HPLC-MS/MS identification of positionally isomeric benzo[c]phenanthrene diol epoxide adducts in duplex DNA

LC-MS and LC-MS/MS analyses were used to investigate the chemoselectivity o f the carcinogenic dial 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'-T(1)A(2)G(3)T(4)C(5)A(6)A(7)G (8)G(9)G(10)C(11)A(12)-3', contains a base (corresponding to A(7)) which is a hot spot for mutagenesis in the hprt gene induced by the carcinogenic (R ,S,S,R)-enantiomer of benzo[alpha ]pyrene 7,8-diol 9,10-epoxide, and an adj acent base (corresponding to As) which gave no mutations with this diol epo xide. Modified oligonucleotides were generated by reaction of(-)BcPh DE-2 w ith both the single-stranded and duplex forms of the dodecamer. Multiple pu rine targets in both strands led to the formation of complex reaction mixtu res of regioisomeric BcPh DE-modified oligonucleotides, which were partiall y separated by reverse phase HPLC on a polystyrene-divinylbenzene column. O n-line LC-MS data allowed facile distinction between adducts on the two str ands 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 A(6), A(7), and G(8). Interestingly, the "h ot spot" A(7W) as about 3 times more reactive with the BcPh DE than the "co ld spot" A(6). Adduct formation from the single-stranded dodecamer was less selective, and resulted in more extensive alkylation of G residues.