Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B_2a and Biological Amines

Abstract

Aflatoxin B has been shown to bind to proteins through a dialdehyde intermediate under physiological conditions. The proposed structure of this adduct has been published showing a Schiff base interaction, but adequate verification using structural elucidation instrumental techniques has not been performed. In this work, we synthesized the aflatoxin B amino acid adduct under alkaline conditions, and the formation of a new product was determined using high performance liquid chromatography-time-of-flight mass spectrometry. The resulting accurate mass was used to generate a novel proposed chemical structure of the adduct in which the dialdehyde forms a pyrrole ring with primary amines rather than the previously proposed Schiff base interaction. The pyrrole structure was confirmed using H,C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation NMR and tandem mass spectrometry. Reaction kinetics show that the reaction is overall second order and that the rate increases as pH increases. Additionally, this study shows for the first time that aflatoxin B dialdehyde forms adducts with phosphatidylethanolamines and does so through pyrrole ring formation, which makes it the first aflatoxin-lipid adduct to be structurally identified. Furthermore, oxidation of the pyrrole adduct produced a product that was 16 m/z heavier. When the aflatoxin B-lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and H NMR spectrum that match aflatoxin B-lysine, which suggest the transformation of the pyrrole ring to a pyrrolin-2-one ring. These data give new insight into the fate and chemical properties of biological adducts formed from aflatoxin B as well as possible interferences with known aflatoxin B exposure biomarkers.

Chronic and Acute Toxicities of Aflatoxins: Mechanisms of Action

“…The mutagenic effects of AFB1 have been the focus of most studies since the discovery of this aflatoxin and were ascribed mainly to the intermediate metabolite AFB1-exo-8,9 epoxide (AFBO) [13]. As a highly unstable molecule, AFBO reacts with cellular macromolecules, including nucleic acids, proteins, and phospholipids, to induce various genetic, metabolic, signalling, and cell structure disruptions [14][15][16]. However, increased evidence is being built up demonstrating equally dramatic or higher effects of AFB1 on cell function and integrity through the induction of oxidative stress (OS) [17][18][19].…”

“…In particular, aflatoxin-protein adducts have been the most frequently associated with acute intoxication, as this blocks protein synthesis, especially the enzymes involved in vital functions, such metabolic pathways, protein synthesis, DNA replication and repair, and immune response (Figure 1). Additionally, there is increasing evidence that aflatoxin-phospholipid adducts and ROS-induced LPO are the main reasons for the disruption of the integrity and function of the membranes of the cells, mitochondria, and endoplasmic reticulum [14,20], as depicted in Figure 2. Moreover, severe DNA fragmentation upon exposure to high doses of aflatoxins is another major effect of acute aflatoxicosis (Figure 1), as was observed in the testicular tissues of mice injected with a daily dose of 20 µg AFB1/kg bw for 21 days [62].…”

“…According to the authors, AF-dhd derives from aflatoxin-exo 8,9-epoxide and forms the aflatoxin-albumin adducts via aflatoxin-aldehyde bypassing the formation of aflatoxin-dialcohol of the detoxification pathway [13]; and the more rapidly and abundantly AF-dhd is formed, the higher is the mortality rate. Moreover, the metabolism of AFB2 a as a dietary contaminant or as an AFB1-phase I metabolite was also suggested to be involved in acute toxicity; apart from the formation of aflatoxin-albumin adducts, AFB2 a was also reported to bind covalently with cellular proteins and phospholipids, yielding lipidand protein-adducts, possibly leading to acute aflatoxicosis [14].…”

The Toxic Effects of Aflatoxin B1: An Update

“…This method was able to completely converse AFB1-contaminated corn in 20 min. This result advocates for adding amino acids to the previous acidification treatments to form stable, nontoxic forms of AFB1 [137].…”

“…The applications of high temperatures (80-120°C) together with acidification such as HCl, citric acid, and lactic acid were also reported to have high degrading potential (85-100%). The addition of citric acid can help degrade 98% of AFB1 at 100°C in 20 min [135][136][137]. The combination of heat and acidification is attractive with the fact that those two methods separately have already been widely used in food manufacturing industry, so that no specialized or expensive equipment is needed additionally.…”