DNA adducts formed from 4-hydroxytamoxifen are more mutagenic than those formed by alpha-acetoxytamoxifen in a shuttle vector target gene replicated in human Ad293 cells

The drug tamoxifen, used to treat breast cancer, causes liver cancer in rats and endometrial cancer in women. Tamoxifen forms liver DNA adducts in both short- and long-term dosing of rodents, and DNA adducts have also been reported in tissues of women undergoing tamoxifen therapy. It is not known if the induction of endometrial cancer in women is through these DNA adducts or through the estrogenic nature of the drug. In this study, we have investigated the mutagenicity of two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM). These form the same DNA adducts as those found in tamoxifen-treated rats. The two compounds were used to treat the pSP189 plasmid containing the supF gene, which was replicated in Ad293 cells before being screened in indicator bacteria. Plasmid reacted with 4-OHtamQM was more likely to be mutated (2-7-fold increase) than that reacted with alpha-acetoxytamoxifen, despite having a lower level of DNA damage (12-20-fold less), as assayed by (32)P-postlabeling. The two compounds induced statistically different mutation spectra in the supF gene. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC -->TA transversions while GC-->AT transitions were formed in 4-OHtamQM-treated plasmid. 4-OHTamQM-treated DNA induced a larger proportion of multiple mutations and large deletions compared to alpha-acetoxytamoxifen. Sites of mutational hotspots were observed for both compounds. In conclusion, the quantitatively minor DNA adduct of tamoxifen (dG-N(2)-4-hydroxytamoxifen) is more mutagenic than the major tamoxifen DNA adduct (dG-N(2)-tamoxifen).     

Comparison of the mutagenic activity of the benzene metabolites, hydroquinone and para-benzoquinone in the supF forward mutation assay: a role for minor DNA adducts formed from hydroquinone in benzene mutagenicity

Benzene, a ubiquitous environmental pollutant and occupational hazardous chemical, is a recognised human leukaemogen and rodent carcinogen. The mechanism by which benzene exerts its carcinogenic effects is to date unknown but it is considered that mutations induced by benzene-DNA adducts may play a role. The benzene metabolite, para-benzoquinone (p-BQ) following reaction in vitro with DNA, forms four major adducts, which include two adducts on 2'-deoxyguanosine 3'-monophosphate (dGp). Reaction of DNA with the benzene metabolite hydroquinone (HQ) results in only one major DNA adduct, which corresponds to one of the dGp adducts formed following reaction with p-BQ. The mutagenicity of the adducts formed from these two benzene metabolites was investigated using the supF forward mutation assay. Metabolite-treated plasmid (pSP189) containing the supF gene was replicated in human Ad293 cells before being screened in indicator bacteria. Treatment with 5-20 mM p-BQ gave a 12 to 40-fold increase in mutation rate compared to 5-20 mM HQ treatment, a result reflected in the level of DNA modification observed (8 to 26-fold increase compared to HQ treatment). Treatment with p-BQ gave equal numbers of GC --> TA transversions and GC --> AT transitions, whereas treatment with HQ gave predominantly GC-->AT transitions. The spectra of mutations achieved for the two individual treatments were shown to be significantly different (P = 0.004). A combination of both treatments also resulted in a high level of GC --> AT transitions and a synergistic increase in the number of multiple mutations, which again predominated as GC --> AT transitions. Sites of mutational hotspots were observed for both individual treatments and one mutational hotspot was observed in the multiple mutations for the combined treatment. These results suggest that the dGp adducts formed from benzene metabolite treatment may play an important role in the mutagenicity and myelotoxicity of benzene.     

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots

Tamoxifen elevates the risk of endometrial tumours in women and alpha-(N(2)-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with alpha-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N(2)-tamoxifen and dG-N(2)-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both alpha-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for alpha-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only alpha-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC-->TA transversions. Application of the LwPy53 algorithm to the alpha-acetoxytamoxifen spectrum predicted strong GC-->TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N(2)-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.     

Mutagenesis by site-specific arylamine adducts in plasmid DNA: enhancing replication of the adducted strand alters mutation frequency

Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by greater than 7-fold to 2.9% and of the AAF adduct by greater than 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA- strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigating DNA adduct-targeted mutagenicity of tamoxifen: preferential formation of tamoxifen-DNA adducts in the human p53 gene in SV40 immortalized hepatocytes but not endometrial carcinoma cells

Tamoxifen is a widely used drug for chemotherapy and chemoprevention of breast cancer worldwide. Tamoxifen therapy is, however, associated with an increased incidence of endometrial cancer. The carcinogenicity of tamoxifen is ascribed to its genotoxic and estrogen agonist effects. We investigated DNA adduct-targeted mutagenicity of tamoxifen as a function of its genotoxicity in the cII transgene in Big Blue mouse embryonic fibroblasts and mapped the formation of tamoxifen-induced DNA adducts in the p53 tumor suppressor gene in SV40 immortalized human hepatocytes and human endometrial carcinoma cells. We used the terminal transferase-dependent polymerase chain reaction for mapping of DNA adducts in the cII and p53 genes. We utilized a lambda phage-based assay and DNA sequencing for determining cII mutant frequency and mutation spectrum, respectively. Tamoxifen treatment yielded polymerase-blocking DNA adducts at multiple nucleotide positions along the cII transgene. The treatment significantly and dose-dependently increased the cII mutant frequency (p < 0.01), leaving a unique mutation spectrum (p < 0.0001) and a signature mutation of G:C --> T:A transversions (p < 0.03), relative to the control. Tamoxifen treatment of the immortalized human hepatocytes but not endometrial carcinoma cells, even in the presence of an external activation system, i.e., rat liver S9 mix, induced DNA adducts at specific codons along exons 6 and 8 of the p53 gene. These data suggest a proficient metabolic activation of tamoxifen in human liver and an inefficient activation and/or efficient detoxification of tamoxifen in human endometrium. Because the liver is essentially a mitotically quiescent organ, tamoxifen-DNA adduction in the liver may, at least partially, prevent its reactants from reaching highly proliferative organs via, e.g., circulating blood. Thus, tamoxifen-DNA adduction in the liver may not have as significant biological consequences as it might have in highly proliferative organs. Our findings favor an involvement of a nongenotoxic mechanism in tamoxifen-associated human endometrial cancer.     

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.     

A single site-specific trans-opened 7,8,9,10-tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct induces mutations at multiple sites in DNA

Site-specific mutagenicity of trans-opened adducts at the exocyclic N(2)-amino group of guanine by the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R,10S)-enantiomers of a benzo[a]pyrene 7,8-diol 9,10-epoxide (7-hydroxyl and epoxide oxygen are trans, BPDE-2) has been determined in Chinese hamster V79 cells and their repair-deficient counterpart, V-H1 cells. Four vectors containing single 10S-BPDE-dG or 10R-BPDE-dG adducts positioned at G(0) or G(-1) in the analyzed 5'-ACTG(0)G(-1)GA sequence of the non-transcribed strand were separately transfected into the cells. Mutations at each of the seven nucleotides were analyzed by a novel primer extension assay using a mixture of one dNTP complementary to the mutated nucleotide and three other ddNTPs and were optimized to quantify levels of a mutation as low as 1%. Only G --> T mutations were detected at the adducted sites; the 10S adduct derived from the highly carcinogenic (+)-diol epoxide was 40-50 and 75-140% more mutagenic than the 10R adduct in V79 and V-H1 cells, respectively. Importantly, the 10S adducts, but not the 10R adducts, induced separate non-targeted mutations at sites 5' to the G(-1) and G(0) lesions (G(0) --> T and C --> T, respectively) in both cell lines. Neither the T 5' to G(0) nor sites 3' to the lesions showed mutations. Non-targeted mutations may enhance overall mutagenicity of the 10S-BPDE-dG lesion and contribute to the much higher carcinogenicity and mutagenicity of (+)-BPDE-2 compared with its (-)-enantiomer. Our study reports a definitive demonstration of mutations distal to a site-specific polycyclic aromatic hydrocarbon adduct.     

Non-oxidative mechanisms are responsible for the induction of mutagenesis by reduction of Cr(VI) with cysteine: role of ternary DNA adducts in Cr(III)-dependent mutagenesis

Intracellular reduction of carcinogenic Cr(VI) generates Cr-DNA adducts formed through the coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the role of Cr(III)-DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction of Cr(VI) caused a strong oxidation of 2', 7'-dichlorofluoroscin (DCFH) and extensive Cr-DNA binding but no DNA breakage. Cr-DNA adducts induced unwinding of supercoiled plasmids and structural distortions in the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189 plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition of replication. Blocking of Cr(III)-DNA binding by occupation of DNA phosphates with Mg(2+) or by sequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored a normal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversal procedure returned mutation frequency to background levels. The mutagenic responses at the different phases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the number and the spectrum of Cr(III)-DNA adducts that were formed. Ternary cysteine-Cr(III)-DNA adducts were approximately 4-5 times more mutagenic than binary Cr(III)-DNA adducts. Although intermediate reaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactive to damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations. The majority of mutations occurred at the sites where G had adjacent purine in the 3' or 5' position. Overall, our results present the first evidence that Cr(III)-DNA adducts play the dominant role in the mutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.     

Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot. A new probe for frameshift mutagenesis

N-2-acetylaminofluorene (AAF), a potent rat liver carcinogen, binds primarily to the C-8 position of guanine residues. In a bacterial forward mutation assay, more than 90% of the mutations induced by -AAF adducts are frameshift mutations located at specific sites: the so-called mutation hot spots. We are particularly interested in a class of -2 frameshift mutations occurring within a specific sequence, the NarI sequence. The NarI site, GGCGCC, contains three guanine residues that are approximately equally reactive toward -AAF substitution. To study further the mechanism by which mutations are induced by -AAF adducts at this site, we designed a new plasmid probe. In this paper we describe the construction and the effectiveness of this probe, pSM14, which provides a simple phenotypic test for detecting frameshift mutations within the NarI site. The construction and the characterization of plasmids with a single -AAF adduct in each of the three positions of the NarI site are also described. The strategy of construction that was used involves the ligation of oligonucleotides containing a single adduct in a NarI site into a gapped-duplex pSM14 plasmid. Plasmids that have successfully integrated the oligonucleotides by ligation at both the 5' and the 3' ends were purified by centrifugation on CsCl gradients. These constructs have been used in single adduct mutation studies.     

Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)glutathione. Evidence for a role of S-(2-N7-guanyl)ethyl)glutathione as a mutagenic lesion formed from ethylene dibromide.

The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.     

Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.     

Genotoxicity and mutagenicity of chromium(VI)/ascorbate-generated DNA adducts in human and bacterial cells

Reduction of carcinogenic Cr(VI) by vitamin C generates ascorbate-Cr(III)-DNA cross-links, binary Cr(III)-DNA adducts, and can potentially cause oxidative DNA damage by intermediate reaction products. Here, we examined the mutational spectrum and the importance of different forms of DNA damage in genotoxicity and mutagenicity of Cr(VI) activated by physiological concentrations of ascorbate. Reduction of Cr(VI) led to a dose-dependent formation of both mutagenic and replication-blocking DNA lesions as detected by propagation of the pSP189 plasmids in human fibroblasts. Disruption of Cr-DNA binding abolished mutagenic responses and normalized the yield of replicated plasmids, indicating that Cr-DNA adducts were responsible for both mutagenicity and genotoxicity of Cr(VI). The absence of DNA breaks and abasic sites confirmed the lack of a significant production of hydroxyl radicals and Cr(V)-peroxo complexes in Cr(VI)-ascorbate reactions. Ascorbate-Cr(III)-DNA cross-links were much more mutagenic than smaller Cr(III)-DNA adducts and accounted for more than 90% of Cr(VI) mutagenicity. Ternary adducts were also several times more potent in the inhibition of replication than binary complexes. The Cr(VI)-induced mutational spectrum consisted of an approximately equal number of deletions and G/C-targeted point mutations (51% G/C --> T/A and 30% G/C --> A/T). In Escherichia coli cells, Cr(VI)-induced DNA adducts were only highly genotoxic but not mutagenic under either normal or SOS-induced conditions. Lower toxicity and high mutagenicity of ascorbate-Cr(III)-DNA adducts in human cells may result from the recruitment of an error-prone bypass DNA polymerase(s) to the stalled replication forks. Our results suggest that phosphotriester-type DNA adducts could play a more important role in human than bacterial mutagenesis.     

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide

Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+/-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the (32)P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.     

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.     

Ability of adducts formed in a shuttle vector by reactive metabolites of 1-nitropyrene and benzo(a)pyrene to induce mutations when the plasmid replicates in human cells

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF), was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into human cell line 293, allowed to replicate, and the progeny plasmids rescued and assayed for the frequency of supF mutants. The agents tested were the 7,8-diol-9,10-epoxide of benzo(a)pyrene (BPDE) and 1-nitrosopyrene (1-NOP). With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies 15 to 25 times higher than the background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately four times more mutagenic than 1-NOP, which binds principally at the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule, but it could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least three times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base substitutions. DNA sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmid and 60 from 1-NOP-treated plasmid indicated that 70% to 80% contained a single base substitution, 5%-10% had two base substitutions, and 4%-10% had small insertions or deletions (one or two basepairs). The majority (83%) of the base substitutions in mutants from BPDE- or 1-NOP-treated plasmid were transversions, mainly G.C----T.A. Each carcinogen produced its own spectrum of mutations.     

Site-specific tamoxifen-DNA adduct formation: lack of correlation with mutational ability in Escherichia coli.

We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.     

Mutagenic properties of estrogen quinone-derived DNA adducts in simian kidney cells

DNA damage caused by catechol estrogens has been shown to play an etiologic role in tumor formation. Catechol estrogens are reactive to DNA and form several DNA adducts via their quinone forms. To explore the mutagenic properties of 2-hydroxyestrogen-derived DNA adducts in mammalian cells, N(2)-(2-hydroxyestrogen-6-yl)-2'-deoxyguanosine and N(6)-(2-hydroxyestrogen-6-yl)-2'-deoxyadenosine adducts induced by quinones of 2-hydroxyestrone, 2-hydroxyestradiol, or 2-hydroxyestriol were incorporated site-specifically into the oligodeoxynucleotides ((5)(')TCCTCCTCXCCTCTC, where X is dG, dA, 2-OHE-N(2)-dG, or 2-OHE-N(6)-dA). The modified oligodeoxynucleotides were inserted into single-stranded phagemid vectors followed by transfection into simian kidney (COS-7) cells. Preferential incorporation of dCMP, the correct base, was observed opposite all 2-OHE-N(2)-dG adducts. Only targeted G --> T transversions were detected; the highest mutation frequency (18.2%) was observed opposite the 2-OHE(2)-N(2)-dG adduct, followed by 2-OHE(1)-N(2)-dG (4.4%) and 2-OHE(3)-N(2)-dG (1.3%). When 2-OHE-N(6)-dA adducts were used, preferential incorporation of dTMP, the correct base, was observed. Targeted mutations representing A --> T transversions were detected, accompanied by small numbers of A --> G transitions. The highest mutation frequencies were observed with 2-OHE(1)-N(6)-dA and 2-OHE(3)-N(6)-dA (14.5 and 14.1%, respectively), while 2-OHE(2)-N(6)-dA exhibited a mutation frequency of only 6.0%. No mutations were detected with vectors containing unmodified oligodeoxynucleotides. Thus, 2-OHE quinone-derived DNA adducts are mutagenic, generating primarily G --> T and A --> T mutations in mammalian cells. The mutational frequency varied depending on the nature of the 2-OHE moiety.     

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.     

Comparing the frequency and spectra of mutations induced when an SV-40 based shuttle vector containing covalently bound residues of structurally-related carcinogens replicates in human cells

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF) was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into the human embryonic kidney cell line 293 and allowed to replicate. The progeny plasmids were rescued and assayed for the frequency of supF mutants by being used to transform indicator bacteria carrying an amber mutation in the beta-galactosidase gene. The agents tested were the 7,8-diol-9,10-epoxide of benzo[a]pyrene (BPDE); 1-nitrosopyrene (1-NOP); N-acetoxy-2-acetylaminofluorene (N-AcO-AAF); and its trifluoro-derivative (N-AcO-F3-AAF) which yields deacetylated adducts. With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies as high as 20 x 10(-4) to 40 x 10(-4), with a background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid, BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately 4 times more mutagenic than 1-NOP, N-AcO-AAF and N-AcO-F3-AAF, which bind principally or exclusively to the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule. It could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least 3 times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of 35 mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base-substitutions. DNA-sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmids and 60 from 1-NOP-treated plasmids indicated that 60% and 80%, respectively, contained a single base-substitution, 5-10% had two base-substitutions, and 4-10% had small insertions or deletions (one or two base pairs).(ABSTRACT TRUNCATED AT 400 WORDS)