Formation of benzo[a]pyrene metabolite-nucleoside adducts in isolated perfused rat and mouse liver and in mouse lung slices.

The formation of benzo[a]pyrene metabolite-nucleoside adducts in perfused rat and mouse liver and in mouse lung slices was studied by Sephadex LH20 chromatography. In liver from β-naphthoflavone-pretreated rats, four different deoxyribonucleoside complexes were observed; these are tentatively attributed to DNA modification by the 7,8-diol-9, 10-epoxide(s), secondary metabolites of benzo[a]pyrene quinones, the 4,5-oxide, and secondary metabolites of benzo[a]pyrene phenols. The diol-epoxide-deoxyribonucleoside adduct was also detected in perfused liver and in lung slices from 3-methylcholanthrene-treated genetically responsive C57BL/6N mice, whereas no adducts were detectable in such samples from 3-methylcholan-threne-treated genetically nonresponsive DBA/2N mice. In perfused liver of phenobarbital-pretreated rats, the 4,5-oxide-deoxyribonucleoside adduct was present. These results suggest that some of the benzo[a]pyrene metabolite-nucleoside complexes generated by microsomes and deproteinized DNA in vitro also occur in the intact rodent liver and lung tissues.Furthermore, complexes with the diol-epoxide(s) were observed with RNA from perfused liver of β-naphthoflavone-treated, but not from untreated or phenobarbital-treated rats. Complexes between ribonucleoside(s) and the diol-epoxide(s) were also found in perfusedliver or lung slices from genetically responsive but not from genetically nonresponsive mice.     

Synthesis and characterization of site-specific and stereoisomeric fjord dibenzo[a,l]pyrene diol epoxide-N(6)-adenine adducts: unusual thermal stabilization of modified DNA duplexes

The fjord polycyclic aromatic hydrocarbon compound dibenzo[a,l]pyrene (DB[a,l]P) is significantly more tumorigenic than the bay region benzo[a]pyrene in animal model systems. The molecular origins of the unusually strong genotoxic properties of DB[a,l]P and its fjord region diol epoxide metabolites are of great interest and are believed to be related to the structural characteristics of the DNA adducts formed. Site-specifically modified oligonucleotides were prepared by reacting the single adenine residue in 5'-d(CTCTCACTTCC) (I) with the racemic fjord diol epoxide r11,t12-dihydrodiol-t13,14-epoxide-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (anti-DB[a,l]PDE) in aqueous solutions. Four different oligonucleotides I with the single adenosine residues involving a covalent bond between the C14 position of DB[a,l]PDE and N(6)-dA are identified and purified. The CD spectra of the mononucleotide adducts are similar to those of Li et al. [Li et al. (1999) Chem. Res. Toxicol. 12, 758] who characterized DB[a,l]PDE-N(6)-dA adducts by a combination of CD and NMR methods. The stereochemical properties of each of the four DB[a,l]PDE-modified oligonucleotides were assigned on the basis of a combination of empirical CD rules and other approaches and differ from those of Li et al. The thermal melting points, T(m), of the unmodified duplex of I with its complementary strand (IC), T(m) = 43.8 +/- 0.5 degrees C, were compared with the same duplexes containing stereoisomeric anti-DB[a,l]PDE-N(6)-dA lesions. The T(m) of duplexes I.IC containing lesions with R absolute configurations at C14 of the DB[a,l]PDE residues are greater by 6-8 degrees C, while those with S configuration are lower by 6-10 degrees C. Similar effects are observed with adducts in the same sequence context derived from the fjord PAH anti-diol epoxides of benzo[g]chrysene, while duplexes containing lesions derived from benzo[c]phenanthrene diol epoxides with 1R and 1S configurations exhibit unchanged T(m) values. In contrast, the T(m) values of duplexes with lesions derived from the bay region benzo[a]pyrene diol epoxides (B[a]PDE) in the same sequence are lower by 12 degrees (10R adducts) and by 19 degrees (10S adducts). The greater thermal stabilities of duplexes with fjord PAH-N(6)-dA lesions relative to those with bay region B[a]PDE-N(6)-dA adducts, are correlated with lower susceptibilities of excision by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849]. The implications of these relationships are discussed in terms of present knowledge of the conformations of fjord and bay region PAH diol epoxide-N(6)-dA lesions in double stranded DNA.     

Site-specific mutagenesis in Escherichia coli by N2-deoxyguanosine adducts derived from the highly carcinogenic fjord-region benzo[c]phenanthrene 3,4-diol 1,2-epoxides

Although there have been numerous studies of site-specific mutagenesis by dGuo adducts of benzo[a]pyrene diol epoxides (B[a]P DEs), the present study represents the first example of site-specific mutagenesis by dGuo adducts of the highly carcinogenic benzo[c]phenanthrene 3,4-diol 1,2-epoxides (B[c]Ph DEs). The eight adducts that would result from cis- and trans-opening at C-1 of four optically active isomers of B[c]Ph DEs by the N(2)-amino group of dGuo were incorporated into 5'-TTCGAATCCTTCCCCC (context III) and 5'-GGGGTTCCCGAGCGGC (context IV) at the underlined site. These modified oligonucleotides along with unmodified controls were ligated into single-stranded M13mp7L2, which were then used to transfect SOS-induced Escherichia coli. Upon replication of the lesions in each of the two sequence contexts, mutational analysis of the progeny was performed by differential hybridization. For the 16 adducts, the mutation frequencies varied over 2 orders of magnitude with a reasonably even distribution (0.4-1% for three adducts, 1-2% for six adducts, 3-7.4% for five adducts, and one adduct each at 11 and 39%). For all but this last adduct, the mutation frequency for a given B[c]Ph DE adduct was less than for its B[a]P analogue with the same stereochemistry in the same sequence. For the vectors containing adducts with S configuration at the site of attachment of the hydrocarbon to the dGuo base, the main base substitution was G --> T followed by G --> A. In contrast, for the vectors containing adducts with R configuration, the main base substitution was G --> A. The most notable observation in the present study is the low frequency of mutations induced by the B[c]Ph DE-dGuo adducts relative to their B[a]P counterparts. A possible structural basis for this difference is proposed.     

Pyrrolizidine alkaloid-induced alterations in benzo[alpha]pyrene metabolism and binding of benzo[alpha]pyrene metabolites to deoxyribonucleic acid

Pretreatment of rats by oral administration of jacobine, a pyrrolizidine alkaloid and inducer of epoxide hydrolase, produced a marked shift in hepatic microsomal metabolism in vitro of benzo[alpha]pyrene. The formation of 9-hydroxybenzo[alpha]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[alpha]pyrene was decreased whereas the formation of 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrene was increased following jacobine treatment. This shift in the ratio of benzo[alpha]pyrene metabolites was accompanied by a significant reduction in DNA binding. Addition of purified epoxide hydrolase to control or jacobine microsomes produced a similar decrease in total DNA binding. Chromatography of benzo[alpha]pyrene metabolite-DNA nucleoside adducts showed a marked reduction in four peaks and the elimination of one peak with microsomes from jacobine-treated rats.     

Stereoselective release of polycyclic aromatic hydrocarbon-deoxyadenosine adducts from DNA by the 32P postlabeling and deoxyribonuclease I/snake venom phosphodiesterase digestion methods

The restricted ability of deoxyribonuclease I/snake venom phosphodiesterase digestion to liberate deoxyadenosine (dA) nucleotide adducts of polycyclic aromatic hydrocarbons from DNA, first observed by Dipple and Pigott with the bay-region diol epoxide adducts of 7,12-dimethylbenz[a]anthracene, has been observed with the dA adducts of benz[a]anthracene and benzo[c]phenanthrene diol epoxides. The micrococcal nuclease/spleen phosphodiesterase digestion used in the original 32P postlabeling procedure developed by Randerath to determine DNA adducts also failed to liberate dA nucleotide adducts quantitatively. Thus either method can potentially lead to an underestimation of the extent to which dA has been modified in DNA. The two digestion procedures exhibit systematic and mostly opposite stereoselectivity in the pattern of which dA adducts are resistant to digestion, which suggest that these adducts may have preferred orientations within modified DNA that are determined by whether they have the R or S configuration at C-1, the point of attachment between the exocyclic amino group of dA and the hydrocarbon; this in turn is dictated by the configuration about the precursor benzylic epoxide carbon and the cis versus trans nature of epoxide opening during adduct formation.     

Covalent binding of benzo[a]pyrene diol epoxide to DNA of mouse skin: in vivo persistence of adducts formation

In the first 9 d after topical application of a single dose of benzo[a]pyrene to the dorsal skin of C3H mice, the half-lives of benzo[a]pyrene diol epoxide-DNA adducts and of DNA were determined to be approximately 5 d. These data indicate that, in proliferating mouse skin, benzo[a]pyrene diol epoxide-DNA lesions are not repaired, but are diluted from the genome at a rate equivalent to DNA turnover (i.e., replication versus degradation). Subsequent to this initial period, benzo[a]pyrene diol epoxide-DNA adduct removal continues, but at a much reduced rate. At 30 d posttreatment with benzo[a]pyrene, approximately 15% of the adducts are still detectable; however, their half-lives had increased to 30 d. Similar experiments with a hairless mouse showed that, although the amount of adduct formation was lower initially, the kinetics of adduct disappearance and persistence were essentially the same as found with the C3H mouse. The data obtained in this work are consistent with the hypothesis that benzo[a]pyrene diol epoxide adducts persist in a subpopulation of skin cells long after their disappearance by DNA turnover would predict.     

Role of cytochrome p4501 family members in the metabolic activation of polycyclic aromatic hydrocarbons in mouse epidermis

Polycyclic aromatic hydrocarbons (PAHs) are known to be activated by the cytochrome P450 (P450) 1 family. However, the precise role of individual P4501 family members in PAH bioactivation remains to be fully elucidated. We therefore investigated the formation of PAH-DNA adducts in the epidermis of Cyp1a2(-/-), Cyp1b1(-/-), and Ahr(-/-) knockout mice. A panel of different PAHs was used, ranging in carcinogenic potency. Mice were treated topically on the dorsal skin with the following tritium-labeled PAHs: dibenzo[a,l]pyre-ne (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[g]chrysene (B[g]C), and benzo[c]phenanthrene (B[c]P). At 24 h after treatment, mice (two male and two female mice per group) were sacrificed, and epidermal DNA was isolated and hydrolyzed with DNase I; subsequently, DNA adducts were quantitated by liquid scintillation counting. In the DB[a,l]P-treated mice, levels of DNA adducts were significantly lower in Cyp1a2(-/-) and Cyp1b1(-/-) mice by 57 and 46%, respectively, as compared to wild-type (WT) mice (C57BL/6 background). The levels of DB[a,l]P DNA adducts formed in Ahr(-/-) mice were 26% lower, but this was not statistically significant. The levels of DMBA-DNA adducts in Cyp1a2(-/-) mice were not different than the WT mice but were significantly lower in Cyp1b1(-/-) and Ahr(-/-) mice by 64 and 52%, respectively. DMBA-DNA adduct samples were further analyzed by HPLC following further digestion to deoxyribonucleosides. HPLC analysis of individual DMBA-DNA adducts revealed differences in the ratio of syn-DMBA-diol epoxide- to anti-DMBA-diol epoxide-derived adducts in the Ahr(-/-) and Cyp1b1(-/-) mice. The ratio of syn-/anti-derived adducts in WT mice was 0.49. This ratio was 0.23 in the Cyp1b1(-/-) mice and 0.87 in the Ahr(-/-) mice. In contrast to the results with DB[a,l]P and DMBA, the levels of B[a]P-, DB[a,h]A-, B[g]C-, and B[c]P-DNA adducts were significantly lower in Ahr(-/-) mice by 73, 75, 50, and 81%, respectively, as compared to WT mice but were not significantly lower in the Cyp1a2(-/-) or Cyp1b1(-/-) mice. Collectively, these and other results support a role for both P4501A1 and P4501B1 in the bioactivation of DMBA; P4501A2, P4501B1, and possibly P4501A1 in the bioactivation of DB[a,l]P; and P4501A1 in the bioactivation of B[a]P, DB[a,h]A, B[g]C, and B[c]P in mouse epidermis. Furthermore, in the metabolic activation of DMBA in mouse epidermis, P4501B1 shows a preference for the formation of syn-DMBA-diol epoxide adducts, whereas P4501A1 shows a preference for the formation of anti-DMBA-diol epoxide adducts.     

Preparation, isolation, and characterization of Dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence line-narrowing spectroscopy.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3'-dAMP or 3'-dGMP with either (+/-)-anti- or (+/-)-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13, 14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (+/-)-trans and (+/-)-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (+/-)-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (+/-)-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (+/-)-syn-trans-DB[a,l]PDE-dGMP adducts, a pair of (+/-)-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDE-dGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.     

Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo[a]pyrene adducts at N(6) of deoxyadenosine

We have previously investigated the mutations induced on replication in Escherichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabolites of benzo[a]pyrene (B[a]P) by alkylation of a specific deoxyadenosine (dAdo) residue at N(6). Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these adducts. To determine how the presence or absence of these hydroxyl groups affects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen. Transfection of the adducted M13 constructs into SOS-induced Escherichia coli consistently gave fewer infective centers than the control construct, with viabilities ranging from 8.4 to 44.9% relative to control. In general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution mutations induced. For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported diol epoxide adducts in the same sequence. Remarkably, this (9S,10R)-adduct with cis orientation of the dAdo residue and the 9-OH group gave the highest mutational frequency of all the B[a]P adducts studied in this sequence, including the diol epoxide adducts. With the present adducts, A --> T transversions predominated, with smaller numbers of A --> G transitions and even fewer A --> C transversions.     

Characterization of covalently modified deoxyribonucleosides formed from dibenz[a,j]anthracene in primary cultures of mouse keratinocytes

Identification of various deoxyribonucleoside adducts formed in primary cultures of mouse keratinocytes exposed to dibenz[a,j]anthracene (DB[a,j]A) is presented. A preliminary analysis of the DNA adducts formed from 7-methyldibenz[a,j]anthracene (7MeDB[a,j]A) also is presented. Cultures of keratinocytes obtained from dorsal skins of female SENCAR mice were exposed to 0.5 microgram of tritium-labeled hydrocarbons/mL of medium for 24 h. The total DNA binding was 2.23 +/- 0.54 and 5.28 +/- 0.97 pmol of hydrocarbon/mg of DNA for DB[a,j]A and 7MeDB[a,j]A, respectively. These binding values represented the radioactivity associated with the modified deoxyribonucleosides separated from the normal deoxyribonucleosides on Sephadex LH-20 columns following enzymatic digestion of isolated DNA. Treatment of keratinocytes with DB[a,j]A produced adduct peaks corresponding to marker adducts derived from trans addition of both deoxyguanosine as well as deoxyadenosine residues to the (+) enantiomer of the anti-diol epoxide where the deoxyadenosine adducts were predominant. In addition, DNA adduct peaks corresponding to markers of trans and cis addition, respectively, of deoxyguanosine and deoxyadenosine to the (+)-syn-diol epoxide were also noted in these chromatograms. A major DNA adduct in cells exposed to DB[a,j]A was tentatively identified as resulting from the addition of deoxyadenosine to DB[a,j]A-5,6-oxide. Several other later eluting DNA adduct peaks, not corresponding to any of the marker adducts, were also present in these chromatograms. In comparison, when cells were exposed to the more biologically potent 7-methyl analogue, at least 12 DNA adduct peaks were consistently observed in HPLC chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Model adducts of benzo[a]pyrene and nucleosides formed from its radical cation and diol epoxide.

Reference adducts formed by reaction of deoxyribonucleosides with the ultimate carcinogenic forms of benzo[a]pyrene (BP), BP radical cation and BP diol epoxide, are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of BP in the presence of dG or dA produces adducts from BP radical cation. When 8 equiv of charge are consumed, four adducts are formed with dG: 7-(BP-6-yl)Gua, 8-(BP-6-yl)Gua, N2-(BP-6-yl)dG and 3-(BP-6-yl)dG. With 2 equiv of charge, however, only 7-(BP-6-yl)Gua and 8-(BP-6-yl)dG (BP-6-C8dG) are formed. Anodic oxidation of BP-6-C8dG affords 8-(BP-6-yl)Gua. Anodic oxidation of BP in the presence of dA produces 7-(BP-6-yl)Ade. Reaction of BP diol epoxide with dG yields 10-(guanin-7-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP, whereas reaction with dA affords three adducts, 10-(adenin-7-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP and two isomers of 10-(deoxyadenosin-N6-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP . On the basis of comparative kinetic studies among adducts of aromatic hydrocarbons and dG or G, only BP-6-C8dG easily loses the sugar moiety, providing a basis for a mechanism of hydrolysis of the glycosidic bond.     

Biological monitoring exposure of workers from plant producing carbon electrodes: quantification of benzo[a]pyrene DNA-adducts in leukocytes, by a 32P-postlabelling method and an immunoassay.

The levels of benzo[a]pyrene were monitored for blood DNA-benzo[a]pyrene adducts in 17 workers from a plant producing carbon electrodes, with high exposure to benzo[a]pyrene (575-902-1149 ng m(-3)). Two different techniques, a 32P-postlabelling method and a competitive immunoassay using polyclonal antibodies obtained from rabbits immunised with DNA modified by benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide were used. For each worker, urinary 1-hydroxypyrene, a potential indicator of exposure to polycyclic aromatic hydrocarbons, was measured. The effect of tobacco by urinary cotinine measurement was also considered. The postlabelling and immunoassay detection limits for DNA-benzo[a]pyrene adducts were respectively 0.15 and 10 fmol 50 microg(-1) of DNA. The results obtained by the two methods demonstrated a good detection of DNA-benzo[a]pyrene adducts, but no direct relationship between the quantity of adducts and the concentration of benzo[a]pyrene in air-borne was noted in the studied plant. The levels of DNA-benzo[a]pyrene adducts obtained by immunoassay were significantly higher than those obtained by the 32P-postlabelling (P < 0.001). For several workers, variations due to professional or non professional factors must be taken into account in interpreting the results. In conclusion, the two methods used proved very efficient in determining DNA-benzo[a]pyrene adducts, and may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.     

Identification of an in vivo chrysene diol epoxide adduct in human hemoglobin

Human blood samples were analyzed by fluorescence and mass spectrometry for the presence of polycyclic aromatic hydrocarbon metabolites covalently bound to hemoglobin. Globin was prepared by HCl-acetone precipitation and enzymatically digested. The polycyclic aromatic alcohols generated from ester adducts during digestion were concentrated by monoclonal antibody immunoaffinity chromatography and separated by C-18 HPLC. Analysis of collected fractions by room temperature fluorescence spectroscopy revealed the presence of the anti-tetrahydrotetrols of benzo[a]pyrene and another component possessing a phenanthrene chromophore. Gas chromatographic-mass spectral analysis of the HPLC fractions revealed that the latter compound was r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene, arising from a hemoglobin-chrysene diol epoxide adduct.     

Structural differentiation of diastereomeric benzo[ghi]fluoranthene adducts of deoxyadenosine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay

The product ion formation characteristics of four diastereomeric deoxyadenosine adducts formed by the reaction of the syn and anti diastereomers of trans-3,4-dihydroxy-5,5a-epoxy-3,4,5,5a-tetrahydrobenzo[ghi]fluoranthene are studied by matrix-assisted laser desorption ionization and postsource decay (PSD) to determine fragmentation pathways that may permit differentiation of their structures. The two adducts derived from each diol-epoxide with DNA differ in structure based on the cis/trans arrangement of the 3'-hydroxyl group on the benzo[ghi]fluoranthene (B[ghi]F) and the adenine base bound to the B[ghi]F 5a carbon. The two adduct diastereomers with the cis adenine-3'-hydroxyl configuration produce product ions at m/z 394 and m/z 510 formed by the loss of water that are not observed in the PSD spectra of the two trans isomers. The data suggest a mechanism of water loss that is initiated by a hydrogen-bonding interaction between the charge-bearing proton on the N1 atom and the 3'-hydroxyl oxygen on the polycyclic aromatic hydrocarbon (PAH). Fragmentation is initiated by the transfer of the adenine N1 proton from the nitrogen to the PAH 3'-hydroxyl oxygen and inductive cleavage of the C3-O(3) bond to form a benzylic carbocation on B[ghi]F. The proposed mechanism is supported by semiempirical molecular modeling calculations.     

Studies of the mechanisms of adduction of 2'-deoxyadenosine with styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides

The mechanism of adduction of 2'-deoxyadenosine by styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides has been explored using (15)N(6)-labeled adenine nucleosides. The extent of reaction at N1 versus N(6) was evaluated by (1)H NMR of the N(6) adducts after allowing Dimroth rearrangement to occur. Products arising from attack at N1 followed by Dimroth rearrangement exhibited a small two-bond (1)H-(15)N coupling constant (N1-H2 J approximately 13 Hz); products from direct attack exhibited a much larger one-bond (1)H-(15)N coupling constant (J approximately 90 Hz). In the case of styrene oxide, all of the N(6) beta adduct arose by initial attack at N1, whereas the majority (70-80%) of the N(6) alpha adducts came from direct attack. The styrene oxide reaction was also studied with a self-complementary oligodeoxynucleotide (24-mer) containing nine (15)N(6)-labeled adenine residues. NMR examination of the N(6) alpha- and beta-styrene oxide adducts isolated after enzymatic degradation of the 24-mer gave very similar results, indicating that N1 attack can occur readily even with a duplexed oligonucleotide. With the PAH dihydrodiol epoxides, only naphthalene dihydrodiol epoxide exhibited significant initial reaction at N1 (50%). No detectable rearranged product was seen in reactions with benzo[a]pyrene dihydrodiol epoxide or non-bay or bay region benz[a]anthracene dihydrodiol epoxide; interestingly, a small amount of N1 attack (5-7%) was seen in the case of benzo[c]phenanthrene dihydrodiol epoxide. It appears that initial attack at N1 is only a significant reaction pathway for epoxides attached to a single aromatic ring.     

Marked differences in base selectivity between DNA and the free nucleotides upon adduct formation from Bay- and Fjord-region diol epoxides

Distributions of adducts formed from each of the four optically active isomers of 3,4-dihydroxy-1,2-epoxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene and of 7,8-dihydroxy-9,10-epoxy-7,8, 9,10- tetrahydrobenzo[a]pyrene (BcPh and BaP diol epoxides) on reaction with an equimolar mixture of deoxyadenosine and deoxyguanosine 5'-monophosphates were compared with the known adduct distributions from these diol epoxides (DEs) upon reaction with calf thymus DNA in vitro. In the presence of an equimolar (100 mM total) mixture of dAMP and dGMP, the efficiency of formation of all types of adducts relative to tetraols is comparable for both the BaP ( approximately 40-60%) and BcPh ( approximately 30-40%) diol epoxides. This is in contrast to the partitioning between tetraols and adducts observed with DNA, where the BcPh DEs form adducts much more efficiently than the BaP DEs. Preference for trans versus cis ring opening by the exocyclic amino groups of the free nucleotides in the dAMP/dGMP mixture is greater for the DE diastereomer in which the benzylic hydroxyl group and the epoxide oxygen are trans (DE-2). This is qualitatively similar to the preferences for trans versus cis adduct formation on reaction of these isomers with DNA, as well as trans versus cis tetraol formation on their acid hydrolysis. For the BcPh DE isomers, competitive reaction between dGMP and dAMP gives 40-62% of the total exocyclic amino group adducts as dA adducts. A similar distribution of dG versus dA adducts had previously been observed on reaction of the BcPh DEs with DNA, except in the case of (+)-3(R),4(S)-dihydroxy-1(R),2(S)-epoxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene, which gives approximately 85% dA adducts on reaction with DNA. With the BaP DEs, 60-77% of the exocyclic amino group adducts formed upon competitive reaction with the free nucleotides are derived from dGMP. The observed dG selectivity of these BaP DEs is much smaller with the nucleotide mixture than it is with DNA, leading to the conclusion that DNA structure has a much larger modifying effect on the base selectivity of the BaP relative to the BcPh DEs.     

Characterization of DNA adducts derived from (+/- )-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4- tetrahydrodibenz[a,j]anthracene and (+/- )-7-methyl-trans-3,4-dihydroxy- anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene

Structural characterizations of the DNA adducts derived from reaction of the racemic bay region anti-diol epoxides of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene with calf thymus DNA are presented. Quantities of adducts necessary for spectroscopic characterization were obtained from reactions of the respective diol epoxides with individual deoxyribonucleotides. Both hydrocarbon diol epoxides showed similar adduct profiles upon reaction with calf thymus DNA in vitro which were composed mainly of three deoxyguanosine and four deoxyadenosine adducts. No significant modification of pyrimidine bases in DNA was detected with either of the diol epoxides. Approximately 3 times more deoxyguanosine than deoxyadenosine residues in the DNA were found to be modified by both diol epoxides. The DNA reactions showed very similar stereo- and enantioselectivities with both diol epoxides. The stereochemistries of addition of the purine bases to the diol epoxides were determined from analysis of the NMR spectra of individual adducts. The predominant adducts formed were products of trans addition of the exocyclic amino group of purines to the diol epoxides. The enantiomeric nature of the various adducts was determined from reaction of the individual deoxyribonucleotides with the pure (+)-anti-diol epoxide of dibenz[a,j]anthracene. The major deoxyguanosine and deoxyadenosine adducts from reactions with DNA were found to arise from the (+)-enantiomer of both hydrocarbon diol epoxides. The high reactivities of both diol epoxides (24-38%) with DNA in solution are consistent with the high tumor-initiating activity exhibited by the diol epoxide of dibenz[a,j]anthracene relative to the parent hydrocarbon.     

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

3'-H-phosphonate synthesis of chiral benzo[a]pyrene diol epoxide adducts at N(2) of deoxyguanosine in oligonucleotides

A synthetic route to oligonucleotides containing N(2)-deoxyguanosine adducts at C-10 of the enantiomeric 7,8-diol 9,10-epoxides of 7,8,9,10-tetrahydrobenzo[a]pyrene in which the epoxide oxygen and the 7-hydroxyl group are trans is described. The present adducts result from the trans addition of N(2) of deoxyguanosine to the epoxide at C-10. Our synthesis proceeds via preparation of the 3'-H-phosphonate of a suitably protected deoxyguanosine N(2)-adduct. The blocking groups consisted of O(6)-allyl on the deoxyguanosine, acetates on the 7-, 8-, and 9-hydroxyl groups of the hydrocarbon moiety, and dimethoxytrityl on the 5'-hydroxyl group of the sugar. These blocking groups are well suited to oligonucleotide synthesis on solid supports. The free 3'-hydroxyl group of this nucleoside adduct was readily converted to its 3'-H-phosphonate with diphenyl phosphite in pyridine in high yield for both the 10R and 10S isomers. For synthesis of oligonucleotides, the first several nucleotides were incorporated onto the solid support with an automated synthesizer using standard phosphoramidite chemistry. The adducted deoxyguanilic acid residue was introduced as the H-phosphonate in a manual step (80% yield), followed by completion of the sequence on the synthesizer. Although a 10-fold excess of the 3'-H-phosphonate was used in the manual coupling step, as much as 70% of the reactant could be recovered. The 3'-H-phosphonate of the protected 10S nucleoside adduct was converted to the unblocked nucleotide adduct, various salts of which failed to form crystals suitable for X-ray analysis. Although submilligram quantities of this compound have been formed as mixed diastereomers by direct reaction of deoxyguanylic acid with racemic diol epoxide, the present study represents the first actual synthesis of the major DNA adduct formed from benzo[a]pyrene in mammals as its 3'-phosphate.