The Nonmutagenic (R)- and (S)--(N 6 -Adenyl)styrene Oxide Adducts Are Oriented in the Major Groove and Show Little Perturbation to DNA Structure † Christophe Hennard, ‡ Jari Finneman, § Constance M. Harris, Thomas M. Harris, and Michael P. Stone* Department of Chemistry and Center in Molecular Toxicology, Vanderbilt UniVersity, NashVille, Tennessee 37235 ReceiVed March 20, 2001; ReVised Manuscript ReceiVed June 11, 2001 ABSTRACT: Conformations of (R)--(N 6 -adenyl)styrene oxide and (S)--(N 6 -adenyl)styrene oxide adducts at position X 6 in d(CGGACXAGAAG)‚d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, were refined from 1 H NMR data. These were designated as the -R(61,2) and -S(61,2) adducts. A total of 533 distance restraints and 162 dihedral restraints were used for the molecular dynamics calculations of the -S(61,2) adduct, while 518 distances and 163 dihedrals were used for the -R(61,2) adduct. The increased tether length of the -adducts results in two significant changes in adduct structure as compared to the corresponding R-styrenyl adducts [Stone, M. P., and Feng, B. (1996) Magn. Reson. Chem. 34, S105-S114]. First, it reduces the distortion introduced into the DNA duplex. For both the -R(61,2) and -S(61,2) adducts, the styrenyl moiety was positioned in the major groove of the duplex with little steric hindrance. Second, it mutes the influence of stereochemistry at the R-carbon such that both the -R(61,2) and -S(61,2) adducts exhibit similar conformations. The results were correlated with site-specific mutagenesis experiments that revealed the -R(61,2) and -S(61,2) adducts were not mutagenic and did not block polymerase bypass. Styrene is a mutagen in prokaryotes (1-3) and eukaryotes (4). It is of concern as a potential human mutagen (5-9). Styrene genotoxicity results from cytochrome P 450 -mediated metabolism to the ultimate carcinogenic species, styrene oxide (SO) 1 (10-17). SO induces sister chromosome ex- change and aberrations in human lymphocytes in vitro (18, 19). Adducts of SO at guanine O 6 and guanine N 2 were identified in human cells (20-23). Increased levels of the guanine O 6 adduct, a potential biomarker for styrene exposure, were observed in lamination workers chronically exposed to styrene in the plastics industry (24). Molecular analysis of mutations at the hypoxanthine-guanine phospho- ribosyl transferase (hprt) gene in peripheral blood lympho- cytes suggested that they occurred at both guanine and adenine sites, and were predominantly base pair substitutions (25). The occurrence of guanine O 6 adducts did not strongly correlate with the frequency of hprt mutations (24). This suggested that the guanine O 6 SO adducts were weakly mutagenic. Alternatively, they were perhaps not the source of the mutations. Thus, the relationship between styrene- induced DNA damage and mutagenesis remains to be established (23). The reactivity of styrene oxide with DNA is complex. This electrophile reacts in vitro to form adducts at a number of nucleophilic sites for both deoxyguanosine and deoxy- adenosine (26, 27). In principle, reaction may proceed via either the R- or -carbons of the epoxide. The -adducts at adenine N 6 arise solely by a mechanism involving attack on the oxirane by the N1 position of deoxyadenosine, followed by Dimroth rearrangement (28, 29). This contrasts with the R-adducts at adenine N 6 which primarily undergo direct reaction between the exocyclic amino group and the R-carbon atom of the oxirane (27, 29). Alternatively, the imino nitrogen of deoxyadenosine can react with the R-carbon, to yield the R-N1 adduct, followed by Dimroth rearrangement to the corresponding R-N 6 adduct (28). Cells containing activated oncogenes often contain muta- tions in ras (30). Mutations within codon 61 cause oncogene activation (31). The ras61 oligodeoxynucleotide 5′-d(CG- GACAAGAAG)-3′‚5′-d(CTTCTTGTCCG)-3′ (32) provides a model with which to simultaneously examine site-specific mutagenesis of styrene oxide adenine N 6 lesions (33), replication bypass of adenine N 6 lesions in vitro (34, 35), and the solution structures of adenine N 6 lesions (36-40). These studies are facilitated by large-scale production of site- specifically modified oligodeoxynucleotides (41). Previous studies were performed on the R-adenine N 6 adducts of styrene oxide. The R-R(61,2) adduct provided a replication block to a variety of polymerases. The R-S(61,2) lesion was weakly mutagenic, yielding low levels of A f G transitions (33). This was the most frequently observed SO-induced hprt mutation in human lymphocytes (25), which † This work was supported by NIH Grants ES-05509 (T.M.H.) and ES-05355 (M.P.S.). Funding for the NMR spectrometer was supplied by NIH Grant RR-05805 and the Vanderbilt Center in Molecular Toxicology (Grant ES-00267). The National Magnetic Resonance Facility at Madison was funded by the University of Wisconsin, NSF Grants DMB-8415048 and BIR-9214394, NIH Grants RR-02301, RR- 02781, and RR08438, and the USDA. * To whom correspondence should be addressed. Phone: (615) 322- 2589. Fax: (615) 343-1234. E-mail: stone@toxicology.mc.vanderbilt.edu. ‡ Current address: European Patent Office, Munich, Germany. § Current address: Pfizer, Inc., Groton, CT 06340. 1 Abbreviations: DSS, sodium 4,4-dimethyl-4-silapentanesulfonate; EDTA, ethylenediaminetetraacetic acid; HPLC, high-pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ioniza- tion time-of-flight mass spectrometry; NMR, nuclear magnetic reso- nance; NOE, nuclear Overhauser enhancement; NOESY, two-dimen- sional NOE spectroscopy; SO, styrene oxide; TPPI, time-proportional phase increment; TOCSY, total homonuclear correlated spectroscopy; 1D, one-dimensional; 2D, two-dimensional. 9780 Biochemistry 2001, 40, 9780-9791 10.1021/bi010564v CCC: $20.00 © 2001 American Chemical Society Published on Web 07/25/2001