Kinetics and Mechanism of Hydrolysis of Aflatoxin B₁ exo-8,9-Epoxide and Rearrangement of the Dihydrodiol

Abstract

Aflatoxin B1 (AFB1) is thought to play a large role in human liver cancer in some parts of the world, and the mechanism of genotoxicity is generally considered to involve the DNA adduct formed at the guanyl N7 atom. The exo epoxide, the genotoxic isomer formed by human cytochrome P450 3A4, has been known to be very unstable in H2O (t 1/2 < 10 s). The rates of hydrolysis of AFB1 exo epoxide have been determined as a function of pH using stopped flow kinetics. The spontaneous reaction with solvent is faster than previously suspected, with a t 1/2 ∼1 s when either absorbance or fluorescence kinetic traces are measured at ambient temperature. An acid-catalyzed reaction with a bimolecular rate constant of 2 × 103 M-1 s-1 is operative below about pH 5 and elevates the rate. The hydrolysis product, AFB diol, reversibly converts to a furofuran-ring-opened oxyanionic AFB1 α-hydroxydialdehyde (AFB dialdehyde) under slightly basic conditions. When AFB diol was treated with base in DMSO, conversion to AFB dialdehyde was accompanied by dehydration of the remaining alcohol group. In aqueous solution, the AFB diol:AFB dialdehyde equilbrium has been characterized and the pK a (8.2) is much higher than previously suggested; the base-catalyzed bimolecular rate of 2.3 × 103 M-1 s-1 results in a very slow rate of conversion at physiological pH. Multiphasic conversion of AFB dialdehyde back to AFB diol has a slow rate-limiting step at 0.01 s-1 and is nearly quantitative. The dehydrated AFB dialdehyde formed in DMSO did not form a detectable ring closed derivative.

Protection Against Aflatoxin B1-Induced Cytotoxicity by Expression of the Cloned Aflatoxin B1-Aldehyde Reductases Rat AKR7A1 and Human AKR7A3

“…Although we did not measure the expression of GSTs in this particular study, previous studies have demonstrated that the level of GSTs in these cells are negligible (20). In the case of AFB 1 dihydrodiol, this metabolite can undergo a slow, base-catalyzed ring opening to a dialdehyde (7,31) that is capable of forming Schiff base adducts with protein amino groups, particularly lysine (16). Previous studies suggest that this type of reaction could be involved in the acute toxicity of AFB 1 (12).…”

Quantitative Comparison of Aflatoxin B1 Serum Albumin Adducts in Humans by Isotope Dilution Mass Spectrometry and ELISA

“…AFB 1 is oxidized by cytochrome P450s to yield a variety of metabolites, and its toxicology has been reviewed (4). The major reactive metabolite is the exo-AFB 1 8,9-epoxide, which if not detoxified, can bind to double-stranded DNA to form the promutagenic AFB 1 -N 7 -guanine adduct or, following hydrolysis to the AFB 1 -dihydrodiol, with proteins such as albumin (5)(6)(7)(8). Both urinary AFB 1 -N 7 -guanine and serum AF-albumin levels are correlated with dietary intake of AFs (8)(9)(10)(11)(12)(13).…”

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts

“…Metabolic activation of AFB 1 by the cytochrome P450 system generates a highly reactive intermediate, AFB 1 -8,9-epoxide, capable of covalent binding to DNA (Essigman et al, 1982;Eaton and Gallagher, 1994;Johnson et al, 1996). The principal adduct formed is 8, 9 -dihydro -8 -(N7 -guanyl) -9 -hydroxya¯atoxin B 1 (AFB 1 -N7-Gua), both in vitro (Essigman et al, 1977) and in vivo (Lin et al, 1977;Croy et al, 1978).…”