Electron transfer reactions between nucleic acid residues in DNA and strong oxidants are often the critical initial steps that initiate oxidative, irreversible DNA damage. Employing laser Ñash photolysis transient absorption spectroscopic techniques, we investigated the characteristics of electron transfer reactions in aqueous solutions between the 2@-deoxynucleoside 5@-monophosphates, dGMP, dAMP, dCMP and dTMP and a representative one-electron oxidant. The latter was a radical cation of a pyrene derivative with enhanced water-solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The BPT radical cation was BPT~`, generated by intense nanosecond laser pulse (308 or 355 nm, 50È70 mJ pulse~1 cm~2) by a non-linear consecutive two-photon absorption process. No electron transfer reactions were observed with dAMP, dTMP and dCMP, consistent with their unfavorable redox potentials. However, efficiently oxidized dGMP BPT~ẁ ith a rate constant M~1 s~1, which is smaller than the di †usion-controlled value by a k b \ 1.7 ^0.1) ] 109 factor of only D3. The dGMP(-H) neutral radicals formed on time scales of a few microseconds, were ĩdentiÐed by their characteristic transient absorption spectrum mn). The rate constant of electron (j max D 310 transfer from dGMP to was smaller in by a factor of D1.5 than in This kinetic isotope BPT~`D 2 O H 2 O. e †ect indicates that the electron transfer reaction from dGMP to is accompanied by the deprotonation BPT~ò fand therefore appears to be a proton-coupled electron transfer reaction. dGMP~`,

Equilenin, an important component of a widely prescribed hormone replacement formulation for postmenopausal women, is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN). The oxidized o-quinone derivative of 4-OHEN is known to form cyclic covalent adducts with DNA [Bolton, J. (1998) Chem. Res. Toxicol. 11, 1113] in vitro and in vivo. The characteristics of 4-OHEN-DNA adduct formation were investigated with the oligonucleotides 5'-d(CCATCGCTACC) (I), its complementary strand 5'-d(GGTAGCGATGG) (II), one rich in C and the other in G, and the duplexes I.II. The identities of the modified bases were elucidated in terms of four stereoisomeric 4-OHEN-2'-deoxynucleoside standards described earlier [Shen et al. (2001) Chem. Res. Toxicol. 11, 94; Embrechts et al. J. Mass Spectrom. 36, 317). The reactions of 4-OHEN with C are favored overwhelmingly in both single-stranded I and II with no guanine adducts observed in either case, and only minor proportions of A adducts were detected in sequence II. However, guanine adducts are observed in oligonucleotides that contain only G and unreactive T residues. The relative levels of cyclic covalent adducts observed in single-stranded I, II, and duplex I.II are approximately 54:21:5, with only the end C groups in I modified in the I.II duplex. When 4-OHEN is reacted with calf thymus DNA, the reaction yield of cyclic adducts is more than approximately 10(3)-fold lower than in I. The cyclic 4-OHEN adducts lead to a pronounced thermal destabilization of duplexes I.II. Overall, cyclic adduct formation is markedly dependent on the sequence context and secondary structure of the DNA. The latter effect is attributed to the poor accessibilities of 4-OHEN to the reactive nucleotide Watson-Crick hydrogen-bonding interface in the interior of the duplex. In the single-stranded oligonucleotides I and II, the strikingly different selectivities of adduct formation are attributed to the formation of noncovalent preassociation complexes that favor reaction geometries with C, rather than with A or G. Finally, the levels of several typical biomarkers of oxidative DNA damage (including 8-oxo-2'-deoxyguanosine) are formed in I in aqueous solutions with a yield at least 10 times smaller than the yield of cyclic 4-OHEN-dC adducts under identical reaction conditions.

The carcinogenic and mutagenic benzo[a]pyrenediol epoxide derivative 7r,8t-dihydroxy-9r,10/-epoxy-7,8,9,-10-tetrahydrobenzo[a] pyrene (BPDE) binds via its C-10 position predominantly to the exocyclic amino group of guanine residues in native DNA. In such DNA adducts, the fluorescence of the pyrenyl moieties is strongly quenched by physicochemical interaction with the DNA bases. Using nanosecond time scale transient absorption techniques,

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Oxidatively generated damage to DNA induced by a pyrenyl photosensitizer residue (Py) covalently attached to a guanine base in the DNA sequence context 5′-d(CAT [G 1 Py ]CG 2 TCCTAC) in aerated solutions was monitored from the initial one-electron transfer, or hole injection step, to the formation of chemical end-products monitored by HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with 355 nm laser pulses, thus producing the radical cation Py •+ and hydrated electrons; the latter are trapped by O 2 , thus forming the superoxide anion O 2•− . The decay of the Py •+ radical is correlated with the appearance of the G •+ /G(-H) • radical on microsecond time scales, and O 2•− combines with guanine radicals at G 1 to form alkali-labile 2,5-diamino-4H-imidazolone lesions (Iz 1 Py ). Product formation in the modified strand is smaller by a factor of 2.4 in double-stranded than in singlestranded DNA. In double-stranded DNA, hot piperidine-mediated cleavage at G 2 occurs only after G 1 Py , an efficient hole trap, is oxidized thus generating tandem lesions. An upper limit of hole hopping rates, k hh < 5×10 3 s −1 from G 1•+ -Py to G 2 can be estimated from the known rates of the combination reaction of the G(-H) • and O 2•− radicals. The formation of Iz products in the unmodified complementary strand in the duplex is ~ 10 times smaller than in the modified strand. The formation of tandem lesions is observed even at low levels of irradiation corresponding to "single-hit" conditions when less than ~ 10% of the oligonucleotide strands are damaged. A plausible mechanism for this observation is discussed.

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The hydrophobic interactions of bulky polycyclic aromatic hydrocarbons with nucleic acid bases and the formation of noncovalent complexes with DNA are important in the expressions of the mutagenic and carcinogenic potentials of this class of compounds. The fluorescence of the polycyclic aromatic residues can be employed as a probe of these interactions. In this work, the interactions of the (+)-trans stereoisomer of the tetraol 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), a hydrolysis product of a highly mutagenic and carcinogenic diol epoxide derivative of benzo[a]pyrene, were studied with 2'-deoxynucleosides in aqueous solution by fluorescence and UV spectroscopic techniques. Ground-state complexes between BPT and the purine derivatives 2'-deoxyguanosine (dG), 2'-deoxyadenosine (dA), and 2'-deoxyinosine (dI) are formed with association constants in the range of approximately 40-130 M(-1). Complex formation with the pyrimidine derivatives 2'-deoxythymidine (dT), 2'-deoxycytidine (dC), and 2'-deoxyuridine (dU) is significantly weaker. Whereas dG is a strong quencher of the fluorescence of BPT by both static and dynamic mechanisms (dynamic quenching rate constant k(DYN) = [2.5 +/- 0.4] x 10(9) M(-1)s(-1), which is close to the estimated diffusion-controlled value of approximately 5 x 10(9) M(-1)s(-1), both dA and dI are weak quenchers and form fluorescence-emitting complexes with BPT. The pyrimidine derivatives dC, dU, and dT are efficient dynamic fluorescence quenchers (k(DYN) approximately [1.5-3.0] x 10(9) M (-1)s(-1), with a small static quenching component due to complex formation evident only in the case of dT. None of the four nucleosides dG, dA, dC and dT are dynamic quenchers of BPT in the triplet excited state; the observed lower yields of triplets are attributed to the quenching of single excited states of BPT by 2'-deoxynucleosides without passing through the triplet manifold of BPT. Possible fluorescence quenching mechanisms involving photoinduced electron transfer are discussed. The strong quenching of the fluorescence of BPT by dG, dC and dT accounts for the low fluorescence yields of BPT-native DNA and of pyrene-DNA complexes.

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