Detection and Quantification of Specific DNA Adducts by Liquid Chromatography−Tandem Mass Spectrometry in the Livers of Rats Given Estragole at the Carcinogenic Dose

Abstract

Estragole (ES) is a natural constituent of several herbs and spices that acts as a carcinogen in the livers of rodents. Given that the proximal electrophilic form of ES with a reactive carbocation is generated by cytochrome P450 and a sulfotransferase metabolizing pathway, there is a possibility that the resultant covalent adducts with DNA bases may play a key role in carcinogenesis. The existence of ES-specific deoxyguanosine (dG) and deoxyadenosine (dA) adducts has already been reported with the precise chemical structures of the dG adducts being confirmed. In the present study, we examined ES-specific dA adduct formation using LC-ESI/MS after the reaction of dA with 1'-acetoxy-ES produced by a sulfotransferase metabolic pathway mimic. Although two peaks were observed in the LC-ESI/MS chromatogram, the identification of ES-3'-N(6)-dA as the measurable peak was determined by NMR analysis. To confirm ES-specific dG and dA adduct formation in vivo, an isotope dilution LC-ESI/MS/MS method applicable to in vivo samples for ES-3'-N(6)-dA together with the two major dG adducts, that is, ES-3'-C8-dG and ES-3'-N(2)-dG, was developed using selected ion recording. The limit of quantification was 0.2 fmol on column for ES-3'-C8-dG and ES-3'-N(2)-dG and 0.06 fmol on column for ES-3'-N(6)-dA, respectively. Using the developing analytical method, we attempted to measure adduct levels in the livers of rats treated with ES at a possible carcinogenic dose (600 mg/kg bw) for 4 weeks. ES-3'-C8-dG, ES-N(2)-dG, and ES-3'-N(6)-dA were detected at levels of 3.5 ± 0.4, 4.8 ± 0.8, and 20.5 ± 1.6/10(6) dG or dA in the livers of ES-treated rats. This quantitative data and newly developed technique for adduct observation in vivo might be helpful for ES hepatocarcinogenesis investigations.