Reaction of Aflatoxin B₁ Oxidation Products with Lysine

Abstract

Aflatoxin (AF) B(1) exo-8,9-epoxide hydrolysis yields AFB(1) dihydrodiol, which undergoes base-catalyzed rearrangement to, and is in equilibrium with, AFB(1) dialdehyde. We investigated the reaction of AFB(1) dialdehyde with albumin to generate a Lys adduct, previously characterized by others [Sabbioni, G., Skipper, P. L., Büchi, G., and Tannenbaum, S. R. (1987) Carcinogenesis8, 819-824; Sabbioni, G. (1990) Chem.-Biol. Interact. 75, 1-15]. Pronase digestion of bovine albumin serum treated with AFB(1) dialdehyde and HPLC yielded the adduct, identified by its characteristic UV and mass spectra. The structure of the Lys-AFB(1) dialdehyde adduct is concluded to be (S)-alpha-amino-2,3-dihydro-2-oxo-4-(1,2,3,4-tetrahydro-7-hydroxy-9-methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-hexanoic acid, structure B of the former paper and 8 of the latter, based on work with the methylamine adduct described in the following paper in this issue [Guengerich, F. P., Voehler, M., Williams, K. M., Deng, Z., and Harris, T. M. (2002) Chem. Res. Toxicol. 15, 793-798]. The time course of product formation at varying concentrations of AFB(1) dialdehyde could be described by complexation with albumin with a K(d) of 1.5 mM and a first-order reaction rate with the N6-amino group of Lys of 0.033 min(-)(1). The reaction of AFB(1) dialdehyde with N(2)-acetylLys was monitored by UV spectroscopy and yielded a final spectrum similar to that of the described Lys adduct. Kinetic analysis of the changes at pH 7.2 was best described with a scheme involving equilibrium of the dialdehyde with dihydrodiol and a rate-limiting reaction of AFB(1) dialdehyde with the N6 atom of N(2)-acetylLys, with an apparent second-order rate constant of 2.6 x 10(3) M(-)(1) min(-)(1), followed by putative carbinolamine formation and rearrangement, collectively described by a first-order rate constant of 7.6 min(-)(1). Competition experiments with the hydrolysis of AFB(1) exo-8,9-epoxide indicate that N2-acetylLys also reacts with the epoxide at pH 7.2 (k = 350 M(-)(1) min(-)(1)) and 9.5 (k = 1.8 x 10(3) M(-)(1) min(-)(1)). This reaction might contribute to the formation of protein Lys adducts, depending upon the local concentration of free or protein Lys. Mass spectral analysis of trypsin digests of bovine serum albumin modified with AFB(1) dialdehyde indicated selective modification of Lys455 and Lys548. Collectively, these results provide more insight into the mechanism of formation of AFB(1) dialdehyde-protein adducts and indicate that the formation of Lys adducts is a moderately efficient process. The binding of AFB(1) dialdehyde to albumin or the protonation of the N6-amino group retards the reaction with Lys residues.

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).…”

“…Production of a C-6a monoalcohol, corresponding to reduction of the C-6a carbon of the dialdehyde, occurred at a much lower rate and the formation of the dialcohol was not rapid in these reactions (17). In a subsequent study, Guengerich and coworkers (16) clearly established a role of AFB 1 dialdehyde in the formation of protein adducts. Based on the rates determined for the various reactions, these author proposed that starting with AFB 1 -8,9-epoxide, the reactivity of the dialdehyde is linear over several hours.…”

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

“…Knowledge of AFB 1 metabolism suggests that additional protein adducts should be formed, and the differences in AF-albumin adduct concentration measured by ELISA and IDMS are therefore likely to be due to the presence of uncharacterized AF adducts. Spontaneous formation of the hemiacetal AFB 2a from AFB 1 or AFB 2 can be expected to yield protein adducts through reactions similar to that producing AF-lysine (19,20,28). Although significant human exposure to AFG 1 occurs, and this toxin can form both DNA and albumin adducts in rats (29), AFG 1 -lysine adduct formation has not been shown in humans.…”

“…The only AFB 1 adduct structurally identified to date in enzymatically digested plasma albumin is AFB 1 -lysine (AF-lysine; refs. 14,19,20). This adduct has been measured by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection and more recently by isotope dilution mass spectrometry (IDMS; refs.…”

The ToxBank Data Warehouse: Supporting the Replacement of In Vivo Repeated Dose Systemic Toxicity Testing

“…It is activated by oxidation to an epoxide, which is subsequently hydrolyzed to a vicinal dialdehyde, which in turn forms bidentate adducts with amines. [56,57] This toxicant also targets alkylation of DNA nitrogens, but the study of this latter reactivity is outside the current scope of the SEURAT-1 project.…”