The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Abstract

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3, N 4 -ethenocytosine and the G/T mismatch

“…In this laboratory, it was unexpected that the human APNG could not only cleave A but with greater efficiency than the original substrate, 3-methyladenine (23). A second recent example is the repair of bulky p-benzoquinone modified dA, dC, and dG by human and Escherichia coli 5Ј-AP endonucleases (32,42,46), inasmuch as the p-benzoquinone moiety adds two additional rings to the bases (47).…”

Repair of exocyclic DNA adducts: rings of complexity

“…(92) Initially, such work by Singer, Hang and colleagues was directed toward testing whether the known glycosylase(s) excising etheno adducts would also act on these structurally related but bulkier adducts. (36,96,97) Although they were not Figure 3. Structures of six-membered exocyclic M 1 G and PdG derivatives.…”

“…Author Proof A found to be excised by the glycosylases, (36,96) it nevertheless led to the finding of a novel human repair activity efficiently cleaving DNA containing pBQ-C. (96) This activity was further purified to apparent homogeneity and surprisingly found to be identical to the major human AP endonuclease (APE1, also termed as HAP1, APEX, and Ref-1). (97) Thus, pBQ-C is a ''new'' substrate for an ''old'' enzyme.…”

Role of Biotransformation Studies in Minimizing Metabolism-Related Liabilities in Drug Discovery

“…This observation has two important consequences: first, quinones, quinone imines, and quinone methides are successively easier to oxidize because electronegativity decreases in the order O>N>C (83), and second, the conjugation of quinones with GSH and other thiols cannot generally be viewed as a detoxication step, since the thioether is itself electron-donating and may facilitate redox cycling or covalent reactivity (84)(85)(86)(87)(88). The redox cycle between a quinone and hydroquinone triggers the formation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and the hydroxyl radical, leading to cellular damage and oxidative stress (89)(90)(91). A structural alert for bioactivation vulnerability is the combination of a 6-membered heterocycle, and some combination of alkyl, amino, or hydroxyl substituents that are situated ortho or para to each other.…”