Exposure to aristolochic acids (AAs) from Aristolochia plants is one of the major global causes of nephropathy, renal failure and urothelial cancer, including Balkan endemic nephropathy (BEN). The high incidence of BEN on the Balkan Peninsula is assumed to result from consumption of Aristolochia clematitis L. seeds co-harvested with crops. Here we show that AAs are long-lived soil contaminants that enter wheat and maize plants by root uptake, with strong pH-dependence. Soil and crops from Serbian farms in areas endemic for A. clematitis were found to be extensively contaminated with AAs, with contamination strongly correlated with local incidence of BEN. The persistence of AAs as soil contaminants suggests that weed control for A. clematitis plants is needed to reduce the incidence of BEN and aristolochic acid nephropathy, and that systematic surveys of soil and crop AA levels would identify high-risk regions and it is imperative to research soil remediation methods.

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Aristolochic acid released from decaying Aristolochia clematitis weed is contaminating soil and food crops in Eastern Europe and is one of the major causes to Balkan endemic nephropathy. Measures should be taken to prevent people from being exposed to these highly potent phytotoxins. Research needs to develop remediation methods.

Aristolochic acids (AAs) are nitrophenanthrene carboxylic acids naturally produced by Aristolochia plants. These plants were widely used to prepare herbal remedies until AAs were observed to be highly nephrotoxic and carcinogenic to humans. Although the use of AA-containing Aristolochia plants in herbal medicine is prohibited in countries worldwide, emerging evidence nevertheless has indicated that AAs are the causative agents of Balkan endemic nephropathy (BEN), an environmentally derived disease threatening numerous residents of rural farming villages along the Danube River in countries of the Balkan Peninsula. This perspective updates recent findings on the identification of AAs in food as a result of the root uptake of free AAs released from the decayed seeds of Aristolochia clematitis L., in combination with their presence and fate in the environment. The potential link between AAs and the high prevalence of chronic kidney diseases in China is also discussed.

Emerging evidence suggests that cross-links formed by reacting DNA lesions with proteins may play a significant role in the pathophysiology of human cancer and degenerative diseases. The goal of this study was to develop a method involving liquid chromatography-tandem mass spectrometry (LC–MS/MS) coupled with the stable isotope-dilution method to quantify DNA–protein cross-link (DPC). A novel type of cross-link involving a S-glycosidic linkage formed by reacting an abasic site in DNA with the cysteine residues in protein was targeted in this study. The method entails hydrolysis of the cross-link to a 2′-deoxyribose-cysteine adduct, addition of isotopically labeled internal standard, and quantitation by LC–MS/MS analysis. The accuracy and precision of the method were evaluated with a synthetic peptide containing the cross-link. The validated method was then applied to quantitate the levels of the DNA–protein cross-link in vitro and in HeLa cells exposed to alkylating agent methylmethanesulfonate (MMS). The analysis detected dosage-dependent formation of the cross-link in both purified DNA (6.0 ± 0.6 DPC per 106 nt μM–1 MMS) and in human cells (7.8 ± 1.2 DPC per 106 nt mM–1 MMS). With the abasic site being one of the most common DNA lesions produced continuously by multiple pathways, the results provide significant new knowledge for better understanding the potential biological implications of its associated DNA–protein cross-link.

Rationale Over the past six decades, residents of farming villages in multiple countries of the Balkan peninsula have been suffering from a unique type of chronic renal disease, Balkan endemic nephropathy (BEN). It was speculated that environmental pollution by aristolochic acids (AAs) produced naturally by Aristolochia clematitis L., a weed that grows in the area, was causing the disease. However, the human exposure pathway to this class of phytotoxin remains obscure. Knowledge of the sink and stability of AAs in the environment would assist in the formulation of policy reducing exposure risk. Methods Using our newly developed liquid chromatography/tandem mass spectrometry method of high sensitivity and selectivity, we analysed over 130 soil samples collected from cultivation fields in southern Serbia for the presence of AAs. The environmental stability of AAs was also investigated by incubating soil samples spiked with AAs at various temperatures. Results The analysis detected AA‐I in over two‐fifths of the tested samples at sub‐μg/kg to μg/kg levels, with higher concentrations observed in more acidic farmland soil. Furthermore, analysis of soil samples incubated at various temperatures revealed half‐lives of over 2 months, indicating that AAs are relatively resistant to degradation. Conclusions Cultivation soil in southern Serbia is being extensively contaminated with AAs released from the decomposition of A. clematitis weeds. Since AAs are resistant to degradation, it is possible that AAs could have been taken up by root absorption and transported to the edible part of food crops. Prolonged exposure to AA‐contaminated food grown from polluted soil could be one of the main aetiological mechanisms of BEN observed in the area.

N 6 -Formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational N 6formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentrationdependent formation of FLys in cells exposed to formaldehyde and Fe 2+ -EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe 2+ -EDTA produced FLys at a frequency of 20.2 and 4.1 per 10 4 lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed Escherichia coli cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time-and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic N 6 -formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of N 6 -formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.

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