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Asari Radix et Rhizoma (ARR) is a traditional Chinese herbal medicine derived from the genus Asarum in the Aristolochiaceae family, which has been widely used for years. Aristolochic acids in it significantly restrict the clinical application of ARR due to its nephrotoxic and carcinogenic properties, of which aristolochic acid I (AA I) is the most representative. The present paper describes two 2D-LC systems modulated by a sample loop and a trap column respectively for the detection of trace aristolochic acid I in ARR. An Inertsil ODS-3 C18 column and a Luna 3u Phenyl-Hexyl column were separately utilized in the first-dimension (1D) and second-dimension (2D) chromatography. A mixed-mode column with reversed-phase and ion-exchange was selected for trap column modulation of 2D-LC system. Method validation results showed that these two 2D-LC methods performed well in terms of repeatability, linearity, intra/interday precision, stability, and accuracy, which were demonstrated qualified for the simultaneous determination of trace AA I in ARR. The AA Ⅰ contents in three batches of ARR were 0.466∼0.812 μg/g by the two 2D-LC methods, which were below the limit requirement in the Chinese Pharmacopoeia. The comprehensive comparison between the two methods showed that a significant solute focusing effect was obtained by trap column modulation, which led to four-time lower LOD compared with loop modulation, while the loop-modulation-based method achieved higher analytical efficiency when analyzing a large batch of samples., 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 © 2024. Published by Elsevier B.V.)

Aristolochic acids are one of the major compounds in aristolochia plants, which are nephrotoxic and carcinogenic. A method was established for the detection and identification of aristolochic acids and their DNA adducts in four different herbs using ultra-high performance liquid chromatography-ion mobility quadrupole time-of flight mass spectrometry. Solid phase extraction conditions were optimized to improve the sensitivity of the experiment by using 40 mg of C 18 as adsorbent and 100 μL ethanol as elution solvent. At a collision energy of 10-40 eV, these compounds and cleavage patterns were precisely identified and analyzed by secondary fragmentation and collision cross section values. The obtained mass spectrometry data were then analyzed by targeted metabolomics, including principal component analysis, partial least squares-discriminant analysis and hierarchical clustering analysis, and importing the samples in the established model, the confidence values can reach 0.61 and 0.76. All in all, this method can provide a useful tool for the detection of aristolochic acids and deoxyribonucleic acid adducts. In conclusion, this method was successfully used for the detection and identification of aristolochic acids and their DNA adducts., Competing Interests: Declaration of competing interest The authors (Ying Shi, Yi-Wen Wu, Qian-Xue Shen, Jun Cao) 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 © 2024. Published by Elsevier B.V.)

Aristolochic acids (AAs) naturally occurring in the herbal genus Aristolochia are associated with a high risk of kidney failure, multiple tumors and cancers. However, approaches with high selectivity and rapidity for measuring AAs in biological samples are still inadequate. Inspired by the mechanism of AAs-induced nephrotoxicity, we designed a hybrid magnetic polymer-porous agarose (denoted as MNs@SiO 2 M@DNV-A), mimicking the effect of basic and aromatic residues of organic anion transporter 1 (OAT1) for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in the plasma. The monomers of vinylbenzyl trimethylammonium chloride (VBTAC), N-vinyl-2-pyrrolidinone (NVP) and divinylbenzene (DVB) were employed to construct the polymer layer, which provided a selective adsorption for AAs by multiple interactions. The porous agarose shell contributed to remove interfering proteins in the plasma samples. A magnetic solid-phase extraction (MSPE) based on the proposed composite enhanced the selectivity toward AA I and AA II in the plasma samples. In combination of HPLC analysis, the proposed method was proved to be applicable to fast and specific quantification of AAs in blood samples, which was characterized by a good linearity, high sensitivity, acceptable recovery, excellent repeatability and satisfactory reusability., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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), which are a group of nitrophenanthrene carboxylic acids formed by Aristolochia plant, have become an increasing serious threat to humans due to their nephrotoxicity and carcinogenicity. Fast and accurate approaches capable of simultaneous sensing of aristolochic acids (I-IV) are vital to avoid intake of such compounds. In this research, the novel ratiometric fluorescence zinc metal-organic framework and its nanowire have been prepared. The two different coordination modes (tetrahedral configuration and twisted triangular bipyramidal configuration) within zinc metal-organic framework lead to the significant double emissions. The ratiometric fluorescence approach based on nanowire provides a broader concentration range (3.00 × 10 -7 ~1.00 × 10 -4 M) and lower limit of detection (3.70 × 10 -8 M) than that based on zinc metal-organic framework (1.00 × 10 -6 ~1.00 × 10 -4 M, 5.91 × 10 -7 M). The RSDs of the results are in the range 1.4-3.5% (nanowire). The density functional theory calculations and UV-Vis absorption verify that the sensing mechanism is due to charge transfer and energy transfer. Excellent spiked recoveries for AAs(I-IV) in soil and water support that nanowire is competent to simultaneously detect these targets in real samples, and the proposed approach has potential as a fluorescence sensing platform for the simultaneous detection of AAs (I-IV) in complex systems., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Air pollution, tobacco smoke, and red meat are associated with renal cell cancer (RCC) risk in the United States and Western Europe; however, the chemicals that form DNA adducts and initiate RCC are mainly unknown. Aristolochia herbaceous plants are used for medicinal purposes in Asia and worldwide. They are a significant risk factor for upper tract urothelial carcinoma (UTUC) and RCC to a lesser extent. The aristolochic acid (AA) 8-methoxy-6-nitrophenanthro-[3,4- d ]-1,3-dioxolo-5-carboxylic acid (AA-I), a component of Aristolochia herbs, contributes to UTUC in Asian cohorts and in Croatia, where AA-I exposure occurs from ingesting contaminated wheat flour. The DNA adduct of AA-I, 7-(2'-deoxyadenosin- N 6 -yl)-aristolactam I, is often detected in patients with UTUC, and its characteristic A:T-to-T:A mutational signature occurs in oncogenes and tumor suppressor genes in AA-associated UTUC. Identifying DNA adducts in the renal parenchyma and pelvis caused by other chemicals is crucial to gaining insights into unknown RCC and UTUC etiologies. We employed untargeted screening with wide-selected ion monitoring tandem mass spectrometry (wide-SIM/MS 2 ) with nanoflow liquid chromatography/Orbitrap mass spectrometry to detect DNA adducts formed in rat kidneys and liver from a mixture of 13 environmental, tobacco, and dietary carcinogens that may contribute to RCC. Twenty DNA adducts were detected. DNA adducts of 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and AA-I were the most abundant. The nitrophenanthrene moieties of 3-NBA and AA-I undergo reduction to their N -hydroxy intermediates to form 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. We also discovered a 2'-deoxycytidine AA-I adduct and dA and dG adducts of 10-methoxy-6-nitro-phenanthro-[3,4- d ]-1,3-dioxolo-5-carboxylic acid (AA-III), an AA-I isomer and minor component of the herbal extract assayed, signifying AA-III is a potent kidney DNA-damaging agent. The roles of AA-III, other nitrophenanthrenes, and nitroarenes in renal DNA damage and human RCC warrant further study. Wide-SIM/MS 2 is a powerful scanning technology in DNA adduct discovery and cancer etiology characterization.

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 MoS 2 layer was grown in situ on their surface using a hydrothermal method. The synthesized MoS 2 -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 MoS 2 used to modify the BHCs and the pH of the electrolyte. Under optimal conditions, the MoS 2 -BHC-based sensor presented excellent AA detection performance. The linear concentration ranges of the MoS 2 -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 MoS 2 -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 MoS 2 -BHC-based sensors can be used as effective platforms for detecting AA in traditional Chinese herbs.

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 acids (AAs) are a family of natural compounds with AA I and AA II being known carcinogens, whose bioactivation causes DNA adducts formation. However, other congeners have rarely been investigated. This study aimed to investigate genotoxicity of AA IVa, which differs from AA I by a hydroxyl group, abundant in Aristolochiaceae plants. AA IVa reacted with 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) to form three dA and five dG adducts as identified by high-resolution mass spectrometry, among which two dA and three dG adducts were detected in reactions of AA IVa with calf thymus DNA (CT DNA). However, no DNA adducts were detected in the kidney, liver, and forestomach of orally dosed mice at 40 mg/kg/day for 2 days, and bone marrow micronucleus assay also yielded negative results. Pharmacokinetic analyses of metabolites in plasma indicated that AA IVa was mainly O-demethylated to produce a metabolite with two hydroxyl groups, probably facilitating its excretion. Meanwhile, no reduced metabolites were detected. The competitive reaction of AA I and AA IVa with CT DNA, with adducts levels varying with pH of reaction revealed that AA IVa was significantly less reactive than AA I, probably by hydroxyl deprotonation of AA IVa, which was explained by theoretical calculations for reaction barriers, energy levels of the molecular orbits, and charges at the reaction sites. In brief, although it could form DNA adducts in vitro, AA IVa was non-genotoxic in vivo, which was attributed to its low reactivity and biotransformation into an easily excreted metabolite rather than bioactivation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aristolochic acid (AA) has been reported to cause a series of health problems, including aristolochic acid nephropathy and liver cancer. However, AA-containing herbs are highly safe in combination with berberine (Ber)-containing herbs in traditional medicine, suggesting the possible neutralizing effect of Ber on the toxicity of AA. In the present study, in vivo systematic toxicological experiments performed in zebrafish and mice showed that the supramolecule self-assembly formed by Ber and AA significantly reduced the toxicity of AA and attenuated AA-induced acute kidney injury. Ber and AA can self-assemble into linear heterogenous supramolecules (A-B) via electrostatic attraction and π-π stacking, with the hydrophobic groups outside and the hydrophilic groups inside during the drug combination practice. This self-assembly strategy may block the toxic site of AA and hinder its metabolism. Meanwhile, A-B linear supramolecules did not disrupt the homeostasis of gut microflora as AA did. RNA-sequence analysis, immunostaining, and western blot of the mice kidney also showed that A-B supramolecules almost abolished the acute nephrotoxicity of AA in the activation of the immune system and tumorigenesis-related pathways.

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.

Aristolochic acid Ⅰ is a nephrotoxic compound and exist in some traditional Chinese medicines at trace level. Up to now, specific enrichment of aristolochic acid Ⅰ remains important procedure and key problem in its analysis. In this study, melamine was proposed as the recognition unit and grafted on the surface of metal-organic framework to fabricate a specific material for aristolochic acid Ⅰ. This material was prepared by using a two-step strategy and the preparation process was optimized. The physical and chemical properties were investigated using scanning electron microscopy, Fourier-transfer infrared spectroscopy, X-ray diffraction and nitrogen adsorption-desorption techniques. Adsorption properties were evaluated by binding experiments. The melamine modified material exhibited a uniform morphology, high specific surface area (460.20 m 2 g -1 ), high adsorption capacity (25.57 mg g -1 ), fast mass transfer rate and excellent selectivity. Further, a specific and sensitive method was established by using this material as adsorbent of mini-solid phase extraction. The limit of detection was as low as 0.02 μg mL -1 . Therefore, melamine modified metal-organic framework is an ideal adsorbent for the recognition and enrichment of aristolochic acid Ⅰ., 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 © 2021. Published by Elsevier B.V.)

A novel aristololactam alkaloid, dasymalactam A ( 1 ), together with nine known analogues ( 2-10 ), were isolated from the roots of Dasymaschalon rostratum . Their structures were elucidated by IR, NMR and MS spectrums and comparisons with data reported in the literature. All compounds demonstrated weak cytotoxicity against Hela, MCF-7, A-549, MGC-803, and COLO-205 human cancer cell lines.

A molecularly imprinted ratiometric fluorescent probe (MIRF probe) was synthesized for the determination of aristolochic acid I (AAI) based on the Schiff-base fluorescent compound N,N'-bis(o-carboxybenzylidene)-p-4,4'-diaminobiphenyl (BDDB). The BDDB was immobilized in the silica nanoparticle (BDDB@SiO 2 ) as an internal standard material. The blue-emitting BDDB@SiO 2 and the yellow-emitting carbon quantum dots (y-CDs) were wrapped in the molecularly imprinted polymer (MIP) to provide a reliable reference signal at 440 nm and a fluorescent response signal at 530 nm at the excitation wavelength of 365 nm, respectively. In the preparation of the MIP of the MIRF probe, 4-vinylbenzoic acid as the functional monomer and AAI as the template molecule were used. An imprinting factor of 2.25 was obtained. Under the optimum conditions, the fluorescent response signal at 530 nm was quenched gradually by AAI in the range 1.0 to 120.0 μmol/L, while the reference signal at 440 nm remained unchanged. The limit of detection was 0.45 μmol/L, and the fluorescent color of the MIRF probe changed gradually from yellow to green to blue, which illustrated that the developed probe had a specific AAI recognition ability, a good anti-interference ability, and a sensitively visual determination ability. The probe was successfully applied to the AAI determination in traditional Chinese medicine (TCM) Asarum. The results showed that it had satisfactory recoveries (95.5-107.3%) and low relative standard deviations (2.0%). Furthermore, this method has a potential for the onsite naked eye determination of AAI in TCM samples.Graphical abstract.

Exposure to aristolochic acid I and II (AAI and AAII) has been implicated in aristolochic acid nephropathy and urothelial carcinoma. The toxicological effects of AAs are attributed to their ability to form aristolacatam (AL)-purine DNA adducts. Among these lesions, the AL-adenine (ALI-N 6 -A and ALII-N 6 -A) adducts cause the "signature" A → T transversion mutations associated with AA genotoxicity. To provide the currently missing structural basis for the induction of these signature mutations, the present work uses classical all-atom molecular dynamics simulations to examine different (i.e., preinsertion, insertion, and postextension) stages of replication past the most abundant AA adduct (ALI-N 6 -A) by a representative lesion-bypass DNA polymerase (Dpo4). Our analysis reveals that, before dNTP incorporation (i.e., preinsertion step), ALI-N 6 -A adopts a nearly planar conformation at the N 6 -linkage and the ALI moiety intercalates within the DNA helix. Since this conformation occupies the dNTP binding site, the same planar lesion conformation results in a significant distortion of the polymerase active site at the insertion step and therefore replication will likely not be successful. However, if ALI-N 6 -A undergoes a small conformational change to introduce non-planarity at the N 6 -linkage during the insertion step, minimal distortion occurs in the Dpo4 active site upon incorporation of dATP. This insertion and subsequent extension would initially lead to A:A mismatches and then result in A → T transversion mutations during the second round of replication. In contrast, if a large conformation flip of the ALI moiety occurs at the insertion step to reorient the bulky moiety from an intercalated position into the major groove, dTTP (non-mutagenic) incorporation will be favored. Molecular dynamics (MD) simulations on postextension complexes reveal that damaged DNA will likely further rearrange during later replication steps to acquire a base-displaced intercalated conformation that is similar to that previously reported for (unbound) ALI-N 6 -A adducted DNA, with the exception of slight non-planarity at the lesion site. Overall, our results provide a structural explanation for both the successful non-mutagenic lesion bypass and the preferential misincorporation of dATP opposite ALI-N 6 -A and thereby rationalize the previously reported induction of A → T signature transversion mutations associated with AAs. This work should thereby inspire future biochemical experiments and modeling studies on the replication of this important class of DNA lesions by related human translesion synthesis polymerases.

Aristolochic acids (AAs), the major components in Aristolochia manshuriensis Kom stems (AMK), may cause Chinese herb nephropathy during clinical application. Therefore, it is necessary to distinguish AMK from other herbs and Chinese medicines using AAs with high purity as standards. So, an efficient method for separation and purification of AAs is required because of their similar structures. In this study, six AAs with purities of >98% were obtained by pH-zone-refining counter-current chromatography (PZRCCC) in a single run. The optimum two-phase solvent system was petroleum ether-ethyl acetate-methanol-water (3:7:3:7, v/v). Triethylamine (10 mM) was added to the aqueous mobile phase and trifluoroacetic acid (10 mM) to the organic stationary phase. As a result, 9.7 mg aristolochic acid IIIa, 12.0 mg aristolochic acid IVa, 32.2 mg aristolochic acid II, 103.7 mg aristolochic acid I, 24.6 mg aristolic acid II, and 26.1 mg aristolic acid I were obtained from 800 mg AAs crude extract. The elution order of AAs during PZRCCC separation corresponded with the pK a values and hydrophobicities of the target compounds. PZRCCC is an efficient method for isolation of AAs with similar structures., Competing Interests: Declaration of Competing Interest We declare that we have no conflicts exists in the submission of this manuscript. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. And the manuscript is approved by all authors for publication., (Copyright © 2019. Published by Elsevier B.V.)

An understanding of the fate of organic compounds originating from plants in soil is crucial for determining their persistence and concentrations in the environment. Aristolochic acids are believed to be the causal agents that induce Balkan endemic nephropathy by food contamination through soil adsorption of humic acids, major components of soil. Aristolochic acids are active chemicals in Aristolochia plant species found in endemic villages. In this article, molecular structure interactions between 18 structures of aristolochic acids with an inserted humic acid structure were studied. These structures were optimized in vacuo and by periodic box simulation with water solvate using the computational molecular mechanics MM+ method with HyperChem software. The QSPR models were used for correlation of the relationship between the hydrophobicity values of 18 AA structures coupled with a HA structure by MM+ and QSAR+ properties. Computational hydrophobicity values were considered dependent variables and were related to the structural features obtained by molecular and quantum mechanics calculations by multiple linear regression approaches. The obtained model was validated, and the results indicated differing hydrophobicity between the MM+ and QSAR+ properties., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

"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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aristolochic acid I is a toxic compound found in the genus of Aristolochia plants, which are commonly used as herbal cough treatment medicines. To remove the aristolochic acid I in extract efficiently and selectively, a molecularly imprinted polymer composed of ethylimidazole ionic liquid-based zeolitic imidazolate framework-67 was synthesized and used as the adsorbent. Under the conditions optimized by the software design expert, the sorbent showed highest adsorption amount of 34.25 mg/g in methanol/water (95:5, v/v) at 39°C for 138 min. The sorbent was then applied to solid phase extraction to isolate aristolochic acid I from the extract of the herbal plant Fibraurea Recisa Pierre. 0.043 mg/g of aristolochic acid I was obtained after the loading, washing, and elution processes. The limit of detection of 2.41 × 10 -5  mg/mL and good recoveries provided evidence for the accuracy of this method., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: In humans, bitterness perception is mediated by ~25 bitter taste receptors present in the oral cavity. Among these receptors three, TAS2R10, TAS2R14 and TAS2R46, exhibit extraordinary wide agonist profiles and hence contribute disproportionally high to the perception of bitterness. Perhaps the most broadly tuned receptor is the TAS2R14, which may represent, because of its prominent expression in extraoral tissues, a receptor of particular importance for the physiological actions of bitter compounds beyond taste., Methods: To investigate how the architecture and composition of the TAS2R14 binding pocket enables specific interactions with a complex array of chemically diverse bitter agonists, we carried out homology modeling and ligand docking experiments, subjected the receptor to point-mutagenesis of binding site residues and performed functional calcium mobilization assays., Results: In total, 40 point-mutated receptor constructs were generated to investigate the contribution of 19 positions presumably located in the receptor's binding pocket to activation by 7 different TAS2R14 agonists. All investigated positions exhibited moderate to pronounced agonist selectivity., Conclusions: Since numerous modifications of the TAS2R14 binding pocket resulted in improved responses to individual agonists, we conclude that this bitter taste receptor might represent a suitable template for the engineering of the agonist profile of a chemoreceptive receptor., General Significance: The detailed structure-function analysis of the highly promiscuous and widely expressed TAS2R14 suggests that this receptor must be considered as potentially frequent target for known and novel drugs including undesired off-effects., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tobacco smoking contributes to about 50% of the bladder-cancer (BC) cases in the United States. Some aromatic amines in tobacco smoke are bladder carcinogens; however, other causal agents of BC are uncertain. Exfoliated urinary cells (EUCs) are a promising noninvasive biospecimen to screen for DNA adducts of chemicals that damage the bladder genome, although the analysis of DNA adducts in EUCs is technically challenging because of the low number of EUCs and limiting quantity of cellular DNA. Moreover, EUCs and their DNA adducts must remain viable during the time of collection and storage of urine to develop robust screening methods. We employed RT4 cells, a well-differentiated transitional epithelial bladder cell line, as a cell-model system in urine to investigate cell viability and the chemical stability of DNA adducts of two prototypical bladder carcinogens: 4-aminobiphenyl (4-ABP), an aromatic amine found in tobacco smoke, and aristolochic acid I (AA-I), a nitrophenanthrene found in Aristolochia herbaceous plants used for medicinal purposes worldwide. The cell viability of RT4 cells pretreated with 4-ABP or AA-I in urine exceeded 80%, and the major DNA adducts of 4-ABP and AA-I, quantified by liquid chromatography-mass spectrometry, were stable for 24 h. Thereafter, we successfully screened EUCs of mice treated with AA-I to measure DNA adducts of AA-I, which were still detected 25 days following treatment with the carcinogen. EUCs are promising biospecimens that can be employed for the screening of DNA adducts of environmental and dietary genotoxicants that may contribute to the development of BC.

The ethanolic extract from the stem bark of Goniothalamus marcanii Craib was shown in preliminary brine shrimp lethality data having good cytotoxic activity. Further bioassay guided isolation was done by means of solvent partition, chromatography and precipitation to provide four isolated compounds: a novel compound 1 with the core structure of 1-azaanthraquinone moiety referred as marcanine G; as well as compounds 2-4 with known aristolactam structures namely, piperolactam C, cepharanone B and taliscanine. These compounds were characterised by spectroscopic techniques. The assessment of cytotoxicity was established on an SRB assay using doxorubicin as a positive control. Marcanine G (1) was considered the most active compound indicating the IC 50 values of 14.87 and 15.18 μM against human lung cancer cells (A549) and human breast cancer cells (MCF7), respectively. However, 2 showed mild activity with the IC 50 values of 83.72 and 82.32 μM against A549 and MCF7 cells, respectively.

Phospholipase A 2 (PLA 2 ) catalyzes the hydrolysis of phospholipids into arachidonic acid and lysophospholipids. Arachidonic acid is modified by cyclooxygenases into active compounds called eicosanoids that act as signaling molecules in a number of physiological processes. Excessive production of eicosanoids leads to several pathological conditions such as inflammation. In order to block the inflammatory effect of these compounds, upstream enzymes such as PLA 2 are valid targets. In the present contribution, molecular dynamic analysis was performed to evaluate the binding of diclofenac, 9-hydroxy aristolochic acid (9-HAA) and indomethacin to PLA 2 . Obtained results revealed that 9-HAA could form a more stable complex with PLA 2 when compared to diclofenac and indomethacin. Furthermore, analysis of intermolecular binding energy components indicated that hydrophobic interactions were dominant in binding process. On the basis of obtained data, inhibitors bearing fused rings with hydrogen acceptor/donor substituent(s) interacted with His48 and Asp49 residues of the active site. More affinity toward PLA 2 might be envisaged through negatively charged moieties via interaction with Trp31, Lys34 and Lys69.

Background: Natural aristolochia alkaloids have attracted the attention of both chemists and biologists from the stand point of their structural and pharmacological aspects. Many of the compounds isolated in this group are potent tumor inhibitors. These are divided into nitrophenanthrinic acid, phenanthrene lactams and isoquinoline alkaloids. A number of structure-activity studies have been performed on aristolochia alkaloids. Of particular interest is the molecule with the β-D-glucoside moiety that has similarity to the clinical anticancer agent daunomycin., Objective: The anticancer activity of aristololactam-β-D-glucoside has been thought to be due to its DNA and RNA binding activities among other actions. In this article we focus on the physicochemical property of this alkaloid and the structural and functional aspects of its binding to different nucleic acid and protein structures., Methods: This review highlights a large number of biophysical studies employing various analytical techniques like absorbance, fluorescence, circular dichroism, thermal melting, viscosity, IR study, isothermal calorimetry and differential scanning calorimetry., Result: The detailed binding mechanism in terms of the structural and thermodynamic aspects at the molecular level has been discussed., Conclusion: This review enables to assess the high potential of developing aristololactam-β-Dglucoside and related alkaloids as therapeutic agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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.

Study on anticancer agents that act via stabilization of telomeric G-quadruplex DNA has emerged as novel and exciting field for anticancer drug discovery. The interaction of carbohydrate containing anticancer alkaloid aristololactam-β-D-glucoside (ADG) with human telomeric G-quadruplex DNA sequence was characterized by different biophysical techniques. The binding parameters were compared with daunomycin (DAN), a well-known chemotherapeutic drug. The Scatchard binding isotherms revealed noncooperative binding for both with the binding affinity values of (1.01 ± 0.05) × 10 6 and (1.78 ± 0.18) × 10 6 M -1 for ADG and DAN, respectively. Circular dichroism, ferrocyanide quenching study, anisotropy study, thiazole orange displacement, optical melting, differential scanning calorimetry study, and molecular docking study suggest significant stacking and stabilizing efficiency of ADG with comparison to DAN. The energetics of the interaction for ADG and DAN revealed that both reactions were predominantly entropy driven. Negative heat capacity values were obtained from the temperature dependence of the enthalpy change. The standard molar Gibbs energy change exhibited only marginal alterations with temperature suggesting the occurrence of enthalpy-entropy compensation. These findings indicate that ADG can act as a stabilizer of telomeric G-quadruplex DNA and thereby can be considered as a potential telomerase inhibitor., (Copyright © 2017 John Wiley & Sons, Ltd.)

Antidesma ghaesembilla is an important medicinal and food plant in many Asian countries. Ten substances could be isolated from the dichloromethane and methanol extract: sitostenone ( 3 ), daucosterol ( 4 ), chavibetol ( 5 ), asperphenamate ( 6 ), protocatechuic acid ( 7 ), vanillic acid-4- O - β -D-glucoside ( 8 ), 1- O - β -D-glucopyranosyl-3- O -methyl-phloroglucinol ( 9 ), and aristolic acid II-8- O - β -D-glucoside ( 10 ), and two new aristolic acid derivatives, 10-amino-5,7-dimethoxy-aristolic acid II (= 6-amino-9,11-dimethoxyphenanthro[3,4- d ]-1,3-dioxole-5-carboxylic acid; 1 ) and 5,7-dimethoxy-aristolochic acid II (= 9,11-dimethoxy-6-nitrophenantro[3,4- d ]-1,3-dioxole-5-carboxylic acid; 2 ). Exposure to humans of some of these compounds is associated with a severe disease today known as aristolochic acid nephropathy. Therefore, the traditional usage of this plant has to be reconsidered carefully., Competing Interests: Conflict of Interest: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues (AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I (AA-I) and aristolochic acid II (AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-QqQ-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts., (Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Four new aristolochic acid derivatives aristchamic A (1), aristchamic B (2), aristochamic C (3a), aristchamic D (3b) and one new aristolactam aristolactam-CV (4), together with 10 known compounds (5-14), were isolated from the rhizomes of Aristolochia championii. Their structures were assigned by detailed analysis of MS and NMR spectroscopic data. All of the isolated compounds were evaluated for their cytotoxic activities against HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cell. Compound 1 exhibited significant cytotoxic activity against HCT-116, HepG2, BGC-823, and NCI-H1650, with IC 50 values of 0.50, 7.37, 2.66, and 0.75μM, respectively., (Copyright © 2017. Published by Elsevier B.V.)

A rapid interference-free spectrofluorometric method combined with the excitation-emission matrix fluorescence and the second-order calibration methods based on the alternating penalty trilinear decomposition (APTLD) and the self-weighted alternating trilinear decomposition (SWATLD) algorithms, was proposed for the simultaneous determination of nephrotoxic aristolochic acid I (AA-I) and aristololactam I (AL-I) in five Chinese herbal medicines. The method was based on a chemical derivatization that converts the non-fluorescent AA-I to high-fluorescent AL-I, achieving a high sensitive and simultaneous quantification of the analytes. The variables of the derivatization reaction that conducted by using zinc powder in acetose methanol aqueous solution, were studied and optimized for best quantification results of AA-I and AL-I. The satisfactory results of AA-I and AL-I for the spiked recovery assay were achieved with average recoveries in the range of 100.4-103.8% and RMSEPs <0.78ngmL -1 , which validate the accuracy and reliability of the proposed method. The contents of AA-I and AL-I in five herbal medicines obtained from the proposed method were also in good accordance with those of the validated LC-MS/MS method. In light of high sensitive fluorescence detection, the limits of detection (LODs) of AA-I and AL-I for the proposed method compare favorably with that of the LC-MS/MS method, with the LODs <0.35 and 0.29ngmL -1 , respectively. The proposed strategy based on the APTLD and SWATLD algorithms by virtue of the "second-order advantage", can be considered as an attractive and green alternative for the quantification of AA-I and AL-I in complex herbal medicine matrices without any prior separations and clear-up processes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 molecularly imprinted polymers (MIP) were prepared via aqueous RAFT precipitation polymerization, with aristolochic acid I (AAI) as the template molecule, AA as the functional monomer, EGDMA as the cross-linker, AIBN as the chain initiator, CTP as the chain transfer agent and 80% (g/g) DMF-aqueous solution as the porogen. The differential UV-vis spectra revealed that a cooperative hydrogen-bonding complex between AAI and AA might be formed at the molar ratio of 1:3 in prepolymerized system. The synthesized MIPs were characterized by FTIR spectra, solid UV-visible absorption spectra, nitrogen adsorption-desorption isotherms and scanning electron microscope, which proved that the MIPs and NIPs have the similar chemical structures and the adding of AAI could affect the size and morphology of the microspheres. UV-visible absorption spectra and High-performance liquid chromatography (HPLC) was used to investigate the adsorption and recognition properties of the MIPs. The Scatchard isotherm model described that the binding sites independently acted. The Langmuir isotherm model suggested an excellent imprinting effect owing to the presence of a large number of specific binding sites on the MIP. The Freundlich model indicated that the AAI could be readily absorbed by MIP. Selective absorption of the template molecule was demonstrated in presence of its analogous compounds, benzoic acid and nitrobenzene. The recycling experiments implied that the MIP could be reused to further selective recognition and separation to AAI for six times at least. MIP was developed for removal of AAI from the Aristolochia manshuriensis extraction. The results indicated that 25mg of MIP could remove the AAI below the HPLC detection limits (6.47ngmL -1 ) from 5.0mL of the extraction (CAAI=0.0018mgmL -1 ) with the recovery of AAI to 91.50% (n=3, SD=4.24%). Therefore, it is clearly revealed that the MIP can be a useful tool to remove toxic compounds from natural products., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The genotoxicity and cytotoxicity of aristolochic acids is well documented, and the Aristolochiaceae plant family has been widely used in China and India for medical purposes. However, the mechanisms of aristolochic acid I (AAI) in treatment and toxicity remain to be fully elucidated. According to the theory of traditional Chinese medicine (TCM), the spleen is responsible for transportation and transformation, in which a substance is transformed, absorbed and distributed in the body. In the present study, rats were randomized into a blank group without spleen deficiency and a spleen deficiency group to investigate the metabolism of AAI. The results showed that the concentration of AAI was higher in the spleen deficiency group, compared with that of the blank group. To further elucidate this process, the expression of organic anion transporting peptide (oatp)2a1 in the rats of the two groups were examined following oral administration of AAI. It was observed that the mRNA level of oatp2a1 in the small intestine of the blank+AAI 60 min group was downregulated, compared with that in the blank group. Compared with the mRNA level of oatp2a1 in the spleen deficiency group, the expression levels in the lung and liver were downregulated in the spleen deficiency+AAI 5 min group, whereas expression levels in the kidney in the spleen deficiency+AAI 60 min group were upregulated. Based on the above results, it was hypothesized that the expression of oatp2a1 may be one of the mechanisms of AAI metabolism in rats. In TCM, the spleen and certain functions of the small intestine, are important in AAI metabolism, and affect the toxicity of AAI. In addition, the lung, liver and kidney may also be involved in spleen deficiency syndrome in rats.

Translesion DNA synthesis (TLS) operates when replicative polymerases are blocked by DNA lesions. To investigate the mechanism of mammalian TLS, we employed a plasmid bearing a single 7-(deoxyadenosine-N 6 -yl)-aristolactam I (dA-AL-I) adduct, which is generated by the human carcinogen, aristolochic acid I, and genetically engineered mouse embryonic fibroblasts. This lesion induces A to T transversions at a high frequency. The simultaneous knockouts of the Polh, Poli and Polk genes did not influence the TLS efficiency or the coding property of dA-AL-I, indicating that an unknown DNA polymerase(s) can efficiently catalyze the insertion of a nucleotide opposite the adduct and subsequent extension. Similarly, knockout of the Rev1 gene did not significantly affect TLS. However, knockout of the Rev3l gene, coding for the catalytic subunit of polζ, drastically suppressed TLS and abolished dA-AL-I to T transversions. The results support the idea that Rev1 is not essential for the cellular TLS functions of polζ in mammalian cells. Furthermore, the frequency of dA-AL-I to T transversion was affected by a sequence context, suggesting that TLS, at least in part, contributes to the formation of mutational hot and cold spots observed in aristolochic acid-induced cancers., Competing Interests: statement The authors declare that there are no conflicts of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants., (Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

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&#95;F352A). The TASK-2&#95;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&#95;T108C current but did not affect TASK-2&#95;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.)

"Chiniy-trèf" is a traditional medicinal preparation used in Martinique, French West Indies, for the prevention of all kinds of attempted poisoning and hex. It is produced by the maceration in alcohol (mostly rum) of larvae (caterpillars) of the butterfly Battus polydamas ssp. cebriones, feeding on the leaves of Aristolochia trilobata. Aristolochic acids I and II that are well-known nephrotoxic and carcinogenic substances were identified on two samples of "chiniy-trèfl" by chromatographic methods., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

Aristolochic acid (AA) is a natural bioactive substance found in Chinese herbs that induce toxicity during ovarian maturation of animals and humans. Apoptosis is induced by various types of damage and governs the progression of biological cell removal that controls the equilibrium between cell growth and death. However, the AA toxicity mechanism during testis maturation in mouse has not been elucidated and was thus the focus of the present study. This study used TM4 Sertoli cells and an ICR mouse model, both of which were injected with aristolochic acid I (AAI) for 4 weeks. Testis dimensions and weight were surveyed to define AAI cytotoxicity in the mice testis. The MTT assay was used to analyze the cytotoxicity of AAI in TM4 Sertoli cells. An apoptosis expression mediator was analyzed through Western blotting, while the measure of apoptosis-induced cell death of TM4 Sertoli cells and testis tissues was analyzed by the TUNEL assay. We found that AAI strongly inhibits survival in TM4 cells and that AAI significantly activated apoptosis-induced cell death in TM4 Sertoli cells and mice testis tissue. In addition, AAI suppressed the expression of B-cell lymphoma 2 (Bcl-2), a factor related to anti-apoptosis. It markedly improved pro-apoptotic protein expression, including Bcl-2-associated X protein, poly(ADP-ribose) polymerase, and caspase-3 and -9. Furthermore, we observed that AAI significantly reduced the size and weight of mouse testis. Moreover, germ cells and somatic cells in testis were markedly damaged by AAI. In addition, we found that AAI significantly inhibits ERK1/2 and Akt activation in TM4 Sertoli cells and testis tissue. The data obtained in this study indicate that AAI causes severe injury for the period of testis development by impeding apoptosis related to the Akt and ERK1/2 pathway.

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.)