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.

MoS2 nanosheets grown on bowl-shaped hollow carbon spheres as an efficient electrochemical sensor for ultrasensitive determination of nephrotoxic aristolochic acids in Chinese traditional herbs.

Aristolochic acid, a substance in herbs, is highly nephrotoxic, so it is crucial to develop an assay that can rapidly and accurately analyze its content. In this study, bowl-shaped hollow carbon spheres (BHCs) were synthesized using a complex template method, and a MoS2 layer was grown in situ on their surface using a hydrothermal method. The synthesized MoS2-BHCs were used to fabricate an electrochemical sensor for the ultrasensitive and highly selective detection of aristolochic acids (AAs). The optimal conditions for AA detection were determined by tailoring the amount of MoS2 used to modify the BHCs and the pH of the electrolyte. Under optimal conditions, the MoS2-BHC-based sensor presented excellent AA detection performance. The linear concentration ranges of the MoS2-BHC-based sensor for the detection of AA were 0.05-10 µmol L-1 and 10-80 µmol L-1, and the limit of detection of the sensor was 14.3 nmol L-1. Moreover, the MoS2-BHC-based sensor detected AA in Aristolochia and Asarum sieboldii samples. The results were consistent with high-performance liquid chromatography data, demonstrating the satisfactory recovery and accuracy of the sensor. Therefore, we believe that MoS2-BHC-based sensors can be used as effective platforms for detecting AA in traditional Chinese herbs.

Rapid identification and detection of aristolochic acids in the herbal extracts by Raman spectroscopy.

Herbs containing aristolochic acids (AAs) have already been proven to be highly carcinogenic and nephrotoxic. In this study, a novel surface-enhanced Raman scattering (SERS) identification method was developed. Ag-APS nanoparticles with a particle size of 3.53 ± 0.92 nm were produced by combining silver nitrate and 3-aminopropylsilatrane. The reaction between the carboxylic acid group of aristolochic acid I (AAI) and amine group of Ag-APS NPs was used to form amide bonds, and thus, concentrate AAI, rendering it easy to detect via SERS and amplified to obtain the best SERS enhancement effect. Detection limit was calculated to be approximately 40 nM. Using the SERS method, AAI was successfully detected in the samples of four Chinese herbal medicines containing AAI. Therefore, this method has a high potential to be applied in the future development of AAI analysis and rapid qualitative and quantitative analysis of AAI in dietary supplements and edible herbs.

Rapid simultaneous determination of thirteen aristolochic acids analogs in Aristolochiaceae plants by Ultra-High-Performance liquid Chromatography- tandem mass spectrometry in dynamic multiple reaction monitoring mode.

Asarum and Aristolochia are two large genera of Aristolochiaceae plants containing typical toxicant aristolochic acid analogs(AAAs), AAAs can be deemed as toxicity markers of Aristolochiaceae plants. Based on the least AAAs in dry roots and rhizomes of Asarum heterotropoides, Asarum sieboldii Miq and Asarum sieboldii var, all of which are enrolled in the Chinese pharmacopeia up to now. AAAs distribution in Aristolochiaceae plants, especially Asarum L. plants, is still obscure and controversial due to few AAAs measured, unverified species of Asarum, and complicated pretreatment in analytical samples making the results more challenging to reproduce. In the present study, a simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. The sample was prepared by extracting Asarum and Aristolochia powder with methanol, and the supernatant was analyzed using the Agilent 6410 system on an ACQUITY UPLC HSS PFP column with gradient elution of water and acetonitrile, containing 1% v/v formic acid (FA) each, at a flow rate of 0.3 mL/min. The chromatographic condition provided good peak shape and resolution. The method was linear over the specific ranges with the coefficient of determination (R2) > 0.990. Satisfactory intra- and inter-day precisions were achieved with RSD less than 9.79%, and the average recovery factors obtained were in the range of 88.50%～105.49%%. The proposed method was successfully applied for simultaneous quantification of the 13 AAAs in 19 samples from 5 Aristolochiaceae species, especially three Asarum L. species enrolled in the Chinese Pharmacopoeia. Except Asarum heterotropoides, the results supported that the Chinese Pharmacopoeia (2020 Edition) adopting the root with rhizome as medicinal parts of Herba Asari instead of the whole herb for drug safety by providing scientific data.

Broad-specificity indirect competitive enzyme-linked immunosorbent assay for aristolochic acids: Computer-aided hapten design and molecular mechanism of antibody recognition.

Long-term dietary exposure of aristolochic acids (AAs)-contaminated food proved to be one of the main culprits of Endemic Nephropathy, renal failure; and urothelial cancer. The antibodies utilized in immunoassays for AAs suffer from low affinity and failure of recognition to the family of AAs. This study, we prepared a broad-specificity monoclonal antibody (mAb) 5H5 with highly and uniform affinity for AAs by help of computational chemistry fully exposing the AAs common structures of methoxy and hydroxyl groups. The mAb 5H5 exhibited half inhibitory concentrations of AAA, AAB, AAC, AAD were 0.03, 0.06, 0.05, 0.03 ng/mL. To explain the broad-specificity profile of mAb 5H5, molecular docking was performed. Results shown that multiple conformations of AAs can be flexibly oriented in the spacious cavity of single-chain variable fragment antibody (scFv) 5H5 and the specific hydron bonds were formed by ASN62 and GLY64 of scFV 5H5 to the nitro group of AAs which gave an explanation of the high cross-reactivity of mAb 5H5. The ELISA based on the broad-specificity mAb 5H5with detection limits of 0.04-0.11 µg/kg and 0.02-0.06 µg/kg for four AAs in flour and soil samples, respectively. The study provided a promising method for the family of AAs in environmental and food samples.

Aristolochic acid-containing Chinese herbal medicine and upper urinary tract urothelial carcinoma in Taiwan: a narrative review.

PURPOSE: The high incidence of upper urinary tract urothelial carcinoma (UTUC) in Taiwan is largely due to exposure to aristolochic acid (AA), a principal component of Aristolochia-based herbal medicines. Here we systematically review the molecular epidemiology, clinical presentation and biomarkers associated with AA-induced UTUC. METHODS: This is a narrative review. Medline, Embase, and Web of Science were searched from inception to December 31, 2021. Studies evaluating the association, detection, and clinical characteristics of AA and UTUC were included. RESULTS: A nationwide database revealed 39% of the Taiwanese population had been exposed to AA-containing herbs between 1997 and 2003. Epidemiological reports revealed AA posed a significantly higher hazard for renal failure and UTUC in herbalists and the general population who ingested AA-containing herbs. The presence of aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, located predominantly on the non-transcribed DNA strand, with a strong preference for deoxyadenosine in a consensus sequence (CAG), was observed in many UTUC patients. Clinically, AA-related UTUC patients were characterized by a younger age, female gender, impaired renal function and recurrence of contralateral UTUC. To date, there are no preventive measures, except prophylactic nephrectomy, for subjects at risk of AA nephropathy or AA-related UTUC. CONCLUSION: AA exposure via Aristolochia-based herbal medicines is a problem throughout Taiwan, resulting in a high incidence of UTUC. Aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, can be used as biomarkers to identify AA-related UTUC. AA-related UTUC is associated with a high recurrence rate of contralateral UTUC.

Rapid and robust analysis of aristolochic acid I in Chinese medicinal herbal preparations by surface-enhanced Raman spectroscopy.

The use of Chinese herbs containing aristolochic acid can induce the exchange of adenine and thymine in gene mutations and even cause liver cancer. To eliminate the harm of aristolochic acids (AAs) to humans, a rapid and robust method of AAs screening is a prerequisite. In this work, a facile and robust Surface-enhanced Raman spectroscopy (SERS) method was used for the qualitative and quantitative detection of AAs in Chinese medicinal herbal preparations based on the mandelic acid modified Ag nanoparticles SERS substrate. Qualitative and quantitative SERS detection of Aristolochic acid I (AAI) was achieved with a good linear relationship ranging from 0.2 - 120.0 µM and a limit of detection (LOD) of 0.06 µM. The proposed method demonstrates a refined strategy for sensitivity analysis of AAs with the advantages of easy operation, time-saving, high sensitivity, and molecular specificity, making it a preferred platform for the screening of AAI in regular inspections of herbal products and regulatory supervision of the supply chain.

[Risk assessment, safe medication and scientific supervision of traditional Chinese medicine containing aristolochic acids--toxicity is different among aristolochic acids, and detection and control of aristolochic acid â /â ¡ is critical].

The safety problem of traditional Chinese medicine containing aristolochic acid is of great concern in China and abraod, which poses a challenge in clinical application and supervision. There are many types of aristolochic acid analogues(AAAs) and 178 have been reported. According to the structure, they are classified into aristolochic acids(AAs) and aristololactams(ALs). The toxi-city is remarkably different among AAAs of different types. For example, AA-Ⅰ has strong nephrotoxicity and carcinogenicity, and the toxicity of AA-Ⅱ is lower than that of AA-Ⅰ. Besides, AA-Ⅳa and AA-Ⅰa are considered to have no obvious nephrotoxicity and carcinogenicity. The types and content of AAAs are significantly different among traditional Chinese medicines derived from different Aristolochiaceae species. For example, Asari Radix et Rhizoma and Aristolochiae Herba mainly consist of AAAs without obvious toxicity(such as AA-Ⅳa). The content of AAAs in compound preparations is related to the proportions of the medicinals and the processing method. The content of AA-Ⅰ in some compound preparations is very low or below the detection limit. Therefore, the author concludes that AAAs of different types have different toxicity, but not all AAAs has nephrotoxicity and carcinogenicity. Moreover, the toxicity of traditional Chinese medicines containing AAAs should not be generalized and AA-Ⅰ and AA-Ⅱ should be emphasized. In this paper, it is suggested that traditional Chinese medicine containing AAAs should be used rationally and research, analysis, and toxicological study of AAAs species and content should be strengthened. In addition, limit standards of AA-Ⅰ and AA-Ⅱ should be formulated and science-based supervision should be performed.

Analysis of Polycyclic Aromatic Hydrocarbons and Phthalate Esters in Soil and Food Grains from the Balkan Peninsula: Implication on DNA Adduct Formation by Aristolochic Acid I and Balkan Endemic Nephropathy.

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.

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.

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.

Health risk associated with the oral consumption of "Chiniy-tref", a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

Electromembrane extraction of aristolochic acids: New insights in separation of bioactive ingredients of traditional Chinese medicines.

Aristolochic acid (AA) I and AA II, which have been classified as carcinogenic to human and have been proven to be nephrotoxic, are bioactive ingredients of many traditional Chinese medicines (TCMs). Thus, development of an efficient approach for separation and determination of AA I and AA II in biological samples and herbal plants is of significance. Herein, electromembrane extraction (EME) was for the first time used to separate AA I and AA II. It is noted that also for the first time 1-decanol was discovered and used as an efficient SLM solvent for EME of acidic compounds. The proposed EME system was used to extract AA I and AA II from urine samples (recovery≥68%). The approach of EME combined with LC-MS (EME-LC/MS) was evaluated using urine samples. The linear range for AA I and AA II was 10-1000 ng mL-1 (R2≥0.9970), and the limits of detection (LOD, S/N = 3) for AA I and AA II were 2.7 and 2.9 ng mL-1, respectively. Finally, this EME-LC/MS approach was employed to discover AA I and AA II in the herbal plants. In addition, using standard addition method, AA I in Aristolochicaceae-Liao Asarum (ALA) and Radix Aristolochice (RA) were 0.23 and 2044.13 µg g-1, and AA II in ALA and RA were 0 and 338.48 µg g-1, respectively. The repeatability of EME-LC/MS at all cases for both urine samples and herbal plants was below 15% (RSD-value). We believe that EME would be a useful tool to isolate bioactive ingredients of TCMs from complex samples for different purposes.

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.

Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway.

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.

[Risk control of traditional Chinese medicines containing aristolochis acids (AAs) based on influencing factors of content of AAs].

Aristolochic acids (AAs) widely exist in such plants as Aristolochia and Asarum. The renal toxicity of AAs as well as its carcinogenicity to urinary system have been widely known. In 2003 and 2004, China prohibited the use of Aristolochiae Radix, Aristolochiae Manshuriensis Caulis and Aristolochiae Fangchi Radix, and required administering other AAs-containing medicines in accordance with the regulations for prescription drugs. In this paper, we retrieved literatures on the content determination of AAs in recent 10 years in China. It suggested that the AAs content is lower in Asarum herb, especially in its roots and rhizomes, and most of which do not show detectable amount of AA-I. Some of traditional Chinese medicines show fairly small amount of detectable AA-I. The AAs content in Aristolochia herb (including Fructus Aristolochiae, kaempfer dutchmanspipe root) is relatively high; however, there are fewer literatures for studying the content determination of AAs in Chinese patent medicines. There were many factors affecting AAs content, including the parts used, origins, processing methods, extraction process. It suggested that we should pay attention to the toxicity of Chinese medicines containing AAs and use these decoction pieces and traditional Chinese medicines cautiously. In addition, basic studies for the origins, processing methods and extraction process of Chinese patent medicines containing AAs, as well as supervision and detection of AAs content in traditional Chinese medicinal materials, decoction pieces and Chinese patent medicines shall be strengthened for reducing medication risk and guaranteeing clinical medication safety.

Aristolochic Acid in the Etiology of Renal Cell Carcinoma.

BACKGROUND: Aristolochia species used in the practice of traditional herbal medicine contains aristolochic acid (AA), an established human carcinogen contributing to urothelial carcinomas of the upper urinary tract. AA binds covalently to genomic DNA, forming aristolactam (AL)-DNA adducts. Here we investigated whether AA is also an etiologic factor in clear cell renal cell carcinoma (ccRCC). METHODS: We conducted a population-based case-control study to investigate the linkage between Aristolochia prescription history, cumulative AA consumption, and ccRCC incidence in Taiwan (5,709 cases and 22,836 matched controls). The presence and level of mutagenic dA-AL-I adducts were determined in the kidney DNA of 51 Taiwanese ccRCC patients. The whole-exome sequences of ccRCC tumors from 10 Taiwanese ccRCC patients with prior exposure to AA were determined. RESULTS: Cumulative ingestion of more than 250 mg of AA increased risk of ccRCC (OR, 1.25), and we detected dA-AL-I adducts in 76% of Taiwanese ccRCC patients. Furthermore, the distinctive AA mutational signature was evident in six of 10 sequenced ccRCC exomes from Taiwanese patients. CONCLUSIONS: This study strongly suggests that AA contributes to the etiology of certain RCCs. IMPACT: The current study offers compelling evidence implicating AA in a significant fraction of the RCC arising in Taiwan and illustrates the power of integrating epidemiologic, molecular, and genetic data in the investigation of cancer etiology. Cancer Epidemiol Biomarkers Prev; 25(12); 1600-8. ©2016 AACR.

Balkan endemic nephropathy: an update on its aetiology.

Balkan endemic nephropathy (BEN) is a unique, chronic renal disease frequently associated with upper urothelial cancer (UUC). It only affects residents of specific farming villages located along tributaries of the Danube River in Bosnia-Herzegovina, Croatia, Macedonia, Serbia, Bulgaria, and Romania where it is estimated that ~100,000 individuals are at risk of BEN, while ~25,000 have the disease. This review summarises current findings on the aetiology of BEN. Over the last 50 years, several hypotheses on the cause of BEN have been formulated, including mycotoxins, heavy metals, viruses, and trace-element insufficiencies. However, recent molecular epidemiological studies provide a strong case that chronic dietary exposure to aristolochic acid (AA) a principal component of Aristolochia clematitis which grows as a weed in the wheat fields of the endemic regions is the cause of BEN and associated UUC. One of the still enigmatic features of BEN that need to be resolved is why the prevalence of BEN is only 3-7 %. This suggests that individual genetic susceptibilities to AA exist in humans. In fact dietary ingestion of AA along with individual genetic susceptibility provides a scenario that plausibly can explain all the peculiarities of BEN such as geographical distribution and high risk of urothelial cancer. For the countries harbouring BEN implementing public health measures to avoid AA exposure is of the utmost importance because this seems to be the best way to eradicate this once mysterious disease to which the residents of BEN villages have been completely and utterly at mercy for so long.

Quantitation of Aristolochic Acids in Corn, Wheat Grain, and Soil Samples Collected in Serbia: Identifying a Novel Exposure Pathway in the Etiology of Balkan Endemic Nephropathy.

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.

Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals with cancer risk.