Identification of metabolites and adducts of aristolochic acid (AA) in mice: potential biomarkers of exposure to AA-containing herbs

• Main Authors: Po-Chi Hsu, 許博期
• Other Authors: Su-Yin Chiang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/40822691345868483359

碩士 === 中國醫藥大學 === 中國醫學研究所 === 96 === Aristolochic acid (AA), a probable human carcinogen, is associated with the urothelial carcinoma in patients with Aristolochic Acid Nephropathy. In vivo metabolism study is important in further understanding the nephrotoxic and carcinogenic effects of AA since AA needs metabolic activation to exert its toxic effects. So far, only limited data on in vivo metabolism of AA has been reported. The purpose of this study was to identify the urine metabolites and adducts of AA in mice as potential biomarkers for AA exposure. Male C3H/He mice (5-6 weeks old) were treated by gavage with a single dose of 30 and 50 mg/kg AA (including AAI and AAII), and then the urine samples were collected in metabolic cages from 0 to 16 h after dosing and stored at -20 degree until analysis. The urine samples were subjected to solid phase extraction before analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the structure elucidation and quantification of AA metabolites. Several metabolites were identified in mouse urine by LC-ESI-MS/MS, including aristolactams, demethylation of AAI (AAIa), glucuronide and/or acetyl conjugations of AAIa. The urinary metabolites may provide useful information about the metabolic activation of AA in vivo. Aristolactam, one of the major active metabolites of AA, was synthesized and quantified to reveal that aristolactam I and II were 13.57 ± 5.78 ug/ml and 3.68 ± 0.91 ug/ml at first day after AA exposure, respectively. The genotoxicity in AA-exposed C3H mice was dose-dependently increased by the micronuclei test, a marker of chromosome damage. After AA exposure, the urine total protein to creatinine ratio was elevated and the pathological examined revealed extensive necrosis of the renal tubules. Moreover, the immunohistochemical expression of Neutral endopeptidase (NEP), a marker for proximal tubular damage, was dramatically decreased. Our data unveil that AA undergoes extensive phase II metabolism. The use of urinary AA metabolites as biomarkers for recent exposure to AA-containing herbs is currently underway.