Detection of DNA Adducts Derived from the Reactive Metabolite of Furan, cis-2-Butene-1,4-dial

Abstract

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.

Formation of 1,4-Dioxo-2-butene-Derived Adducts of 2‘-Deoxyadenosine and 2‘-Deoxycytidine in Oxidized DNA

“…γ-Radiation is known to oxidize all positions in deoxyribose (24), but the spectrum of products differs from the enediynes and bleomycin (3,8,25). Adduct levels were determined by LC-MS/MS using the method recently established in our laboratory (18). The treated DNA was reacted with O-benzylhydroxylamine, which forms a stable oxime with the aldehydic functionality of the adducts (Scheme 2).…”

“…(Scheme 2). The dC adduct does not undergo the final dehydration (18). Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry (LC-ESI/MS/MS) with selected reaction monitoring for the O-benzyloxime derivatives of the adducts.…”

“…The DNA adducts were isolated by solid phase extraction (Strata-X 30 mg cartridges, Phenomenex, Torrence, CA) with sequential water, 20% methanol, and 100% methanol washes (1 mL each). The methanol wash, which contained the adducts, was concentrated to approximately 30 μL under reduced pressure for analysis by capillary LC-MS (18). Capillary LC-MS analyses were performed with an Agilent capillary LC system linked to a ThermoFinnigan Quantum triple quadrupole mass spectrometer with an electrospray ionization source in positive mode as previously described (18).…”

“…The amount of adduct in each sample was determined from the product of the area ratio (unlabeled/labeled) and the amount of internal standard added, as described in detail elsewhere (18). The concentration of DNA in each sample was determined by measuring the concentration of dG in each DNA hydrolysate by HPLC with UV detection (21).…”

“…We have demonstrated that both of these electrophilic isomers react with nucleophiles in DNA in vitro to form novel, stable oxadiazabicyclo(3.3.0)octaimine adducts of dC, dG and dA under biologically relevant conditions (15-17) (Scheme 2). The dC and dA adducts were also detected in DNA from bacteria exposed to mutagenic concentrations of cis-1,4-dioxo-2-butene, indicating that these adducts are likely to be mutagenic (18,19).…”