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.
Humans ; Retrospective Studies ; Incidence ; Carcinoma, Hepatocellular ; Liver Neoplasms/epidemiology* ; Kidney Diseases/chemically induced* ; Aristolochic Acids/adverse effects*

OBJECTIVE: To investigate the protective effects of Schisandra chinensis oil (SCEO) against aristolochic acid I (AA I)-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, AA I group, and AA I +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 AA I groups were treated with sodium carboxymethyl cellulose. Mice of all groups except for the control group were injected intraperitoneally with AA I (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 µ g/mL) was added 12 h before treatment with AA I (40 µ mol/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 AA I-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 µ g/mL inhibited apoptosis and ROS generation (P<0.05 or P<0.01). CONCLUSIONS SCEO can alleviate AA I-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.
Animals ; Apoptosis ; Aristolochic Acids/toxicity* ; Cytochrome P-450 CYP1A1/metabolism* ; Cytochrome P-450 CYP1A2/metabolism* ; Glutathione/metabolism* ; Kidney/drug effects* ; Kidney Diseases/drug therapy* ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Plant Oils/therapeutic use* ; Protective Agents/therapeutic use* ; Reactive Oxygen Species/metabolism* ; Schisandra ; Superoxide Dismutase/metabolism*

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 μm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.
Animals ; Aristolochic Acids ; Chromatography, High Pressure Liquid ; DNA Adducts ; Liver ; Mice ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

OBJECTIVE: To evaluate the changes in renal oxygenation in rats with acute aristolochic acid nephropathy using blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) at 7.0T. METHODS: Wistar rats were randomly divided into AAN group (=18) and control group (=6) for intraperitoneal injections of AAI at 40 mg/kg and PEG400, respectively, on a daily basis for 6 consecutive days. All the control rats and 6 rats from AAN group underwent BOLD MRI scan before and at 2, 4, and 6 days after the initial injection for measuring renal cortical and medullary R2 values. At each of the 4 time points, 3 rats in AAN group were sacrificed for histological evaluation; the control rats were examined at 6 days after the initial injection. RESULTS: The cortical and medullary R2 values of the rats in AAN group on days 4 and 6 were significantly higher than those in the control group ( < 0.05). In AAN group, the cortical R2 values showed no obvious changes on day 2 as compared with the baseline values, but increased significantly on day 4 ( < 0.05) and day 6 ( < 0.01); the medullary R2 values increased progressively and were significantly higher than the baseline values on day 4 ( < 0.01) and day 6 ( < 0.01). In the control group, no significant changes were detected in either cortical or medullary R2 values throughout the experiment. CONCLUSIONS BOLD MRI allows non-invasive measurement of renal oxygenation levels in rats with AAN. The increase of renal cortical and medullary R2 values, and particularly the latter, indicates a lowered renal oxygenation level, which provides potentially useful information for clinical decisions.
Animals ; Aristolochic Acids ; Kidney ; Kidney Diseases ; metabolism ; Magnetic Resonance Imaging ; Oxygen ; Random Allocation ; Rats ; Rats, Wistar

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.
Aristolochic Acids ; toxicity ; Carcinogens ; toxicity ; Carcinoma, Hepatocellular ; chemically induced ; genetics ; China ; Environment ; Humans ; Liver Neoplasms ; chemically induced ; genetics ; Mutation ; Taiwan ; Tobacco ; toxicity ; Vinyl Chloride ; toxicity

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.
Aristolochia ; chemistry ; Aristolochic Acids ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Fruit ; chemistry ; Molecular Structure ; 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 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.
Aristolochiaceae ; chemistry ; Aristolochic Acids ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Molecular Structure ; Tandem Mass Spectrometry ; methods

OBJECTIVETo explore the correlation between Pi and Shen by observing the relationship between the metabolism of aristolochic acid (AA) and mRNA and protein expression levels of organic anion transporting polypeptide (oatp) superfamily member 2a1 and 2 b1 (oatp2al and oatp2bl) in renal, small intestinal, and large intestinal tissues of Pi deficiency syndrome (PDS) model rats.

METHODSTotally 46 Sprague-Dawley (SD) rats were randomly divided into four groups, i.e., the blank group (n = 12), the PDS group (n = 22), the AA-I group (n = 6), and the PDS AA-I group (n = 6). PDS model was established by subcutaneously injecting Reserpine at the daily dose of 5 mg/kg for 16 successive days. Carotid intubation was performed in 6 rats selected from the blank group and the PDS group. Pharmacokinetics of AA-I were detected at 5, 15, 30, 45, and 60 min after gastrogavage of AA-I. AA-I concentrations in renal, small intestinal, and large intestinal tissues of 10 rats selected from the PDS group were determined. Normal saline was administered to 6 rats selected from the PDS group and the blank group by gastrogavage. Renal, small intestinal, and large intestinal tissues were collected in the AA-I group and the PDS AA-I group at 60 min after gastrogavage of AA-I. mRNA and protein expression levels of oatp2a1 and oatp2b1 in each tissue were detected using real-time polymerase chain reaction (RT- PCR) and Western blot.

RESULTSCompared with the blank group, plasma concentrations of in vivo AA-I were obviously higher in the PDS group at 15, 30, 45, and 60 min after gastrogavage of AA-I with statistical difference (P < 0.05). Plasma concentrations of AA-I were obviously decreased at 60 min after gastrogavage of AA-I; AA-I concentrations in renal and large intestinal tissues were elevated; AA-I concentrations in small intestinal tissues were obviously reduced in the PDS group. There was no statistical difference in mRNA expression levels of oatp2a1 and oatp2b1 in the aforesaid three tissues of rats between the blank group and the PDS group. Compared with the blank group, mRNA expression levels of oatp2a1 and oatp2b1 decreased in small intestinal tissues of the AA-I group, and the mRNA expression level of oatp2a1 in large intestinal tissues significantly decreased (P < 0.05, P < 0.01). Compared with the PDS group, mRNA expression levels of oatp2a1 and oatp2b1 increased in renal tissues of the PDS AA-I group (P < 0.05); mRNA expression levels of oatp2b1 increased in large intestinal tissues of the PDS AA-I group (P < 0.05).

CONCLUSIONSThe difference in AA-I metabolism might be associated with changed expression levels of oatp2a1 and oatp2b1 in renal, small intestinal, and large intestinal tissues under Pi deficiency induced loss of transportation. Shen and Dachang played important roles in substance metabolism under Pi deficiency state, which proved Pi-Shen correlated in Chinese medical theories.

Animals ; Anions ; Aristolochic Acids ; metabolism ; Drugs, Chinese Herbal ; Kidney ; Medicine, Chinese Traditional ; Organic Cation Transport Proteins ; metabolism ; Peptides ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

This paper was aim to screen microorganisms with attenualed efficiency for Chinese medicine containing aristolochic acid A by liquid-state fermentation. Twelve Chinese medicine were detected by UPLC and aristolochic acid A was only founded in four species of Aristolochia, those were Caulis Aristolochiae Manshuriensis, Aristolochiae Radix, Aistolochia Contorta Bunge and Herba Aristolochiae Mollissima,but not in the others. With the four Chinese medicine containing aristolochic acid A as raw material, ten microorganisms were tested, and the content of aristolochic acid A was detected by UPLC. The results showed that one microorganism can decrease content of aristolochic acid A in all those four Chinese medicine.
Aristolochic Acids ; analysis ; metabolism ; Bacteria ; metabolism ; Biotransformation ; Drugs, Chinese Herbal ; analysis ; metabolism ; Fungi ; metabolism ; Plants, Medicinal ; chemistry ; microbiology

The renal toxicity and mutagenicity of aristolochic acid (AA) as well as its carcinogenicity on upper urinary tract transitional epithelial cells have been widely known. Since 2003, drug regulatory departments have successively cancelled the quality standards for AA-containing medicines such as Aristolochiae Radix, Aristolochiae Manshuriensis Caulis and Aristolchiae Fangchi Radix, and adopted measures for strengthening regulation and revising package insert or quality standards for other AA-containing medicines, including Aristolochia Cinnabarina Radix, Aristolochiae Fructus, Aristolochiae Mollissimae Herba, in order to control its safety risk. In recent years, domestic and foreign studies on AA have mainly involved action mechanism and clinical performance of AA toxicity, early-stage diagnosis and treatment method. In this paper, authors gave a brief summary and evaluation on risk factors for using AA-containing medicines, and offered measures and suggestions for preventing and controlling AA toxicity.
Animals ; Aristolochia ; adverse effects ; chemistry ; Aristolochic Acids ; analysis ; therapeutic use ; toxicity ; Drug Therapy ; Drug-Related Side Effects and Adverse Reactions ; epidemiology ; etiology ; Drugs, Chinese Herbal ; analysis ; therapeutic use ; toxicity ; Humans