A comparison of the intercalative binding of nonreactive benzohpyrene metabolites and metabolite model compounds to dna

The need for large-scale direct synthesis of stereochemically defined and site-specific benzo[alpha]pyrenediol epoxide-oligodeoxyribonucleotide adducts for detailed NMR and other biochemical and physicochemical studies has necessitated a better understanding of variables that lead to an enhancement of the reaction yields. It is shown that, in aqueous solution, the formation of noncovalent hydrophobic complexes between 7r, 8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[alpha] pyrene (BPDE) and the single-stranded oligonucleotide 5'-d(CCATCGCTACC) precedes the covalent binding reaction of BPDE with the single deoxyguanosine residue. The yield of covalent reaction products (involving reaction of BPDE at the C10 position with the exocyclic amino group of the dG residue) increases with increasing DNA concentration and tends toward saturation at oligonucleotide single-strand concentrations above approximately 3 mM. The addition of NaCl (0.3 M) also tends to enhance the adduct reaction yields. However, organic solvents such as tetrahydrofuran in the reaction mixtures (10-40%) decrease the stabilities of the noncovalent complexes, which in turn leads to reductions in the yields of covalent BPDE-dG oligonucleotide adducts. The efficiencies of formation of hydrophobic complexes were probed by fluorescence and UV absorption techniques using the BPDE tetrol hydrolysis product 7,8,9,10-tetrahydroxytetrahydrobenzo[alpha]pyrene as a model system.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Role of Hydrophobic Effects in the Reaction of a Polynuclear Aromatic Diol Epoxide with Oligodeoxynucleotides in Aqueous Solutions

Pirogov

Shafirovich

Kolbanovskiy

et al. 1998

“…Results from earlier investigations ( 6 , 7 , 29 , 31 ) have shown that Stern−Volmer quenching constants ( K SV ) obtained from measurements of the fluorescence quenching of BP and BA derivatives by double-stranded DNA are equal to intercalation association constants ( K A ) when the fluorescence lifetimes of the metabolites measured, with and without DNA, are equal. This occurs if the fluorescence quenching is static, and if the fluorescence quantum yields of the intercalated BP or BA derivatives are negligible compared to those of the unbound molecules ( , ). Figure shows Stern−Volmer plots, measured in 2.0 mM Na + , for the fluorescence quenching of the model compounds, BP78D, BP45D, BA34D, and BA56D, by ctDNA.…”

Section: Resultsmentioning

confidence: 99%

“…The quenching of the diol fluorescence by ctDNA required that, in measurements with DNA, a subtraction procedure be employed to correct for scattered light. In this procedure, the decay profile of a ctDNA blank was subtracted from the profile measured for a diol with DNA ( , ). Lifetime measurements with ctDNA were carried out at DNA concentrations of 0.10, 0.20, 0.50, and 0.50 mM for BP78D, BP45D, BA34D, and BA56D, respectively.…”

Section: Methodsmentioning

confidence: 99%

Influence of Na⁺ on DNA Reactions with Aromatic Epoxides and Diol Epoxides: Evidence That DNA Catalyzes the Formation of Benzo[a]pyrene and Benz[a]anthracene Adducts at Intercalation Sites

Fernando

Huang

Milliman

et al. 1996

Reactions of the benzo[a]pyrene (BP) and benz[a]anthracene (BA) metabolites, (+/-)-trans-7 8-dihydroxy-anti-9, 10-epoxy-7, 8, 9, 10-tetrahydro-BP (BPDE), (+/-)-trans-3, 4-dihydroxy-anti- 1,2-epoxy-1,2,3,4-tetrahydro-BA (BADE), (+/-)-BP-4,5-oxide (BPO), and (+/-)-BA-5, 6-oxide (BAO), were examined under pseudo-first-order conditions at varying Na+ (2.0-100 Mm) and native calf thymus DNA (ctDNA) concentrations. In 0.2 mM ctDNA and 2.0 mM Na+, at a pH of 7.3 most BPDE, BADE, BPO, and BAO (87-95%) undergo DNA catalyzed hydrolysis or rearrangement. For BPDE and BPO, overall, pseudo-first-order rate constants, k, in 2.0 mM Na+ and 0.2 mM ctDNA are 21-72 times larger than values obtained without DNA. For BADE and BAO, the rate constants are less strongly influenced by DNA; k values in 0.2 mM ctDNA are only 9-12 times larger than values obtained without DNA. Kinetic data for BPDE, BPO, BADE, and BAO and DNA intercalation association constants (KA) for BP and BA diols which are model compounds indicate that KA values for BPDE and BPO in 2.0 mM Na+ are 6.6-59 times larger than those of BADE and BAO. The greater DNA enhancement of rate constants for BPDE and BPO, versus BADE and BAO, correlates with the larger KA values of the BP metabolites. DNA adducts, which account for less than 10% of the yields, also form. For BPDE in 0.20 mM ctDNA, k decreases 5.1 times as the Na+ concentration increases from 2.0 to 100 mM. Nevertheless, the DNA adduct level remains constant over the range of Na+ concentrations examined. These results provide evidence that, for BPDE in 0.20 mM DNA and 2.0 mM Na+, ctDNA adduct formation follows a mechanism which is similar to that for DNA catalyzed hydrolysis. The pseudo-first-order rate constant for adduct formation, kAd, given approximately by kAd approximately equal to (kcat,AdKA[DNA])/(1 + KA[DNA]), where kcat,Ad is a catalytic rate constant. for BADE, BPO, and BAO, the influence of varying DNA and Na+ concentrations on k values is similar to that for BPDE, and provides evidence that the formation of adducts follows the same rate law.

“…Collection of fluorescence lifetime data for each of the oligomer adducts required between 3 and 4 h. A previously described blank subtraction procedure was used to obtain reliable decay profiles (2,5,6). The temperatures at which the fluorescence decay profiles were determined are given in the figures.…”

Section: Methodsmentioning

confidence: 99%

Multiple Fluorescence Lifetimes for Oligonucleotides Containing Single, Site-Specific Modifications at Guanine and Adenine Corresponding to Trans Addition of Exocyclic Amino Groups to (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R,10S)-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

Lebreton¹,

Huang²,

Fernando³

et al. 1995

Fluorescence decay profiles of four oligonucleotide duplexes, [table: see text] ((+)- and (-)-trans-1) and [table: see text] ((+)- and (-)-trans-2), in which an exocyclic amino group of deoxyadenosine (A*) or deoxyguanosine (G*) has been alkylated by trans opening at C-10 of the epoxide group of either the (+)-(R,S,S,R)- or (-)-(S,R,R,S)-enantiomer of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE in which the benzylic 7-hydroxy group and the epoxide oxygen are trans), exhibit more than one fluorescence lifetime. Decay profiles of the oligomers, measured at 15 degrees C with excitation and emission wavelengths of 335 and 400 nm, respectively, have been analyzed using a triple-exponential decay law. Results for (+)- and (-)-trans-1 and -2 have been compared with results for the modified, single-stranded oligonucleotides ((+)- and (-)-trans-SS-1, and (+)- and (-)-trans-SS-2) and for the cis and trans opened products formed on alkylation at the 6-amino group of 2'-deoxyadenosine 5'-phosphate by (+)-(R,S,S,R)-BPDE ((+)-trans- and (+)-cis-A). The profiles of (+)-trans- and (+)-cis-A are well represented by single-exponential decay laws with lifetimes of 86 and 110 +/- 3 ns, respectively. For the single- and double-stranded oligomer adducts, which exhibit at least three fluorescence lifetimes, two of the lifetimes are short (0.5-14 +/- 1 ns) and one is long (35-59 +/- 3 ns). The fluorescence lifetimes and the amplitudes of the long-lived components in the decay profiles of the double-stranded oligomer adducts are generally smaller than those for the corresponding single-stranded adducts. The data provide evidence that the double-stranded oligomer adducts exist as multiple conformations. Previously reported NMR results suggest that the short lifetime fluorescence components are due to major adduct conformations in which the pyrenyl group is intercalated ((+)- and (-)-trans-1) or lies in the minor groove ((+)- and (-)-trans-2). The observation of long lifetime fluorescence species for the double-stranded oligomers is consistent with the presence of minor conformations (approximately 1-5%) in which the double-stranded oligomer either is locally denatured or is a mixture of locally denatured double-stranded conformations and equilibrium concentrations of single-stranded oligomers.