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Chem. Res. Toxicol. 2000, 13, 1342-1348
HPLC-MS/MS Identification of Positionally Isomeric Benzo[c]phenanthrene Diol Epoxide Adducts in Duplex DNA Andreas Harsch,† Jane M. Sayer,‡ Donald M. Jerina,*,‡ and Paul Vouros*,† Department of Chemistry and Barnett Institute, Northeastern University, Boston, Massachusetts 02115, and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received June 30, 2000
LC-MS and LC-MS/MS analyses were used to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (-)-(1R,2S,3S,4R)-1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [(-)-(R,S,S,R)-BcPh DE-2], on reaction in vitro with an oligonucleotide dodecamer derived from the HPRT gene. The sequence of this dodecamer, 5′-T1A2G3T4C5A6A7G8G9G10C11A12-3′, contains a base (corresponding to A7) which is a hot spot for mutagenesis in the hprt gene induced by the carcinogenic (R,S,S,R)-enantiomer of benzo[a]pyrene 7,8-diol 9,10-epoxide, and an adjacent base (corresponding to A6) which gave no mutations with this diol epoxide. Modified oligonucleotides were generated by reaction of (-)BcPh DE-2 with both the single-stranded and duplex forms of the dodecamer. Multiple purine targets in both strands led to the formation of complex reaction mixtures of regioisomeric BcPh DE-modified oligonucleotides, which were partially separated by reverse phase HPLC on a polystyrene-divinylbenzene column. On-line LC-MS data allowed facile distinction between adducts on the two strands of the duplex, and MS/MS analysis permitted unambiguous assignment of the major sites of modification in the regioisomeric, adducted strands. In the duplex, these sites were at A6, A7, and G8. Interestingly, the “hot spot” A7w as about 3 times more reactive with the BcPh DE than the “cold spot” A6. Adduct formation from the singlestranded dodecamer was less selective, and resulted in more extensive alkylation of G residues.
Introduction Bay-region diol epoxide (DE)1 metabolites of polycyclic aromatic hydrocarbons are potent mutagens, which alkylate DNA to yield ring-opened adducts predominantly at the exocyclic amino groups of deoxyadenosine (dA) and deoxyguanosine (dG) (Figure 1) (1). Mutational spectra obtained after exposure of cells to these agents have shown a strong dependency of the mutational frequencies on the sequence context of the purine targets. A striking example involves a sequence from the transcribed strand of the coding region of the hprt gene in Chinese hamster V79 cells, TAGTCA579A578GGGCA, in which the two indicated dA residues differ markedly in their extents of mutation after treatment of the cells with the carcinogenic (+)-(7R,8S,9S,10R)-isomer of benzo[a]pyrene 7,8diol 9,10-epoxide [(+)-BaP DE-2]. A578 was determined to be the hottest spot for base substitution mutations induced by low, noncytotoxic doses of (+)-BaP DE-2, whereas the adjacent A579 gave no mutations (2). Multiple factors, including sequence effects on replication (3, 4) and on repair (5-7) of specifically modified nucleotide * Corresponding authors. † Northeastern University. ‡ The National Institutes of Health. 1 Abbreviations: BcPh, benzo[c]phenanthrene; BaP, benzo[a]pyrene; DE, diol epoxide; DE-2, diol epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans; ESI, electrospray ionization; ss, single-stranded; ds, double-stranded; PSDVB, polystyrene-divinylbenzene; TIC, total ion current; CID, collision-induced dissociation.
Figure 1. Structures of (-)-(1R,2S,3S,4R)-1,2,3,4-tetrahydrobenzo[c]phenanthrene 3,4-diol 1,2-epoxide [(-)-BcPh DE-2] and its adducts in DNA formed by trans ring opening by the exocyclic amino groups of dG and dA.
residues, may contribute to this selectivity. However, possible chemical selectivity for adduct formation in a particular sequence environment represents the first
10.1021/tx000140m CCC: $19.00 © 2000 American Chemical Society Published on Web 11/16/2000
HPLC-MS/MS of Adducted DNA
point where sequence differences may be manifested in the complex process leading to mutations. In vitro investigation of chemical selectivity in DNA sequences containing multiple target bases competing for adduct formation poses a challenging analytical problem, since the resultant mixtures of positional isomers from short oligonucleotides are generally difficult to separate (8). Maxam-Gilbert sequencing (methylation of the modified oligonucleotide followed by depurination and base-induced strand cleavage) allows assignment of the position of the modified residues based on the decreased electrophoretic mobility of oligonucleotide fragments containing the hydrocarbon moiety relative to the native fragments. Although this method has been used successfully for oligonucleotides containing BaP-dG adducts (8), its generality for dA adducts and/or adducts derived from other hydrocarbons has not been demonstrated. More importantly, Maxam-Gilbert sequencing cannot provide any structural information about the adduct. A potentially more general approach using off-line ESImass spectrometry for sequencing of purified, positionally isomeric oligonucleotide adducts has recently been described (9, 10). However, this approach requires off-line separation using solvent systems incompatible with ESIMS analysis, isolation of the target compounds, and subsequent removal of the LC solvent additives by solid phase extraction (SPE) or ion exchange. This multistep process is cumbersome and time-consuming, and may also result in critical sample loss. Interfacing LC separations with on-line MS and MS/MS analysis eliminates the need for isolation of individual analytes, minimizing sample handling and losses and improving the throughput of the assay. While on-line capillary electrophoresisMS/MS analysis of isomeric modified oligonucleotides has previously been carried out (11), there are no reports of on-line LC-MS or LC-MS/MS analysis of modified, isomeric oligonucleotides. Herein, we describe the on-line LC-MS and LC-MS/MS analysis of positional isomers of DE-modified oligonucleotides. LC-MS analysis allowed simultaneous profiling of both strands in a duplex oligonucleotide, and LC-MS/MS experiments permitted unambiguous identification of multiple, positionally isomeric adducted oligonucleotides. In the study presented here, LC-MS/MS was employed to obtain information about the relative reactivity of a DE with the two central dA residues in both singlestrand and duplex forms of the hprt-derived 12-mer, 5′T1A2G3T4C5A6A7G8G9G10C11A12-3′. A6 corresponds to A579 and A7 to A578 in the HPRT gene, which were cold and hot spots, respectively, for mutations induced by (+)-BaP DE-2. In this work, (-)-(1R,2S,3S,4R)-1,2-epoxy-3,4dihydroxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [(-)(R,S,S,R)-BcPh DE-2] (Figure 1), which has the same (R,S,S,R)-absolute configuration in the cyclohexene ring as the carcinogenic (+)-BaP DE-2, was chosen to obtain a measurable extent of adduct formation at the two dA residues. This BcPh DE, unlike its (+)-BaP DE-2 analogue, is highly efficient in covalent binding to DNA relative to hydrolysis to tetraols [∼70% as compared with 13-15% for (+)-BaP DE-2] (12, 13) and is also dAselective [∼65% of exocyclic amino group adducts are at dA as compared to
