Aflatoxin B1 adducts with d(ATCGAT)2 and d(ATGCAT)2: Do methylation and hydroxymethylation influence reaction stoichiometry?
Whitley, Brent Tyler
Aflatoxin B1 (AFB1), a carcinogenic mycotoxin produced by the common agricultural contaminant A. flavus, has been implicated in the high rates of hepatocellular carcinoma observed in some developing countries. Upon ingestion, AFB1 is metabolized by liver monooxygenases to AFB1-exo-8,9-epoxide prior to reacting with DNA. AFB1 epoxide preferentially reacts at CpG islands, where it is attacked by the N7 atom of dG. The primary lesion found in vitro is trans-8,9-dihydro-(N7-guanyl)-9-hydroxy AFB1. The stoichiometry of this reaction has been characterized for the two oligonucleotide sequence isomers 5’-ATCGAT-3’ and 5’-ATGCAT-3’. In the sequence 5’-ATCGAT-3’, the AFB1 adduct occupies the 5’ side of dG, blocking the addition of a second equivalent of epoxide to the complementary strand. This results in a 1:1 AFB1:oligonucleotide limiting stoichiometry in the formation of the 5’-ATC(AFB)GAT-3’. However, a second equivalent of epoxide is free to bind to the complementary strand of 5’-ATGCAT-3’, giving a 2:1 stoichiometry. Since CpG islands are frequently subject to epigenetic regulation, it is important to know whether the reaction stoichiometry is altered by cytosine methylation or hydroxymethylation. In this work, 5’-[AT(5-methyl-dC)GAT]2-3’, 5’-[ATG(5-methyl-dC)AT]2-3’, 5’-[AT(5-hydroxymethyl-dC)GAT]2-3’, and 5’-[ATG(5-hydroxymethyl-dC)AT]2-3’ were each reacted with an excess of AFB1 epoxide and analyzed by HPLC. The equilibrium concentrations of the unreacted oligonucleotide and the adduct were used to calculate the limiting stoichiometry of the reaction. Methylation and hydroxymethylation did not influence reaction stoichiometry.