Characterization of N1- and N6-adenosine adducts and N1-inosine adducts formed by the reaction of butadiene monoxide with adenosine: evidence for the N1-adenosine adducts as major initial products

Abstract

1,3-Butadiene is a known human mutagen and possible human carcinogen; however, the molecular mechanisms of its activity are poorly understood. We have previously shown that the primary metabolite, butadiene monoxide (BM), reacts with guanosine to form N1-, N2-, and N7-guanosine adducts. In this study we characterize the reaction of BM with adenosine; ten adducts identified as diastereomeric pairs of N1-(1-hydroxy-3-buten-2-yl)adenosine,N1-(2-hydroxy-3-buten-1 -yl)adenosine, N6-(1-hydroxy-3-buten-2-yl)adenosine,N6-(2-hydroxy-3-buten-+ ++1yl)adenosine, and N1-(1-hydroxy-3-buten-2-yl)inosine are characterized. The N6-adenosine and N1-inosine adducts were characterized by their UV spectra, 1H NMR, FAB/MS, and stability studies. The N6-adenosine and N1-inosine adducts were stable for up to 168 h at 37 degrees C in phosphate buffer (pH 7.4). The N1-adenosine adducts, which were unstable at pH 7.4 at 37 degrees C (half-life of 7 and 9.5 h for the two regioisomers), were characterized by their UV spectra and their ability to undergo the Dimroth rearrangement to yield the corresponding N6-adenosine adducts, or undergo deamination to yield the corresponding N1-inosine adducts. Upon the reaction of BM with adenosine in phosphate buffer (pH 7.4) at 37 degrees C, the N1-adenosine adducts were the first to be detected, with the N6-adenosine and N1-inosine adducts. showing a lag in formation possibly due to the time needed for rearrangement/deamination. Reaction of adenosine with an excess of BM in phosphate buffer (pH 7.4) at 37 degrees C, followed by extraction of the reaction mixture with ethyl ether to remove excess unreacted BM and incubation at 80 degrees C for 1 h, resulted in complete conversion of N1-adenosine adducts to the corresponding N6-adenosine and N1-inosine adducts. Under these conditions, adduct formation exhibited pseudo-first-order kinetics, with the combined N6-adenosine adducts being formed 3-fold more favorably than the combined N1-inosine adducts. When incubations were carried out at lower BM concentrations, the N6-adenosine adducts remained the major detectable adducts at all concentrations. These results show that adenosine, in addition to guanosine, can lead to multiple adducts when incubated with BM, and may be useful in development of biomarkers for exposure to 1,3-butadiene. Characterization of the N1-adenosine adducts and their rearrangement/deamination products may also contribute to the understanding of mutagenic and carcinogenic mechanisms of 1,3-butadiene.