Identification of DNA Adducts Derived from Riddelliine, a Carcinogenic Pyrrolizidine Alkaloid

• Published in: Chemical research in toxicology 2003-09, Vol.16 (9), p.1130-1137
• Main Authors: Chou, Ming W, Jian, Yan, Williams, Lee D, Xia, Qingsu, Churchwell, Mona, Doerge, Daniel R, Fu, Peter P
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - biosynthesis; Adenine Nucleotides - chemistry; Administration, Oral; Animals; Carcinogens - chemistry; Carcinogens - isolation & purification; Carcinogens - metabolism; Cattle; DNA - chemistry; DNA - genetics; DNA Adducts - biosynthesis; DNA Adducts - chemistry; Guanine Nucleotides - biosynthesis; Guanine Nucleotides - chemistry; Isomerism; Liver - drug effects; Liver - metabolism; Micrococcal Nuclease - chemistry; Micrococcal Nuclease - metabolism; Monocrotaline - analogs & derivatives; Monocrotaline - metabolism; Phosphorus Radioisotopes - chemistry; Pyrrolizidine Alkaloids - adverse effects; Pyrrolizidine Alkaloids - chemistry; Pyrrolizidine Alkaloids - isolation & purification; Pyrrolizidine Alkaloids - metabolism; Rats; riddelliine; riddelline; Spectrometry, Mass, Electrospray Ionization; Spleen - drug effects; Spleen - metabolism; Thymine Nucleotides - biosynthesis; Thymine Nucleotides - chemistry; Thymus Gland - chemistry; Thymus Gland - drug effects; Thymus Gland - metabolism
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx030018y

Riddelliine is a naturally occurring carcinogenic pyrrolizidine alkaloid that produces liver tumors in experimental animals. Riddelliine requires metabolic activation to dehydroriddelliine and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) to exert its toxicity. Previously, 32P-postlabeling HPLC was used to detect a set of eight DHP-derived adduct peaks from DNA modified both in vitro and in vivo. Among these DHP-derived DNA adducts, two were identified as epimers of DHP-2‘-deoxyguanosine 3‘-monophosphate. In this study, the remaining adducts have been characterized as DHP-modified dinucleotides. A series of dinucleotides, TpGp, ApGp, TpCp, ApCp, TpAp, ApAp, TpTp, and ApTp, were obtained by enzymatic digestion of calf thymus DNA with micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) followed by HPLC separation and structural identification by negative ion electrospray tandem mass spectrometry (ES/MS/MS). Incubation of individual dinucleotides with DHP produced DHP-modified dinucleotide adducts that were also characterized using LC-ES/MS/MS. A parallel analysis of the isolated DHP-modified dinucleotides using 32P-postlabeling recapitulated the series of unidentified adduct peaks that we previously reported from DHP-modified calf thymus DNA in vitro and rat liver DNA in vivo. Intact calf thymus DNA was also reacted with DHP and then digested by MN/SPD under the same conditions. The adduct profile obtained from LC-ES/MS/MS analysis was similar to that observed from the isolated dinucleotides. Structural analysis using LC-ES/MS/MS showed that DHP bound covalently to both 3‘- and 5‘-guanine, -adenine, and -thymine bases (but not cytosine) of dinucleotides to produce two or more isomers of each DHP−dinucleotide adduct. By comparing adduct formation at dissimilar bases within individual dinucleotides, the relative reactivity of DHP with individual bases was determined to be guanine > adenine ∼ thymine. Identification of the entire set of DHP-derived DNA adducts further validates the conclusion that riddelliine is a genotoxic carcinogen and enhances the applicability of these biomarkers for assessing carcinogenic risks from exposure to pyrrolizidine alkaloids.