Natural products from taxa; Aristolochic acids (AA) analog

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 (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/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 · 106 M−1 for HSA/AA-I versus 8.4 and 9.0 · 105 M−1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 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 [2].

---

Aristolochic acid (AA) is known to be a potent mutagen and carcinogen. Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of AA, differ from each other by a single methoxy group. However, their individual mutagenic characteristics in vivo are unclear. In the present study, we compared their DNA adduct formation and mutagenicities in the gpt delta transgenic mouse kidney. The dA-AAI, dG-AAI, dA-AAII and dG-AAII were identified in the kidney two days after intragastric administration of AAI or AAII at 5mg/kg. The concentration of DNA adducts formed by AAII was approximately 2.5-fold higher than that formed by AAI (p<0.05). The mutant frequency induced by AAII was nearly two-fold higher than that induced by AAI (p<0.05) following administration of 5mg/kg AAI or AAII, five times per week for six weeks. Investigation of the mutation spectra showed no statistically significant difference between AAI- and AAII-treated mice (p>0.05). A:T to T:A transversion was the predominant type of mutation in both treated groups, the GC-associated mutation rates, however, differed between the AAI and AAII treatments. The in vivo metabolic pathways of AAI and AAII are different, and this may affect their mutagenicity. In the present study, we measured the levels of AAI and AAII in the kidney and plasma of gpt delta transgenic mice at multiple time points after a single intragastric dose of 1 or 5mg/kg of either component. Our results showed that the levels of AAII in both kidney and plasma were considerably higher than those of AAI (p<0.01). The present study indicated that AAII showed more carcinogenic risk than AAI in vivo, and this may be, at least partly, the result of its increased levels in kidney and plasma [1].
In vivo in gpt delta transgenic mouse kidney: after intragastric administration of Aristolochic acid II at 5 mg/kg for two days, DNA adducts dA-AAII and dG-AAII were detected. The total DNA adduct concentration (sum of dA-AAII and dG-AAII) was approximately 2.5-fold higher than that formed by AAI (p < 0.05). [1]
After six weeks of treatment (5 mg/kg, five times per week), the mutant frequency of the gpt gene in kidney was 32.00 × 10⁻⁶ (13.5-fold higher than vehicle control, p < 0.05), which was nearly twice that induced by AAI at the same dose (p < 0.05). [1]
Mutation spectrum analysis: A:T to T:A transversion was the predominant mutation type in AAII-treated mice (30.9%). G:C to A:T transitions accounted for 21.8% (with only 2 of 12 transitions occurring at CpG sites). The overall mutation pattern in AAII-treated mice showed no significant difference compared to spontaneous mutants (p > 0.05). [1]
The 5'-TGG-3' sequence was identified as a potential mutation hotspot. [1]

Animal protocol for DNA adduct detection: Eight-week-old male gpt delta transgenic mice (n=5 per group) were treated intragastrically with Aristolochic acid II at 5 mg/kg for two days. Mice were killed 24 h after last treatment, kidneys removed and stored at -40°C. [1]
Animal protocol for mutant frequency: Eight-week-old male gpt delta transgenic mice were divided into groups: vehicle control (n=5), AAI 1 mg/kg (n=4), AAI 5 mg/kg (n=4), Aristolochic acid II 1 mg/kg (n=4), and Aristolochic acid II 5 mg/kg (n=4). Compounds were dissolved in 1% NaHCO₃ and administered intragastrically five days per week for six consecutive weeks. One week after the last treatment, mice were killed and kidneys frozen at -80°C. [1]
Animal protocol for plasma and kidney concentration measurement: Eight-week-old male gpt delta transgenic mice received a single intragastric dose of Aristolochic acid II at 1 or 5 mg/kg. Tail vein blood samples were collected at 5, 10, 20, 30, 60, 90, 120, and 180 min after dosing. Bilateral kidneys were collected at 30 and 60 min. [1]

Plasma levels of Aristolochic acid II in gpt delta transgenic mice were much higher than those of AAI at the same dose (1 or 5 mg/kg) at multiple time points (p < 0.01). At 5 mg/kg, AAII plasma levels remained detectable up to 180 min, whereas AAI became undetectable after 90 min. [1]
Kidney levels of Aristolochic acid II at 30 and 60 min after a single intragastric dose (1 or 5 mg/kg) were much higher than those of AAI at the same dose (p < 0.01). [1]

Aristolochic acid II is a known or suspected human carcinogen and nephrotoxin. It induces DNA adducts (dA-AAII and dG-AAII) and causes mutations predominantly A:T to T:A transversions in the kidney of gpt delta transgenic mice. The carcinogenic risk of AAII in vivo is higher than that of AAI, partly due to its increased levels in kidney and plasma. [1]

[1]. Comparison of the mutagenicity of aristolochic acid I and aristolochic acid II in the gpt delta transgenic mouse kidney. Mutat Res. 2012 Mar 18;743(1-2):52-8.

[2]. Structural and mechanistic insights into the transport of aristolochic acids and their active metabolites by human serum albumin. J Biol Chem. 2024 May 22;300(7):107358.

Aristolochic acid B is an aristolochic acid, a phenanthrene-1-carboxylic acid with methylenedioxy groups at positions 3 and 4 and a nitro group at position 10. It possesses carcinogenic, metabolic, mutagenic, nephrotoxic, and toxic properties. It is a C-nitro compound belonging to the aristolochic acid class of compounds, and is an aromatic ether, cyclic acetal, monocarboxylic acid, and organic heterotetracyclic compound. Aristolochic acid II has been reported in Aristolochia tuberosa, Stephania tetrandra, and other organisms with relevant data. See also: Aristolochia tuberosa root (partial).

---

Aristolochic acid II is only nitroreduced to form aristolactam II (ALII), whereas AAI can also undergo O-demethylation. This difference in metabolic pathways may affect their mutagenicity. The methoxy group in AAI affects DNA adduct stability; dG-AAII fits better into the DNA minor groove than dG-AAI. The predominant mutation type induced by AAII is A:T to T:A transversion, but G:C to A:T transitions occur at a higher frequency than with AAI (21.8% vs 11.8%). [1]

C16H9NO6

311.249

311.042

475-80-9

108168

Light yellow to yellow solid powder

1.6±0.1 g/cm3

592.2±50.0 °C at 760 mmHg

270-272℃

311.9±30.1 °C

0.0±1.8 mmHg at 25°C

1.787

3.5

1

6

1

23

505

0

MEEXETVZNQYRSP-UHFFFAOYSA-N

InChI=1S/C16H9NO6/c18-16(19)10-6-12-15(23-7-22-12)14-9-4-2-1-3-8(9)5-11(13(10)14)17(20)21/h1-6H,7H2,(H,18,19)

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

IIBRN-0329754; LS-102940; IIBRN-0329754; Aristolochic acid; BCCRIS6497; Q100503; LS-102940; Aristolochic acid B; Aristolochic acid II; 475-80-9; CCRIS 6497; UNII-BB72D5PU2Y; EINECS 207-499-6; BB72D5PU2Y; BRN 0329754; CCRIS 6497; Q-100503

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 : ~13.89 mg/mL (~44.63 mM)

Solubility in Formulation 1: ≥ 1.39 mg/mL (4.47 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 13.9 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.

---

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