow) of AAs, which resulted in a dramatically higher hydrophobicity-driven plant uptake efficiency of AAs into food crops in endemic areas, characterized by higher acidity levels, compared to non-endemic areas. We believe the results of this study have significantly unraveled the mystery surrounding the uneven distribution of BEN incidence.

While to date investigations provided convincing evidence on the role of aristolochic acids (AAs) in the etiology of Balkan endemic nephropathy (BEN) and upper urothelial cancer (UUC), the exposure pathways by which AAs enter human bodies to cause BEN and UUC remain obscure. The goal of this study is to test the hypothesis that environmental pollution by AAs and root uptake of AAs in the polluted soil may be one of the pathways by which AAs enter the human food chain. The hypothesis driving this study was that the decay of Aristolochia clematitis L., a AA-containing herbaceous plant that is found growing widespread in the endemic regions, could release free AAs to the soil, which could be taken up by food crops growing nearby, thereby transferring this potent human nephrotoxin and carcinogen into their edible parts. Using the highly sensitive and selective high-performance liquid chromatography coupled with fluorescence detection method, we identified and quantitated in this study for the first time AAs in corn, wheat grain, and soil samples collected from the endemic village Kutles in Serbia. Our results provide the first direct evidence that food crops and soil in the Balkans are contaminated with AAs. It is possible that the presence of AAs in edible parts of crops originating from the AA-contaminated soil could be one of the major pathways by which humans become exposed to AAs.

While to date investigations provided convincing evidence on the role of aristolochic acids (AAs) in the etiology of Balkan endemic nephropathy (BEN) and upper urothelial cancer (UUC), the exposure pathways by which AAs enter human bodies to cause BEN and UUC remain obscure. The goal of this study is to test the hypothesis that environmental pollution by AAs and root uptake of AAs in the polluted soil may be one of the pathways by which AAs enter the human food chain. The hypothesis driving this study was that the decay of Aristolochia clematitis L., a AA-containing herbaceous plant that is found growing widespread in the endemic regions, could release free AAs to the soil, which could be taken up by food crops growing nearby, thereby transferring this potent human nephrotoxin and carcinogen into their edible parts. Using the highly sensitive and selective high-performance liquid chromatography coupled with fluorescence detection method, we identified and quantitated in this study for the first time AAs in corn, wheat grain, and soil samples collected from the endemic village Kutles in Serbia. Our results provide the first direct evidence that food crops and soil in the Balkans are contaminated with AAs. It is possible that the presence of AAs in edible parts of crops originating from the AA-contaminated soil could be one of the major pathways by which humans become exposed to AAs.

Aristolochic acids (AAs) are nephrotoxic and carcinogenic derivatives found in several Aristolochia species. To date, the toxicity of AAs has been inferred only from the effects observed in patients suffering from a kidney disease called "aristolochic acid nephropathy" (AAN, formerly known as "Chinese herbs nephropathy"). More recently, the chronic poisoning with Aristolochia seeds has been considered to be the main cause of Balkan endemic nephropathy, another form of chronic renal failure resembling AAN. So far, it was assumed that AAs can enter the human food chain only through ethnobotanical use (intentional or accidental) of herbs containing self-produced AAs. We hypothesized that the roots of some crops growing in fields where Aristolochia species grew over several seasons may take up certain amounts of AAs from the soil, and thus become a secondary source of food poisoning. To verify this possibility, maize plant (Zea mays) and cucumber (Cucumis sativus) were used as a model to substantiate the possible significance of naturally occurring AAs' root uptake in food chain contamination. This study showed that the roots of maize plant and cucumber are capable of absorbing AAs from nutrient solution, consequently producing strong peaks on ultraviolet HPLC chromatograms of plant extracts. This uptake resulted in even higher concentrations of AAs in the roots compared to the nutrient solutions. To further validate the measurement of AA content in the root material, we also measured their concentrations in nutrient solutions before and after the plant treatment. Decreased concentrations of both AAI and AAII were found in nutrient solutions after plant growth. During this short-term experiment, there were much lower concentrations of AAs in the leaves than in the roots. The question is whether these plants are capable of transferring significant amounts of AAs from the roots into edible parts of the plant during prolonged experiments.