Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin

Article pubs.acs.org/crt

Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B2a and Biological Amines Blake R. Rushing* and Mustafa I. Selim Department of Pharmacology & Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27834, United States S Supporting Information *

ABSTRACT: Aflatoxin B2a 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 B2a 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 1H, 13C, 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 B2a 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 B2a-lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and 1H NMR spectrum that match aflatoxin B1lysine, 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 B2a as well as possible interferences with known aflatoxin B1 exposure biomarkers.

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INTRODUCTION Aflatoxins are a class of mycotoxins produced primarily by the fungi Aspergillus f lavus and Aspergillus parasictus.1 These compounds are known to contaminate a wide variety of food and plant products such as corn, wheat, peanuts,2 rice,3 tobacco products,4 and cereals5 as well as dried foods, spices, and medicinal plants.6,7 Of the aflatoxins produced by these fungi, ingestion of aflatoxin B1 (AFB1) is the greatest cause of concern due to its well characterized ability to cause the development of hepatocellular carcinoma (HCC).8−11 This occurs as a result of bioactivation of AFB1 by P450 enzymes into the reactive intermediate aflatoxin-exo-8,9-epoxide (AFBO), which then binds to guanine residues on DNA and causes point mutations.12,13 It is estimated that up to 28% of all HCC cases are caused by AFB1 consumption particularly in regions where hepatitis B infection is prevalent as well as those that lack rigorous food safety regulations, which make exposure to this mycotoxin a large concern for global human health.14 Acute exposure to AFB1 can cause additional health effects such as hemorrhagic liver necrosis, edema, bile duct proliferation, immunosuppression, and stunted growth.15−17 Because of this, many methods have been developed in an attempt to detoxicate © 2017 American Chemical Society

contaminated foods to mitigate or prevent these toxicities. However, issues such as incomplete removal, the formation of toxic byproducts, or the use of sophisticated infrastructures have prevented many of these methods from being incorporated in many countries.18 Organizations such as the FDA and USDA consider AFB1 as an unavoidable contaminant due to the toxin’s ability to be produced during pre- and postharvest events such as transportation and storage.19 Because of this, controlling exposure to AFB1 can be difficult, particularly in countries where regulatory bodies are either limited or nonexistent. As a result, it is estimated that approximately 4.5 billion people per year, mostly in developing countries, are at risk for uncontrolled exposure of aflatoxin.16 Aflatoxin B2a (AFB2a) is a hemiacetal form of AFB1 and was first discovered as a metabolic product of AFB1.20 Production of AFB2a has been shown to occur in microsomal fractions of the liver with NADPH as a cofactor and is increased when animals are pretreated with phenobarbital, which suggests that it is a P450-dependent metabolic pathway.21−23 Conversion to AFB2a Received: January 4, 2017 Published: May 17, 2017 1275

DOI: 10.1021/acs.chemrestox.7b00002 Chem. Res. Toxicol. 2017, 30, 1275−1285

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Chemical Research in Toxicology

from Sigma-Aldrich (St. Louis, MO). Synthesized aflatoxin B2a was characterized according to methods previously published by our lab.31 High Performance Liquid Chromatography/Time of Flight Mass Spectrometry (HPLC/TOFMS) conditions. The HPLC/ TOFMS system used in this study consisted of an Agilent 1200 series high performance liquid chromatograph (HPLC) equipped with an Agilent 1200 series Diode Array Detector (DAD), which was coupled to an Agilent 6220 time-of-flight mass spectrometer (TOFMS) with a dual electrospray ionization interface. The HPLC column used was an Agilent Zorbax Eclipse Plus C18 column (3.5 μm, 2.1 × 150 mm2). Mobile phases consisted of LC−MS grade water (solvent A) and a 1:1 mix of acetonitrile and methanol (solvent B). Formic acid (1% v/v) was added to both mobile phase solvents. Solvent programming was as follows: 20% B at 0 min, ramp to 75% B at 4 min, and ramp to 100% B at 9 min. Column temperature was held at 35 °C. The diode-array detector was set to collect absorbance between 220 and 900 nm. The TOFMS was run in positive ionization mode. The drying gas (nitrogen) for the ESI was held at 335 °C at a rate of 10 L/min. The nebulizer pressure was 35 psi, and the voltage capillary was kept at 3300 V. HPLC/TOFMS data were collected and analyzed using Agilent MassHunter Data Acquisition software. The mass spectrometer was tuned and calibrated each day before sample analysis. Standards of each compound were run daily before analysis to verify retention times and accurate masses. Blanks were run in between samples to avoid sample carryover. Adduction of AFB2a to Amino Acids. To synthesize the adduct and confirm the site of interaction, 50 μL solutions of AFB2a (1500 nM) were dried to completion using an Organomation N-EVAP analytical evaporator heated to 40 °C and redissolved in 50 μL of alanine or N-acetylalanine (5.5 mM in 1 M sodium bicarbonate). The diode-array detector on the HPLC was set to measure at 365 nm to aid in the detection of reaction products. Online TOFMS scan range was set to monitor [M + H]+ values of potential products, and extracted ion chromatograms (EICs) were then used for confirmation based on retention times as compared to the UV chromatograms and accurate mass data. To test the ability of the other amino acids to react, individual amino acid solutions were prepared in 1 M sodium bicarbonate (200− 250 mM). A 100 μL aliquot of each amino acid solution was added to tubes containing dried AFB2a (100 μL of 900 nM in acetonitrile). After mixing, each solution was transferred to separate HPLC vials run on the HPLC/TOFMS immediately for analysis. Samples were prepared in reduced light conditions to prevent photodegradation of aflatoxins. Accurate mass error (ppm) error of the [M + H]+ ions was calculated using the following equation:

has been shown to account for up to 50% of AFB1 metabolism in some species.22 Additionally, AFB2a has been shown to be a dietary contaminant most notably in eggs from poultry, which suggests dietary consumption may be a major route of exposure of AFB2a.24 A study involving 161 individuals at Lagos, Nigeria detected AFB2a as the most prominent form of aflatoxin in human urine occurring in 32.7% of all samples, which suggested that the occurrence of AFB2a in humans can be quite significant.25 Furthermore, AFB2a formation from AFB1 has been shown to occur nonenzymatically under acidic conditions providing an alternative method of production that does not require the presence of hepatic enzymes.26 During the production of AFB2a, the 8,9-double bond of AFB1 is hydrated, removing the site of bioactivation that is associated with mutagenicity. Ames’ test results have shown that this chemical modification severely reduces its mutagenicity, which suggests that AFB2a is far less mutagenic than AFB1.27 While in vivo toxicological data on AFB2a are limited, it has been shown to have reduced activity as compared to AFB1 in terms of its LD50 and its ability to induce bile duct hyperplasia, as well as its capability to form adducts with DNA.28 The reduced mutagenicity of AFB2a as well as its ability to be produced nonenzymatically has spurred interest in the development of treatment methods that involve the formation of AFB2a as a way to detoxicate contaminated foods, mainly through the use of acidic compounds.29−31 One underexplored characteristic of AFB2a that may result in additional toxicities is its ability to spontaneously bind to proteins. AFB2a initially exists as a hemiacetal, but when introduced to higher pH values, the hemiacetal ring opens into a dialdehyde.32 Work performed by Ashoor and Chu concluded that this dialdehyde binds to amine residues on amino acids or proteins likely through the formation of reversible Schiff bases using thin-layer chromatography and UV-spectroscopy; however, the authors note that the structure of the adduct was unable to be confirmed using mass spectrometry.33 As such, adequate structural characterization has not been performed on the AFB 2a adduct, likely due to limitations of the instrumentation at that time. Additionally, AFB2a has been shown to bind to pancreatic DNase and inhibit its function,34 and it also binds to microsomal proteins, potentially altering metabolic function.22 Interestingly, species that are more susceptible to AFB1 toxicities show a high production of AFB2a, which may in part explain their susceptibility.23,35−37 Further information on how this binding occurs and the effects it has on macromolecules is greatly lacking, which is important for determining the safety of AFB2a as a major metabolite and its potential as a detoxification product. The aim of this study is to identify the structure of AFB2a adducts, some key biological molecules that may be involved in the formation of adducts, and any oxidation products that may be formed from this adduct.

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⎛ M − Mt ⎞ 6 E=⎜ e ⎟(10 ) ⎝ Mt ⎠ where E = error in ppm, Me = experimentally determined mass of the [M + H]+ ion, and Mt = theoretical mass of the [M + H]+ ion. Purification and NMR of Adduct. A large stock of AFB2a was synthesized by incubating 40 mg of AFB1 in 0.1 M HCl at 60 °C for 4 h. The resulting pale yellow solution was lyophilized, and to it, 1 mL of a 100 mg/mL L-alanine solution in 1 M sodium bicarbonate was added. The solution immediately turned a dark yellow color, which indicated a rapid conversion. The AFB2a-alanine adduct was purified using a Bio-Rad BioLogic DuoFlow HPLC equipped with a Vydac C18 prep column (22 × 250 mm2, 10 μm) with water and acetonitrile as solvents. Confirmation of the collected adduct was performed by HPLC/TOFMS analysis. The purified fraction was lyophilized and then redissolved in 0.6 mL of deuterium oxide. The resulting solution was filtered into an NMR tube using a 0.45 μm filter prior to analysis. Spectra for 1H, 13C, DEPT-135, COSY, HSQC, and HMBC NMR were obtained using a Bruker Advance 400 NMR (400 MHz). Data were collected using TopSpin 3.2 software. Quadrupole Time-of-Flight (QTOF) Mass Spectrometry. Solutions containing 20 μg/mL of AFB1, AFB2a, and AFB2a-alanine were prepared for direct injection using a Waters Acquity LC with a Waters Micromass QTOF for analysis. QTOF parameters were as

EXPERIMENTAL SECTION

Chemicals. Aflatoxin B1, LC−MS grade solvents (acetonitrile, methanol, isopropanol, and water), formic acid, sodium bicarbonate, hydrochloric acid (HCl), sodium hydroxide (NaOH), acylase I from porcine kidney (≥ 2000 units/mg), L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, Lhistidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, Lvaline, Nα-acetyl-L-lysine, oxone (monopersulfate), deuterium oxide, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased 1276


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Figure 1. (a) UV chromatograms and (b) EICs of AFB2a in the presence of alanine (tops) or N-acetylalanine (bottoms) as well as (c) the TOF mass spectrum of AFB2a-alanine. UV chromatogram was monitored at 365 nm. EICs consisted of the theoretical [M + H]+ adduct masses of AFB2a (C17H14O7), AFB2a‑alanine (C20H17NO7), and AFB2a-N-acetylalanine (C22H19NO8): m/z 331.0812, 384.1078, and 426.1183 respectively. Calculated ppm error for part c = 0 ppm. analyzed by HPLC/TOFMS every 20 min over 200 min to measure concentrations of AFB2a and AFB2a-alanine at each time point. To determine the reaction order with respect to alanine, AFB2a was held constant at a concentration of 1500 nM while alanine was added at concentrations of 5.5, 11, and 22 mM (pH 9). Reaction mixtures were measured every 6 min due to the increased reaction rate. The HPLC gradient programming was modified to accommodate the shorter time intervals. As mentioned previously, the rate for each mixture was calculated by taking the slope of the linear portion of the concentration versus time graph. Each level was repeated in triplicate. Adduction of AFB2a to Phospholipids. An aliquot of 50 μL of AFB2a (150 mM in acetonitrile) was dried and redissolved in 50 μL of a 1 M sodium bicarbonate solution. To this, 50 μL of a DPPE or DPPC in chloroform (3.5 mM) was added. The two layers were mixed by sonication for 5 min until the organic layer turned a dark yellow color. The organic layer was then separated and analyzed by HPLC/ TOFMS for EICs of the corresponding adduct masses for both phospholipids. The HPLC methods were changed to include isopropanol as solvent B with neither solvent A nor B containing formic acid. Solvent programming was as follows: 5% B at 0 min, ramp to 65% B at 5 min, ramp to 100% B at 25 min, hold at 100% B for an additional 5 min. The TOFMS was set to analyze in negative mode. Oxidation of the Aflatoxin B2a Adduct. A concentrated solution of AFB2a-alanine was synthesized by drying 100 μL of AFB2a (1.5 μg/ mL in acetonitrile) and redissolving in 50 μL of an L-alanine solution (10 mg/mL) in 1 M sodium bicarbonate. To this, 50 μL of a 2 mM solution of monopersulfate (oxone) in deionized water was added. The solution was measured by HPLC/TOFMS over 24 h in positive mode. The oxidation product was identified using the diode array detector (347 nm), and the proposed structure was determined using the corresponding accurate mass value.

follows: capillary was set to 3000 V, sample cone was set to 35 V, extraction cone was set to 4 V, source temperature was held at 80 °C, desolvation temperature was set to 150 °C, desolvation nitrogen gas flow was 500 L/h, and the microchannel plate detector (MCP) was set to 2600 V. Flow rates for infusions were performed at 0.05 mL/min, and 10 μL was injected for analysis. AFB1 and AFB2a were analyzed in positive mode with a collision induced dissociation (CID) energy of 20 eV. AFB2a-alanine was analyzed in negative mode with a CID of 15 eV. Effect of pH on AFB2a-Adduct Conversion on Rate. Solutions of alanine (5.5 mM) were prepared and adjusted to various pH levels (3, 5, 7, 9, and 11) using 1 M HCl and 1 M NaOH. Five aliquots of 100 μL of AFB2a (375 nM) were dried using a nitrogen evaporator and then redissolved in 100 μL of alanine solutions at each pH level. Each reaction mixture was immediately injected into the HPLC/TOFMS to measure the concentration of AFB2a and alanine in the solution. Injections were then repeated at regular time intervals to obtain a linear portion of a concentration versus time graph. Injections every 20 min over 200 min were determined to be suitable for this analysis. Rates for each pH value were determined by taking the slope of the linear portion of each concentration versus time graph. Each pH level was repeated in triplicate. A 100% conversion from AFB2a to the adduct was assumed when the AFB2a peak was undetectable and therefore was used to quantify the adduct. Statistical analysis involved the use of a one-way ANOVA followed by Students’ t tests. Determination of Reaction Order. Using a nitrogen evaporator, 50 μL of AFB2a solutions in acetonitrile of various concentrations (375, 750, and 1500 nM) was dried to completion and then redissolved in 50 μL of an aqueous alanine solution (5.5 mM, pH 9) to determine the reaction order with respect to AFB2a. After the addition of the alanine solution, each concentration level was immediately 1277


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Scheme 1. Mechanism of AFB2a Formation and Subsequent Binding to Proteins along with the Proposed Resulting Adduct Structures

The similarities of the oxidized AFB2a adduct were then compared to the AFB1-lysine adduct from the literature. Since AFB1 was found to adduct to the ε amino group on lysine, AFB2a (5 mg) was dissolved in a solution of Nα-acetyllysine (3 mL at 10 mg/mL in phosphate buffered saline) along with 200 μL of 1 M sodium bicarbonate to adduct AFB2a to the ε amine. After a complete conversion to AFB2aNα-acetyllysine, 50 mg of acylase I was added to the mixture, which was then incubated for 72 h at 37 °C. The resulting AFB2a-lysine (ε) was purified by the HPLC fractionation method described earlier. After lyophilization, AFB2a-lysine (ε) was dissolved in 100 μL of a 10 mM monopersulfate solution and allowed to react at room temperature for 3 h. The resulting oxidation product was analyzed by HPLC/TOFMS and QTOF (both in positive mode). CID of oxidized AFB2a-lysine (ε) was at 25 eV. Purification and 1H NMR were performed according to methods described earlier.

acetylalanine, which produced no reaction products (Figure 1a,b). The reaction conditions were repeated with all 20 common amino acids. With the exception of proline, every amino acid produced a product that contained an accurate mass value corresponding to the equation given previously within ±5 ppm error (Table S1). The adducts displayed a wide range of retention times indicating large differences in polarities, and they all showed a strong absorption at 347 nm. Lysine, due to having a second primary amine group, formed two isomeric peaks with different retention times (Nα and Nε adducts). Accurate mass values of the adducts all followed the formula mentioned earlier. Because the masses of the reaction products did not match the previously proposed Schiff base structure, a new pyrrole adduct structure was proposed, which accommodated the 1:1 AFB2a/amino acid observation (Scheme 1). Since the masses of all the 19 amino acid adducts followed this same formula, alanine was chosen as a representative amino acid for further analysis to confirm the pyrrole structure (compound IV) and obtain kinetic properties of the reaction between AFB2a and amines. Confirmation of Pyrrole Formation. Approximately 40 mg of the adduct was synthesized from an AFB1 standard and purified using preparative HPLC. Because of low organic solubility of the adduct, D2O was chosen as the solvent for the adduct to undergo NMR analysis. 1H NMR spectra of the AFB2a-alanine adduct showed the formation of three proton peaks downfield (δ 6.48, 6.93, 7.13) in addition to the benzylic proton (Figure 2a). These three peaks correspond to the three protons found within the pyrrole ring of the proposed structure. Proton c, located in the 2 position as indicated in the figure, was shown to undergo hydrogen−deuterium exchange with the solvent after a prolonged period of time, which could be reversed by redissolving in H2O. 13C NMR spectra showed a multitude of peaks in the aromatic region

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RESULTS Identification of Products Formed between AFB2a and Amino Acids. To characterize the binding between AFB2a and amino acids, alanine was chosen to react with AFB2a. Because of the dependence of the formation of the AFB2a dialdehyde, which occurs at higher pH levels, the amino acid was dissolved in 1 M sodium bicarbonate to give an alkaline reaction mixture. The diode-array detector connected to the HPLC was used to detect any UV absorption signals of any remaining AFB2a as well as any related products. By monitoring at 365 nm, the reaction mixture of AFB2a and alanine showed a prominent product peak with a longer retention time as compared to AFB2a (Figure 1a). Upon analyzing the mass spectrum of this peak, the base peak of m/z 384.1078 corresponded to the [M + H]+ adduct of (mass of AFB2a) + (mass of alanine) − 2H2O (Figure 1b,c). The maximum absorption of AFB2a changed from 365 to 347 nm after adduction to alanine as determined by the online DAD. The site of interaction on the amino acid was confirmed by repeating these conditions with N1278


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Figure 2. (a) 1H NMR and (b) 13C NMR of AFB2a-alanine taken at 400 MHz. The proton peak at 4.70 ppm is due to HOD resonance from the solvent (deuterium oxide). A reduction in the intensity proton peak c was observed after prolonged periods in the solvent (D2O) due to hydrogen− deuterium exchange. The 13C peak at 69.5 ppm is due to a solvent impurity. Nonpyrrole carbons and protons also agree with 1H and 13C assignments from previous works.39,43

and indicate that they are all in close proximity to each other, which support the hypothesis of pyrrole formation. AFB2a-alanine: 1H NMR (D2O, 400 MHz): δ 1.63 (d, 3H), 2.14 (s, 2H), 2.79 (s, 2H), 3.56 (s, 3H), 3.61 (overlapped, 1H), 5.87 (s, 1H), 6.38 (d, 1H), 6.81 (s, 1H), 7.02 (s, 1H). The peak at δ 4.70 was due to residual HOD. AFB2a-alanine: 13C NMR (D2O, 400 MHz): δ 18.1, 28.4, 35.2, 55.1, 59.5, 99.5, 99.65, 107.0, 107.9, 109.9, 113.4, 119.0, 120.7, 154.5, 159.5, 161.5, 176.0, 178.7, 180.3, 205.5. QTOF mass spectrometry was used to obtain MS/MS spectra of AFB1, AFB2a, and AFB2a-alanine to track changes in chemical features through analyzing mass fragments. MS/MS spectra of AFB1 showed a fragmentation pattern similar to previously published work with major fragments at m/z 285, 270, 257, 243, 229, and 201 from the parent ion (m/z 313) (Figure 4a).38 The fragmentation pattern produced by the parent ion of AFB2a (m/z 331) showed an initial fragment that corresponded of a loss of H2O giving the same structure as

(Figure 2b). A DEPT-135 spectra showed four positive peaks in the aromatic regions, which corresponded to the CH carbons found on the pyrrole and benzene rings (Figure S1). COSY NMR showed a strong link in the aromatic system between the three pyrrole protons indicating they are close in proximity. The proposed 5-membered pyrrole ring would adequately allow all three protons to interact with each other as shown in the COSY spectrum (Figure 3). Of additional note is the water peak that contributed to an increased amount of background signal, which included an interaction with the methyl protons of alanine. Also, proton peak c can also be seen here without any hydrogen−deuterium exchange. HSQC NMR confirmed the carbon peaks that were associated with the pyrrole protons (Figure S2), and HMBC NMR indicated that the pyrrole protons were strongly linked with these surrounding pyrrole carbons (Figure S3). These 1D and 2D NMR spectra clearly indicate the presence of these aromatic protons and carbons 1279


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Figure 3. COSY NMR spectrum of AFB2a-Alanine taken at 400 MHz. The three pyrrole protons designated in Figure 2 show an expected aromatic coupling system.

fragmentation of the pyrrole ring (Figure 4c). Further details concerning structural assignments to mass fragments of AFB1, AFB2a, and AFB2a-alanine can be viewed in Scheme S1. Kinetics of AFB2a Binding to Amines. Two factors were tested for their effects on reaction rate: pH of the reaction mixture and concentrations of each reactant. For pH, solutions of 5.5 mM of alanine were made in deionized water and adjusted to a final pH of 3, 5, 7, 9, and 11. These solutions were added to dried AFB2a (100 μL of 375 nM), and the resulting reaction mixtures were measured at regular time intervals for the formation of AFB2a-alanine over 200 min. Rates were calculated by taking the slope of the linear portion of the concentration versus time graph. The results demonstrate that adduction did not occur until pH 5, and then increased five-fold when the pH was increased to 7. The rate at pH 7 was further increased by nine-fold when the pH was increased to 9. The change in rate from pH 9 to pH 11 was much less pronounced, with the rate increasing by a factor of 1.14 (Figure 5). These results show that adduction is greatly enhanced in mild alkaline solutions, although it does also occur in mildly acidic conditions. The effect of reactant concentration was also measured by changing the concentration of one species while the other was kept constant. Linear portions of the concentration versus time graph were again used to calculate the reaction rate. Increasing the concentration of AFB2a by a factor of 2 also changed the magnitude of the rate by a factor of 2 when the concentration of alanine was held constant. Likewise, this was also true when

Figure 4. CID spectra of the [M + H]+ parent ions for (a) AFB1 (m/z 313.1) and (b) AFB2a (m/z 331.1) or the [M-H]− parent ion for (c) AFB2a-alanine (m/z 384.1). Structural assignments to mass fragments can be found in Scheme S1.

AFB1, and subsequent fragments were of identical masses to the spectra given by AFB1 (Figure 4b) confirming the formation of the hemiacetal intermediate. The MS/MS spectra of the [MH]− parent ion of AFB2a-alanine (m/z 382) showed an initial loss of CO2 from alanine giving a fragment of m/z 338, as well as fragments m/z 311 and 245, which correspond to 1280


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chloroform and a sodium bicarbonate buffer to achieve an alkaline condition to facilitate binding. Higher concentrations of AFB2a and lipids were used to visually determine by color in which layer the adduct was present. Analysis by HPLC/ TOFMS confirmed the presence of the adduct in the organic layer as seen by the EIC peak of the corresponding [M-H]− of AFB2a-DPPE as well as the disappearance of the AFB2a peak. DPPC showed no color change, and a search of the corresponding [M-H]− did not produce a product peak for the adduct, while the AFB2a peak remained, which indicated the absence of a reaction (Figure 6a,b). The mass spectrum of the product peak from AFB2a and DPPE gave an [M-H]− ion mass that corresponded to (mass of AFB2a) + (mass of DPPE) − 2H2O (Figure 5c). Oxidation of the AFB2a Amino Acid Adduct. Upon oxidation, the UV chromatogram showed a decrease in the AFB2a-alanine peak along with the formation of a secondary peak with a faster elution time. The new compound had an [M + H]+ of m/z 400.0988, which indicated the addition of oxygen (Figure S4). Because of the reactivity of pyrroles, it is likely that the addition of oxygen occurred on this ring. Neither AFB2a nor alanine underwent this conversion when incubated with monopersulfate alone, which further suggested that this modification occurred at the newly formed pyrrole ring. It was hypothesized that the pyrrole was oxidized to a pyrrolin-2one, possibly giving the same as structure as the AFB1-lysine adduct.39 To analyze the AFB2a-lysine (ε) adduct specifically, AFB2a was reacted with Nα-acetyl-lysine so that the pyrrole would form at the Nε amine group. After deacetylation by acylase I, this gave AFB2a-lysine (ε). This was confirmed by comparing the EIC with that of AFB2a reacted with unprotected lysine. The unprotected lysine gave two isomers at approximately 3.6 and 4.5 min with the expected [M + H]+ adduct mass of m/z 441.1656, whereas the protected lysine adduct only gave a singular peak at 4.5 min, which suggested that only

Figure 5. Effect of pH on the rate of formation of AFB2a-alanine. AFB2a and alanine were mixed together in solutions of various pH values, which were then analyzed by HPLC/TOFMS at regular time intervals. Each pH level was performed in triplicate. Error bars are expressed as standard deviation (n = 3). Groups that contain different letters are statistically different from one another (p < 0.05).

alanine was changed by a factor of 2, while AFB2a’s concentration was held constant (Table S2). These results show that the rate of AFB2a binding with amino acids is first order with respect to each of the reactants, which gives the following rate law equation: rate = k[AFB2a ][alanine]

Binding of AFB2a to Phospholipids. For phospholipids, it was expected that those with a phosphoethanolamine headgroup would be able to form adducts with AFB2a. DPPE was chosen as a model phosphatidylethanolamine to adduct with AFB2a, while DPPC was chosen as a negative control due to the choline group blocking the nitrogen. Because DPPE and DPPC were insoluble in water, a bilayer system was used with

Figure 6. EICs of AFB2a with (a) DPPC or (b) DPPE as well as (c) the TOF mass spectrum of AFB2a-DPPE. EICs consisted of the theoretical [MH]− adduct masses of AFB2a (C17H14O7), AFB2a‑DPPE (C54H84NO13P), and AFB2a-DPPC (C57H90NO13P): m/z 329.0667, 984.5608, 1026.6077, respectively. Calculated ppm error for part c = 0.20 ppm. 1281


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Chemical Research in Toxicology the ε adduct was formed (Figure S5). Upon oxidizing the AFB2a-lysine (ε) adduct, the addition of m/z 16 was observed again to give an accurate mass of 457.1606, which matches the accurate mass of AFB1-lysine adduct (Figure S6). After analysis by QTOF, MS/MS data show the formation of fragment masses from the parent ion m/z 457 such as m/z 411, 394, 382, 376, 366, 328, 247, 148, and 84 from oxidized AFB2a-lysine (ε) (Figure 7). These fragments have been repeatedly shown after

AFB2a-lysine (ε): 1H NMR (D2O, 400 MHz): δ 1.33 (m, 2H), 1.79 (m, 2H), 2.32 (s, 2H), 3.02 (s, 2H), 3.69 (s, 3H), 3.80 (s, 1H), 3.94 (m, 1H). Oxidized AFB2a-lysine (ε): 1H NMR (D2O, 400 MHz): δ 1.65 (m, 2H), 1.88 (m, 2H), 2.42 (s, 2H), 2.94 (s, 2H), 3.48 (t, 2H), 3.74 (s, 3H), 3.94 (overlapped, 1H), 4.20 (s, 2H), 5.96 (s, 1H), 7.15 (s, 1H).

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DISCUSSION By reacting alanine with AFB2a, the accurate mass of the resulting product did not correspond to the proposed Schiff base structure. The accurate mass of this product along with the product of the other 18 amino acids gave an [M + H]+ value that suggests a 1:1 AFB2a to amino acid reaction with a dual condensation. The Schiff base reaction (compound V) would result in a 2:1 amino acid/AFB2a reaction with an [M + H]+ value of m/z 473.1555 for the alanine adduct. Given the amino acids involved in this reaction and the effect of blocking the amine group, we maintain that the site of interaction still occurs at primary amine functional groups. On the basis of the newly observed accurate mass values of the adduct, we suggest that the dialdehyde forms a pyrrole structure through Paal-Knorr synthesis (compound IV) giving a novel structure of the AFB2a amino acid adduct, which is supported by 1H, 13C, COSY, HSQC, and HMBC NMR as well as TOFMS and QTOF data as discussed earlier. AFB1 dialdehyde, which is formed from hydrolysis of AFBO, has been shown to adduct to primary amines of amino acids in a similar manner. It has been demonstrated that the AFB1 dialdehyde binds to amine residues at a 1:1 ratio and condenses to form a five-membered ring, although the AFB1 dialdehyde forms a pyrrolin-2-one ring in contrast to the pyrrole ring as shown with AFB2a.39,44,45 Pyrrole adducts have been shown to be formed with other reactive aldehydes and ketones such as lipid peroxidation products 4-HNE and levuglandin E2 as well as γ-diketones such as 2,5-hexanedione.46−49 Formation of these adducts occurs during oxidative stress conditions and can disrupt protein

Figure 7. CID spectra of the [M + H]+ parent ion of oxidized AFB2alysine (m/z 457.2). The fragment masses match the fragmentation pathway of AFB1-lysine described previously.42

fragmentation of AFB1-lysine, giving further confirmation that they are similar structures.40−42 In comparison to the 1H NMR of the unoxidized AFB2a-lysine (ε) (Figure S7), the 1H NMR data of the oxidized product showed only a singular proton peak downfield of the benzylic proton as well as the formation of an additional singlet at 4.20 ppm (Figure S8). This shows a loss of the pyrrole ring shown in Figure 2, which demonstrates an oxidation in this region. This 1H NMR spectrum of oxidized AFB2a-lysine (ε) shows great similarities to that of the 1H NMR spectra of AFB1-lysine in previous studies, which further supports the formation of this compound.39,43 Furthermore, the appearance of these two peaks at 4.20 and 7.15 ppm also helps distinguish between other possible isomers as depicted by Sabbioni et al.39

Scheme 2. Fate and Intersection of Aflatoxin Dialdehydes and Their Biological Adducts

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Chemical Research in Toxicology function as well as elicit immunoreactivity.47 Although many aldehydes such as 4-HNE and MDA can form Schiff base adducts and cross-link proteins, these adducts have also been shown to be reversible unlike their pyrrole counterparts.49,50 Pyrrole adducts themselves have also been shown to cross-link proteins through polymerization, which occurs during oxidation of the pyrrole ring. A strong example of this is 2,5-hexanedione, which is a known neurotoxicant that binds to proteins through pyrrole ring formation and then autoxidizes to cross-link proteins resulting in neuropathy.46 Interestingly, different substitutions on the pyrrole ring of 2,5-hexanedione have been shown to affect its ability to be oxidized and polymerize. Substitution at the 3 and 4 position with electron donating groups, such as alkyl groups, increases the extent of toxin oxidation and subsequent cross-linking, exacerbating neurotoxic effects.51−53 Because the pyrrole ring of the AFB2a amino acid adduct includes an alkyl substitution at the 3 position, there was a concern for its ability to oxidize and polymerize giving it the ability to cross-link proteins similarly to 2,5-hexanedione. However, oxidation yielded only a product with an [M + H]+ that was 16 m/z heavier than the original compound. It was then hypothesized that the pyrrole of the adduct was being oxidized to a pyrrolin-2-one ring. Confirmation of this was found by analyzing MS/MS fragmentation patterns as well as 1 H NMR spectral data of oxidized AFB2a-lysine (ε) so that it could be compared to AFB1-lysine, a known aflatoxin adduct that contains a pyrrolin-2-one ring. AFB1-lysine is formed by the metabolism of AFB1 and subsequent binding to serum albumin at lysine side chains, which has been found to be a reliable biomarker for aflatoxin exposure. Analysis of this biomarker by mass spectrometry is often done by digesting serum proteins to give free AFB1-lysine, which is then used to quantify AFB1 exposure.54−56 It has been previously thought that this adduct could only be formed from the AFB1 dialdehyde, which is a direct result of epoxide formation, thus making it a marker of genotoxic damage, and is specific to AFB1 exposure.57,58 The results in this study show that this adduct may also be formed when AFB2a adducts to lysine side chains and is exposed to oxidative conditions. This suggests that the AFB1-lysine biomarker may also be the result of AFB2a exposure or metabolic production of AFB2a from AFB1 (Scheme 2). In these cases, the presence of the adduct would not necessarily be indicative of genotoxic damage through AFBO production, which may in part contribute to why this biomarker has been shown in some cases to not correlate significantly with the development of HCC.59 The reaction order of AFB2a binding to amines has also not been previously determined. In this study, the reaction demonstrated that it was first order with respect to AFB2a as well as alanine giving an overall second order reaction, which was also observed with the binding of AFB1 dialdehyde to amino acids.44 The rate of this reaction is complicated by the fact that AFB2a is also present in equilibrium with the dialdehyde and closed-ring form, which changes depending on the pH. This is demonstrated by our finding that the rate constant changed dramatically across the pH values used in this study. To simplify the system, we measured the reaction order of dialdehyde binding at pH 9 so that the dialdehyde form was greatly favored which isolated the final step of this reaction. The binding of aflatoxin to proteins and DNA has been previously established, but to our knowledge, this study is the first to demonstrate a covalent interaction between aflatoxin

and phospholipids. It is expected that these interactions also occur with the AFB1 dialdehyde to allow more opportunities for lipid adduct formation following aflatoxin exposure. Our results show the ability of AFB2a to bind to ethanolamine head groups of phospholipids and demonstrate that irreversible binding between aflatoxin and lipids can occur. This binding of aflatoxin dialdehydes to cell membranes may change the distribution and accumulation of AFB1 and AFB2a in tissues and contribute to additional toxicities. Until more comprehensive toxicological studies emerge, the potential of AFB2a as a definitive detoxification product remains unclear. In the present study, we demonstrate that AFB2a readily forms pyrrole adducts with macromolecules such as proteins and phospholipids. Because this type of interaction is very stable and considered irreversible in biological systems, AFB2a could have many widespread cellular toxicities. This binding of AFB2a may contribute to some of the nonmutagenic effects of AFB1 due to AFB2a’s ability to be formed spontaneously (under acidic conditions) or though metabolism of AFB1. Additionally, we have found that oxidized AFB2a-lysine (ε) may interfere with the quantification of the AFB1-albumin adduct when determining AFB1 exposure or genotoxic damage. The results presented in this study help to elucidate the factors affecting the mechanism of toxicity and the biological fate of AFB2a and its adducts.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00002. Detection of oxidized AFB2a-alanine after monopersulfate addition; preferential formation of AFB2a-lysine (ε) using Nα-acetyllysine; detection of oxidized AFB2a-lysine (ε) after monopersulfate addition; 1H NMR of AFB2a-lysine (ε); 1H NMR of oxidized AFB2a-lysine (ε); accurate mass and molecular formula table for all AFB2a amino acid adducts; effect of changing AFB2a or alanine on rate of AFB2a-alanine formation; assigned chemical structures of AFB1, AFB2a, and AFB2a-alanine MS/MS fragments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (704) 2829838. ORCID

Blake R. Rushing: 0000-0002-8662-3270 Funding

This work was funded internally by the Brody Brothers Foundation of the Brody School of Medicine at East Carolina University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Drs. Colin Burns, Subodh Dutta, and Brian Love for their assistance in NMR sample preparation and operations. 1283


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ABBREVIATIONS AFB1, aflatoxin B1; AFB2a, aflatoxin B2a; AFBO, aflatoxin-exo8,9-epoxide; COSY, correlation spectroscopy; DPPE, 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; EIC, extracted ion chromatogram; HCC, hepatocellular carcinoma; HMBC, heteronuclear multiple bond correlation; HPLC/TOFMS, high performance liquid chromatography-time-of-flight mass spectrometry; HSQC, heteronuclear single quantum correlation; NMR, nuclear magnetic resonance; QTOF, quadrupole time-of-flight

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Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin

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