Synergistic Interaction of Polycyclic Aromatic Hydrocarbons, Phthalate Esters, or Phenol on DNA Adduct Formation by Aristolochic Acid I: Insights into the Etiology of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with chronic exposure to aristolochic acids (AAs) through AA-contaminated food being one of the major etiological mechanisms. However, the bulk of previous research has only focused on investigating the possible roles of individual pollutants in disease development and the etiological mechanism of BEN remains controversial. In this study, we investigated the exposure concentration and duration dependence of coexposure to phthalate esters and lignite coal-derived phenol and polycyclic aromatic hydrocarbons (PAHs) on the metabolism and DNA adduct formation of aristolochic acid I (AAI). Results showed that both the metabolic activation and DNA adduct formation of AAI in cultured human kidney cells were affected by their coexposure to the above-mentioned environmental pollutants. Furthermore, our results suggest that chemicals leached from lignite coal likely played a role by triggering AA-activating enzymes to produce more of the promutagenic DNA adducts, thus further elevating the nephrotoxicity and carcinogenicity of AAs and increasing the risk of BEN. It is believed that the results of this study provide a better understanding of the etiological mechanism of BEN and offer insights into methods and policies to lower the risk of this devastating disease.

Quantitation of Protein Adducts of Aristolochic Acid I by Liquid Chromatography-Tandem Mass Spectrometry: A Novel Method for Biomonitoring Aristolochic Acid Exposure.

Emerging evidence suggests that chronic exposure to aristolochic acids (AAs) is one of the etiological pathways leading to chronic kidney disease (CKD). Due to the traditional practice of herbal medicine and AA-containing plants being used extensively as medicinal herbs, over 100 million East Asians are estimated to be at risk of AA poisoning. Given that the chronic nephrotoxicity of AAs only manifests itself after decades of exposure, early diagnosis of AA exposure could allow for timely intervention and disease risk reduction. However, an early detection method is not yet available, and diagnosis can only be established at the end stage of CKD. The goal of this study was to develop a highly sensitive and selective method to quantitate protein adducts of aristolochic acid I (AAI) as a biomarker of AA exposure. The method entails the release of protein-bound aristolactam I (ALI) by heat-assisted alkaline hydrolysis, extraction of ALI, addition of internal standard, and quantitation by liquid chromatography-tandem mass spectrometric analysis. Accuracy and precision of the method were critically evaluated using a synthetic ALI-containing glutathione adduct. The validated method was subsequently used to detect dose-dependent formation of ALI-protein adducts in human serum albumin exposed to AAI and in proteins isolated from the tissues and sera of AAI-exposed rats. Our time-dependent study showed that ALI-protein adducts remained detectable in rats even at 28 days postdosing. It is anticipated that the developed method will fill the technical gap in diagnosing AA intoxication and facilitate the biomonitoring of human exposures to AAs.

Occurrence and Environmental Stability of Aristolochic Acids in Groundwater Collected from Serbia: Links to Human Exposure and Balkan Endemic Nephropathy.

Aristolochic acids (AAs) have been known as potent nephrotoxins since the use of AA-containing herbal medicines was linked with a series of sporadic renal fibrotic nephropathy cases, and yet an estimated 100 million people worldwide are still at risk today because of continued use of similar medicines. However, a similar nephropathic condition is endemic in the rural Balkan regions (e.g., Serbian farming villages) and AAs were again found to be the causative agents. In the case of this Balkan endemic nephropathy, AAs were found to have originated from a widespread local weed Aristolochia clematitis L. In this study, we tested the hypothesis that AAs released from decomposition of A. clematitis were also being leached into groundwater, thus polluting the drinking water of local residents. We initiated the study by developing a dispersive solid-phase extraction-based sample preparation method for water samples suspected of AA contamination. The validated method was then coupled with a liquid chromatography-tandem mass spectrometric method to measure AAs in groundwater samples collected from Serbia. Our study revealed for the first time that groundwater in Serbia is extensively contaminated with AA-I, at ng/L levels. Results also showed that AAs are long-lived water contaminants, with no observable concentration changes over a 2-month period of sample storage.