NMR Studies on the Subacute Biochemical Effects of Aristolochic Acid on Rat Serum

The subacute effect of aristolochic acid （AA） on rat serum was studied by NMR method. The biochemical effects induced by AA were characterized by an increase in the amounts of creatinine, trimethylamine N-oxide, acetoacetate, acetate and 3-D-hydroxybutyrate and lactate in serum from ^1H NMR spectra. Principal component analysis was used for further comparing the similarities of ^1H NMR spectral profiles of serum from rats treated with AA and model toxins.

Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid （AA） is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2′-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction （SPE）, and further purified by a reversed-phase high performance liquid chromatography （HPLC）. By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity （more than 94% as indicated by HPLC） were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry （LC-ESI-Q-TOF-MS/MS） and LC-Diode array detector-fluorescence （LC-DAD-FL） analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

A NEW ARISTOLOCHIC ACID FROM ARISTOLOCHIA CINNABARINA

A new compound, aristolochoc acid Ⅲ a- 6-0-β-D-glucoside, was isolated along with four known compounds from Aristolochia cinnabarina. The structure of the new,compound was elucidated on the basis of spectral evidences

Chemical profiling of principle active and toxic constituents in herbs containing aristolochic acids

Objective:To clear the amounts of the principal active/toxic components in herbs containing aristolochic acids(HCAAs),which are still used as medicine and/or seasoning in many ethnic minority areas of China.Methods:In this study,six major active and toxic components in HCAAs were extracted with ultrasonic extraction.With 6-O-methyl guanosine as internal standard,the target compounds were analyzed qualitatively and quantitatively by using ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry(UPLC-ESI-MS/MS)with multiple reaction monitoringinformation dependent acquisition-enhanced production ion scanning mode(MRM-IDA-EPI)combined with dynamic background subtraction(DBS)function.Results:The method showed good linearity in the linear range of the six analytes.The limit range of detection was from 0.01 ng/mL to 0.27 ng/mL.All of the detection repeatability,extraction repeatability and accuracy of the method were good.After extraction,the samples remained stable at 15C within 24 h.Six analytes were all found in samples except aristolactam(AL)in sample 2,and the contents varied greatly.The contents of these compounds decreased in fruits,leaves and stems of Aristolochia delavayi successively.Conclusion:This method has the advantages of less sample dosage,simple operation,short analysis cycle,high sensitivity,specificity and accuracy.It laid a good foundation for guiding the safety of HCAAs,the indepth study of pharmacological and toxicological effects and the scientific and standardized processing and compatibility of HCAAs.

No Incidence of Liver Cancer Was Observed in A Retrospective Studyof Patients with Aristolochic Acid Nephropathy

Objective:To assess the risk of aristolochic acid(AA)-associated cancer in patients with AA nephropathy(AAN).Methods:A retrospective study was conducted on patients diagnosed with AAN at Peking University First Hospital from January 1997 to December 2014.Long-term surveillance and follow-up data were analyzed to investigate the influence of different factors on the prevalence of cancer.The primary endpoint was the incidence of liver cancer,and the secondary endpoint was the incidence of urinary cancer during 1 year after taking AA-containing medication to 2014.Results:A total of 337 patients diagnosed with AAN were included in this study.From the initiation of taking AA to the termination of follow-up,39 patients were diagnosed with cancer.No cases of liver cancer were observed throughout the entire follow-up period,with urinary cancer being the predominant type(34/39,87.17%).Logistic regression analysis showed that age,follow-up period,and diabetes were potential risk factors,however,the dosage of the drug was not significantly associated with urinary cancer.Conclusions:No cases of liver cancer were observed at the end of follow-up.However,a high prevalence of urinary cancer was observed in AAN patients.Establishing a direct causality between AA and HCC is challenging.

In situ metabolomics in nephrotoxicity of aristolochic acids based on air flow-assisted desorption electrospray ionization mass spectrometry imaging

Understanding of the nephrotoxicity induced by drug candidates is vital to drug discovery and development.Herein,an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI)was established for direct analysis of metabolites in renal tissue sections.This method was subsequently applied to investigate spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I,a known nephrotoxic drug,aimed to discover metabolites associated with nephrotoxicity.As a result,38 metabolites related to the arginine-creatinine metabolic pathway,the urea cycle,the serine synthesis pathway,metabolism of lipids,choline,histamine,lysine,and adenosine triphosphate were significantly changed in the group treated with aristolochic acid I.These metabolites exhibited a unique distribution in rat kidney and a good spatial match with histopathological renal lesions.This study provides new insights into the mechanisms underlying aristolochic acids nephrotoxicity and demonstrates that AFADESI-MSI-based in situ metabolomics is a promising technique for investigation of the molecular mechanism of drug toxicity.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry

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 MS1 of AAs, the characteristic pseudomolecular ions were [M + NH_4]^+, [M + H]^+, and [M + H - H_2O]^+. However, only [M + H]^+ was found in the MS1 of ALs, which was simpler than that of AAs. Distinct MSn 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.

Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae Fructus, using UHPLC-Q/TOF-MS and UHPLC-Qq Q-MS

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-Qq Q-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-Qq Q-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.

Transitional cell carcinoma associated with aristolochic acid nephropathy： most common cancer in chronic hemodialysis patients in China

Background The research of cancer in patients on hemodialysis （HD） in China has not been reported. The aim of this study was to investigate the clinical and histological features and outcomes of cancer in Chinese HD patients. Methods The study subjects were 49 cancer patients （1.4%） out of 3448 end stage renal disease （ESRD） patients maintained on HD at China-Japan Friendship Hospital from October 1997 to July 2011. Results Urinary tract cancer （74%） was the most common followed by gastrointestinal tract cancer （12%）, breast cancer （6%）, lung cancer （4%）, thyroid cancer （2%）, and hematologic cancer （2%）. Thirty-three patients （67%） had urinary tract transitional cell carcinoma （TCC） and 29 of them had aristolochic acid nephropathy （AAN） as underlying disease. Death occurred in eight patients out of 49, and the survival rate of HD patients with cancer was similar to those without cancer （P=0.120）. Conclusion The urinary tract TCC is the most common cancer in HD patients with AAN in one of the centers of northern China.

Aristolochic Acid-Induced Genotoxicity and Toxicogenomic Changes in Rodents

Aristolochic acid(AA)is a group of structurally related nitrophenanthrene carboxylic acids found in many plants that are widely used by many cultures as traditional herbal medicines.AA is a causative agent for Chinese herbs nephropathy,a term replaced later by AA nephropathy.Evidence indicates that AA is nephrotoxic,genotoxic,and carcinogenic in humans;and it also induces tumors in the forestomach,kidney,renal pelvis,urinary bladder,and lung of rats and mice.Therefore,plants containing AA have been classified as carcinogenic to humans(Group 1)bytheInternational AgencyforResearchonCancer.In our laboratories,we have conducted a series of genotoxicity and toxicogenomic studies in the rats exposed to AA of 0.1–10 mg/kg for 12 weeks.Our results demonstrated that AA treatments induced DNA adducts and mutations in the kidney,liver,and spleen of rats,as well as significant alteration of gene expression in both its target and nontarget tissues.AA treatments altered mutagenesis-or carcinogenesis-related microRNA expression in rat kidney and resulted in significant changes in protein expression profiling.We also applied benchmark dose(BMD)modeling to the 3-month AA-induced genotoxicity data.The obtained BMDL10(the lower 95%confidence interval of the BMD10 that is a 10%increase over the background level)for AA-induced mutations in the kidney of rats was about 7μg/kg body weight per day.This review constitutes an overview of our investigations on AA-induced genotoxicity and toxicogenomic changes including gene expression,microRNA expression,and proteomics;and presents updated information focused on AA-induced genotoxicity in rodents.

Schisandra chinensis Oil Attenuates Aristolochic Acid Ⅰ-Induced Nephrotoxicity in vivo and in vitro

Objective: To investigate the protective effects of Schisandra chinensis oil(SCEO) against aristolochic acid Ⅰ(AAⅠ)-induced nephrotoxicity in vivo and in vitro and elucidate the underlying mechanism.Methods: C57BL/6 mice were randomly divided into 5 groups according to a random number table, including control group, AAⅠ group, and AAⅠ+SCEO(0.25, 0.5 and 1 g/kg) groups(n=5 per group). Pretreatment with SCEO was done for 2 days by oral administration, while the control and AAⅠ groups were treated with sodium carboxymethyl cellulose. Mice of all groups except for the control group were injected intraperitoneally with AAⅠ(5 mg/kg) from day 3 until day 7. Histopathological examination and apoptosis of kidney tissue were observed by hematoxylin and eosin and TdT-mediated dUTP nick-end labeling(TUNEL) staining, respectively. The levels of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), blood urea nitrogen(BUN), and serum creatinine(SCr), as well as renal malondialdehyde(MDA), glutathione, r-glutamyl cysteingl+glycine(GSH), and superoxide dismutase(SOD) were analyzed using enzyme-linked immunosorbent assay(ELISA).Expressions of hepatic cytochrome P450 1A1(CYP1A1), CYP1A2, and nad(p)hquinonedehydrogenase1(NQO1) were analyzed using ELISA, quantitative real-time polymerase chain reaction(qPCR) and Western blot,respectively. In vitro, SCEO(40 μg/mL) was added 12 h before treatment with AAⅠ(40 μmol/mL for 48 h) in human renal proximal tubule cell line(HK-2), then apoptosis and reactive oxygen species(ROS) were analyzed by flow cytometry. Results: SCEO 0.5 and 1 g/kg ameliorated histopathological changes and TUNEL+ staining in the kidney tissues of mice with AAⅠ-induced nephrotoxicity, and reduced serum levels of ALT, AST, BUN and SCr(P<0.01 or P<0.05). SCEO 0.5 and 1 g/kg alleviated the ROS generation in kidney, containing MDA,GSH and SOD(P<0.01 or P<0.05). SCEO 1 g/kg increased the expressions of CYP1A1 and CYP1A2 and decreased NQO1 level in the liver tissues(P<0.01 or P<0.05). Besides, in vitro studies also demonstrated that SCEO 40 μg/mL inhibited apoptosis and ROS generation(P<0.05 or P<0.01). Conclusions: SCEO can alleviate AAⅠ-induced kidney damage both in vivo and in vitro. The protective mechanism may be closely related to the regulation of metabolic enzymes, thereby inhibiting apoptosis and ROS production.

Aristolochic acids exposure was not the main cause of liver tumorigenesis in adulthood

Aristolochic acids(AAs) have long been considered as a potent carcinogen due to its nephrotoxicity. Aristolochic acid I(AAI) reacts with DNA to form covalent aristolactam(AL)-DNA adducts,leading to subsequent A to T transversion mutation, commonly referred as AA mutational signature. Previous research inferred that AAs were widely implicated in liver cancer throughout Asia. In this study, we explored whether AAs exposure was the main cause of liver cancer in the context of HBV infection in China's Mainland. Totally 1256 liver cancer samples were randomly retrieved from 3 medical centers and a refined bioanalytical method was used to detect AAI-DNA adducts. 5.10% of these samples could be identified as AAI positive exposure. Whole genome sequencing suggested 8.41% of 107 liver cancer patients exhibited the dominant AA mutational signature, indicating a relatively low overall AAI exposure rate. In animal models, long-term administration of AAI barely increased liver tumorigenesis in adult mice, opposite from its tumor-inducing role when subjected to infant mice. Furthermore, AAI induced dose-dependent accumulation of AA-DNA adduct in target organs in adult mice, with the most detected in kidney instead of liver. Taken together, our data indicate that AA exposure was not the major threat of liver cancer in adulthood.

Dissection of cellular and molecular mechanisms of aristolochic acid-induced hepatotoxicity via single-cell transcriptomics

Background:Aristolochic acids(AAs),a class of carcinogenic and mutagenic natural products from Aristolochia and Asarum plants,are well-known to be responsible for inducing nephrotoxicity and urothelial carcinoma.Recently,accumulating evidence suggests that exposure to AAs could also induce hepatotoxicity and even hepatocellular carcinoma,though the mechanisms are poorly defined.Methods:Here,we aimed to dissect the underlying cellular and molecular mechanisms of aristolochic acid I(AAI)-induced hepatotoxicity by using advanced single-cell RNA sequencing(scRNA-seq)and proteomics techniques.We established the first single-cell atlas of mouse livers in response to AAI.Results:In hepatocytes,our results indicated that AAI activated NF-κB and STAT3 signaling pathways,which may contribute to the inflammatory response and apoptosis.In liver sinusoidal endothelial cells(LSECs),AAI activated multiple oxidative stress and inflammatory associated signaling pathways and induced apoptosis.Importantly,AAI induced infiltration of cytotoxic T cells and activation of proinflammatory macrophage and neutrophil cells in the liver to produce inflammatory cytokines to aggravate inflammation.Conclusions:Collectively,our study provides novel knowledge of AAs-induced molecular characteristics of hepatotoxicity at a singlecell level and suggests future treatment options for AAs associated hepatotoxicity.

ARISTOLOCHIC ACID NEPHROPATHY: AN UPDATE

Aristolochic acid nephropathy(AAN)is a rapidly progressive interstitial nephritis leading to end-stage renal disease and urothelial malignancy.It was originally reported in Belgium in a group of more than 100 patients who had ingested slimming pills containing powdered root extracts of a Chinese herb,Aristolochia fangchzi.

AristolochicacidI inducedapoptosisinLLC PK1cellsandameliorationoftheapoptoticdamagebycalciumantagonist

To examine the effect of different concentrations of aristolochic acid I (AAI) in inducing apoptosis of cultured porcine renal cell line LLC PK1 and to investigate the relationship between intracellular free calcium concentration ([Ca ++ ]i) and LLC PK1 apoptosis induced by AAI and the influence of a calcium antagonist, lacidipine on apoptosis and [Ca ++ ]i Methods LLC PK1 cells were treated in different groups: a the normal group without treatment; b the group with AAI alone (0 01?g·L 1 , 0 02?g·L 1 , 0 04 ?g·L 1 , 0 08?g·L 1 ); c the group with lacidipine alone (10?ng·L 1 , 10 2 ?ng·L 1 , 10 3?ng·L 1 ); d the group with AAI (0 04?g·L 1 ) plus lacidipine (10?ng·L 1 , 10 2? ng·L 1 , 10 3?ng·L 1 ) Light microscopy, agarose gel electrophoresis, Annexin V Flous apoptosis detection kit and flow cytometry using propidium iodide staining to identify or quantify the apoptosis of LLC PK1 cells Mean [Ca ++ ]i was measured by laser confocus microscopy using Fluo 3/AM staining Results A series of morphologic changes that were characteristic of apoptosis, Annexin V Flous staining positive apoptotic cells and “DNA ladder' were identified in AAI (0 02?g·L 1 -0 08?g·L 1 ) treated LLC PK1 cells Quantitative analysis of apoptotic cells showed that the percentage of apoptotic cells in AAI (0 02?g·L 1 , 0 04?g·L 1 or 0 08?g·L 1 ) group was significantly higher than that in normal group (5 3%, 48 5%, 78 7% vs 2 6%, P <0 001) Mean [Ca ++ ]i was significantly higher in cells treated with AAI (0 04?g·L 1 ) than that in normal cells (58 01±18 89 vs 22 66±4 78, P <0 001) In group treated with AAI plus lacidipine (102?ng·L 1 , 103?ng·L 1 ), mean [Ca ++ ]i was significantly lower than that treated with AAI alone (35 47±12 85, 28 55±10 16 vs 58 01±18 89, P <0 001) And the percentage of apoptotic cells in group treated with AAI plus lacidipine (10 2?ng·L 1 , 10 3?ng·L 1 ) was also significantly lower than that treated with AAI alone (19 0%, 27 8% vs 34 7%, P <0 001) Conclusions High concentrations of AAI may induce apoptosis of LLC PK1 cells The mean [Ca ++ ]i in AAI treated LLC PK1 cells was increased significantly, sugguesting that the increase of [Ca ++ ]i may be related to apoptosis in LLC PK1 cells Lacidipine may decrease the raised mean [Ca ++ ]i levels caused by AAI and the percentage of apoptotic cells, and lacidipine may ameliorate AAI induced apoptotic damage by inhibiting the increase of [Ca ++ ]i in LLC PK1 cells

Identification and determination of the major constituents in traditional Chinese medicine Longdan Xiegan Pill by HPLC-DAD-ESI-MS

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill.The chemical profile of the twelve compounds,including geniposidic acid（1）,geniposide（2）,gentiopicroside（3）,liquiritin（4）,crocin（5）,baicalin（6）,wogonoside（7）,baicalein（8）,glycyrrhizic acid（9）,wogonin（10）,oroxylin A（11）and aristolochic acid A（12）,was acquired using high-performance liquid chromatography-diode array detector coupled with an electrospray tandem mass spectrometer（HPLC-DAD-ESI-MS）.The analysis was performed on a Dikma Platisil ODS C18 column（250 mm×4.6 mm,5 μm）with a gradient solvent system of acetonitrile-0.1% aqueous formic acid.The validation was carried out and the linearities（r〉0.9996）,repeatability（RSD〈1.8%）,intra-and inter-day precision（RSD〈1.3%）,and recoveries（ranging from 96.6% to 103.4%）were acceptable.The limits of detection（LOD）of these compounds ranged from 0.29 to 4.17 ng.Aristolochic acid A,which is the toxic ingredient,was not detected in all the batches of Longdan Xiegan Pill.Furthermore,hierarchical cluster analysis was used to evaluate the variation of the herbal prescription.The proposed method is simple,effective and suitable for the quality control of this traditional Chinese medicine（TCM）.

Asari Radix et Rhizoma consumption lacks relevance for hepatocellular carcinoma in patients:A retrospective cohort study

Objective:Although some studies have linked Asari Radix et Rhizoma(Asari Radix)administration to hepatocellular carcinoma(HCC),few studies have examined the association between the development of HCC and use of Asari Radix among patients in China's Mainland.This study aimed to evaluate the realworld association between Asari Radix and HCC in patients to strengthen the understanding of Asari Radix safety.Methods:A retrospective cohort study among hepatitis B virus(HBV)-monoinfected patients and nonHBV-monoinfected patients were performed.Patients over 18 years of age were eligible for inclusion.Prescription records of inpatients and outpatients were inquired to distinguish Asari Radix users and nonusers.The risk of developing HCC among Asari Radix users and nonusers in the HBV cohort and the non-HBV cohort was analyzed.Results:There were 49500 HBV and 133148 non-HBV patients involved in the two cohorts.Among HBV patients(2901 users;46599 nonusers),the prevalence of HCC in Asari Radix users was lower than that in nonusers(145.70 vs.265.43 per 105).Among non-HBV patients(5042 users;128106 nonusers),the prevalence of HCC in Asari Radix users was lower than that in nonusers(81.62 vs.134.11 per 105).None of the hazard ratios(HRs)of Asari Radix exposure ranging from 1 g to 200 g in the two cohorts showed correlation between Asari Radix exposure and hepatocarcinogenesis.Conclusion:An obvious irrelevancy was found between the consumption of Asari Radix and HCC development both in patients with and in those without HBV infection.Use of Asari Radix under 200 g appears safe in terms of HCC risk in the Chinese population;further prospective studies are needed to confirm our results.

Carcinogens that induce the A：T 〉 T：A nucleotide substitutions in the genome

Recently, Ng et al. reported that the A：T 〉 T：A substitutions, proposed to be a signature of aristolochic acid （AA） exposure, were detected in 76/98 （78%） of patients with hepatocellular carcinoma （HCC） from the Taiwan Province of China, and 47% to 1.7% of HCCs from the Chinese mainland and other countries harbored the nucleotide changes. However, other carcinogens, e.g., tobacco carcinogens 4-aminobiphenyl and 1,3-butadiene, air toxic vinyl chloride and its reactive metabolites chloroethylene oxide, melphalan and chlorambucil, also cause this signature in the genome. Since tobacco smoke is a worldwide public health threat and vinyl chloride distributes globally and is an air pollutant in Taiwan Province, the estimation of the patients＇ exposure history is the key to determine the ＂culprit＂ of the A：T 〉 T：A mutations. Apparently, without estimation of the patients＇ exposure history, the conclusion of Ng et al, is unpersuasive and misleading.