Inhibitory effect of 3-hydroxybenzo(a)pyrene on the mutagenicity and tumorigenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

The 12 isomeric phenols of benzo(a)pyrene were tested for their ability to inhibit the mutagenic activity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [B(a)P 7,8-diol-9,10-epoxide-2], an ultimate mutagenic and carcinogenic metabolite of benzo(a)pyrene. 3-Hydroxybenzo(a)pyrene [3-HO-B(a)P], a major metabolite of benzo(a)pyrene, was the most potent antagonist tested. Approximately 3 nmol of 3-HO-B(a)P, 14 nmol of 10-HO-B(a)P, and 5-8 nmol of 1-, 2-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, and 12-HO-B(a)P inhibited the mutagenic activity of 0.05 nmol of B(a)P 7,8-diol-9,10-epoxide-2 by 50% in Salmonella typhimurium strain TA 100. The importance of the phenolic group for antimutagenic activity was indicated by the lack of antimutagenic activity of benzo(a)pyrene itself. 3-HO-B(a)P also inhibited the mutagenic activity resulting from the metabolic activation of benzo(a)pyrene and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by rat liver microsomes. This inhibition may have resulted from an effect of 3-HO-B(a)P on the metabolic activation of these carcinogens and/or from a direct effect on the action of B(a)P 7,8-diol-9,10-epoxide-2. In a mammalian cell culture system utilizing Chinese hamster V79 cells, 3-HO-B(a)P (8 microM) inhibited the mutagenicity of B(a)P 7,8-diol-9,10-epoxide-2 (0.2 microM) by 50%. Although 3-HO-B(a)P was a potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo(a)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene in S. typhimurium strain TA 100, higher concentrations of 3-HO-B(a)P were needed to inhibit the mutagenicity of the chemically less reactive benzo(a)pyrene 4,5-oxide and the bay-region diol epoxides of benz(a)anthracene, chrysene, and benzo(c)phenanthrene. Both 3-HO-B(a)P and 10-HO-B(a)P accelerated the disappearance of B(a)P 7,8-diol-9,10-epoxide-2 from 1:9 dioxane-water solutions at pH 7 and 25 degrees C. 3-HO-B(a)P, the most effective antimutagen of the B(a)P phenols tested, was much more reactive with the diol epoxide than 10-HO-B(a)P, the least effective antimutagen. The rate constant for the reaction of 3-HO-B(a)P with the diol epoxide exhibited a nonlinear (greater than first-order) dependence on the concentration of the phenol. Evidence was obtained for covalent adduct formation between the diol epoxide and each of the two phenols.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of the enantiomers of the diastereomeric bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides in bacterial and mammalian cells.

The mutagenic activities of the enantiomers of the pair of diastereomeric bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides were evaluated in histidine-dependent strains of Salmonella typhimurium and in an 8-azaguanine-sensitive Chinese hamster cell line. In strains TA 98 and TA 100 of S. typhimurium, the range in mutagenic activity observed for the four optically active isomers was less than 4- and 2-fold, respectively. The diol-epoxide with (1S,2R,3R,4S) absolute configuration and the benzylic hydroxyl group trans to the epoxide oxygen [(+)-diol epoxide-2] was the most active isomer in both strains. The enantiomeric (-)-diol-epoxide-2 isomer, with (1R,2S,3S,4R) absolute configuration identical to that of the exceptionally tumorigenic (+)-diol-epoxide-2 isomers of benzo(a)pyrene, benz(a)anthracene, and chrysene, was the least active isomer in strain TA 98 (27%) and the second most active isomer in strain TA 100 (90%). In Chinese hamster V79 cells (-)-diol-epoxide-2 was the most active of the four benzo(c)phenanthrene isomers, and a 4- to 5-fold range in mutagenic activity was observed. The differences in mutagenic activity between the four bay-region diol-epoxide isomers of benzo(c)phenanthrene in the three test systems are relatively small when compared with results from similar studies with optically active bay-region diol-epoxide isomers of three other polycyclic aromatic hydrocarbons, and may be explicable, in part, by a tendency of the hydroxyl groups of benzo(c)phenanthrene diol-epoxides to adopt comparable pseudodiequatorial conformations.

Mutagenicity of the bay-region diol-epoxides and other benzo-ring derivatives of dibenzo(a,h)pyrene and dibenzo(a,i)pyrene.

The mutagenic activities of dibenzo(a,h)(pyrene, dibenzo(a,i)pyrene, and a total of 11 of their benzo-ring derivatives were evaluated in bacterial and mammalian cells in the absence or presence of a mammalian metabolic activation system. trans-1,2-Dihydroxy-1,2-dihydrodibenzo(a,h)pyrene and trans-3,4-dihydroxy-3,4-dihydrodibenzo(a,i)pyrene, the expected dihydrodiol precursors of bay-region diol-epoxides, were metabolized to products which were more mutagenic to strains TA98 and TA100 of Salmonella typhimurium than were the metabolic products formed from their respective parent hydrocarbons. For each dihydrodiol, replacement of the benzo-ring double bond adjacent to the diol moiety with a single bond resulted in tetrahydrodiol derivatives which could not be metabolically activated, suggesting that one or both diastereomeric bay-region diol-epoxides were the bioactivated metabolites. The authentic bay-region diol-epoxide diastereomers of dibenzo(a,h)pyrene and dibenzo(a,i)pyrene in which the benzylic hydroxyl group and the epoxide oxygen are trans (diol-epoxide 2 series) were highly mutagenic in strains TA98 and TA100 of S. typhimurium and in cultured Chinese hamster V79 cells. Neither diol-epoxide was significantly, if at all, metabolized by epoxide hydrolase. The bay-region diol-epoxide of dibenzo(a,i)pyrene was from 1.5 to 5 times more active as a mutagen than the diol-epoxide of dibenzo(a,h)pyrene, and in strain TA98 of S. typhimurium as well as Chinese hamster V79 cells, it had activity comparable to that of the highly carcinogenic bay-region diol-epoxide of benzo(a)pyrene.

Effect of single benzo[a]pyrene diol epoxide-deoxyguanosine adducts on the action of DNA polymerases in vitro.

Neither Sequenase 2.0 nor Klenow fragment were able to extend 12-mer primers using the eight templates (16-mers) derived by placing each of the four isomeric benzo[a]pyrene diol epoxide-deoxyguanosine adducts at the 13th nucleotide from the 3'-end of two different sequence contexts. Using an 11-mer primer to get a running start did not overcome the adduct induced block of primer extension except for the Klenow fragment and one of the two sequence contexts, indicating primer extension is dependent on both the polymerase and sequence context. In this case, purine nucleoside triphosphates (dATP>dGTP) were incorporated opposite each of the four adducts.

The ratio of deoxyadenosine to deoxyguanosine adducts formed by (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene in purified calf thymus DNA and DNA in V-79 cells is independent of dose.

The hypothesis that the decrease in the proportion of mutations at AT base pairs in Chinese hamster V-79 cells treated with increasing doses of (+)-(R,S,S,R)-benzo[a]pyrene diol epoxide ((+)-BPDE) is due to saturation of A for adduct formation was investigated by comparing the ratio of dA to dG adducts formed at high (0.48 microM) and low (0.04 microM) doses of [3H]-labeled (+)-BPDE. The dA to dG adduct ratio was similar in both calf thymus DNA and the genomic DNA in V-79 cells, and did not change with dose. For the V-79 cells, this ratio was also unaffected by a 24-h post treatment repair incubation.

Combined percutaneous transhepatic and endoscopic placement of biliary stents.

Combined percutaneous transhepatic cholangiography and endoscopic retrograde cholangiography can be used to stent biliary obstruction when attempts at endoscopic stenting have failed. Between January 1987 and August 1991 we performed 35 combined procedures in 31 patients with malignant obstruction. Post stenting serum bilirubin and serum alkaline phosphatase concentration fell after 33 and 29 procedures, respectively. In six studies there was evidence of infection prior to stenting. In spite of the use of prophylactic antibiotics, septic complications developed after eight procedures (23%). Pseudomonas was the most commonly isolated pathogen (46%). Twenty-three patients were discharged, 30-day mortality was 8 (23%) and median survival was 14 weeks (range 0-75 weeks). Seven patients required eight stent changes because of blockage (median patency time 18 weeks; range 7-75 weeks). Use of this technique allows relief of biliary obstruction. Potential infective and bleeding complications must be anticipated.

Effects of pH and salt concentration on the hydrolysis of a benzo[alpha]pyrene 7,8-diol-9,10-epoxide catalyzed by DNA and polyadenylic acid.

The time-dependent absorbance change that occurs when benzo[alpha]pyrene 7,8-diol-9,10-epoxide is added to solutions of calf thymus DNA has been shown, by an unequivocal chromatographic method, to correspond to DNA-catalyzed hydrolysis of the diol-epoxide. At 25 degrees C and mu = 0.10, the kinetics of the reaction of the diol-epoxide with polyadenylic acid or DNA are consistent with preequilibrium formation of a non-covalent complex between the diol-epoxide and the polynucleotide or DNA, followed by hydrolysis of the bound epoxide by a process that is first-order in hydronium ions. Cacodylic acid also catalyzes the hydrolysis of the epoxide bound to polyadenylic acid. The rate of the DNA-catalyzed hydrolysis exhibits little or no enantiomeric selectivity for the diol-epoxide. DNA catalyzed hydrolysis of the diol-epoxide is extraordinarily sensitive to the salt concentration in the reaction medium: the rate of hydrolysis of the bound epoxide at pH 7 is retarded by a factor of approximately 45 in the presence of 0.1 M sodium chloride compared to a 1 mM buffer containing no added salt. Thus, studies of the interactions of DNA with carcinogenic diol-epoxides must take into account the ionic environment of DNA within the cell.

The proximate carcinogen trans-3,4-dihydroxy-3,4-dihydro-dibenz[c,h]acridine is oxidized stereoselectively and regioselectively by cytochrome 1A1, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

Metabolism of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrodibenz[c,h]acridine has been examined with rat liver enzymes. The dihydrodiol is metabolized at a rate of 2.4 nmol/nmol of cytochrome P450 1A1/min with microsomes from 3-methylcholanthrene-treated rats, a rate more than 10-fold higher than that observed with microsomes from control or phenobarbital-treated rats. Major metabolises consisted of a diastereomeric pair of bis-dihydrodiols (68-83%), where the new dihydrodiol group has been introduced at the 8,9-position, tetraols derived from bay region 3,4-diol-1,2-epoxides (15-23%), and a small amount of a phenolic dihydrodiol(s) where the new hydroxy group is at the 8,9-position of the substrate. A highly purified monooxygenase system reconstituted with cytochrome P450 1A1 and epoxide hydrolase (17 nmol of metabolites/nmol of cytochrome P450 1A1/min) gave a metabolite profile very similar to that observed with liver microsomes from 3-methylcholanthrene-treated rats. Study of the stereoselectivity of these microsomes established that the (+)-(3S,4S)-dihydrodiol gave mainly the diol epoxide-1 diastereomer, in which the benzylic 4-hydroxyl group and epoxide oxygen are cis. The (-)-(3R,4R)-dihydrodiol gave mainly diol epoxide-2 where these same groups are trans. The major enantiomers of the diastereomeric bis-dihydrodiols are shown to have the same absolute configuration at the 8,9-position. Correlations of circular dichroism spectra suggest this configuration to be (8R,9R). The (8R,9S)-oxide may be their common precursor.

Covalent bonding of bay-region diol epoxides to nucleic acids.

Although the solution chemistry of diol epoxides is now fairly well understood, a great deal remains to be elucidated regarding their reaction in the presence of DNA. Not only DNA but also small molecules are capable of sequestering diol epoxides in aqueous solutions with equilibrium constants on the order of 10(2)-10(4) M-1. In the case of DNA, at least two major families of complexes are presently recognized, possibly the result of groove binding vs. intercalation. As is the case for diol epoxides free in solution, the complexed diol epoxides undergo solvolysis to tetraols and in some cases possibly to keto diols as well. Fractionation between covalent bonding and solvolysis from within the complex(s) is determined more by the nature of the parent hydrocarbon from which the diol epoxide is derived than any other factor. Studies of a wide variety of alkylating and arylating agents have show that practically every potentially nucleophilic site on DNA can serve as a target for modification. In the case of the diol epoxides, practically all of the modification occurs at the exocyclic amino groups of the purine bases. In contrast to the diol epoxides, other epoxides such as those derived from aflatoxin B1, vinyl chloride, propylene, 9-vinylanthracene, and styrene preferentially bind to the aromatic ring nitrogens N-7 in guanine and N-3 in adenine (cf. Chadha et al., 1989). Molecular modeling as well as the spectroscopic evidence suggests that the hydrocarbon portion of the diol epoxides lies in the minor groove of DNA when bound to the exocyclic 2-amino group of guanine and in the major groove when bound to the exocyclic 6-amino group of adenine. Detailed conformational analysis of adducted DNA should prove to be extremely valuable in developing mechanistic models for the enzymatic processing of chemically altered DNA. At present, the critical lesion or lesions responsible for induction of neoplasia remains obscured by the large number of apparently noncritical adducts which form when polycyclic hydrocarbon diol epoxides bond to DNA.

Reactivity and tumorigenicity of bay-region diol epoxides derived from polycyclic aromatic hydrocarbons.

During the past decade substantial progress has been made in elucidating factors that determine the tumorigenic activity of bay-region diol epoxides, major ultimate carcinogenic metabolites derived from polycyclic aromatic hydrocarbons. Neither high nor low chemical reactivity of the diol epoxides (as measured by rates of uncatalyzed solvolysis) is required for high tumorigenic response. In contrast, aspects of molecular structure such as conformation and absolute configuration strongly influence tumorigenic activity. The role of conformation is illustrated by the observation that those diol epoxides whose hydroxyl groups are pseudoaxial are weak or inactive as tumorigens. Absolute configuration is an important determinant of biological activity of bay-region diol epoxides: in all cases studied to date, the predominantly formed (R,S)-diol-(S,R)-epoxides are generally the most tumorigenic of the four metabolically possible configurational isomers. In the course of investigating the effects of structural factors on tumorigenic activity, we identified the (4R,3S)-diol-(2S,1R)-epoxide of benzo(c)phenanthrene as the most potent tumorigen (in initiation-promotion experiments on mouse skin) of the diol epoxides studied to date. Studies of all four configurationally isomeric diol epoxides derived from benzo(c)phenanthrene led to the striking observation that these diol epoxides exhibit an exceptionally high efficiency of covalent binding, relative to hydrolysis, when allowed to react with calf thymus DNA in aqueous solution. Thus, these diol epoxides should provide an excellent tool for the detailed study of such binding. When the four isomeric benzo(c)phenanthrene diol epoxides are compared, there appears to be no simple correlation between tumorigenic response and either the extent of binding to DNA or the major types of deoxyribonucleoside adducts formed. Deoxyribonucleoside adducts of benzo(c)phenanthrene diol epoxide have also been identified from the DNA of cultured rodent embryo cells after treatment of the cells with tritium-labeled benzo(c)phenanthrene. The distribution of adducts is consistent with predominant metabolic formation of the (4R,3S)-diol-(2S,1R)-epoxide; deoxyadenosine is the major site in the cellular DNA attacked by this epoxide, just as it is in DNA in solution. Further experiments are in progress which we hope will identify more subtle aspects of the DNA binding of benzo(c)phenanthrene diol epoxides that may be uniquely correlated with their tumorigenic activity.

Highly tumorigenic bay-region diol epoxides from the weak carcinogen benzo[c]phenanthrene.

In the four years since its inception, the bay-region theory has proved highly successful in predicting which diol epoxide of a polycyclic aromatic hydrocarbon would have the highest tumorigenic activity. The present studies on benzo[c]phenanthrene have shown this hydrocarbon to be unique. It is the first hydrocarbon for which the bay-region diol epoxide that has its benzylic hydroxyl group and epoxide oxygen cis (isomer-1 series) has significant tumorigenic activity. Additionally, its bay-region diol epoxides are the most tumorigenic diol epoxides yet tested on mouse skin despite their expected and observed very low chemical reactivity. Perhaps some unique feature of the shape of benzo[c]phenanthrene can account for the remarkable biological activity of its bay-region diol epoxides. The high degree of crowding in the bay-region of benzo[c]phenanthrene may be such a contributing factor. It is know, for example, that methyl-substitution in the bay-region but not on the critical benzo-ring enhances the tumorigenic activity of 7,12-dimethylbenzo[a]anthracene relative to 7-methylbenzo[a]anthracene (Newman, 1976), of 5-methylchrysene relative to chrysene (Hecht et al., 1974), and of 11-methylbenzo[a]pyrene relative to benzo[a]pyrene (Iyer et al., 1980). Steric crowding in the bay-region of benzo[c]phenanthrene (Hirshfeld, 1963) and 7,12-dimethylbenzo[a]anthracene has been shown by x-ray crystallography to cause out-of-plane deformation of their aromatic ring systems.

Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase.

Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy. In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20. The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods. Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay. The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment. Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive.

Stereoselectivity of microsomal epoxide hydrolase toward diol epoxides and tetrahydroepoxides derived from benz[a]anthracene.

We have examined the selectivity of rat liver microsomal epoxide hydrolase (EC 3.3.2.3) toward all of the possible positional isomers of benzo-ring diol epoxides and tetrahydroepoxides of benz[a]anthracene, as well as the 1,2-diol 3,4-epoxides of triphenylene. This set includes compounds with no bay region in the vicinity of the benzo-ring, a bay-region diol group, a bay-region epoxide group, and (for the triphenylene derivatives) both a bay-region diol and a bay-region epoxide. In all cases where both the tetrahydroepoxides and the corresponding diol epoxides were examined, there is a large retarding effect of hydroxyl substitution on the rate of the enzyme-catalyzed hydration. When the tetrahydroepoxides are fair or poor substrates (epoxide group in the 1,2-, 8,9-, or 10,11-position), the additional retardation introduced by adjacent hydroxyl groups causes the enzyme-catalyzed hydrolysis of the corresponding diol epoxides to be insignificantly slow or nonexistent. In contrast, a benz[a]anthracene derivative with an epoxide group in the 3,4-position, (-)-tetrahydrobenz[a]anthracene (3R,4S)-epoxide, has been identified as the best substrate known for epoxide hydrolase, with a Vmax at 37 degrees C and pH 8.4 of 6800 nmol/min/mg of protein, and the two diastereomeric (+/-)-benz[a]anthracene 1,2-diol 3,4-epoxides, unlike all the other diol epoxides examined to date, are moderately good substrates for epoxide hydrolase. This novel observation is accounted for by the fact that the very high reactivity of the tetrahydrobenz[a]anthracene 3,4-epoxide system towards epoxide hydrolase is large enough to overcome a kinetically unfavorable effect of hydroxyl substitution. The enantioselectivity and positional selectivity of the enzyme have been determined for the tetrahydro-1,2- and -3,4-epoxides of benz[a]anthracene as well as the 1,2-diol 3,4-epoxides. When the epoxide is located in the 3,4-position, the benzylic carbon is the preferred site of attack, whereas for the enantiomers of the bay-region tetrahydro-1,2-epoxides, the chemically less reactive non-benzylic carbon is preferred. The regio- and enantioselectivity of epoxide hydrolase are discussed in terms of a possible model for the hydrophobic binding site of this enzyme.

Primer extension by various polymerases using oligonucleotide templates containing stereoisomeric benzo[a]pyrene-deoxyadenosine adducts.

Four isomeric benzo[a]pyrene-deoxyadenosine adducts, corresponding to the products of trans opening of the epoxide ring in the four configurationally isomeric benzo[a]pyrene dihydrodiol epoxides by the amino group of deoxyadenosine, were separately introduced into each of two 16-mer sequence contexts. The sequences were from the supF gene, and the site of the adducted adenine was known, for some hydrocarbon dihydrodiol epoxides, to be a hotspot for mutation in Context I and a coldspot for mutation in Context II. Using primers complementary to the 3' ends of these oligonucleotides, the abilities of several polymerases to replicate these templates in vitro were investigated. Each adduct proved to be an effective block to primer extension such that only with high concentrations of exo- Klenow fragment was any bypass of adducts seen. DNA polymerase alpha and HIV-1 reverse transcriptase were blocked 3' to the adduct when the configuration at C10 of the hdyrocarbon was S, and some introduction of thymine opposite the adenine adduct was seen with the R configuration. Incorporation of a nucleotide opposite the adduct occurred more readily with Sequenase and the Klenow fragment, and the mutagenic introduction of adenine was apparent in most cases. This corresponded to the A-->T transversions frequently seen in mutation studies with hydrocarbon dihydrodiol epoxides that react extensively with adenine in DNA. Overall, it was clear that sequence context, adduct stereochemistry, and the choice of polymerase all influenced the polymerization reaction. With these in vitro systems, no major differences correlating with the differing tumorigenicities of the isomeric dihydrodiol epoxides or with the hotspot or coldspot nature of the sequences were detected.

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.

O(6)-allyl protected deoxyguanosine adducts of polycyclic aromatic hydrocarbons as building blocks for the synthesis of oligonucleotides.

We describe a synthetic strategy for the preparation of oligonucleotides using N(2)-alkylated and O(6)-allyl protected deoxyguanosine phosphoramidite building blocks derived from cis- and trans-opened (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and from trans-opened (+/-)-3alpha,4beta-dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene. The appropriately blocked phosphoramidite building blocks were obtained as mixtures of the cis- and trans-opened diol epoxide adducts upon initial reaction of the diol epoxides with O(6)-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine. Key to the present approach is the removal of the O(6)-allyl protecting group utilizing a palladium catalyst prior to release of the constructed oligonucleotide with ammonia from the solid support. The methodology described enables a very convenient access to oligonucleotides containing cis- and trans-N(2)-deoxyguanosine adducts of polycyclic aromatic hydrocarbons in different sequence contexts.

Differences between the mutational consequences of replication of cis- and trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-deoxyguanosine adducts in M13mp7L2 constructs.

The four adducts at N(2) of deoxyguanosine derived from cis-opening at C-10 of four optically active isomers of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene were incorporated into 5'-TTCGAATCCTTCCCCC [context III(G)] and 5'-GGGGTTCCCGAGCGGC [context IV(G)] at the underlined site. The mutagenic consequences of these lesions in each of the two sequence contexts were examined after ligation of the modified oligonucleotides into single-stranded M13mp7L2 and replication of the vector in SOS-induced Escherichia coli. Total frequencies of base substitution mutations ranged between 14 and 48%. The mutation frequencies were generally higher in context IV(G) than in context III(G), and consisted mainly of G-->T followed by G-->C base substitutions. A substantial number of deletions or insertions of one guanine was also found for all adducts in context IV(G), where the adduct is located at the 3'-end of a run of five guanines. The overall frequencies of base substitution mutations induced by cis-opened adducts were substantially higher than those observed with the trans-opened dGuo adducts in the same sequences [Page et al. (1998) Biochemistry 37, 9127-9137]. Although G-->T base substitutions predominated for both the cis- and trans-opened adducts, the cis-opened dGuo adducts generally resulted in a higher proportion of G-->C [particularly in context III(G)] relative to G-->A, whereas the opposite was true for the trans-opened dGuo adducts. The present results along with previous data indicate that mutagenicity is highly dependent on a combination of sequence context and adduct stereochemistry.

HPLC-MS/MS identification of positionally isomeric benzo[c]phenanthrene diol epoxide adducts in duplex DNA.

LC-MS and LC-MS/MS analyses were used to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (-)-(1R,2S,3S,4R)-1,2-epoxy-3,4-dihydroxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene [(-)-(R,S,S,R)-BcPh DE-2], on reaction in vitro with an oligonucleotide dodecamer derived from the HPRT gene. The sequence of this dodecamer, 5'-T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12)-3', contains a base (corresponding to A(7)) which is a hot spot for mutagenesis in the hprt gene induced by the carcinogenic (R,S,S,R)-enantiomer of benzo[a]pyrene 7,8-diol 9,10-epoxide, and an adjacent base (corresponding to A(6)) which gave no mutations with this diol epoxide. Modified oligonucleotides were generated by reaction of (-)-BcPh DE-2 with both the single-stranded and duplex forms of the dodecamer. Multiple purine targets in both strands led to the formation of complex reaction mixtures of regioisomeric BcPh DE-modified oligonucleotides, which were partially separated by reverse phase HPLC on a polystyrene-divinylbenzene column. On-line LC-MS data allowed facile distinction between adducts on the two strands of the duplex, and MS/MS analysis permitted unambiguous assignment of the major sites of modification in the regioisomeric, adducted strands. In the duplex, these sites were at A(6), A(7), and G(8). Interestingly, the "hot spot" A(7)w as about 3 times more reactive with the BcPh DE than the "cold spot" A(6). Adduct formation from the single-stranded dodecamer was less selective, and resulted in more extensive alkylation of G residues.

Incorporation of 4-thiothymidine into DNA by the Klenow fragment and HIV-1 reverse transcriptase.

The 5'-triphosphate of 4-thiothymidine (4S-TTP) is an excellent substrate for the Klenow fragment of Escherichia coli DNA polymerase 1 and HIV-1 reverse transcriptase with values of k(cat)/Km within a factor of approximately 3 of those for TTP. A large UV change (deltaepsilon= -9770 M(-1)cm(-1) at 340 nm) associated with incorporation of 4S-TMP into nucleic acid duplexes makes possible a rapid, continuous spectrophotometric assay of the reaction progress.

Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages.

The catalytic intermediates of DNA topoisomerase I (top1) are cleavage complexes that can relax DNA supercoiling (intramolecular reaction) or mediate recombinations (intermolecular religation). We report here that DNA adducts formed from benzo[a]pyrene bay-region diol epoxides can markedly affect top1 activity. Four oligonucleotide 22-mers of the same sequence were synthesized, each of which contained a stereoisomerically unique benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the 2-amino group of a central 2'-deoxyguanosine residue. These four adducts correspond to either cis or trans opening at C-10 of the (+)-(7R, 8S, 9S, 10R)- or (-)-(7S, 8R, 9R, 10S)-7,8-diol 9,10-epoxides. Their solution conformations in duplex DNA (intercalated and minor-groove bound for the cis and trans opened adducts respectively) can be deduced from previous NMR studies. All four adducts completely suppress top1 cleavage activity at the alkylation site and induce the formation of new top1cleavage complexes on both strands of the DNA 3-6 bases away from the alkylation site. The trans opened adduct from the highly carcinogenic (+)-diol epoxide is the most active in inducing top1 cleavage independently of camptothecin, demonstrating that minor groove alkylation can efficiently poison top1. We also found that this isomer of the diol epoxide induces the formation of top1-DNA complexes in mammalian cells, which suggests a possible relationship between induction of top1 cleavage complexes and carcinogenic activity of benzo[a]pyrene diol epoxides.