NF-κB; AP-1

Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis [1].

Hepatic CYP1A especially CYP1A2 plays an important role in the reduction of aristolochic acid I (AAI) nephrotoxicity. In this study, we investigated the effects of tanshinone I, a strong inducer of Cyp1a, on the nephrotoxicity induced by AAI. Histopathology and blood biochemistry assays showed that tanshinone I could reduce AAI-induced acute kidney injury. Pharmacokinetics analysis revealed that tanshinone I markedly decreased AUC of AAI in plasma and the content of AAI in both liver and kidney, indicating the enhancement of AAI metabolism. Real-time PCR and Western blot analysis confirmed that tanshinone I effectively increased the mRNA and protein levels of hepatic CYP1A1 and CYP1A2 in vivo. Luciferase assay showed that tanshinone I strongly increased the transcriptional activity of CYP1A1 and CYP1A2 in the similar extent. In summary, our data suggested that tanshinone I facilitated the metabolism of AAI and prevented AAI-induced kidney injury by induction of hepatic CYP1A 1/2 in vivo [2].

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Aristolochic acid I (AAI) can induce renal tubular epithelial cells (RTECs) autophagy, which thereby extenuates apoptosis in vitro. In this study, we aimed to determine whether the in vitro data also apply to the AAI-induced pathologic condition in vivo. BALB/c mice were treated with AAI, autophagy inhibitors [3-methyladenine (3MA) or chloroquine diphosphate salt (CQ)], and AAI plus the inhibitors for consecutive 5 days, respectively. Mice were euthanized on day 3 and 5. AAI induced RTECs autophagy was confirmed by electron microscopy and western blot. The results showed induction of apoptotic RTECs and up-regulation of mitochondrial and endoplasmic reticulum stress-related proteins in AAI-treated mice at both of the two time points. There were more apoptotic RTECs in AAI + inhibitor groups, which might be due to increased mitochondrial stress-related proteins (cytochrome C and apoptotic protease activating factor 1, APAF-1). On day 5, severe tubulointerstitial injuries induced by AAI led to a significant decline in kidney function. There were numerous autolysosomes in dying RTECs of the AAI group. Autophagy inhibitors increased AAI-induced RTECs mitochondrial apoptosis by increasing mitochondrial stress-related proteins, but they partially mitigated the AAI-induced severe renal tubulointerstitial injury. These results confirmed that AAI could induce autophagy in RTECs, which prevented apoptosis via mitochondrial pathway in vivo. However, continuous stimulation with AAI induced excess autophagy, which ultimately resulted in AAI-induced cell death. It suggested that apoptosis wasn't the main culprit in acute aristolochic acid nephropathy mice model.[3]

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Aristolochic acid A can be used to induce nephrotoxicity models.
Pharmacokinetic studies have shown that in C57BL/6 male mice, Aristolochic acid A (10 mg/kg, intraperitoneal injection) has a higher concentration in the kidneys than in the liver after 30 minutes of administration. The concentration of Aristolochic acid A in plasma also reached its peak about 30 minutes after administration. The pathogenesis of Aristolochic acid A-induced nephropathy is still unclear. Existing studies have shown that Aristolochic acid A mainly damages renal tubular epithelial cells, renal tubules, and leads to interstitial fibrosis, resulting in nephrotoxicity.

Analysis of BLCAP promoter activity [1]
HEK293 cells (2 × 105), seeded in 6-well plate with serum free medium, were co-transfected with 2 μg of constructed reporter plasmid and 0.05 μg of pSV-β-galactosidase control vector with the aid of Turbofect reagent and then incubated in the CO2 incubator for 6 h prior to replacement with fresh medium containing 10% serum. Twenty-four hours after medium replacement, the transfected HEK293 were treated with various concentrations of Aristolochic acid I (AAI) for 24 h. The total protein extracts of cultures were prepared by lysing the cells with 1× lysis reagent containing 25 mM Tris-phosphate (pH7.8), 2 mM DTT, 2 mM 1, 2-diamino cyclohexane-N′, N′, N′, N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100. After centrifugation (5 min at 10,000 × g), part of the supernatant fluid was subjected to the luciferase assay kit. For the determination of β-galactosidase activity, the other part of protein fluid was incubated with an equal volume of 2× assay buffer (substrate ONPG (O-nitrophenyl-d- galactopyranoside) 1.33 mg/ml, 200 mM sodium phosphate buffer (pH 7.3), 2 mM MgCl2 and 100 mM β-mercaptoethanol) for 30 min at 37 °C. The reaction was terminated by sodium carbonate and the absorbance was read at 420 nm with a Spectramax microplate reader. To account for the differences in transfection rates, the data of luciferase activity were normalized to the β-galactosidase activity.

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Genomic DNA isolation and bisulfite modification [1]
Genomic DNAs from vehicle- or Aristolochic acid I (AAI)-exposed HEK293 cells were isolated using a WelPrep DNA Isolation Kit. Five micrograms of genomic DNA in 25 μl of 0.01 M PBS were denatured with 25 μl of 0.4 N NaOH for 20 min at 55 °C. Followed by the addition of 30 μl of freshly prepared hydroxyquinone (10 mM) and 520 μl of sodium bisulfite (3 M pH 5.0), the sample was incubated at 55 °C for 16 h. During the process, unmethylated cytosine residues were converted to uracil, but the methylated ones remained unchanged. Modified DNA was purified using the DNA Clean-Up Kit and then the reaction was completed with NaOH (0.6 N) treatment for 20 min at room temperature. After ethanol precipitation, the bisulfite-modified DNA samples were resuspended in 10 mM Tris-EDTA and stored at −20 °C.

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Bisulfite genomic sequencing [1]
Bisulfite-modified DNAs obtained from solvent- or Aristolochic acid I (AAI)-treated cultures were amplified with two rounds of PCR. The first round PCR was performed using bsp1F/bsp2R for region 1 fragment (R1) and bsp3F/bsp3R2 for region 2 fragment (R2). For the second round of PCR, nested primers (bsp1F/R, bsp2F/R, and bsp3F/R) were used and the reactions were initiated for 10 min at 95 °C, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products, bsp1, bsp2 and bsp3, were independently cloned into pGEM-T Easy Vectors and the clones from each PCR product were sequenced to assess methylation status at each CpG site.

Viability and cell growth analysis [1]
For viability analysis, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay was performed according to Liu et al. (2005). Briefly, human cell monolayers in 96-well plates were treated with vehicle or various concentrations of Aristolochic acid I (AAI) for designated times (1, 3, 6, and 24 h). Subsequently, cultures were incubated with medium containing 0.5 mg/ml of MTT for 3 h at 37 °C. The absorbance was measured at a wavelength of 570 nm on a microplate reader. For cell growth analysis, HEK293/shLuc or HEK293/shBLCAP (1 × 104) were seeded in a 24-well plate in triplicate and incubated at 37 °C for 7 days. At the designated time, cells from three separated wells were trypsinized and stained with 0.2% trypan blue; the un-stained cells were counted using a hemocytometer to graph the growth curve.

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Migration and invasion assay [1]
HEK293 cells that had been infected with virus containing shLuc (HEK293/shLuc) or shBLCAP plasmid (HEK293/shBLCAP) were cultured in a 24-well plate (3 × 104 cells/well) with ThinCert™ cell culture inserts. For invasion assay, cell inserts would be coated 100 μg/ml matrigel before usage. After 16 h incubation, the cells on the upper surface of insert membrane were removed by cotton swabs and the insert was fixed with methanol for 15 min. The migrated or invaded cells on the lower surface of membrane were stained with 0.01% crystal violate solution and the cell number was counted from 10 random fields under microscope (200×).

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Colony-forming assay [1]
HEK293/shLuc or HEK293/shBLCAP (3 × 104 cells) were mixed with 1 ml of 0.2% soft agar in complete medium and seeded in a 6-well plate containing 1.5 ml of 0.6% soft agar in complete medium. After incubation at 37 °C for 14 days, colonies in the soft agar were stained by 0.005% crystal violate, and those lager than 100 μm in diameter were counted from 10 random fields under microscope (40×).

The acute aristolochic acid nephropathy (AAN) mouse model was established and modified according to Matsui’s reported method. A total of 150 healthy female BALB/c mice, approximately 20 g, were used. Mice were allowed free access to an SPF laboratory diet, distilled water and were housed at stable temperature (25 °C) and humidity with regular light–dark cycles (12:12 h) during the experiments. After 5 days’ acclimatization, weight-matched mice were assigned to six groups at each of two time points (control, 3MA, chloroquine diphosphate salt/CQ, Aristolochic acid I (AAI), 3MA + AAI, CQ + AAI, for either 3 or 5 days). Mice in Aristolochic acid I (AAI) group were injected intraperitoneally with Aristolochic acid I (AAI) (10 mg/kg/day) once daily for 3 or 5 consecutive days. Mice in 3MA + AAI, CQ + AAI groups were injected intraperitoneally with 3MA (30 mg/ml in saline at 37 °C) and CQ (60 mg/ml in saline) respectively, 1 h before AAI injection, once daily for 3 or 5 consecutive days. Control, 3MA and CQ groups were given PEG400 in saline, 3MA, and CQ, respectively. After 3 or 5 days of administration, 12 mice per group at each time point were killed: blood was collected by enucleation and then the mice were killed by craniocervical dislocation. Kidneys were then removed for histological analysis, Western blot, immunohistochemistry, and transmission electron microscopy detection.[3]

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Aristolochic acid I (AAI) powder was dissolved in PEG400 as a stock solution of 20 mg/ml, which was diluted in 10 mg/ml final concentration with saline before use.

Metabolism / Metabolites
Aristolochic acids are absorbed after oral exposure. They are metabolized to aristolactams, which are further metabolized to a cyclic N-acylnitrenium ion, a re-active intermediateforming adducts with purine bases (adenine and guanine) in DNA (dA-AAI, dG-AAI, dA-AAII, and dG-AAII). A number of cytosolic and microsomal enzymes (CYP1A1, CYP1A2, NADPH:CYP reductase, prostaglandin H synthase, DT-diaphorase, xanthine oxidase, cyclooxygenase, and NAD(P)H:quinone oxidore-ductase) are capable of bioactivating aristolochic acids to the reactive form.

Toxicity Summary
The carcinogenic and mutagenic effects associated with the binding of metabolites of ingested aristolochic acid (AA) to DNA have been extensively described in vitro and in vivo, resulting in the classification of AA as a genotoxic carcinogen. AA-derived DNA adducts in renal cortical and urothelial tumor tissue of patients with documented BEN, associated with the dominance of the A:T to T:A transversions in the p53 tumor suppressor gene mutational spectrum. (A15444) AA is a nephrotoxic and carcinogenic compound, which has been demonstrated to be genotoxic and mutagenic both in vitro and in vivo. Toxicity and carcinogenicity of the nephrotoxic compound aristolochic acid between rodents and humans suggest a species-dependent mechanism of action. AA had a comparable effect on the cell cycle in primary human and porcine cells and the rat NRK-52E cell line following 48 h exposure, also corroborated by the reduced 3H-thymidine incorporation in NRK-52E cells. In addition, DNA unwinding, suggestive of enhanced DNA damage, was observed in primary porcine cells.

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Acute Effects
man LDLo intravenous 3 mg/kg/2D-I KIDNEY, URETER, AND BLADDER: CHANGES IN TUBULES (INCLUDING ACUTE RENAL FAILURE, ACUTE TUBULAR NECROSIS) Cancer Chemotherapy Reports., 42(35), 1964
rat LD50 oral 184 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: DYSPNEA Archives of Toxicology., 59(328), 1987 [PMID:3579596]
rat LD50 intravenous 74 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: DYSPNEA Archives of Toxicology., 59(328), 1987 [PMID:3579596]
mouse LD50 oral 55900 ug/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: DYSPNEA Archives of Toxicology., 59(328), 1987 [PMID:3579596] mouse LD50 intraperitoneal 14320 ug/kg Guangxi Yixue. Kuanghsi Medicine., (6)(2), 1981

[1]. Aristolochic acid I interferes with the expression of BLCAP tumor suppressor gene in human cells. Toxicol Lett. 2018 Jul;291:129-137.

[2]. Tanshinone I protects mice from aristolochic acid I-induced kidney injury by induction of CYP1A. Environ Toxicol Pharmacol. 2013 Nov;36(3):850-7.

[3]. Autophagy inhibitors promoted aristolochic acid I induced renal tubular epithelial cell apoptosis via mitochondrial pathway but alleviated nonapoptotic cell death in mouse acute aritolochic acid nephropathy model. Apoptosis. 2014 Aug;19(8):1215-24.

Aristolochic acid A is an aristolochic acid that is phenanthrene-1-carboxylic acid that is substituted by a methylenedioxy group at the 3,4 positions, by a methoxy group at position 8, and by a nitro group at position 10. It is the most abundant of the aristolochic acids and is found in almost all Aristolochia (birthworts or pipevines) species. It has been tried in a number of treatments for inflammatory disorders, mainly in Chinese and folk medicine. However, there is concern over their use as aristolochic acid is both carcinogenic and nephrotoxic. It has a role as a nephrotoxin, a carcinogenic agent, a mutagen, a toxin and a metabolite. It is a monocarboxylic acid, a C-nitro compound, a cyclic acetal, an organic heterotetracyclic compound, an aromatic ether and a member of aristolochic acids.
Aristolochic acid has been reported in Aristolochia tuberosa, Stephania tetrandra, and other organisms with data available.
Aristolochic acids are a family of carcinogenic, mutagenic, and nephrotoxic compounds commonly found in the Aristolochiaceae family of plants, including Aristolochia and Asarum (wild ginger), which are commonly used in Chinese herbal medicine. Aristolochic acid I is the most abundant of the aristolochic acids and is found in almost all Aristolochia species. Aristolochic acids are often accompanied by aristolactams.
See also: Aristolochia fangchi root (part of).

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In conclusion, AAI treatment significantly suppressed the BLCAP expression in kidney and bladder cancer cells and the knock down of BLCAP by siRNA successfully led to the malignant transformation of HEK 293 cells. However, whether BLCAP plays a role in the mediation of AAI-associated carcinogenesis need to be further investigated.[1]

C17H11NO7

341.27

341.053

C, 59.83; H, 3.25; N, 4.10; O, 32.82

313-67-7

313-67-7

2236

Light yellow to orange solid powder

1.6±0.1 g/cm3

615.5±55.0 °C at 760 mmHg

260 °C

326.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.747

3.41

1

7

2

25

550

0

COC1=CC=CC2=C3C(=C(C=C21)[N+](=O)[O-])C(=CC4=C3OCO4)C(=O)O

BBFQZRXNYIEMAW-UHFFFAOYSA-N

InChI=1S/C17H11NO7/c1-23-12-4-2-3-8-9(12)5-11(18(21)22)14-10(17(19)20)6-13-16(15(8)14)25-7-24-13/h2-6H,7H2,1H3,(H,19,20)

8-methoxy-6-nitronaphtho[2,1-g][1,3]benzodioxole-5-carboxylic acid

Aristolochin; Birthwort; NSC-11926; NSC11926; Aristolochic acid; Aristolochic acid A; 313-67-7; Aristolochic acid I; Tardolyt; Aristolochin; Aristolochic acid-I; Birthwort; NSC 11926; NSC-50413; NSC 50413; NSC50413; TR-1736; Aristolochic acid I; Tardolyt ; AristA; Aristolochic acid A

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 16.7~34 mg/mL (48.9~99.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)