Reactions of benzene oxide, a reactive metabolite of benzene, with model nucleophiles and DNA

Abstract

1. Reactivity of benzene oxide (BO), a reactive metabolite of benzene, was studied in model reactions with biologically relevant S- and N-nucleophiles by LC-ESI-MS. 2. Reaction with N-acetylcysteine (NAC) in aqueous buffer solutions gave N-acetyl-S-(6-hydroxycyclohexa-2,4-dien-1-yl)cysteine (pre-phenylmercapturic acid, PPhMA), which was easily dehydrated in acidic solutions to phenylmercapturic acid (PhMA). The yield of PPhMA + PhMA increased exponentially with pH up to 11% in the pH range from 5.5 to 11.4. 3. Primary 6-hydroxycyclohexa-2,4-dien-1-yl (HC) adducts were detected also in reactions of purine nucleosides and nucleotides under physiological conditions. After a vigorous acidic hydrolysis, all HC adducts were converted to corresponding phenyl purines, which were identified as 7-phenylguanine (7-PhG), 3-phenyladenine (3-PhA) and N(6)-phenyladenine (6-PhA). The yield of 7-PhG amounted to 14 ± 5 and 16 ± 7 ppm for 2'-deoxyguanosine and 2'-deoxyguanosine-5'-monophosphate, respectively, that of 6-PhA was 500 ± 70 and 455 ± 75 ppm with 2'-deoxyadenosine and 2'-deoxyadenosine-5'-phosphate, respectively, with only traces of 3-PhA. 4. Reactions with the DNA followed by acidic hydrolysis yielded 26 ± 11 ppm (mean ± SD; n = 9) of 7-PhG as the sole adduct detected. 5. In contrast to the reactions with S-nucleophiles, the reactivity of BO with nucleophilic sites in the DNA is very low and can therefore hardly account for a significant DNA damage caused by benzene.

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“…Previous studies have shown that benzene oxide is relatively unreactive with sulfur nucleophiles and that this pathway occurs at low efficiency compared to rearrangement to phenol at pH 7 [14]. This is also consistent with the studies by Micova and Linhart and with our data indicating the relatively low yields of 7-PhG in reactions of benzene oxide with DNA [8]. …”

“…Applying our method to calf thymus DNA reacted with benzene oxide, we were able to confirm the observations of Micova and Linhart [8] that 7-PhG is produced after treatment of the DNA by either acid or neutral thermal hydrolysis. Our results are consistent with theirs in demonstrating a rather low reactivity of benzene oxide with calf thymus DNA, but direct comparison is difficult because different conditions were used.…”

“…This method provides a level of sensitivity and accuracy expected to be adequate both for the analysis of 7-PhG in reactions of DNA with benzene oxide and for the analysis of DNA from laboratory animals, cells, and humans exposed to benzene. In one previous study, Micova and Linhart described the use of LC-ESI-MS/MS for the analysis of 7-PhG as the only adduct detected in reactions of benzene oxide and calf thymus DNA [8]. It is not clear whether their method would have had sensitivity adequate for analysis of samples produced from benzene exposure in vivo .…”

“…Benzene oxide is known to react with DNA to produce 7-phenylguanine (7-PhG) following the dehydration of the transient hydroxycyclohexadienyl intermediate shown in Scheme 1[7,8]. Other mechanisms of DNA adduct formation from benzene have also been investigated [9–12].…”

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“…Previous reports suggest that product yield increases under basic conditions, which should increase the nucleophilicity of the thiol and increase the stability of BO in solution [29, 30]. After 30 min, the pH was adjusted to ~2 with 1 N HCl (1.2 mL) in order to dehydrate the intermediate, yielding SPG.…”