- Development of a 32p-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro
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Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a 32P-postlabeling/HPLC method for detection of (i) two DHR-3′-dGMP and four DHR-3′-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3′-dGMP, DHR-3′-dAMP, and DHR-3′,5′dG-bisphosphate standards and characterization of their structures by mass and 1H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and 32P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3′,5′-dG-bisphosphate adducts both in vitro and in vivo.
- Yang, Ya-Chen,Yan, Jian,Churchwell, Mona,Beger, Richard,Chan, Po-Cheun,Doerge, Daniel R.,Fu, Peter P.,Chou, Ming W.
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- Pyrrolizidine Alkaloid Secondary Pyrrolic Metabolites Construct Multiple Activation Pathways Leading to DNA Adduct Formation and Potential Liver Tumor Initiation
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Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are hepatotoxic, genotoxic, and carcinogenic phytochemicals. PAs induce liver tumors through a general genotoxic mechanism mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. To date, the primary pyrrolic metabolites dehydro-PAs, their hydrolyzed metabolite DHP, and two secondary pyrrolic metabolites 7-glutathione-DHP (7-GS-DHP) and 7-cysteine-DHP are the known metabolites that can generate these DHP-DNA adducts in vivo and/or in PA-treated cells. Secondary pyrrolic metabolites are formed from the reaction of dehydro-PAs with glutathione, amino acids, and proteins. In this investigation, we determined whether or not more secondary pyrrolic metabolites can bind to calf thymus DNA and to cellular DNA in HepG2 cells resulting in the formation of DHP-DNA adducts using a series of secondary pyrrolic metabolites (including 7-methoxy-DHP, 9-ethoxy-DHP, 9-valine-DHP, 7-GS-DHP, 7-cysteine-DHP, and 7,9-diglutathione-DHP) and synthetic pyrroles for study. We found that (i) many secondary pyrrolic metabolites are DNA reactive and can form DHP-DNA adducts and (ii) multiple activation pathways are involved in producing DHP-DNA adducts associated with PA-induced liver tumor initiation. These results suggest that secondary pyrrolic metabolites play a vital role in the initiation of PA-induced liver tumors.
- Xia, Qingsu,He, Xiaobo,Ma, Liang,Chen, Shoujun,Fu, Peter P.
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p. 619 - 628
(2018/06/11)
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- High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of pyrrolizidine alkaloid-derived DNA adducts in vitro and in viwo
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Pyrrolizidine alkaloid-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids require metabolism to exert their genotoxicity and tumorigenicity. We have determined that the metabolism of a series of tumorigenic pyrrolizidine alkaloids in Vitro or in ViVo generates a common set of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts that are responsible for tumor induction. The identification and quantitation of the DHP-derived DNA adducts formed in ViVo and in Vitro were accomplished previously by 32P-postlabeling/HPLC methodology. In this article, we report the development of a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ES-MS/MS) method to detect DHP-derived DNA adducts formed in Vitro and in ViVo. The method is used to quantify the levels of DHP-2′-deoxyguanosine (dG) and DHP-2′-deoxyadenosine (dA) adducts by multiple reaction monitoring (MRM) analysis in the presence of known quantities of isotopically labeled DHP-dG and DHP-dA internal standards. This HPLC-ES-MS/MS method is accurate and precise. When applied to liver samples from rats treated with the pyrrolizidine alkaloids riddelliine and monocrotaline, the method provided significant new information regarding the mechanism of DNA adduct formation.
- Fu, Peter P.,Chou, Ming W.,Churchwell, Mona,Wang, Yuping,Zhao, Yuewei,Xia, Qingsu,Da Costa, Goncalo Gamboa,Marques, M. Matilde,Beland, Frederick A.,Doerge, Daniel R.
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experimental part
p. 637 - 652
(2011/02/24)
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