Identification and Characterization of 2′-Deoxyadenosine Adducts Formed by Isoprene Monoepoxides in Vitro

Isoprene, the 2-methyl analog of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after reaction of 2′-deoxyadenosine (dAdo1) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1–2′-deoxyinosine (N1-dIno) due to deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-Spectrometry and LC-MS/MS and characterized by 1H and 1H,13C HSQC and NMR experiments. Adducts of IP-1,2-O that were fully identified are: R,S-C1-N6-dAdo, R-C2-N6-dAdo, and S-C2-N6-dAdo; adducts of IP-3,4-O are: S-C3-N6-dAdo, R-C3-N6-dAdo, R,S-C4-N6-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the external and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation, or as biomarkers of isoprene metabolism and exposure

Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites.

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2'-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route

Final refinement statistics for PDT crystallized with 8,9-dihydro-9-oxoimidazo[2,1-<i>b</i>]purine.

a<p><i>R</i> factor  = <i>Σ</i>_hkl||F_obs |−k|F_cal||/<i>Σ</i>_hkl|F_obs| where F_obs and F_cal are observed and calculated structure factors, respectively.</p>b<p>For <i>R</i>_free the sum is extended over a subset of reflections (5%) excluded from all stages of refinement.</p><p>Final refinement statistics for PDT crystallized with 8,9-dihydro-9-oxoimidazo[2,1-<i>b</i>]purine.</p