Hydrocarbon carcinogens induce p53 activity in normal mouse tissue.

Previous studies led to the suggestion that a sub class of hydrocarbon carcinogens have the capacity of damage DNA and evade normal cellular defence mechanisms. To examine the ability of hydrocarbon carcinogens to induce p53 transcriptional activity in normal tissues, we have used transgenic mice in which LacZ transgene is driven by a p53 response element. We demonstrate that transcriptionally active p53 is induced in normal tissue after exposure to putative stealth carcinogens in an identical manner to non-stealth agents.

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.

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.

Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2.

The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.

Diverse chemical carcinogens fail to induce G(1) arrest in MCF-7 cells.

The effect of three reactive potent chemical carcinogens on the passage of MCF-7 cells through the cell cycle was investigated. While these cells, which express wild-type p53, were arrested in G(1) after treatment with actinomycin D (a positive control), treatment with anti-benzo[a]pyrene dihydrodiol epoxide, N-acetoxy-N-2-fluorenylacetamide or N-methyl-N'-nitro-N-nitrosoguanidine, at doses consistent with survival of significant numbers of cells, caused the cells to accumulate in S phase, with little increase in those in G(1). This property of these three reactive potent carcinogens, of diverse chemical types, to induce evasion of G(1) arrest (the stealth property) presumably increases the likelihood of malignant change, because DNA replication continues on a damaged template. This stealth characteristic may be a major contributor to the tumorigenicity of DNA-adducting chemical carcinogens in general.

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene.

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5, 6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at A&z.ccirf;T or G&z.ccirf;C pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide-DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.

Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts.

Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity.

Mutagenicity of benzo[a]pyrene-deoxyadenosine adducts in a sequence context derived from the p53 gene.

Mutations in the human p53 tumor suppressor gene are prominently linked to sporadic cancers in breast, lung and other tissues. Recent research has shown that tobacco-associated cancer in the human lung is related to mutation of the p53 gene mediated by the carcinogen benzo[a]pyrene (BaP), and the mutations are targeted to DNA "hot spots" at specific codons. In order to gain insight into the relation between the structures of the adducts formed by BaP at these sites and their mutagenic activities, we have synthesized site-specifically modified oligo-nucleotide adducts of the active BaP diol epoxide metabolite (anti-BaPDE). This manuscript reports on the mutagenic consequences of replication past anti-BaPDE-deoxyadenosine adducts located within a sequence context related to codon 157 in exon 5 of the p53 gene. In this sequence context, the adduct derived from the carcinogenic 7R,8S-dihydrodiol 9S,10R-epoxide was much more active as a mutagen than the adduct derived from the noncarcinogenic 7S,8R-dihydrodiol 9R,10S-epoxide and the mutation found most frequently was an A-->G transition. Since previous studies in other sequence contexts have yielded somewhat different findings, these studies further emphasize the key role played by sequence context in determining the mutational properties of carcinogen-DNA adducts.

Lack of p53-mediated G1 arrest in response to an environmental carcinogen.

The environmental carcinogen, 5-methylchrysene, is a component of cigarette smoke. Its reactive metabolite, anti-5-methylchrysene-1, 2-dihydrodiol-3,4-epoxide (5-MeCDE) mainly reacts with the N(2)-position of guanine residues in the DNA molecule. In this study, we demonstrate that the tumor suppressor protein p53 is stabilized in response to DNA damage by 5-MeCDE but fails to induce the cells' protective mechanism of G1 arrest in the human breast carcinoma cell line, MCF-7. In contrast, actinomycin D treatment of these cells did lead to G1 arrest. Western analyses revealed that, though both actinomycin D and 5-MeCDE treatment stabilized p53, only trace levels of p21(waf1/cip1) were seen in the latter case. This lack of p21(waf1/cip1) expression in 5-MeCDE-treated cells is attributed to a stealth characteristic of this environmental carcinogen that allows it to damage DNA and still escape the p53-mediated cellular defense mechanism of G1 arrest.

Formation of deaminated products in styrene oxide reactions with deoxycytidine.

The reaction of racemic styrene oxide with deoxycytidine under aqueous conditions was studied. The four principal products isolated were a pair of diastereomeric N(4)-(2-hydroxy-1-phenylethyl)deoxycytidines ( approximately 20% of the products) and a pair of diastereomeric 3-(2-hydroxy-2-phenylethyl)deoxyuridines ( approximately 80% of the products). Reactions with optically active styrene oxides allowed the configurations of the 3-(2-hydroxy-2-phenylethyl)deoxyuridines to be assigned, and these structures were confirmed by an independent synthesis from deoxyuridine. Also, it was possible to tentatively assign the configurations of the N(4)-(2-hydroxy-1-phenylethyl)deoxycytidines that had undergone some racemization during the reaction (the ratio of the retained to inverted configuration of the products was approximately 1:7).

Metabolic activation of 4H-cyclopenta[def]chrysene in human mammary carcinoma MCF-7 cell cultures.

The tumor initiating activities of 4H-cyclopenta[def]chrysene (C[def]C) and its two putative reactive metabolites, trans-1, 2-dihydroxy-anti-3,3a-epoxy-1,2,3, 3a-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-3,3a-DE) and trans-6,7-dihydroxy-anti-8,9-epoxy-6,7,8, 9-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-8,9-DE), were evaluated previously in mice [Amin, S., et al. (1995) Carcinogenesis 16, 2813-2817]. C[def]C-3,3a-DE was the more active inducer of lung tumors and elicited twice as many tumors as C[def]C-8,9-DE. In this study, the route of metabolism of C[def]C to DNA-reactive metabolites in the human mammary carcinoma cell line (MCF-7) was investigated using the 32P-postlabeling assay. The results show that metabolic activation to DNA-binding species proceeds through the formation of both trans-1,2-dihydrodiol and trans-6,7-dihydrodiol metabolites of C[def]C. At a 1 microM dose, adducts from the methylene-bridged (C[def]C-3,3a-DE) and bay region (C[def]C-8,9-DE) dihydrodiol epoxides were detected in comparable amounts. In contrast, the majority of the postlabeled adducts recovered from cells exposed to a 10 microM dose were derived from the bay region dihydrodiol epoxide, C[def]C-8,9-DE. Using markers from reactions of the dihydrodiol epoxides with deoxyguanosine 3'-phosphate and deoxyadenosine 3'-phosphate, it was shown that the major radioactive spots formed with both anti-C[def]C-3,3a-DE and anti-C[def]C-8,9-DE chromatographed with deoxyguanosine adduct markers. Thus, the human cells used in these studies can activate C[def]C to carcinogenic metabolites.

Mutational consequences of replication of M13mp7L2 constructs containing cis-opened benzo[a]pyrene 7,8-diol 9, 10-epoxide-deoxyadenosine adducts.

The four adducts that arise by cis ring opening of the four optically active benzo[a]pyrene diol epoxides by the exocyclic N6-amino group of deoxyadenosine were incorporated synthetically into each of two different oligonucleotide 16-mers, 5'-TTTXGAGTCTGCTCCC-3' [context I(A)] and 5'-CAGXTTTAGAGTCTGC-3' [context II(A)], at the X position. The eight resultant oligonucleotides were separately ligated into bacteriophage M13mp7L2 and replicated in Escherichia coli that had been SOS-induced, and the progeny were analyzed to evaluate the consequences of replication past these adducts. The presence of these adducts reduced plaque yields substantially. However, the progeny obtained exhibited high frequencies of base substitution mutation ranging from 9 to 68%, depending upon the individual adduct and the sequence context in which it was placed. For most of the adducts, A --> T transversion was the mutation found most frequently in either sequence context, and mutation frequencies in context I(A) were always substantially greater than those in context II(A). In context I(A), adducts with an R configuration at the site of nucleoside attachment were more mutagenic than those with an S configuration. In both sequence contexts that were studied, the cis adduct arising from the (7S,8R)-diol (9S,10R)-epoxide was the most mutagenic adduct. These findings clearly show that individual mutation frequencies are determined by the combined effects of both adduct structure and sequence context.

Sequence context effects on mutational properties of cis-opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts in site-specific mutation studies.

Diastereomeric N6-substituted dAdo adducts (cis B[c]PhDE-2/1R and cis B[c]PhDE-2/1S) that correspond to cis-opening at C-1 of the enantiomeric benzo[c]phenanthrene 3,4-diol 1,2-epoxides in which the epoxide oxygen and the benzylic hydroxyl group are trans (DE-2) were synthetically incorporated into oligonucleotide 16-mers. Each adduct was placed at the fourth nucleotide from the 5'-end of each of two different oligonucleotide sequences derived from the E. coli supF gene. Each adduct was also placed in two additional oligonucleotide sequences that were constructed by interchanging the adduct site and the immediately adjacent nucleotides between the two original sequences. These oligonucleotides were designed for use in site-specific mutation studies, with a single-stranded bacteriophage M13mp7L2 vector, to determine if the effects of sequence context on types and frequencies of base substitution mutations are attributable only to nucleotides immediately adjacent to these polycyclic aromatic hydrocarbon diol epoxide-dAdo adducts, or whether more distant nucleotide residues also affect the mutagenic response. In SOS-induced Escherichia coli SMH77, total base substitution mutation frequencies for the cis B[c]PhDE-2/1R-dAdo adduct were relatively low (0.62-5.6%) compared with those for the cis B[c]PhDE-2/1S-dAdo adduct (11.9-56.5%). Depending on sequence context, cis B[c]PhDE-2/1R-dAdo gave predominantly A-->T or a more equal distribution of A-->T and A-->G mutations whereas cis B[c]PhDE-2/1S-dAdo gave either predominantly A-->T or predominantly A-->G base substitutions. Our results clearly indicate that nucleotides that are distal as well as those that are proximal to the adduct site are capable of influencing both the mutation frequency and the distribution of base substitution mutations.

DNA reactions, mutagenic action and stealth properties of polycyclic aromatic hydrocarbon carcinogens (review).

A brief summary of recent research, primarily from the authors' laboratory, on polycyclic aromatic hydrocarbon carcinogens with respect to their DNA adduct formation, the mutational properties of these adducts and the effects of hydrocarbon dihydrodiol epoxide metabolites on the passage of cells through the cell cycle is presented. The concept of stealth properties of potent carcinogens, i.e. their ability to damage DNA without inducing a G1 arrest, is discussed. Also, mutation studies with dihydrodiol epoxide metabolites, the sequence-dependence of site-specific mutation, as well as the selectivity of hydrocarbon-DNA adduct formation are summarized.

DNA adduct levels associated with p53 induction and delay of MCF-7 cells in S phase after exposure to benzo[g]chrysene dihydrodiol epoxide enantiomers.

Optically active isomers of a mammary carcinogen, anti-benzo[g]chrysene 11, 12-dihydrodiol 13, 14-epoxide, react to different extents with DNA and generate DNA adducts that differ in their stereochemistry. In the study reported here, the effect of these two enantiomers on the progress of human breast carcinoma MCF-7 cells through the cell cycle was investigated. Each enantiomer caused the cells to accumulate in the S phase, but a higher dose of the benzo[g]chrysene 11S, 12R-dihydrodiol 13R, 14S-epoxide than of its enantiomer was required to induce this effect. Similarly, induction of p53 also required a higher dose of benzo[g]chrysene 11S, 12R-dihydrodiol 13R, 14S-epoxide. Postlabeling studies indicated that the latter enantiomer also caused less modification of MCF-7 cell DNA for a given level of exposure than did benzo[g]chrysene 11R, 12S-dihydrodiol 13S, 14R-epoxide. These results suggest that p53 induction and delay in the S phase are similarly related to DNA binding and that a level of binding of the order of 1 adduct per 10(5) nucleotides is associated with these effects.

Separation and identification of positively charged and neutral nucleoside adducts by capillary electrochromatography-microelectrospray mass spectrometry.

Capillary electrochromatography (CEC) is shown to be capable of separating mixtures containing both positively charged and neutral styrene oxide-adenosine adducts. In a study of the mechanism of deamination of positively charged 1-(2-hydroxy-1-phenylethyl) adenosine using 18O-labeled water, possible contamination of the chromatographically purified deamination product, 1-(2-hydroxy-1-phenylethyl) inosine, with the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine was observed. Because the deamination product and the presumed contamination have the same molecular weights and similar structures, CEC-microelectrospray mass spectrometry (CEC-microESI/MS) was used to confirm the presence and identity of the suspected impurity. A trace amount of the positively charged 1-(2-hydroxy-1-phenylethyl) adenosine, which could not be observed by either HPLC-UV or CEC-UV, was detected by CEC-microESI/MS. This discriminatory ability of CEC-microESI/MS is attributed to the fact that positive ion mode ESI-MS is a more sensitive detector for a positively charged compound than a UV detector, and that the combination of electroosmotic and electrophoretic flows and hydrophobic interactions with the stationary phase contributes to the separation of the positively charged compound. As a result, the positively charged compound was observed to elute much earlier and with much sharper peaks than the neutral compounds for which electroosmotic flow is the only "pumping" force for the solvent.

Deamination and Dimroth rearrangement of deoxyadenosine-styrene oxide adducts in DNA.

In reactions between styrene oxide and the ring nitrogen at the 1-position of deoxyadenosine, the epoxide is opened at both the alpha- (benzylic) and beta-carbons. The 1-substituted nucleosides formed are unstable and subsequently undergo either Dimroth rearrangement to give N6-substituted deoxyadenosines or deamination to give 1-substituted deoxyinosines. alphaN6-Substituted compounds are also formed from direct reaction at the exocyclic nitrogen. Kinetic experiments revealed that relative rates of deamination of 1-substituted deoxyadenosine-styrene oxides and 1-substituted adenosine-styrene oxides were similar. However, the rate of Dimroth rearrangement in beta1-substituted adenosine-styrene oxides was approximately 2.3-fold greater than that of beta1-substituted deoxyadenosine-styrene oxides and approximately 1.5-fold greater in alpha1-substituted adenosine-styrene oxides relative to alpha1-substituted deoxyadenosine-styrene oxides. Analysis of the products formed from reactions of styrene oxide with [3H]deoxyadenosine and [3H]deoxyadenosine incorporated into native and denatured DNA showed that the double-helical DNA structure reduced the levels of adducts formed 5-fold relative to denatured DNA but did not present a complete barrier to formation of either N6-substituted deoxyadenosine- or 1-substituted deoxyinosine-styrene oxide adducts in native DNA. Additionally, in denatured and native DNA the product distributions were altered in favor of formation of beta1-substituted deoxyinosine-styrene oxide adducts with respect to reactions of the nucleoside. The ratio of retained to inverted configuration of alphaN6-substituted products was higher in DNA than in nucleoside reactions. These experiments indicate that in addition to the N6-position, the ring nitrogen at the 1-position of deoxyadenosine is available, to some extent, for reaction in native DNA. In styrene oxide-DNA reactions, formation of 1-substituted adenines can lead to deaminated products where both Watson-Crick hydrogen-bonding sites are disrupted.

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.

Sequence context profoundly influences the mutagenic potency of trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-purine nucleoside adducts in site-specific mutation studies.

The postoligomerization method was used to prepare oligonucleotide 16-mers that contained dAdo or dGuo adducts, derived from trans opening of each enantiomer of the two diastereomeric benzo[a]pyrene 7,8-diol 9,10-epoxides, in two sequence contexts. These 16 oligonucleotides, along with the four corresponding oligonucleotides containing unsubstituted purines, were ligated into single-stranded DNA from bacteriophage M13mp7L2 and transfected into Escherichia coli SMH77. The mutagenic effects of replication past these adducts were then evaluated. The various adduct isomers induced point mutations at different frequencies and with different distributions of mutation types, as was anticipated. However, sequence context had the most substantial effects on mutation frequency. A high frequency of deletions of a single guanine was found in a context where the dGuo adduct was at the 3'-end of a run of five guanines, whereas no single base deletion was found in the other context studied, 5'-CGA-3'. Mutation frequencies in constructs containing dAdo adducts were much higher in a 5'-TAG-3' context (37-58%, depending on the individual isomer) than in a 5'-GAT-3' context (5-20%), and for a given adduct, mutation frequency was up to 10-fold higher in the former sequence than in the latter. These findings indicate that sequence context effects need more thorough evaluation if the goal of understanding the mechanism through which DNA adducts lead to mutation is to be achieved.

Effect of cadmium exposure on background and anti-5 methylchrysene-1,2-dihydrodiol 3,4-epoxide-induced mutagenesis in the supF gene of pS189 in human Ad293 cells.

Cadmium is a toxic environmental contaminant that is carcinogenic in humans and rodents. Although cadmium has proven to be mutagenic in a variety of assay systems, exactly how cadmium achieves gentoxicity is poorly understood. To define the mechanism(s) underlying the mutagenicity and comutagenicity of cadmium, human Ad293 cells were exposed to subtoxic doses of the metal and transfected with untreated or anti-5-methylchrysene-3,4-dihydrodiol 1,2-epoxide (5-MCDE)-treated pS189 shuttle vector. Alterations in the frequency, types, and distribution of mutations were subsequently assessed in the supF gene of pS189 that was replicated in Ad293 cells and screened in indicator bacteria. Doses of 0.5 and 1 microM CdCl2 increased the mutation frequency of untreated pS189 by approximately 4- and 8-fold, respectively, with no apparent effect on the types of mutations generated. In contrast, host-cell exposure to cadmium had little or no effect on the frequency, types, or distribution of mutations generated with 5-MCDE-treated pS189. These results indicate that cadmium increases mutagenesis of untreated pS189 by affecting a process that is not involved in mutagenesis of the 5-MCDE-treated vector. Although it is not clear exactly how host-cell exposure to cadmium increases background mutagenesis, presumably, the mutagenic effect does not involve cadmium interaction with the cellular machinery used to replicate past bulky DNA lesions.