Deoxyguanosine forms a bis-adduct with E,E-muconaldehyde, an oxidative metabolite of benzene: implications for the carcinogenicity of benzene

Abstract

Benzene is employed in large quantities in the chemical industry and is an ubiquitous contaminant in the environment. There is strong epidemiological evidence that benzene exposure induces hematopoietic malignancies, especially acute myeloid leukemia, in humans, but the chemical mechanisms remain obscure. E,E-Muconaldehyde is one of the products of metabolic oxidation of benzene. This paper explores the proposition that E,E-muconaldehyde is capable of forming Gua-Gua cross-links. If formed in DNA, the replication and repair of such cross-links might introduce structural defects that could be the origin of the carcinogenicity. We have investigated the reaction of E,E-muconaldehyde with dGuo and found that the reaction yields two pairs of interconverting diastereomers of a novel heptacyclic bis-adduct having a spiro ring system linking the two Gua residues. The structures of the four diastereomers have been established by NMR spectroscopy and their absolute configurations by comparison of CD spectra with those of model compounds having known configurations. The final two steps in the formation of the bis-nucleoside (5-ring → 6-ring → 7-ring) have significant reversibility, which is the basis for the observed epimerization. The 6-ring precursor was trapped from the equilibrating mixture by reduction with NaBH(4). The anti relationship of the two Gua residues in the heptacyclic bis-adduct precludes it from being formed in B DNA, but the 6-ring precursor could readily be accommodated as an interchain or intrachain cross-link. It should be possible to form similar cross-links of dCyt, dAdo, the ε-amino group of lysine, the imidazole NH of histidine, and N termini of peptides with the dGuo-muconaldehyde monoadduct.

Figure 1

Numbering and ring designations for the bis-adduct of dGuo with E,E-muconaldehyde.

Figure 2

HPLC chromatogram showing the reaction of dGuo with muconaldehyde in phosphate buffer (0.05 M, pH 7.4) after 7 weeks at ambient temperature.

Figure 3

Effect of pH on the reaction of dGuo with muconaldehyde at ambient temperature. Reaction mixtures contained dGuo (5.8 µM) and muconaldehyde (2.9 µM) in potassium phosphate buffer (0.05 M) at pH 6.5, 7.5 and 8.5. Chromatogram shows reaction mixtures after 72 h.

Figure 4

¹H Chemical shift and coupled networks in compounds 1a–d. Spectra acquired in DMSO-d₆/D₂O (~90:10, v/v).

Figure 5

¹H chemical shift and coupled networks in compounds 2ac and 2bd. Spectra recorded in anhydrous DMSO-d₆. Chemical shifts (δ) in ppm. Addition of D₂O caused disappearance of the exchangeable protons, which are indicated in red.

Figure 6

¹H spectra of 2ac and 2bd were recorded in anhydrous DMSO-d₆ ¹³C spectra in DMSO-d₆/D₂O. Chemical shifts (δ) in ppm n.o. = not observed.

Figure 7

Chemical shift differences between the M3 and M5 geminal protons of 2ac and 2bd. Spectra were recorded in anhydrous DMSO-d₆.

Figure 8

NOE experiments with 2ac and 2bd conducted in DMSO-d₆/D₂O (92:8, v/v).

Figure 9

CD spectra of reduced bis-nucleosides 4a–d obtained at ambient temperature in water-methanol (1:1, v/v) mixtures.

Figure 10

Depiction of the helical relationship of Gua residues in 4a and 4b versus 4c and 4d.

Scheme 1

Metabolic activation of benzene.

Scheme 2

Adducts of Z,Z- and E,E-muconaldehyde characterized by Golding’s group. ^,

Scheme 3

Deglycosylation of bis-nucleosides 1a–d to form the analogous bis-bases 2a–d and reduction of the bis-bases with NaBH₄ to form 3a–d.

Scheme 4

Equilibria of bis-nucleoside 1a with 1d and of 1b with 1c, reduction of 1a–d to bis-nucleosides 4a–d with NaBH₄, and configurational assignments of 1a–d and 4a–d.

Scheme 5

Proposed mechanism for formation of bis-nucleosides 1a–d.

Scheme 6

Reversible opening of ring C of bis-nucleosides 1 to form imines 12 and carbinolamines 13.

Scheme 7

Proposed reaction of the dGuo-muconaldehyde monoadduct with the ε-amino group of lysine to form DNA-protein cross-links.