Reaction of aflatoxin B1 exo-8,9-epoxide with DNA: kinetic analysis of covalent binding and DNA-induced hydrolysis

Abstract

The exo isomer of aflatoxin B1 (AFB1) 8,9-epoxide appears to be the only product of AFB1 involved in reaction with DNA and reacts with the N7 atom of guanine via an SN2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in H2O (0.6 s-1 at 25 degrees C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA (Kd = 0.43 mg ml-1, or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 s-1. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 s-1 and half-maximal velocity at a DNA concentration of 1.8 mg ml-1 (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 s-1 at pH 6.2 with 0.17 mg DNA ml-1). The same rate enhancement effect was observed with poly[dA-dT].poly[dA-dT], in which the epoxide can intercalate but not form significant levels of N7 purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.

Figure 1

Reaction of AFB₁ exo-8,9-epoxide with H₂O and DNA (3, 4).

Figure 2

Kinetic modeling of DNA adduct yields to obtain kinetic parameters. Yields of AFB diol obtained with 18 μM AFB₁ exo-8,9-epoxide and varying concentrations of DNA were fit to the model: where A = AFB₁ exo-8,9-epoxide, D = AFB diol, B = DNA, C = DNA–AFB adduct, and k₀ was determined to be 0.60 s⁻¹ (4). K_d and k_cat were estimated by reiterative fitting with a KINSIM program (11) to give values of C that are consistent with the experimentally determined values (of C and D) found with varying concentration of A and B (12). The solid line is that for K_d = 0.43 mg ml⁻¹ (1.4 mM monomer equivalents) and k_cat = 35 s⁻¹.

Figure 3

Spectral changes during reaction of AFB₁ exo-8,9-epoxide with H₂O and DNA. The spectra are at equal intervals over a 10-s span and are reconstructed from individual kinetic traces at 5-nm intervals. The arrows indicate the direction of absorbance change during the course of the reaction. (Insets) Selected individual kinetic traces for the wavelengths indicated. The rate of the reaction with H₂O is 0.6 s⁻¹ (A), and the rate of the reaction with DNA (0.10 mg ml⁻¹= 0.32 mM monomer equivalents) is 1.9 s⁻¹ (B).

Figure 4

Rate of reaction of AFB₁ exo-8,9-epoxide with DNA measured by fluorescence changes. (Inset) Rate of fluorescence decrease upon reaction in the presence of DNA (0.10 mg⋅ml⁻¹ = 0.32 mM monomer equivalents). The observed pseudo-first order single exponential rate is 1.9 s⁻¹. These data were fit to a quadratic equation for a hyperbola represented by the line.

Figure 5

Fluorescence changes associated with AFB₁ exo-8,9-epoxide interaction with DNA. A solution of the epoxide [7 μM in (CH₃)₂CO] was mixed with 10 volumes of either (a) 10 mM sodium EDTA buffer (pH 6.2) or (b) the same buffer containing DNA under the same conditions (temperature, reagent sample, buffer, instrument parameters, and others). The solid lines show exponential fits. (A) Hydrolysis control reaction k_obs = 0.59 ± 0.01 s⁻¹; + 0.10 mg⋅ml⁻¹ dsDNA (= 0.32 mM monomer equivalents), k_obs = 0.82 ± 0.02 s⁻¹ (39% difference). (B) Hydrolysis control reaction k_obs = 0.77 ± 0.01 s⁻¹; + poly d(AT) 0.20 mg⋅ml⁻¹, k_obs = 1.03 ± 0.03 s⁻¹ (33% difference). (C) Hydrolysis control reaction k_obs = 0.62 ± 0.01 s⁻¹, + 0.15 mg⋅ml⁻¹ ssDNA, k_obs = 0.89 ± 0.02 s⁻¹ (40% difference).