Animals:** Male C57BL/6 mice (6 weeks old, 18-22 g) were obtained from Shanghai Laboratory Animal Center. All animal experiments were approved by the Shanghai Animal Care and Use Committee [Certificate No. SCXK (Shanghai) 2002-0010]. [3]
* **Treatment Groups:** Mice were divided into three groups (n=15 per group): (1) AAI group: received corn oil (CO) i.p. daily for 3 days, followed by a single i.p. injection of 10 mg/kg AAI in saline 24 h after last CO injection; (2) BNF+AAI group: received 80 mg/kg BNF in CO i.p. daily for 3 days, followed by a single i.p. injection of 10 mg/kg AAI 24 h after last BNF injection; (3) Control group: received CO i.p. daily for 3 days, followed by a single i.p. injection of saline 24 h after last CO injection. [3]
* **Sample Collection:** Blood samples were collected by tail bleeding at various time points after AAI injection (20 μL each) in heparin-coated capillaries. Tissues (liver and kidney) were harvested at 30 min post-AAI for metabolite analysis, or at 24 h after last BNF/CO injection for microsome preparation and RNA extraction. For histopathology and serum biochemistry, mice were sacrificed at 3, 7, and 14 days after AAI/saline injection. [3]
* **Serum Biochemistry:** Blood urea nitrogen and serum creatinine were measured using an automatic HITACHI Clinical Analyzer Model 7080. [3]
* **Histopathology:** Kidneys were fixed in 10% formalin, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin. Immunostaining for α-SMA was performed using anti-mouse α-SMA antibody (1:800) and avidin-biotin-peroxidase complex method. TUNEL assay was performed using Roche Diagnostics kit according to supplier's instructions. [3]
* **HPLC Analysis:** AAI and its metabolites (AAIa and ALI) were quantified using an HP1100 HPLC system with UV detection at 250 nm. Separation used a Welchorn XB-C18 column with isocratic mobile phase of methanol:0.1% acetic acid in water (7:3) at 0.8 mL/min. Calibration curves, linear ranges, precision, and recovery were established for serum, liver, and kidney samples. [3]
* **Real-Time RT-PCR:** Total RNA was isolated using UNIQ-10 column & TRIZOL kit. cDNA was synthesized using Cloned AMV Reverse Transcriptase. Real-time PCR used TaKaRa Ex Taq R-PCR kit with gene-specific primers for CYP1A1, CYP1A2, CPR, and β-actin. PCR was monitored for 45 cycles with annealing at 60°C. Relative amounts were normalized to β-actin mRNA. [3]
* **Western Blotting:** Microsomal proteins (30 μg) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against CYP1A1 (1:100), CYP1A2 (1:1000), and CPR (1:1000). Signal was detected using ECL system. [3]

[1]. Ishida T, Takechi S. β-Naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor, disrupts zinc homeostasis in human hepatoma HepG2 cells. J Toxicol Sci. 2019;44(10):711-720.

[2]. α- and β-Naphthoflavone synergistically attenuate H2O2-induced neuron SH-SY5Y cell damage. Exp Ther Med. 2017 Mar;13(3):1143-1150.

[3]. β-Naphthoflavone protects mice from aristolochic acid-I-induced acute kidney injury in a CYP1A dependent mechanism. Acta Pharmacologica Sinica 30.11 (2009): 1559-1565.

β-naphthoflavone is an extended flavonoid compound formed by the fusion of a benzene ring with the f-side of a flavonoid. It is an aryl hydrocarbon receptor agonist. It is an extended flavonoid compound, an organic heterocyclic tricyclic compound, and a naphtho-γ-pyranone. β-naphthoflavone, also known as 5,6-benzoflavonoid, is a potent agonist of aryl hydrocarbon receptors and can induce cytochrome P450 (CYP) and uridine 5'-bisphosphate glucuronyl transferase (UGT). It may be a chemopreventive agent. β-naphthoflavone is a polycyclic aromatic hydrocarbon consisting of a flavonoid group attached to a benzene ring. β-naphthoflavone is an inducer of hepatic cytochrome P450 CYP1A1 and CYP1A2. A polycyclic aromatic hydrocarbon compound that can induce P4501A1 and P4501A2 cytochromes. (Proc Soc Exp Biol Med 1994 Dec:207(3):302-308)

C19H12O2

272.3

272.083

6051-87-2

2361

White to light yellow solid powder

1.3±0.1 g/cm3

460.9±45.0 °C at 760 mmHg

185-189 °C

215.8±22.3 °C

0.0±1.1 mmHg at 25°C

1.695

4.79

0

2

1

21

433

0

OUGIDAPQYNCXRA-UHFFFAOYSA-N

InChI=1S/C19H12O2/c20-16-12-18(14-7-2-1-3-8-14)21-17-11-10-13-6-4-5-9-15(13)19(16)17/h1-12H

3-phenylbenzo[f]chromen-1-one

beta-Naphthoflavone beta-NF

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~25 mg/mL (~91.81 mM)

Solubility in Formulation 1: 1 mg/mL (3.67 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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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Solubility in Formulation 2: ≥ 1 mg/mL (3.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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