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 mainland China. 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.

Schisandra chinensis Oil Attenuates Aristolochic Acid I-Induced Nephrotoxicity in vivo and in vitro / 中国结合医学杂志

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.

Monitoring and quantitative analysis of trace aristolochic acid I in a Qing-Fei-Pai-Du decoction using liquid chromatography-mass spectrometry / 药学学报

The dosage of asarum in Qing-Fei-Pai-Du decoction (QFPD) is twice the dosage prescribed by the Chinese Pharmacopoeia. Due to the potential toxicity of aristolochic acid I (AAI), a limited component in asarum, the possibility that its dosage also exceeds the dosage prescribed by the Chinese Pharmacopoeia had aroused wide concern. In this study, the UHPLC-Q TOF method was used to determine the presence of AAI in QFPD. A UHPLC-QQQ method was then established to determine the content of AAI in QFPD, a reflux extract of asarum and an ultrasonic extract of 70% methanol of asarum. The results showed that the amount of AAI in the three samples was approximate 1.5, 3.2 and 9.0 μg respectively with equivalent dosages of asarum (6 g). All were obviously lower than the maximum daily limit stipulated in the Chinese Pharmacopoeia (30 μg). Therefore, we concluded that the content of AAI could be effectively reduced by using a Chinese herbal compound decoction and optimization of asarum. This assay is not only convenient, rapid, sensitive and reproducible for the trace detection of AAI in Chinese herbal compound decoction, but also useful for the rational application of asarum in QFPD.

Effects of aristolochic acid I and/or hypokalemia on tubular damage in C57BL/6 rat with aristolochic acid nephropathy

BACKGROUND/AIMS: This study was designed to investigate the roles of aristolochic acid I (AA-I) and hypokalemia in acute aristolochic acid nephropathy (AAN). METHODS: After an adaptation period (1 week), a total of 40 C57BL/6 mice (male, 8 weeks old) were divided into four groups: I (control group), II (low potassium [K] diet), III (normal K diet with administration of AA-I [10 mg/kg weight]), and IV (low K diet with AA-I). After collecting 24 hours of urine at 2 weeks, the mice were sacrificed, and their blood and kidneys were obtained to perform immunochemical staining and/or Western blot analysis. RESULTS: Proteinuria, glycosuria, and increased fractional excretion of sodium and K were prominent in groups III and IV (p < 0.05). Diffuse swelling and poor staining of collecting duct epithelial cells were evident in the medullas of group II. Typical lesions of toxic acute tubular injury were prominent in the cortices of groups III and IV. Α-Smooth muscle actin (α-SMA) was higher in the cortices of the mice in groups III and IV versus group II (p < 0.05), and higher in the medullas of group IV than groups I and III (p < 0.05). E-cadherin was higher in the cortices of groups III and IV compared to group I (p < 0.05). The F4/80 value was higher in the cortices and medullas of groups II, III, and IV compared to group I (p < 0.05), particularly in the case of group II. CONCLUSIONS: AA-I can induce acquired Fanconi syndrome in the acute stage of AAN. Macrophages appear to play a key role in the pathogenesis of AAN and hypokalemic nephropathy. It remains uncertain whether hypokalemia plays any role in AAN and hypokalemia.

Chrysosplenetin, in the absence and presence of artemsininin, alters breast cancer resistance protein-mediated transport activity in Caco-2 cell monolayers using aristolochic acid I as a specific probe substrate

ABSTRACT The present study describes the impact of chrysosplenetin, in the absence and presence of artemisinin, on in vitro breast cancer resistance protein-mediated transport activity in Caco-2 cell monolayers using aristolochic acid I as a specific probe substrate. We observed that novobiocin, a known breast cancer resistance protein active inhibitor, increased Papp (AP-BL) of aristolochic acid I 3.13 fold (p < 0.05) but had no effect on Papp (BL-AP). Efflux ratio (PBA/PAB) declined 4.44 fold (p < 0.05). Novobiocin, consequently, showed a direct facilitation on the uptake of AAI instead of its excretion. Oppositely, both artemisinin and chrysosplenetin alone at dose of 10 µM significantly decreased Papp (BL-AP) instead of Papp (AP-BL). Chrysosplenetin alone attenuated the efflux ratio, which was suggestive of being as a potential breast cancer resistance protein suppressant. Oddly, Papp (BL-AP) as well as efflux ratio were respectively enhanced 2.52 and 2.58 fold (p < 0.05), when co-used with artemisinin and chrysosplenetin in ratio of 1:2. The potential reason remains unclear; it might be relative to binding sites competition between artemisinin and chrysosplenetin or the homodimer/oligomer formation of breast cancer resistance protein bridged by disulfide bonds, leading to an altered in vitro breast cancer resistance protein-mediated efflux transport function.

HPLC Fingerprint of Fructus Aristolochiae and Honey Processed Products from Different Habitats / 中国药学杂志

OBJECTIVE: To establish the HPLC fingerprints of Fructus Aristolochiae and honey-fried products from different habitats, compare and analyze their difference in chemical components. METHODS: Using an Agilent ZORBAX SB-C18 column (4.6 mm×250 mm, 5 μm), HPLC method was performed to collect the fingerprints. The mobile phase A and B were acetonitrile and 1% acetic acid, respectively. Gradient elution was performed at the flow rate of 1.0 mL·min-1 and column temperature of 30℃. The detection was carried out at 250 nm. Similarity Evaluation System for Chromatographic Fingerprint of TCMs (Ver. 2012) and SPSS22.0 was used in cluster analysis and principal component analysis (PCA). RESULTS: The fingerprints of 14 batches of Fructus Aristolochiae samples, before and after process, were established by HPLC. The reduction rate of AA I, a poisonous component, was over 36%. Except for S4 (0.822), S18 (0.771) and S19 (0.639), the similarities of Fructus Aristolochiae and processed products were over 0.9. The non-processing samples were classified into four groups by cluster analysis. As PCA shown, the five principal components including AA I had a contributing rate of 84.914% to cumulative sum of squares, illustrating the chemical similarity and difference of samples from different areas. CONCLUSION: The results may provide reference for Fructus Aristolochiae processing and standardization of its detoxification technology.

Study on differential expression of miRNA in serum of rats after renal injury induced by aristolochic acid I / 中草药

Objective: To screen the miRNA differential expression profile in serum of rats after renal injury induced by aristolochic acid I, (AAI) to identify the target of miRNA in the mechanism of aristolochic acid nephropathy (AAN), and to make further study on the mechanism of AAN. Methods: Eighteen male SD rats were randomly divided into two groups, control group and AAI group (2.25 mg/kg). Rats were ip injected with AAI once daily for 14 d. On day 14, blood collected was used for the determination of miRNA in serum by miRNA microarray; Renal pathological changes were examined by HE staining. The miRNA that expressed significantly different would be verified by real time quantitative PCR (qPT-PCR). Target genes were predicted using bioinformatics as well as Pathway analysis did. Results: HE staining revealed that kidneys were damaged with different degrees. By significance analysis of microarray based on microarray screening, significantly different expression of five miRNA (miR-10a-3p, miR-207, miR-3594-3p, miR-874-3p, and miR-9a-3p) which were up-regulated were obtained, while there were no miRNAs which were down-regulated. qPT-PCR approved the results as well. It was suggested that 16 genes, such as Rhebl1 and Usp10, might be the target genes. Pathway analysis showed a greater correlation between MAPK signaling pathways. Conclusion: The differential expressed miRNAs obtained by gene analysis might be related with AAI and further studies on the mechanism of some miRNA would be a new target of gene therapy and provide an effective basis for the further studies of bioinformatics.