Synthesis and Identification of Major Metabolites of Environmental Pollutant Dibenzo[<i>c,mno</i>]chrysene

Dibenzo[c,mno]chrysene commonly known as naphtho[1,2-a]pyrene (N[1,2-a]P) is an environmental pollutant, recently identified in coal tar extract, in air-borne particulate matter, in marine sediment, and in cigarette-smoke condensate. We recently reported an efficient synthesis of N[1,2-a]P and examined its in vitro metabolism by male Sprague Dawley rat liver S9 fraction, which resulted in a number of dihydrodiol and phenolic metabolites. The synthesis of 10-hydroxy-N[1,2-a]P and fjord region N[1,2-a]P trans-9,10-dihydrodiol, which were identified among the various metabolites, was assigned earlier by comparing with the synthetic standards. The other major metabolites were separated by HPLC and, based on the 1H NMR analysis, were tentatively suggested to be the two K-region dihydrodiols, that is, N[1,2-a]P trans-4,5-dihydrodiol (6) and N[1,2-a]P trans-7,8-dihydrodiol (7), and the hydroxy derivatives of N[1,2-a]P. To unequivocally assign the structure to each of the peaks and to have them in larger amounts for toxicological studies, we have now synthesized the two K-region dihydrodiols and the 1-/3-hydroxy-N[1,2-a]P, short-listed based on the proton NMR of the collected peaks. The K-region dihydrodiols 6 and 7 were prepared by the treatment of N[1,2-a]P with OsO4 to give a mixture of cis dihydrodiols 2 and 3, followed by pyridinium chlorochromate-assisted oxidation to quinones 4 and 5, and finally reduction with NaBH4 to afford the dihydrodiols 6 and 7 with the desired trans stereochemistry. The 1-hydroxy-N[1,2-a]P (22) and 3-hydroxy-N[1,2-a]P (23) were synthesized using a photochemical approach. As expected, all the synthesized dihydrodiol and phenolic derivatives of N[1,2-a]P identified with those obtained from in vitro metabolism enabling the assignment of all the major metabolites

Fluorescence Characteristics of Site-Specific and Stereochemically Distinct Benzo[<i>a</i>]pyrene Diol Epoxide−DNA Adducts as Probes of Adduct Conformation

Spectroscopic fluorescence quenching techniques are described for distinguishing the conformational characteristics of adducts derived from the binding of the benzo[a]pyrene metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz[a]pyrene) to the exocyclic amino groups of guanine ([BP]-N 2-dG) and adenine ([BP]-N 6-dA) in double stranded oligonucleotides. These methods are calibrated by comparing the fluorescence quenching and UV absorbance characteristics of different, stereoisomeric anti-[BP]-N 2-dG adducts of known adduct conformations, previously established by high-resolution NMR techniques. It is shown that intercalative adduct conformations can be distinguished from solvent-exposed adduct conformations, e.g., adducts in which the pyrenyl residues are positioned in the minor groove. These low resolution fluorescence methods are at least 4 orders of magnitude more sensitive than the high-resolution NMR techniques; the fluorescence methods are useful for distinguishing adduct conformations when either small amounts of material are available or the NMR signals are of such poor quality that high-resolution structures cannot be determined. This methodology is illustrated using a variety of anti-BPDE-modified oligonucleotides of varying adduct conformations. It is shown that the 10S (+)-trans-anti-[BP]-N 6-dA adduct in an oligonucleotide duplex containing an N-ras protooncogene sequence, believed to be conformationally heterogeneous and disordered, is significantly more exposed to the solvent environment than the stereoisomeric, intercalated 10R adduct [Zegar et al. (1996) Biochemistry 35, 6212]. These differences suggest an explanation for the greater efficiencies of excision of the 10S adduct (relative to the 10R adduct) by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849]

A Highly Abbreviated Synthesis of Dibenzo[<i>def,p</i>]chrysene and Its 12-Methoxy Derivative, a Key Precursor for the Synthesis of the Proximate and Ultimate Carcinogens of Dibenzo[<i>def,p</i>]chrysene

Dibenzo[def,p]chrysene (DBC) (1), is by far the most mutagenic and toxic polycyclic aromatic hydrocarbon identified. Its metabolic activation leads to trans-11,12-dihydroxy-11,12-dihydro-DBC (2), which is further metabolized to the ultimate metabolite, anti-trans-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydro-DBC (3), that binds to DNA causing mutations and ultimately tumor induction. We report a facile route for the syntheses of DBC (1) and its 12-methoxy derivative (12-methoxy-DBC) (13), a key intermediate for the synthesis of 2 and 3, using a Suzuki cross-coupling approach

Primer Length Dependence of Binding of DNA Polymerase I Klenow Fragment to Template−Primer Complexes Containing Site-Specific Bulky Lesions^†

The binding of the benzo[a]pyrene metabolite anti-BPDE (r7,t8-dihydroxy-t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene) to the N2 group of 2‘-deoxyguanosine residues (dG*) is known to adversely affect the Michaelis−Menten primer extension kinetics catalyzed by DNA Pol I and other polymerases. In this work, the impact of site-specific, anti-BPDE-modified DNA template strands on the formation of Pol I (Klenow fragment, KF)/template−primer complexes has been investigated. The 23-mer template strand 5‘-d(AAC G*C-1 T-2 ACC ATC CGA ATT CGC CC), I (dG* = (+)-trans- and (−)-trans-anti-BPDE-N2-dG), was annealed with primer strands 18, 19, or 20 bases long. Complex formation of these template−primer strands with KF- (exonuclease-free) at different enzyme concentrations was determined using polyacrylamide gel mobility shift assays in the absence of dNTPs. The lesion dG* causes an increase in the dissociation constants, Kd, of the monomeric, 1:1 KF-/DNA template−primer complexes by factors of 10−15 when the 3‘-end base of the primer strand is positioned either opposite dG*, or opposite dC-1 in I, and the shapes of the binding isotherms are sigmoidal. The sigmoidal shapes are attributed to the formation of dimeric 2:1 KF-/DNA template−primer complexes. In contrast, when the 3‘-end of the primer strand extends only to dT-2 in I, the Kd of 1:1 complexes is increased by factors of only 2−3, the shapes of the binding isotherms are hyperbolic and nonsigmoidal and are similar to those observed with the unmodified control, and monomeric KF-/DNA complexes are dominant. The impact of bulky lesions on polymerase/DNA complex formation in polymerase-catalyzed primer extension reactions needs to be taken into account in interpreting the site-specific Michaelis−Menten kinetics of these reactions

Bending and Circularization of Site-Specific and Stereoisomeric Carcinogen−DNA Adducts^†

The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino group of guanine residues in DNA in vivo and in vitro. While the (−)-7S,8R,9R,10S enantiomer, (−)-anti-BPDE, also reacts with DNA to form similar covalent N2-deoxyguanosyl adducts, this diol epoxide is nontumorigenic and its mutagenic activities are different from those of (+)-anti-BPDE. In this work, T4 ligase-induced cyclization methods have been employed to demonstrate that the (+)-anti-[BP]-N2-dG lesions (G*) cause significantly greater amounts of bending and circularization of the one-base overhang undecamer duplex 5‘-d(CACAT[G*]TACAC)·d(TGTACATGTGG) than the stereoisomeric oligonucleotide duplex with G* = (−)-anti-[BP]-N2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotides, the ratio of circular to linear DNA multimers reaches values of 8−9 for circle contour sizes of 99−121 base pairs, while for the (−)-anti-[BP]-N2-dG-modified DNA this ratio reaches a maximum value of only ∼1 at 154−176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90−92% of the observed circular ligation products range from 30 to 51° per (+)-trans-anti-[BP]-N2-dG lesion and from 20 to 40° per (−)-trans-anti-[BP]-N2-dG lesion. In the case of unmodified DNA, the probability of circular product formation is at least 1 order of magnitude less efficient than in the BPDE-modified sequences and about 90% of the circular products exhibit bending angles in the range of 14 −19°. In the most abundant circular products observed experimentally, the bending angles are 40° and 26 ± 2° per (+)-anti-[BP]- or (−)-anti-[BP]-modified 11-mer; these values correspond to a net contribution of 21−26° and 5−19°, respectively, to the observed overall bending per lesion. The coexistence of circular DNA molecules of different sizes and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends, which permit efficient cyclization, especially in the case of (+)-trans-[BP]-N2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N2-dG lesion in the 11-mer duplex 5‘-d(CACAT[G*]TACAC)·d(GTGTACATGTG) indicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the duplex on the 5‘-side of the modified guanosine residue, which may be due to bending enhanced base pair opening and bending induced by the bulky carcinogen residue. The implications of base sequence-dependent flexibilities and conformational mobilities of anti-[BP]-N2-dG lesions on DNA replication and mutation are discussed

Synthesis, Microsome-Mediated Metabolism, and Identification of Major Metabolites of Environmental Pollutant Naphtho[8,1,2-<i>ghi</i>]chrysene

Naphtho[8,1,2-ghi]chrysene, commonly known as naphtho[1,2-e]pyrene (N[1,2-e]P) is a widespread environmental pollutant, identified in coal tar extract, air borne particulate matter, marine sediment, cigarette smoke condensate, and vehicle exhaust. Herein, we determined the ability of rat liver microsomes to metabolize N[1,2-e]P and an unequivocal assignment of the metabolites by comparing them with independently synthesized standards. We developed the synthesis of both the fjord region and the K-region dihydrodiols and various phenolic derivatives for metabolite identification. The 12-OH-N[1,2-e]P, fjord region dihydrodiol 14 and diol epoxide 15 were synthesized using a Suzuki cross-coupling reaction followed by the appropriate manipulation of the functional groups. The K-region trans-4,5-dihydrodiol (18) was prepared by the treatment of N[1,2-e]P with OsO4 to give cis-dihydrodiol 16, followed by pyridinium chlorochromate oxidation to quinone 17, and finally reduction with NaBH4 to afford the dihydrodiol 18 with the desired trans stereochemistry. The 9-OH-N[1,2-e]P (30) and N[1,2-e]P trans-9,10-dihydrodiol (32) were also synthesized following a Suzuki cross-coupling approach starting from 1,2,3,6,7,8-hexahydropyrene-4-boronic acid. The metabolism of N[1,2-e]P with rat liver microsomes led to several dihydrodiol and phenolic metabolites as assessed by the HPLC trace. The 11,12-dihydrodiol and 4,5-dihydrodiol were identified as major dihydrodiol metabolites. The synthesized 9,10-dihydrodiol, on the other hand, did not match with any of the peaks in the metabolism trace. Among the phenols, only 12-OH-N[1,2-e]P was identified in the metabolism. The other phenolic derivatives synthesized, that is, the 4-/5-, 9-, 10-, and 11-hydroxy derivatives, were not detected in the metabolism trace. In summary, N[1,2-e]P trans-11,12-dihydrodiol was the major metabolite formed along with N[1,2-e]P 4̅,5-trans-dihydrodiol and 12-OH-N[1,2-e]P on exposure of rat liver microsomes to N[1,2-e]P. The presence of N[1,2-e]P in the environment and formation of fjord region dihydrodiol 14 as a major metabolite in in vitro metabolism studies strongly suggest the role of N[1,2-e]P as a potential health hazard

Base Sequence Dependence of in Vitro Translesional DNA Replication past a Bulky Lesion Catalyzed by the Exo^- Klenow Fragment of Pol I^†

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo-) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N2-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5‘-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5‘-...XG*Y..., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G*, with A > G). Depending on X or Y, full lesion bypass differed by factors of ∼1.5−5 (∼0.6−3.0% bypass efficiencies). A downstream T flanking G* on the 5‘-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template−primer intermediates are particularly dominant (∼5.0−6.0% efficiencies) with an upstream flanking C, while a 3‘-flanking T lowers the levels of deletion products (∼0.5−2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3‘-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching ∼90%) was found with Z = T in the case of sequences with 3‘-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4−6 bases from G*) with the 3‘-terminal primer base and its 5‘-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis

Microwave-Assisted Suzuki Cross-Coupling Reaction, a Key Step in the Synthesis of Polycyclic Aromatic Hydrocarbons and Their Metabolites

A highly efficient and general method for Suzuki cross-coupling reaction en route to the synthesis of polycyclic aromatic hydrocarbons (PAHs) and their metabolites has been developed. Microwave irradiation of aryl bromides 1 and boronic acids (2 and 3) using polyurea microencapsulated palladium catalyst (Pd EnCat 30) gave the coupling adducts 4 and 5 in excellent yields in just 20 min compared to ∼24 h under thermal conditions, corresponding to a ∼72-fold increase in reaction rate

Convenient Syntheses of Dibenzo[<i>c,p</i>]chrysene and Its Possible Proximate and Ultimate Carcinogens: In Vitro Metabolism and DNA Adduction Studies

Dibenzo[c,p]chrysene (DB[c,p]C) is the only hexacyclic polycyclic aromatic hydrocarbon having two fjord regions, both in different chemical environments. Its environmental presence and relative tumorigenic potency are not known due to the lack of synthetic standards. We report here the synthesis of dibenzo[c,p]chrysene (1), its proximate carcinogens, i.e., trans-1,2-dihydroxy-1,2-dihydro-DB[c,p]C (2) and trans-11,12-dihydroxy-11,12-dihydro-DB[c,p]C (3), and possible ultimate carcinogens, i.e., anti-trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-DB[c,p]C (4) and anti-trans-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydro-DB[c,p]C (5). The syntheses of 1 and the appropriately methoxy-substituted DB[c,p]C (12 and 27), key intermediates for the synthesis of its proximate and ultimate metabolites, were tried first using a Suzuki cross-coupling reaction. However, the cyclization of olefins (10 and 11) gave poor yields of the desired products. An alternate method was thus developed employing a photochemical approach. The in vitro metabolism of DB[c,p]C was established with the S9 fraction of liver homogenate from phenobarbital/β-naphthoflavone-induced Sprague−Dawley rats. The major dihydrodiol formed was identified as the fjord region 11,12-dihydroxy-11,12-dihydro-DB[c,p]C, while the major and minor phenols were identified as 11-hydroxy-DB[c,p]C and 12-hydroxy-DB[c,p]C, respectively. Further, the DNA adduction studies with the calf thymus DNA led to a mixture of dA and dG adducts for both fjord region diol epoxides (4 and 5). Interestingly, the dA to dG ratio for 1,2-dihydroxy-3,4-epoxide was much higher (3.2) compared to that of 11,12-dihydroxy-13,14-epoxide (0.5)

Method for the Rapid Detection and Molecular Characterization of DNA Alkylating Agents by MALDI-TOF Mass Spectrometry

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) may cause DNA adduct formation. While these are commonly detected by the 32P-postlabeling assay, this method is not informative on the chemical nature of the alkylating agent. Here we report a simple and reliable method that employs MALDI-TOF-MS with 2,5-dihydroxybenzoic acid (DHB) matrix layer (ML) sample preparations for the detection and structural characterization of PAH-DNA adducts. The method involves the enzymatic digestion of DNA to 2′-deoxynucleotides followed by solid phase extraction to remove salt and other contaminants prior to MALDI-MS analysis. By collision induced dissociation (CID) structurally relevant fragments are obtained to permit characterization of the alkylating molecules and the adducted nucleotide. Next to guanosine, adenosine and cytidine adducts formed from reactions with (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) are identified at a sensitivity of anti-benzo[c]chrysene-9,10-diol-11,12-epoxide (B[c]ChDE) further document the versatility and usefulness of the method. When compared with the 32P-postlabeling assay MALDI-MS only indentified deoxycytidine as well nucleoside and dinucleotides adducts. Therefore, this sensitive method enables molecular specification and characterization of adducted nucleotides and of the alkylating agent, and thus, provides comprehensive information that is beyond the 32P-postlabeling assay