Fabrication of a Simple and Efficient HPLC Reduction Column for Online Conversion of Aristolochic Acids to Aristolactams Prior to Sensitive Fluorescence Detection.

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.

Effects of Diet on Aristolochic Acid-DNA Adduct Formation: Implications for Balkan Endemic Nephropathy Etiology.

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.

Polyurethane Foam Face Masks as a Dosimeter for Quantifying Personal Exposure to Airborne Volatile and Semi-Volatile Organic Compounds.

Airborne volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs) are commonly quantitated by collecting the analytes on solid sorbent tubes or passive air samplers, followed by solvent extraction and instrumental analysis, or by grab bag/canister measurements. We report herein a user-friendly sampling method by breathing through polyurethane foam (PUF) face masks to collect airborne VOCs and SVOCs for chemical analysis. Specifically, dibasic esters, phthalate esters, polycyclic aromatic hydrocarbons, linalool, and nicotine trapped on PUF masks were quantitated by gas chromatography-mass spectrometry analysis as model VOCs and SVOCs. Results showed that the amount of these model VOCs and SVOCs trapped on PUF masks is proportional to the exposure duration. After cross-validation by parallel sampling using XAD-2 packed sorbent tubes, the method was used to quantitate VOCs and SVOCs in a variety of indoor and outdoor environments with varying air concentrations of analytes, temperature, humidity, and wind speed. Because air pollution is considered a major cause of many human diseases and premature deaths and the developed PUF mask sampling method showed high trapping efficiencies for both VOCs and SVOCs, it is believed that the developed sampling method will find wide application in assessing air pollution-associated disease risks with possible extension to more classes of VOCs and SVOCs when coupled with suitable instrumental detection methods.

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.

Analysis of aristolochic acids in Houttuynia cordata by liquid chromatography-tandem mass spectrometry.

Houttuynia cordata (H. cordata) is a popular vegetable in Asian countries and is also used extensively as herbal medicine in treating various diseases. H. cordata contains aristolactams, which have a similar Chinese name as aristolochic acids (AAs); hence, an emerging concern in the greater China region has arisen about the potential linkage between H. cordata and aristolochic acid nephropathy (AAN). However, only a single study has tested for the presence of AAs in H. cordata samples, and the analysis was limited by the analytical sensitivity of the method. Thus, further analysis of AAs in H. cordata using analytical method of higher sensitivity is needed to alleviate public anxiety over the use of this popular vegetable. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to analyze H. cordata samples for the natural existence of aristolochic acid I (AA-I) and aristolochic acid II (AA-II), which are the most carcinogenic and nephrotoxic compounds in the AA family. After evaluating the method performance by fortifying blank samples with three concentrations of AAs, the validated method was applied to identify AA-I and AA-II in both fresh and sun-dried H. cordata samples (n = 20) collected from different cities in China. The LC-MS/MS method achieved method detection limits (MDLs) as low as 2 ng/g of AAs in H. cordata. Analysis of the collected fresh and sun-dried H. cordata samples revealed that AA-I and AA-II either do not exist naturally in H. cordata or exist at concentrations below the MDLs. Therefore, it is not very likely that consumption of H. cordata will result in AAN because AA-I and AA-II, the nephrotoxic and carcinogenic culprits of AAN, are not produced naturally in the plant or are produced at levels that do not pose a risk of AAN.

Determination of Aristolochic Acids in Vegetables: Nephrotoxic and Carcinogenic Environmental Pollutants Contaminating a Broad Swath of the Food Supply and Driving Incidence of Balkan Endemic Nephropathy.

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.

Remediation of aristolochic acid-contaminated soil by an effective advanced oxidation process.

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.

Quantitation of DNA Adducts of Aristolochic Acids in Repair-Deficient Cells: A Mechanistic Study of the DNA Repair Mechanism.

Accumulating evidence has revealed that nephrotoxic and carcinogenic aristolochic acids (AAs) released from decaying Aristolochia clematitis L. weeds are soil and food grain contaminants in the Balkan Peninsula, while AA toxicity has been linked to induced DNA damage. In this study, we investigated the DNA repair mechanism that excises the aristolactam-DNA adducts in gene-knockout Escherichia coli cells. These results demonstrated that cell lines deficient in nucleotide excision repair (NER) machinery accumulated higher adduct levels, indicating that NER is the major mechanism responsible for the repair of these lesions. Furthermore, data also revealed the involvement of base excision repair enzymes in repairing the lesions but with lower contribution than NER.

Identifying Cysteine, N-Acetylcysteine, and Glutathione Conjugates as Novel Metabolites of Aristolochic Acid I: Emergence of a New Detoxification Pathway.

There is accumulating evidence that Balkan endemic nephropathy (BEN) is an environmental disease caused by aristolochic acids (AAs) released from the decomposition of Aristolochia clematitis L., an AA-containing weed that grows abundantly in the Balkan Peninsula. AA exposure has also been associated with carcinoma development in the upper urinary tract of some patients suffering from BEN. It is believed that an aristolactam-nitrenium ion intermediate with a delocalized positive charge produced in the hepatic metabolism of AAs binds to DNA and the resulting DNA adduct is responsible for initiating the carcinoma development process. In this study, we demonstrated for the first time that the aristolactam-nitrenium ion intermediate will also react with endogenous aminothiols, for example, cysteine, N-acetylcysteine, and glutathione in vitro, and in rats, producing phase II-conjugated metabolites in a dosage-dependent manner. It is highly possible that this conjugation process consumes and ultimately deactivates this carcinogenic intermediate and acts as an important, but previously unreported, detoxification mechanism of AAs. Results also showed AAs, phase I metabolites, and the aminothiol-conjugated metabolites are rapidly eliminated from AA-exposed rats. Furthermore, we found evidence that AA exposure induced oxidative stress in rats, as indicated by the glutathione depletion in rat serum samples.

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.

Liquid chromatography-tandem mass spectrometry analysis of aristolochic acids in soil samples collected from Serbia: Link to Balkan endemic nephropathy.

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.

Quantitation of N6-Formyl-lysine Adduct Following Aristolochic Acid Exposure in Cells and Rat Tissues by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method.

N6-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 N6-formylation 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 concentration-dependent formation of FLys in cells exposed to formaldehyde and Fe2+-EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe2+-EDTA produced FLys at a frequency of 20.2 and 4.1 per 104 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 N6-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 N6-formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.

Development of a novel liquid chromatography-tandem mass spectrometric method for aristolochic acids detection: Application in food and agricultural soil analyses.

Prolonged dietary exposure to AA-contaminated food crops cultivated in contaminated farmland was shown to be one of the main culprits of Balkan endemic nephropathy. Ultra-sensitive methods for the detection of AA at trace levels are important for surveillance purposes. We report the development of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for AA-detection. One striking feature of the method is that the Zn/H+-induced nitroreduction convert AA into their respective aristolactams, which dramatically enhances their analytical sensitivity. The method was applied to quantitate AA in food grains and soil samples collected from farming villages of Serbia, and flour samples purchased from supermarkets. To the best of our knowledge, this is the first report of detecting AA in food ingredients available in supermarkets, which is alarming because it reveals AA poses insidious threats not only to residents of rural farming villages, but also to people residing in urban areas.

Aristolochic Acids: Newly Identified Exposure Pathways of this Class of Environmental and Food-Borne Contaminants and its Potential Link to Chronic Kidney Diseases.

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.

Quantification of a Novel DNA-Protein Cross-Link Product Formed by Reacting Apurinic/Apyrimidinic Sites in DNA with Cysteine Residues in Protein by Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

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.

Quantitation of DNA Adducts in Target and Nontarget Organs of Aristolochic Acid I-Exposed Rats: Correlating DNA Adduct Levels with Organotropic Activities.

Chronic exposure to aristolochic acids (AAs) from Aristolochia plants is one of the major causes of nephropathy and cancer of the kidney and forestomach. However, the organotropic activities of AAs remain poorly understood. In this study, using LC-MS/MS coupled with a stable isotope-dilution method, we rigorously quantitated for the first time the organ-specific dosage- and time-dependent formation of DNA-AA adducts in the tumor target and nontarget organs of AA-I-treated rats. The results support the proposal that the DNA adduct level is a major contributor to the observed organotropic activities of AAs.

Aristolochic Acids as Persistent Soil Pollutants: Determination of Risk for Human Exposure and Nephropathy from Plant Uptake.

Exposure to aristolochic acids (AAs) from Aristolochia plants is one of the major global causes of nephropathy, including Balkan endemic nephropathy (BEN); renal failure; and urothelial cancer. The high incidence of BEN on the Balkan Peninsula is assumed to result from consumption of Aristolochia clematitis L. seeds coharvested 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, systematic surveys of soil and crop AA levels would identify high-risk regions, and it is imperative to research soil-remediation methods.

Etiology of Balkan Endemic Nephropathy: An Update on Aristolochic Acids Exposure Mechanisms.

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.

Cooking methods employing natural anti-oxidant food additives effectively reduced concentration of nephrotoxic and carcinogenic aristolochic acids in contaminated food grains.

Emerging evidence suggests that aristolochic acids (AA) produced naturally by a common weed Aristolochia clematitis in the cultivation fields is contaminating the food products in Balkan Peninsula and acting as the etiological agent in the development of Balkan endemic nephropathy. In this study, we investigated the combined use of natural anti-oxidative "food additives" and different cooking methods to find a solution for the widespread contamination of AA in food products. The results indicated that the addition of healthy dietary supplements (such as cysteine, glutathione, ascorbic acid, citric acid and magnesium) during cooking, is a highly efficient method in lowering the concentration of AA in the final food products. Because previous observation indicated one of the toxicological mechanisms by which AA exert its toxicity is to induce oxidative stress in internal organs, it is anticipated that these added anti-oxidants will also help to attenuate the nephrotoxicity of AA.

Single-Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate-Dependent Catalysis in Vitamin†K Biosynthesis.

MenD, or (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase, uses a thiamine diphosphate (ThDP)-dependent tetrahedral Breslow intermediate rather than a canonical enamine for catalysis in the biosynthesis of vitamin K. By real-time monitoring of the cofactor chemical state with circular dichroism spectroscopy, we found that a new post-decarboxylation intermediate was formed from a multistep process that was rate limited by binding of the α-ketoglutarate substrate before it quickly relaxed to the characterized tetrahedral Breslow intermediate. In addition, the chemical steps leading to the reactive post-decarboxylation intermediates were not affected by the electrophilic substrate, isochorismate, whereas release of the product was found to limit the whole catalytic process. Moreover, these intermediates are likely kinetically stabilized owing to the low biological availability of isochorismate under physiological conditions, in contrast to the tight coupling of enamine formation with binding of the electrophilic acceptor in some other ThDP-dependent enzymes. Together with the unusual tetrahedral structure of the intermediates, these findings strongly support a new ThDP-dependent catalytic mode distinct from canonical enamine chemistry.