Quantification of Urinary Aflatoxin B₁ Dialdehyde Metabolites Formed by Aflatoxin Aldehyde Reductase Using Isotope Dilution Tandem Mass Spectrometry

Abstract

The aflatoxin B 1 aldehyde reductases (AFARs), inducible members of the aldo-keto reductase superfamily, convert aflatoxin B 1 dialdehyde derived from the exo- and endo-8,9-epoxides into a number of reduced alcohol products that might be less capable of forming covalent adducts with proteins. An isotope dilution tandem mass spectrometry method for quantification of the metabolites, C-8 monoalcohol, dialcohol, and C-6a monoalcohol, was developed to ascertain their possible role as urinary biomarkers for application to chemoprevention investigations. This method uses a novel (13)C 17-aflatoxin B 1 dialcohol internal standard, synthesized from (13)C 17-aflatoxin B 1 biologically produced by Aspergillus flavus. Chromatographic standards of the alcohols were generated through sodium borohydride reduction of the aflatoxin B 1 dialdehyde. This method was then explored for sensitivity and specificity in urine samples of aflatoxin B 1-dosed rats that were pretreated with 3 H-1,2-dithiole-3-thione to induce the expression of AKR7A1, a rat isoform of AFAR. One of the two known monoalcohols and the dialcohol metabolite were detected in all urine samples. The concentrations were 203.5 +/- 39.0 ng of monoalcohol C-6a/mg of urinary creatinine and 10.0 +/- 1.0 ng of dialcohol/mg of creatinine (mean +/- standard error). These levels represented about 8.0 and 0.4% of the administered aflatoxin B 1 dose that was found in the urine at 24 h, respectively. Thus, this highly sensitive and specific isotope dilution method is applicable to in vivo quantification of urinary alcohol products produced by AFAR. Heretofore, the metabolic fate of the 8,9-epoxides that are critical for aflatoxin toxicities has been measured by biomarkers of lysine-albumin adducts, hepatic and urinary DNA adducts, and urinary mercapturic acids. This urinary detection of the alcohol products directly contributes to the goal of mass balancing the fate of the bioreactive 8,9-epoxides of AFB 1 in vivo.

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

“…Using isotope dilution tandem mass spectroscopy, it was estimated that the reduction products AFB 1 -C6 monoalcohol and C6,C8-dialcohol (Fig. 14) account for 8.4% of the administered AFB 1 dose that was found in urine of rats pretreated with aflatoxin reductase inducer 3H-1,2-dithiole-3-thione (D3T) (Johnson et al, 2008). Thus, reduction by AKR7 family proteins represents an important metabolic step in the detoxification of AFB 1 .…”

Bioinformatic Identification of Aldo-Keto Reductase from Newly Isolated Arthrobacter nicotianae strain PR and Its Phylogenetic Analysis Among Soil Bacteria

“…The presence of AKR in A. nicotianae strain PR suggests that this strain may have been exposed to harsh environment similar to that of marine conditions during its early phylogenetic diversification period. In some animals, the expression of AKR is induced by an exposure to aflatoxin [24]. Even though the metabolic triggering of superfamily of enzymes like AKR in those higher eukaryotes has the capability to detoxify such aflatoxin B-1 dialdehyde, its parallel metabolic process in prokaryotes are yet to be identified.…”

Aspergillus parasiticus from wheat grain of Slovak origin and its toxigenic potency

“…LC-MS/MS was carried out according to Sulyok et al (2007). The jars containing 50 ml of a synthetic minimal liquid medium, Adye et Mateles (A&M; Johnson et al 2008) modified by KCl, were inoculated with 100 µl of the spore suspension and cultivated still at 25°C in dark for 7 days. The whole volume was extracted with an equal volume of ethyl acetate on a horizontal shaker (approx.…”