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Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSA MYR ) and its AA complexes (HSA MYR /AA-I and HSA MYR /AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 10 6  M -1 for HSA/AA-I versus 8.4 and 9.0 · 10 5  M -1 for HSA/AA-II. Fluorescence quenching of the HSA Trp 214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents 1,2 . TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications 3 . Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and G i1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Aristolochic acids (AAs) are naturally occurring genotoxic carcinogens linked to Balkan endemic nephropathy and aristolochic acid nephropathy. Aristolochic acid I and II (AA-I and AA-II) are the most abundant AAs, and AA-I has been reported to be more genotoxic and nephrotoxic than AA-II. This study aimed to explore metabolic differences underlying the differential toxicity. We developed a novel microdialysis sampling coupled with solid-phase extraction liquid chromatography-tandem mass spectrometry (MD-SPE-LC-MS/MS) to simultaneously study the toxicokinetics (TK) of AA-I and AA-II and their corresponding aristolactams (AL-I and AL-II) in the blood of Sprague Dawley rats co-treated with AA-1 and AA-II. Near real-time monitoring of these analytes in the blood of treated rats revealed that AA-I was absorbed, distributed, and eliminated more rapidly than AA-II. Moreover, the metabolism efficiency of AA-I to AL-I was higher compared to AA-II to AL-II. Only 0.58% of AA-I and 0.084% of AA-II was reduced to AL-I and AL-II, respectively. The findings are consistent with previous studies and support the contention that differences in the in vivo metabolism of AA-I and AA-II may be critical factors for their differential toxicities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Previous reports demonstrated that aristolochic acids (AAs) exposure-induced nephrotoxicity, mutations, and tumorigenesis are mainly due to aristolochic acid I (AAI). Notably, the chemical structure of aristolochic acid IVa (AAIVa), which exists at higher levels in many Aristolochiaceae herbs, is extremely similar to AAI. In lack of toxicological data, it is unknown whether AAIVa exposure leads to aristolochic acid nephropathy (AAN), mutations, and tumorigenesis as of AAI. To answer these questions, mice were administered AAIVa by single or repeated long-term gavage, while AAI was used as a positive control. We found that single gavage of 40 mg/kg of AAIVa exhibited no obvious toxicity. Also, there were no tumors or death in mice administrated with 1 and 10 mg/kg of AAIVa for 6 months followed by a 12-month recovery time. There were no noteworthy alterations in gene mutation frequency in the kidney, liver, and stomach between the AAIVa and control mice. Fascinatingly, AA-associated mutational signatures, adenine-to-thymine (A>T) transversions, were absent in AAIVa-treated mice. Nonetheless, 10 mg/kg of AAIVa triggered lymphocytic infiltration and slight fibrous hyperplasia in the kidney at the 6 th month; however, these were alleviated at the 12 th and 18 th months. On the contrary, AAI (positive control) caused severe diffuse fibrosis, tubular atrophy, necrosis, tumors in the forestomach and kidney, and death after the 6 th month. It seems that long-term AAIVa exposure induced mild renal lesions could be due to the activation of the canonical or noncanonical transforming growth factor-β (TGFβ) pathway. Overall, these findings suggest that the mutagenicity and carcinogenic risk of AAIVa are very low., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aristolochic acids are known for nephrotoxicity, and implicated in multiple cancer types such as hepatocellular carcinomas demonstrated by recent studies. Natural products that are analogues to aristolochic acids have been constantly isolated from organisms; a larger chemical space of these compounds and a wider coverage of biological sources should be determined in consideration of the potential hazard of aristolochic acid analogues and the wide distribution of their biological sources in the nature. Therefore, we carried out an in silico research of naturally occurring aristolochic acid analogues and their biological sources, as a supplement to existing studies. The result shows a chemical space of 238 naturally occurring aristolochic acid analogues that are present in 175 species of biological sources including 44 traditional medicines. With the computational estimation for toxicity and the implication in hazard assessment of a biological source with the presence of aristolochic acid analogues, we propose that additional awareness should be raised to the public for avoidance of toxic species, especially those that are used as herbal medicines and easily accessible.

Aristolochic acid analogues (AAAs), naturally existing in herbal Aristolochia and Asarum genera, were once widely used in traditional pharmacopeias because of their anti-inflammatory properties, but lately they were identified as potential nephrotoxins and mutagens. A method for rapid characterization of AAAs in serum was developed using ion mobility spectrometry coupled with mass spectrometry (IMS-MS). Five AAAs, containing four aristolochic acids and one aristolactam, were separated and identified within milliseconds. AAAs were separated in gas phase based on the difference of their ion mobility (K 0 ), and then identified based on their K 0 values, m/z, and product ions from MS/MS. Quantitative analysis of AAAs was performed using an internal standard with a satisfactory sensitivity. Limits of detection (signal-to-noise = 3) and quantification (signal-to-noise = 10) were 1-5 ng/mL and 3-8 ng/mL, respectively. The method was validated and successfully applied to the pharmacokinetics study of AAAs in rats, offering a promising way for fast screening and evaluation of AAAs in biological samples.

Aristolochia triangularis Cham., is one of the most frequently used medicinal plant in Southern Brazil. Preparations containing the leaves and/or stems are traditionally used as anti-inflammatory, diuretic, as well as antidote against snakebites. This study screened A. triangularis extracts, fractions and isolated compounds for different bioactivities. A weak antiproliferative activity against human lung cancer cell line (A549) was observed only for chloroform fraction obtained from stems (CFstems - CC50: 2.93 µg/mL). Also, a moderate antimicrobial activity against Staphylococcus aureus was detected just for chloroform fraction obtained from leaves (CFleaves -13-16 mm inhibition zone). Additionally, two semi-purified fractions (CFstems-4 and CFleaves-4) selectively inhibited HSV-1 replication (IC50 values of 0.40 and 2.61 µg/mL, respectively), while only CFleaves showed promising results against Leishmania amazonensis. Fractionation of extracts resulted in the isolation of one neolignan (-) cubebin and one lignan (+) galbacin. However, these compounds are not responsible for the in vitro bioactivities herein detected. The presence of aristolochic acid I and aristolochic acid II in the crude ethanol extract of stems (CEEstems) and leaves (CEEleaves) was also investigated. The HPLC analysis of these extracts did not display any peak with retention time or UV spectra comparable to aristolochic acids I and II.

The TGF- β and Wnt/ β -catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF- β has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF- β signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF- β type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF- β receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF- β receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/ β -catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF- β receptor impaired β -catenin activity in vitro and in vivo Genetically restoring β -catenin activity in proximal tubules lacking the TGF- β receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF- β receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of β -catenin activity or an indirect effect of β -catenin interacting with other growth factors. In conclusion, blocking TGF- β and β -catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD., (Copyright © 2017 by the American Society of Nephrology.)

Aristolochic acid (AA) is a plant alkaloid that causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases frequently associated with upper urothelial cancer (UUC). This review summarizes the significance of AA-derived DNA adducts in the aetiology of UUC leading to specific A:T to T:A transversion mutations (mutational signature) in AAN/BEN-associated tumours, which are otherwise rare in individuals with UCC not exposed to AA. Therefore, such DNA damage produced by AA-DNA adducts is one rare example of the direct association of exposure and cancer development (UUC) in humans, confirming that the covalent binding of carcinogens to DNA is causally related to tumourigenesis. Although aristolochic acid I (AAI), the major component of the natural plant extract AA, might directly cause interstitial nephropathy, enzymatic activation of AAI to reactive intermediates capable of binding to DNA is a necessary step leading to the formation of AA-DNA adducts and subsequently AA-induced malignant transformation. Therefore, AA-DNA adducts can not only be utilized as biomarkers for the assessment of AA exposure and markers of AA-induced UUC, but also be used for the mechanistic evaluation of its enzymatic activation and detoxification. Differences in AA metabolism might be one of the reasons for an individual's susceptibility in the multi-step process of AA carcinogenesis and studying associations between activities and/or polymorphisms of the enzymes metabolising AA is an important determinant to identify individuals having a high risk of developing AA-mediated UUC., Competing Interests: The authors declare no conflict of interest.

Background: Aristolochiae Fructus (AF) and honey-fried Aristolochiae Fructus (HAF) have been used in China for a long time as anti-tussive and expectorant drugs. Few clinical cases have been reported to be associated with the toxicity of AF and HAF, although relatively high amounts of aristolochic acids (AAs) have been found in them. Our previous experiments have verified from the chemical changes and from traditional toxicology that honey-processing can significantly reduce the toxicity of AF. To further elucidate the detoxification mechanism of honey-processing, comparative pharmacokinetics of AAs in AF and HAF are performed in this study., Methods: An HPLC-MS/MS (high-performance liquid chromatography-tandem mass spectrometry) method was developed and validated for the determination of AA I, AA II, AA C, AA D and 7-OH AA I in rat plasma. The multi-component pharmacokinetics of AAs in AF and HAF extracts were investigated after the oral administration of three doses to rats. The relative pharmacokinetic parameters were compared systematically., Results: The five AAs shared a similar nonlinear PK (pharmacokinetic) process. They involve rapid absorption and elimination, and they were fit into a two-compartmental open model. Some significant pharmacokinetic differences were observed between the AF and HAF groups: the C max and AUC values of AA I and AA II in the AF groups were much higher than those of the HAF groups., Conclusions: Honey-frying technology can reduce the toxicity of AF by significantly decreasing the absorption of AA I and AA II. The PK parameters obtained in this work could provide valuable references for the toxicity research and clinical use of Aristolochiaceae herbs, including AF and HAF. Process diagram of comparative pharmacokinetics study.

The term "aristolochic acid nephropathy" (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) as part of traditional phytotherapies (formerly known as "Chinese herbs nephropathy"), or by the environmental contaminants in food (Balkan endemic nephropathy). It is frequently associated with urothelial malignancies. Although products containing AA have been banned in most of countries, AAN cases remain regularly reported all over the world. Moreover, AAN incidence is probably highly underestimated given the presence of AA in traditional herbal remedies worldwide and the weak awareness of the disease. During these two past decades, animal models for AAN have been developed to investigate underlying molecular and cellular mechanisms involved in AAN pathogenesis. Indeed, a more-in-depth understanding of these processes is essential to develop therapeutic strategies aimed to reduce the global and underestimated burden of this disease. In this regard, our purpose was to build a broad overview of what is currently known about AAN. To achieve this goal, we aimed to summarize the latest data available about underlying pathophysiological mechanisms leading to AAN development with a particular emphasis on the imbalance between vasoactive factors as well as a focus on the vascular events often not considered in AAN.

The association between urothelial carcinoma (UC) and subsequent ESRD incidence has not been confirmed. This was a population-based study using claims data from the Taiwan National Health Institutes from 1998 to 2010. The study cohort consisted of 26,017 patients with newly diagnosed UC and no history of ESRD, and the comparison cohort consisted of 208,136 matched enrollees without UC. The incidence of ESRD was ascertained through cross-referencing with a registry for catastrophic illnesses. Cox proportional hazard regression analysis was used to estimate the risk of ESRD associated with UC and UC subtype. A total of 979 patients (3.76%) from the UC group and 1,829 (0.88%) from the comparison group developed ESRD. Multivariable analysis indicated that compared with the comparison group, the hazard ratios (HRs) for ESRD were 7.75 (95% confidence interval [CI]: 6.84 to 8.78) and 3.12 (95% CI: 6.84 to 8.78) in the cohort with upper urinary tract UC (UT-UC) and bladder UC (B-UC), respectively. In addition, there were significantly increased risks for ESRD in UC patients receiving and not receiving nephrouretectomies or aristolochic acids (AA). Moreover, the UC patients receiving segmental ureterectomy and ureteral reimplantation had approximately 1.3-fold and 2.4-fold increased risk for ESRD after control for confounders, respectively. Thus, our data indicate that UT-UC and B-UC independently increased the risk for ESRD in patients after considering about nephrouretectomies or aristolochic acids (AA). In addition, UC patients receiving segmental ureterectomy and ureteral reimplantation had increased risk for ESRD., Competing Interests: The authors have no conflicts of interest to disclose.

Background and Purpose: Aristolochic acid (AristA) is found in plants used in traditional medicines to treat pain. We investigated the action of AristA on TREK and TRESK, potassium (K2P) channels, which are potential therapeutic targets in pain. Balkan endemic nephropathy (BEN) is a renal disease associated with AristA consumption. A mutation of TASK-2 (K2P 5.1) channels (T108P) is seen in some patients susceptible to BEN, so we investigated how both this mutation and AristA affected TASK-2 channels., Experimental Approach: Currents through wild-type and mutated human K2P channels expressed in tsA201 cells were measured using whole-cell patch-clamp recordings in the presence and absence of AristA., Key Results: TREK-1- and TREK-2-mediated currents were enhanced by AristA (100 μM), whereas TRESK was inhibited. Inhibition of TRESK did not depend on the phosphorylation of key intracellular serines but was completely blocked by mutation of bulky residues in the inner pore (F145A_F352A). The TASK-2_T108P mutation markedly reduced both current density and ion selectivity. A related mutation (T108C) had similar but less marked effects. External alkalization and application of flufenamic acid enhanced TASK-2 and TASK-2_T108C current but did not affect TASK-2_T108P current. AristA (300 μM) produced a modest enhancement of TASK-2 current., Conclusions and Implications: Enhancement of TREK-1 and TREK-2 and inhibition of TRESK by AristA may contribute to therapeutically useful effects of this compound in pain. Whilst AristA is unlikely to interact directly with TASK-2 channels in BEN, loss of functional TASK-2 channels may indirectly increase susceptibility to AristA toxicity., (© 2016 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Aristolochic acids (AA) are nephrotoxic agents found in Aristolochia species whose consumption leads to the onset of a progressive tubulointerstitial fibrosis. This AA-nephropathy was first reported during the Belgian outbreak of the 1990's in which more than a hundred patients consumed slimming pills containing an Aristolochia species and Magnolia officinalis. The patients developed an end-stage kidney disease requiring dialysis or transplantation. Magnolol and honokiol are bioactive compounds from M. officinalis known for their potent antioxidant activity. As they can alleviate oxidative stress, we investigated their respective effects on AA-mediated tubulotoxicity using HK-2 cells. Magnolol and honokiol were able to reduce the oxidative stress associated with AA-treatment. Cytotoxicity alleviation was further investigated and overall cell viability measurements unexpectedly revealed that both compounds worsened the survival of AA-treated cells. Flow cytometry analyses of annexin V/PI stained cells indicated that the lignans efficiently prevented AA-induced apoptosis; but favored necrosis. Microscopy observations highlighted extensive vacuolization; other types of cell death, including autophagy, paraptosis or accelerated senescence were excluded. Ki-67 index and cell cycle analysis indicated that both magnolol and honokiol inhibited proliferation by blocking the cell cycle at the G1 phase; they also prevented the AA-induced G2/M arrest., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b₅, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.

In this communication, we report the springtime variation of the composition of aristolochic acids (AAs) in Aristolochia chilensis leaves and stems. The dominant AA in the leaves of all samples, which were collected between October and December, was AA-I (1), and its concentration varied between 212.6±3.8 and 145.6±1.2 mg/kg and decreased linearly. This decrease occurred in parallel with the increase in AA-Ia (5) concentration from 15.9±0.8 mg/kg at the beginning of October to 96.8±7.8 mg/kg in mid-December. Both acids are enzymatically related by methylation-demethylation reactions. Other AAs also showed important variations: AA-II (2) significantly increased in concentration, reaching a maximum in the first two weeks of November and subsequently decreasing in mid-December to approximately the October levels. The principal component in the AA mixture of the stems was also AA-I (1); similar to AA-II (2), its concentration increased beginning in October, peaked in the second week of November and subsequently decreased. The concentrations of AA-IIIa (6) and AA-IVa (7) in the leaves and stems varied throughout the study period, but no clear pattern was identified. Based on the variation of AAs in A. chilensis leaves and stems during the study period, the reduced contents of non-phenolic AAs and increased concentrations of phenolic AAs are likely associated with a decrease in this plant's toxicity during the spring.

Aristolochic acid (AA) is a potent carcinogen and nephrotoxin and is associated with the development of "Chinese herb nephropathy" and Balkan endemic nephropathy. Despite decades of research, the specific mechanism of the observed nephrotoxicity has remained elusive and the potential effects on proteins due to the observed toxicity of AA are not well-understood. To better understand the pharmacotoxicological features of AA, we investigated the non-covalent interactions of AA with proteins. The protein-binding properties of AA with bovine serum albumin (BSA) and lysozyme were characterized using spectrofluorometric and mass spectrometric (MS) techniques. Moreover, the protein-AA complexes were clearly identified by high-resolution MS analyses. To the best of our knowledge, this is the first direct evidence of non-covalently bound protein-AA complexes. An analysis of the spectrofluorometric data by a modified Stern-Volmer plot model also revealed that both aristolochic acid I (AAI) and aristolochic acid II (AAII) were bound to BSA and lysozyme in 1:1 stoichiometries. A significantly stronger protein binding property was observed in AAII than in AAI as evidenced by the spectrofluorometric and MS analyses, which may explain the observed higher mutagenicity of AAII.

Computational modeling is employed to provide a plausible structural explanation for the experimentally-observed differential global genome repair (GGR) propensity of the ALII-N(2)-dG and ALII-N(6)-dA DNA adducts of aristolochic acid II. Our modeling studies suggest that an intrinsic twist at the carcinogen-purine linkage of ALII-N(2)-dG induces lesion site structural perturbations and conformational heterogeneity of damaged DNA. These structural characteristics correlate with the relative repair propensities of AA-adducts, where GGR recognition occurs for ALII-N(2)-dG, but is evaded for intrinsically planar ALII-N(6)-dA that minimally distorts DNA and restricts the conformational flexibility of the damaged duplex. The present analysis on the ALII adduct model systems will inspire future experimental studies on these adducts, and thereby may extend the list of structural factors that directly correlate with the propensity for GGR recognition., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims.

This review summarizes the results found in studies investigating the enzymatic activation of two genotoxic nitro-aromatics, an environmental pollutant and carcinogen 3-nitrobenzanthrone (3-NBA) and a natural plant nephrotoxin and carcinogen aristolochic acid I (AAI), to reactive species forming covalent DNA adducts. Experimental and theoretical approaches determined the reasons why human, Nad(p)h: quinone oxidoreductase (NQO1) and cytochromes P450 (CYP) 1A1 and 1A2 have the potential to reductively activate both nitro-aromatics. The results also contributed to the elucidation of the molecular mechanisms of these reactions. The contribution of conjugation enzymes such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) to the activation of 3-NBA and AAI was also examined. The results indicated differences in the abilities of 3-NBA and AAI metabolites to be further activated by these conjugation enzymes. The formation of DNA adducts generated by both carcinogens during their reductive activation by the NOQ1 and CYP1A1/2 enzymes was investigated with pure enzymes, enzymes present in subcellular cytosolic and microsomal fractions, selective inhibitors, and animal models (including knock-out and humanized animals). For the theoretical approaches, flexible in silico docking methods as well as ab initio calculations were employed. The results summarized in this review demonstrate that a combination of experimental and theoretical approaches is a useful tool to study the enzyme-mediated reaction mechanisms of 3-NBA and AAI reduction.

Background: Balkan Endemic Nephropathy (BEN) is late-onset kidney disease thought to arise from chronic exposure to aristolochic acid, a phytotoxin that contaminates wheat supplies in rural areas of Eastern Europe. It has recently been demonstrated that humans are capable of perceiving aristolochic acid at concentrations below 40 nM as the result of high-affinity interactions with the TAS2R43 bitter taste receptor. Further, TAS2R43 harbors high-frequency loss-of-function mutations resulting in 50-fold variability in perception. This suggests that genetic variation in TAS2R43 might affect susceptibility to BEN, with individuals carrying functional forms of the receptor being protected by an ability to detect tainted foods., Methods: To determine whether genetic variation in TAS2R43 predicts BEN susceptibility, we examined genotype-phenotype associations in a case-control study. A cohort of 88 affected and 99 control subjects from western Bulgaria were genotyped with respect to two key missense variants and a polymorphic whole-gene deletion of TAS2R43 (W35S, H212R, and wt/Δ), which are known to affect taste sensitivity to aristolochic acid. Tests for association between haplotypes and BEN status were then performed., Results: Three major TAS2R43 haplotypes observed in previous studies (TAS2R43-W35/H212, -S35/R212 and -Δ) were present at high frequencies (0.17, 0.36, and 0.47 respectively) in our sample, and a significant association between genotype and BEN status was present (P = 0.020; odds ratio 1.18). However, contrary to expectation, BEN was positively associated with TAS2R43-W35/H212, a highly responsive allele previously shown to confer elevated bitter sensitivity to aristolochic acid, which should drive aversion but might also affect absorption, altering toxin activation., Conclusions: Our findings are at strong odds with the prediction that carriers of functional alleles of TAS2R43 are protected from BEN by an ability to detect and avoid aristolochic acid exposure. Evidence for a positive association between high-sensitivity alleles and BEN status suggests instead that possession of toxin-responsive receptor variants may paradoxically increase vulnerability, possibly by shifting attractive responses associated with low-intensity bitter sensations. The broad-spectrum tuning of the ~25-member TAS2R family as a whole toward xenobiotics points to a potentially far-reaching relevance of bitter responses to exposure-related disease in both individuals and populations.

Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by A:T to T:A transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues.

Background: Interaction of aristololactam-β-D-glucoside and daunomycin with tRNA(phe) was investigated using various biophysical techniques., Methodology/principal Findings: Absorption and fluorescence studies revealed that both the compounds bind tRNA(phe) non-cooperatively. The binding of daunomycin was about one order of magnitude higher than that of aristololactam-β-D-glucoside. Stronger binding of the former was also inferred from fluorescence quenching data, quantum efficiency values and circular dichroic results. Results from isothermal titration calorimetry experiments suggested that the binding of both compounds was predominantly entropy driven with a smaller but favorable enthalpy term that increased with temperature. A large favorable electrostatic contribution to the binding of daunomycin to tRNA(phe) was revealed from salt dependence data and the dissection of the free energy values. The electrostatic component to the free energy change for aristololactam-β-D-glucoside-tRNA(phe) interaction was smaller than that of daunomycin. This was also inferred from the slope of log K versus [Na(+)] plots. Both compounds enhanced the thermal stability of tRNA(phe). The small heat capacity changes of -47 and -99 cal/mol K, respectively, observed for aristololactam-β-D-glucoside and daunomycin, and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak noncovalent interactions. Molecular aspects of the interaction have been revealed., Conclusions/significance: This study presents the structural and energetic aspects of the binding of aristololactam-β-D-glucoside and daunomycin to tRNA(phe).

Phytochemical investigation of 90% ethanol extracts of the roots of Aristolochia fangchi yielded two new aristolochic acid derivatives named Aristolochic Acid F and Aristolochic Acid G, together with three known compounds. Their structures were elucidated by spectral analysis. The cytotoxicity of the isolated compounds was also determined.

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In approximately 50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA-->T and AL-II-dG-->T transversions.

A native female-specific chemoreceptive protein of a swallowtail butterfly [oviposition stimulant binding protein (OSBP)] was shown to specifically bind to aristolochic acid, a main stimulant for oviposition from its host plant. Oviposition stimulants are recognized by chemoreceptive organs of insects. OSBP isolated previously from the chemoreceptive organs was assumed to bind to an oviposition stimulant. Using a highly sensitive fluorescent micro-binding assay, we clarified OSBP bound to aristolochic acid. Three-dimensional molecular modeling revealed the structure of the OSBP-aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant as a ligand in insects.

We compared the short-term toxicity of toxic components of aristolochic acid in rats. Twenty-four female Wistar rats were divided into 4 groups and treated orally every 3-days with 10 mg/kg each of aristolochic acid, aristolochic acid-I and aristolochic acid-II for 19 days. After treatment, the relative ratio of liver and kidney weight to body weight, the concentrations of RBC, hemoglobin and hematocrit in the blood, the levels of aspartate amino transferase, alanine amino transferase, alkaline phosphatase, blood urea nitrogen and creatinine in the plasma, and the levels of urinary urea nitrogen and creatinine in the urine were significantly increased. Body weight of rats and the levels of Na(+), K(+), Ca(2+) in the urine were significantly decreased, especially for groups treated with aristolochic acid and aristolochic acid-II. Pathological examination of liver and kidney also showed cell enlargement and lesions, especially for groups treated with aristolochic acid and aristolochic acid-II. The aristolochic acid exhibited significant toxicity, and the short-term toxicity of aristolochic acid-II and aristolochic acid was similar to each other. Renal but not hepatic failure induced by aristolochic acid could be prevented by pentoxifylline.

In traditional Chinese medicine several Aristolochia species are used. Aristolochia spp. contain a mixture of aristolochic acids (AAs), mainly AA I and AA II which are nephrotoxicants and carcinogens. After AA-related nephropathy (AAN) and urothelial cancer were described in female patients in Belgium following intake of AA-contaminated herbal preparations, herbs with AAs were prohibited worldwide. Confusing nomenclature can cause AA contamination of certain Chinese traditional herbal preparations (THPs). Here we report the results of investigations by the Dutch Food and Consumer Product Safety Authority (VWA) into the presence of AAs in THPs sampled on the Dutch market using a liquid-chromatography--mass spectrometry method. Between 2002 and 2006 we sampled 190 Chinese THPs using recent information on Chinese THPs potentially containing AAs. AA I was found in 25 samples up to a concentration of 1,676 mg/kg. AA II was also found in 13 of these samples up to 444 mg/kg. All 25 positive samples including Mu Tong, Fang Ji, Tian Xian Teng and Xi Xin were part of a group of 68 THPs identified as possibly containing AAs. In a worst-case scenario, use of a sample of Mu Tong with the highest AA content over a 7-day period would result in the same intake levels of AAs which significantly raised the cancer risk in the Belgian AAN cases. Our results show that contaminated THPs still can be found on the market following worldwide publicity. Therefore, it can be concluded that testing of possibly AA-contaminated THPs is still essential.

Endemic (Balkan) nephropathy (EN), a devastating renal disease affecting men and women living in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, is characterized by its insidious onset, invariable progression to chronic renal failure and a strong association with transitional cell (urothelial) carcinoma of the upper urinary tract. Significant epidemiologic features of EN include its focal occurrence in certain villages and a familial, but not inherited, pattern of disease. Our experiments test the hypothesis that chronic dietary poisoning by aristolochic acid is responsible for EN and its associated urothelial cancer. Using (32)P-postlabeling/PAGE and authentic standards, we identified dA-aristolactam (AL) and dG-AL DNA adducts in the renal cortex of patients with EN but not in patients with other chronic renal diseases. In addition, urothelial cancer tissue was obtained from residents of endemic villages with upper urinary tract malignancies. The AmpliChip p53 microarray was then used to sequence exons 2-11 of the p53 gene where we identified 19 base substitutions. Mutations at A:T pairs accounted for 89% of all p53 mutations, with 78% of these being A:T --> T:A transversions. Our experimental results, namely, that (i) DNA adducts derived from aristolochic acid (AA) are present in renal tissues of patients with documented EN, (ii) these adducts can be detected in transitional cell cancers, and (iii) A:T --> T:A transversions dominate the p53 mutational spectrum in the upper urinary tract malignancies found in this population lead to the conclusion that dietary exposure to AA is a significant risk factor for EN and its attendant transitional cell cancer.

Electrophilic attack of aristolactam-nitrenium ion by the C7 position to the exocyclic amino group in the DNA bases led to the formation of the major adducts. In this study, liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied to the study of DNA adducts of aristolochic acid (AA). When DNA (bases and CT-DNA) was incubated with AA, dG-AAI, dG-AAII, dA-AAI, dA-AAII, dC-AAI, and dC-AAII were detected and characterized. The dC adducts of AA were identified for the first time. The soft ionization technology allowed detection of the intact DNA adducts. High-resolution MS and MS-MS capabilities of a quadrupole time-of-flight mass spectrometer were shown to be efficient for DNA adducts analysis. DNA-AA adducts showed characteristic fragmentation patterns in MS-MS analysis. The dissociative loss of 116 Da from the DNA-AA adducts, which resulted from internal hydrogen transfer and cleavage at the C-N glycosidic bond, provided a characteristic fragment for the structural elucidation.

The subacute toxicity of aristolochic acid (AA) was investigated by (1)H NMR spectroscopic and pattern recognition (PR)-based metabonomic methods. Model toxins were used to enable comparisons of the urinary profiles from rats treated with known toxicants and AA at various time intervals. Urinary (1)H NMR spectra were data-processed and analyzed by pattern recognition method. The result of visual comparison of the spectra showed that AA caused a renal proximal tubular and papillary lesion and a slight hepatic impair. Pattern recognition analysis indicated that the renal proximal tubule lesion was the main damage induced by AA, and the renal toxicity induced by AA was a progressive course with the accumulation of dosage by monitoring the toxicological processes from onset, development and part-recovery. These results were also supported by the conventional clinical biochemical parameters.

Column chromatography of the alcoholic extract of Piper betle roots furnished aristololactam A-II and a new phenyl propene, characterized as 4-allyl resorcinol, while the petroleum-ether extract yielded a diketosteroid, viz. stigmast-4-en-3,6-dione. All these compounds were characterized by spectroscopic means. Isolation of these compounds from this source is being reported here for the first time.

Background: Aristolochia species are nephrotoxic and carcinogenic. Recent studies showed that aristolochic acid (AA) could induce acute renal failure and tubular lesions in several species and available evidences demonstrate the unequivocal role of AA in so called Chinese herbs nephropathy., Methods: A series of AA derivatives isolated from Aristolochia spp. were analyzed for their nephrotoxic potential using the neutral red dye exclusion assay in cultures of LLC-PK(1) cells. The structural relationships between AA I and its analogues were compared with their cytotoxic effects to predict structural determinants for AA toxicity. Further, caspase-3 assay was performed on toxic compounds to determine if caspases, the enzymes that play a critical role in apoptosis are involved in AA-induced cytotoxicity., Results: AA I was found to be most toxic followed by AA II, AA VIIIa, and AA Ia in decreasing levels of toxicity. The other compounds, nitrophenanthrene carboxylic acid analogues of AA I, aristolactams, and other derivatives did not exhibit considerable toxicity. The results showed significant relationships between cytotoxicity of AA compounds and the localization of functional groups in their structure. Analogues containing hydroxyl groups diminished cytotoxicity. The demethylated analogues of AA I are markedly less active. The negative impact on cytotoxicity was found on nitroreduction of AA I. AA induced caspase activation was also observed., Conclusion: These cytotoxic data suggest that the nitro and methoxy groups are critical determinants of nephrotoxicologic potency of AA.

[reaction: see text] A variety of substituted isoindolin-1-ones are readily prepared in good to excellent yields under very mild reaction conditions by the reaction of o-(1-alkynyl)benzamides with ICl, I(2), and NBS. In a few cases, substituted isoquinolin-1-ones were obtained as the major product instead. This methodology accommodates various alkynyl amides and functional groups and has been successfully extended to heterocyclic starting materials. This chemistry has been successfully applied to the synthesis of a biologically interesting alkaloid, cepharanone B.

Aristolochic acid (AA), a natural component in some Chinese medicinal plants, is nephrotoxic and carcinogenic, and is involved in a specific type of renal fibrosis, called Chinese herbs nephropathy (CHN). In this paper, we report the electrochemistry of AA and a novel method to detect the species based on the electrochemical studies. The detection limit is estimated to be 1.0x10(-8) M with a linear range from 5.0x10(-8) M to 1.3x10(-6) M. Meanwhile, this method is applicable in detection in real samples, such as Caulis Aristolochiae Manshuriensis (CAM), Radix Aristolochiae (RA), Fructus Aristolochiae (FA) and Radix Stephaniae Tetrandrae (RST). The concentration of AA in the CAM sample was 3.50 +/- 0.12x10(-4) M. Consistent results have been obtained from both the electrochemical approach described here and the previously reported HPLC method.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA metabolism is important in the assessment of an individual's susceptibility to this carcinogen. Using the 32P-postlabeling assay we examined the ability of enzymes of cytosolic samples from 10 different human livers and from one human kidney to activate the major component of the plant extract AA, 8-methoxy- 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI), to metabolites forming adducts in DNA. Cytosolic fractions of both organs generated AAI-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II, indicating a possible demethoxylation reaction of AAI, were identified as AA-DNA adducts formed from AAI by all human hepatic and renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors or selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase. We also determined whether the activities of NQO1 and XO in different human hepatic cytosolic samples correlated with the levels of AAI-DNA adducts formed by the same cytosolic samples. Based on these studies, we attribute most of the activation of AA in human cytosols to NQO1, although a role of cytosolic XO cannot be ruled out. With purified NQO1 from rat liver and kidney and XO from buttermilk, the major role of NQO1 in the formation of AAI-DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was predicted from molecular modeling based on published X-ray structures. The results demonstrate for the first time the potential of human NQO1 to activate AAI by nitroreduction.

Over the last 10 years, numerous cases of intoxications, leading for the most part to end-stage renal failure, have been reported after consumption of slimming regimens made of Chinese herbal preparations. These intoxications were associated with species of the Aristolochia genus, such as Aristolochia fangchi (Aristolochiaceae), known to contain very nephrotoxic and carcinogenic metabolites named aristolochic acids. Several commercial dietary supplements, teas and phytomedicines used as slimming regimens were analysed for their aristolochic acid I content. A preliminary detection of this toxic compound was made by thin-layer chromatography. The presence of aristolochic acid I in these preparations was confirmed by a HPLC/UV-DAD/MS analysis. A quantitative determination of aristolochic acid I was also achieved in the incriminated preparations using both UV and MS detection. Out of 42 analysed preparations, four were found to contain aristolochic acid I and two were suspected to contain aristolochic acid derivatives. Immediate removal of these products from the Swiss market was called for.