6212 Biochemistry 1996 35 6212 6224 Adduction of the Human N ras Codon 61 Sequence with 7S 8R 9R 10S 7 8 Dihydroxy 9 10 epoxy 7 8 9 10 tetrahydrobenzo a pyrene Structural Refinement of the Intercalated SRSR 61 2 7S 8R 9S 10R N6 10 7 8 9 10 Tetrahydrobenzo a pyrenyl 2 deoxyadenosyl Adduct from 1H NMR Irene S Zegar Seong J Kim Tommy N Johansen Pamela J Horton Constance M Harris Thomas M Harris and Michael P Stone Center in Molecular Toxicology and Department of Chemistry Vanderbilt UniVersity NashVille Tennessee 37235 ReceiVed October 17 1995 ReVised Manuscript ReceiVed January 18 1996X The structure of the 7S 8R 9S 10R N6 10 7 8 910 tetrahydrobenzo a pyrenyl 2 deoxyadenosyl adduct at X6 of 5 d CGGACXAGAAG 3 5 d CTTCTTGTCCG 3 derived from trans addition of the exocyclic N6 amino group of dA to 7S 8R 9R 10S 7 8 dihydroxy 9 10 epoxy 7 8 9 10tetrahydrobenzo a pyrene DE2 was determined using molecular dynamics simulations restrained by 369 NOEs from 1H NMR This was named the SRSR 61 2 adduct derived from the N ras protooncogene at and adjacent to the nucleotides encoding amino acid 61 underlined of the p21 gene product NOEs between C5 S R S RA6 and A7 were disrupted as were those between T17 and G18 NOEs between benzo a pyrene and DNA protons were localized on the two faces of the pyrenyl ring The benzo a pyrene H3 H6 protons showed NOEs to T17 CH3 while H1 H2 and H3 showed NOEs to T17 deoxyribose the latter protons and H4 showed NOEs to T17 H2 H2 and to T17 H6 NOEs were observed between H11 and H12 and C5 H1 H2 H2 G18 N1H showed NOEs to both faces of benzo a pyrene Upfield shifts of 2 6 ppm for T17 N3H and 1 8 ppm for G18 N1H 1 ppm for T17 H6 and CH3 and 0 75 ppm for C5 H5 with a smaller shift for C5 H6 and a 1 5 ppm dispersion of the pyrenyl protons suggested that benzo a pyrene intercalated above the 5 face of S R S RA6 The precision of the refined structures was monitored by pairwise root mean square deviations which were 1 5 accuracy was measured by complete relaxation matrix calculations which yielded a sixth root R factor of 8 1 10 2 Interstrand stacking between the pyrenyl ring and the T17 pyrimidine and G18 purine rings was enhanced by the bay ring Changes of 30 and 25 in buckle for C5 G18 and S R S RA6 T17 respectively were calculated as was a 40 change in propeller twist for C5 G18 The rise between C5 G18 and S R S RA6 T17 was calculated to be 7 The work extended the pattern for adenine N6 benzo a pyrene adducts in which the R stereochemistry at C10 predicted 5 intercalation of the pyrenyl moiety ABSTRACT Interest in the polycyclic aromatic hydrocarbon PAH 1 class of chemical carcinogens dates to the Percival Pott observations in the 18th century which linked the occurrence of scrotal cancer in chimney sweeps with occupational exposure to soot The genotoxicity of PAH compounds is This research was supported by the NIH ES 05355 Funding for the AMX 500 NMR spectrometer was supplied by the NIH shared instrumentation program RR 05805 and the Vanderbilt Center in Molecular Toxicology ES 00267 Author to whom correspondence should be addressed Present address Research and Development Center Samsung Fine Chemicals Co Ltd Yusong Munjidong 103 1 Republic of South Korea Present address Department of Medicinal Chemistry The Royal Danish School of Pharmacy Universitetsparken 2 DK 2100 Copenhagen Denmark X Abstract published in AdVance ACS Abstracts April 15 1996 1 Abbreviations BPDE benzo a pyrenediol epoxide DNA deoxyribonucleic acid DSS sodium 4 4 dimethyl 4 silapentanesulfonate EDTA ethylenediaminetetraacetic acid HPLC high pressure liquid chromatography NMR nuclear magnetic resonance NOE nuclear Overhauser enhancement NOESY two dimensional NOE spectroscopy ppm parts per million PAH polycyclic aromatic hydrocarbon PEM potential energy minimization rms root mean square TPPI timeproportional phase increment TOCSY total homonuclear correlated spectroscopy 1D one dimensional 2D two dimensional S0006 2960 95 02473 1 CCC 12 00 linked to metabolic activation into reactive electrophiles which react with DNA Miller 1970 In the case of benzo a pyrene microsomal P450 enzymes reviewed by Guengerich 1992 catalyze formation of diastereomeric 7 8dihydrodiol 9 10 epoxides Sims et al 1974 first synthesized by the Jerina Yagi et al 1975 and the Harvey Beland Harvey 1976 laboratories Benzo a pyrene is metabolized to produce bay region epoxides including 7R 8S 9S 10R 7 8 dihydroxy 9 10 epoxy 7 8 9 10 tetrahydrobenzo a pyrene DE2 and 7S 8R 9R 10S 7 8 dihydroxy 9 10epoxy 7 8 9 10 tetrahydrobenzo a pyrene DE2 2 Evidence that DE2 had the greatest bonding affinity to DNA was obtained from reactions using diastereomeric mixtures of the epoxides with DNA followed by degradation and analysis Weinstein et al 1976 King et al 1976 Identification of the DNA adducts Jeffrey et al 1977 Nakanishi et al 1977 revealed an adduct formed between 2 The empirical designations for these diol epoxides are the anti BPDE or DE2 Benzo a pyrene is also metabolized into two additional diol epoxides 7S 8R 9S 10R 7 8 dihydroxy 9 10 epoxy7 8 9 10 tetrahydrobenzo a pyrene and 7R 8S 9R 10S 7 8 dihydroxy 9 10 epoxy 7 8 9 10 tetrahydrobenzo a pyrene These are the syn BPDE or DE1 which are generally regarded to be less mutagenic and tumorigenic 1996 American Chemical Society SRSR 61 2 Benzo a pyrene Adduct at Adenine N6 the 2 amino group of guanine and C10 of the PAH and derived from DE2 to be the major product Mutagenesis results were consistent with the hypothesis that the diastereomeric diol epoxides represented the ultimate carcinogenic species DE2 was generally the most mutagenic form Newbold Brookes 1976 Huberman et al 1976 There has thus been considerable interest in the structures of benzo a pyrene Cosman et al 1992 1993a 1994a b De Los Santos et al 1992 Fountain Krugh 1995 and related PAH Cosman et al 1995b and styrene oxide Zegar et al 1996 adducts at guanine N2 Minor products in the reaction of various diastereomeric BPDE with DNA have also been observed including adenine N6 adducts Jennette et al 1977 Osborne et al 1981 These are of interest since the extent to which prevalent adduction sites are related to prevalent mutagenic lesions remains unknown Indeed at lower and possibly physiologically relevant dosages increased benzo a pyreneinduced mutagenesis at adenine was reported Wei et al 1991 1993 1994 Therefore correlating the solution structures of benzo a pyrene and additional PAH adducts at adenines with mutations at