Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10–100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption.

Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide–DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant ( k cat / K m ) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.

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1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA crosslinking effects. DBE crosslinks glutathione (GSH) to DNA, notably to sites on 2´-deoxyadenosine and 2´-deoxyguanosine (Cmarik, J. L. et al. (1991) J. Biol. Chem. 267, 6672–6679). Adduction at the N6 position of 2´-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of O6-alkylguanine DNA alkyltransferase (Chowdhury, G., et al. (2013) Angew. Chem. Int. Ed. 52, 12879–12882). We identified and quantified a new adduct, S-[2-(N6-deoxyadenosinyl)ethyl]GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) η, ι, and κ, as well as with Sulfolobus solfataricus Dpo4, Escherichia coli polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols η and ι, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol η and ι showed increased misincorporation opposite the adduct compared to unmodified 2’-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol η confirmed the incorporation of dC opposite S-[2-(N6-deoxyadenosinyl)ethyl]GSH and also showed the production of a −1 frameshift. These results reveal the significance of N6-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.

SummaryFANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of the fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

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Aspirin (acetylsalicylic acid) is a commonly used analgesic. It is rapidly deacetylated both enzymatically and non‐enzymatically to salicylic acid. Salicylic acid can then be further metabolized by conjugation with glycine or glucuronides or by hydroxylation to form 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and 2,5‐dihydroxybenzoic acid (2,5‐DHBA). This oxidation has been suspected to occur from oxygen radicals via Fenton reactions as well as by cytochrome P450 enzymes. We analyzed the formation of 2,3‐DHBA and 2,5‐DHBA in the presence of human liver microsomes by HPLC with fluorescence detection. We were able to determine that microsomal oxidation was much faster for salicylic acid than aspirin. 2,3‐DHBA was found to be the major oxidative product formed from salicylic acid, whereas aspirin could detect only 2,5‐DHBA. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5‐hydroxylation of salicylic acid. Inhibitor studies were able to confirm that all six of these enzymes played a role in the formation of 2,3‐ and 2,5‐DHBA, as well as found that 2A6 and 2B6 played a role in the hydroxylation to 2,5‐DHBA. It was also shown in the inhibitor studies that P450 2E1 was the major enzyme contributing to the in both 3‐ and 5‐hydroxylations.This work was supported in part by National Institutes of Health grant R37 CA090426.