title: Liquid chromatography electrospray-mass spectrometry of urinary aflatoxin biomarkers: characterization and application to dosimetry and chemoprevention in rats.
abstract: A liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the measurement of aflatoxin biomarkers in urine has been developed and validated. The two major aflatoxin-DNA adducts formed in rat tissues, aflatoxin N(7)-guanine and its imidazole ring opened derivative, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-aflatoxin B(1), were detected and quantified in urine by the LC-ESI-MS/MS technique. Other metabolites derived from the conjugation and/or oxidation of aflatoxin B(1) measured in the urine of dosed rats included aflatoxin P(1), aflatoxin P(1)-glucuronide, aflatoxin Q(1), aflatoxin M(1), 8,9-dihydro-8,9-dihydroxy aflatoxin B(1), aflatoxin B(1)-mercapturic acid, the aflatoxin-cysteine glycine adduct derived from the aflatoxin-glutathione conjugate, aflatoxin M(1)P(1) and the aflatoxin B(1)-dialcohol. For in vivo studies to determine the dosimetry of certain aflatoxin metabolites, aflatoxin B(2) was used as an internal standard for recovery since this compound is not naturally produced in rats. In the final method using the internal standard, the coefficient of variation of six replicate analyses of in vivo rat urine samples for aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid, and aflatoxin M(1) was 12.5, 12.8, and 5.8%, respectively. Further, the LC-ESI-MS/MS method to detect aflatoxin N(7)-guanine in in vivo rat urine samples was at least 20-fold more sensitive than prior techniques. Using the LC-ESI-MS/MS technique, the dosimetry, on a weekly basis, of major urinary aflatoxin metabolites was assessed in animals chronically dosed over a 5-week period. Of particular importance was the application of this method to determine the modulation of levels of urinary aflatoxin metabolites by treatment with oltipraz, a chemopreventive agent that can completely ablate aflatoxin hepatocarcinogenesis in the rat. After 1 week, oltipraz administration diminished urinary aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) levels by 83, 92, and 82%, respectively. The magnitude of this reduction was persistent at the day 14, 21, 28, and 35-day time points with the average decrease of aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) being 73, 92, and 90%, respectively. Importantly, even under circumstances where the oltipraz intervention was most efficient in reducing aflatoxin metabolite levels, the LC-ESI-MS/MS method was still sensitive enough to detect the reduced biomarker content. This outcome has important translational implications for the application and analysis of the efficacy of primary and secondary prevention interventions in human populations where ambient exposure levels are low, but the toxicologic hazards of these exposures remain high.
authors.affiliation: Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.
authors.forename: M
authors.initials: M
authors.lastname: Walton
authors.forename: P
authors.initials: P
authors.lastname: Egner
authors.forename: P F
authors.initials: PF
authors.lastname: Scholl
authors.forename: J
authors.initials: J
authors.lastname: Walker
authors.forename: T W
authors.initials: TW
authors.lastname: Kensler
authors.forename: J D
authors.initials: JD
authors.lastname: Groopman
mesh_terms: D000348:Aflatoxins / Q000378:metabolism / Q000652:urine*; D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D015415:Biomarkers; D002273:Carcinogens / Q000378:metabolism; D018890:Chemoprevention; D002853:Chromatography, Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D008297:Male; D011719:Pyrazines / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013057:Spectrum Analysis; D013871:Thiones; D013876:Thiophenes
chemical_list: D000348:Aflatoxins; D016588:Anticarcinogenic Agents; D015415:Biomarkers; D002273:Carcinogens; D011719:Pyrazines; D013871:Thiones; D013876:Thiophenes; C026209:oltipraz