Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[a]pyrene diol epoxides in CHO cells.

The initiation of carcinogenesis by carcinogens such as 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7r,8t-dihydroxy-9,10c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37 degrees C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in greater than 20% survival, and were found to increase linearly with dose. In the DNA-repair-deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10(6) survivors at a dose that resulted in one adduct per 10(6) base pairs) with our previous studies of BPDE-1, we found that BPDE-II was 4-5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts.     

Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[a]pyrene.

Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[a]pyrene, 9r,10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D0's, the DNA-repair-deficient cell lines were found to be 4-5-fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells.     

Stabilization of a reactive, electrophilic carcinogen, benzo[a]pyrene diol epoxide, by mammalian cells

We have studied several features of the interactions of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with a DNA repair-proficient derivative of Chinese hamster ovary cells (CHO), AT3-2, and with a UV-light sensitive mutant, UVL-10, derived from AT3-2. Methods were developed for quantitating the amount of unhydrolysed BPDE-I associated with cells and for purifying DNA from cells under conditions where artificial labeling during preparation is minimized. In both cell types, about 30% of the BPDE-I added to a cell culture is rapidly taken up by the cells and is maintained in a cellular compartment in which the half-life of BPDE-I is about 10-fold longer than in aqueous medium. The kinetics of covalent binding to DNA were measured in both cell types and found to be described well by a single exponential process with a half-life of about 60 min. This is virtually identical to the half-life for intracellular hydrolysis of BPDE-I (57 min), consistent with the suggestion that this intracellular, relatively stable BPDE-I is responsible for binding.     

The aprt heterozygote/hemizygote system for screening mutagenic agents allows detection of large deletions

Frequencies of mutation at the hprt and aprt loci in various CHO cell lines were measured after exposure of the cells to ionizing radiation. In D423 and AA8-16, which are aprt+/- heterozygotes, the ratio of hprt- mutants to aprt- mutants ranged from 0.11 to 0.36. In D422 and AA8-5, which are aprt+/0 cell lines in which only one copy of the gene and its flanking sequences is present these ratios were greater than 5. In contrast, chemical mutagenesis generated mutations at both loci, in all cell lines, at equal frequencies. Southern blot analysis of DNA from hprt- and aprt- mutants of one of the aprt+/- heterozygous lines showed some apparently unaltered genes, some rearrangements and some complete deletions of hprt among hprt- mutants, but only complete deletions of aprt-linked sequences among aprt- mutants. These results strongly suggest that X-ray-induced mutational events are frequently larger than 40 kb (the length of the hprt gene) and that the difference among the frequencies observed at the two loci in the two types of cell lines were due to the presence of essential sequences close the respective target genes. The combined use of these cell lines in screening environmental mutagens should allow qualitative as well as quantitative analysis of the mutagenic potential of environmental agents.     

Hypersensitivity to cell killing and mutation induction by chemical carcinogens in an excision repair-deficient mutant of CHO cells

A strain of Chinese hamster ovary cells that is deficient in nucleotide excision repair, strain UV5, was compared with the normal parental CHO cells in terms of cytotoxicity and mutagenesis after exposure to several chemical carcinogens that are known to produce bulky, covalent adducts in DNA. Induced mutations were measured at the hprt locus using thioguanine resistance and at the aprt locus using azaadenine resistance. The compounds tested that required metabolic activation (using rat or hamster microsomal fractions) were 7,12-dimethylbenz(a)anthracene, 3-methylcholanthrene, benzo(a)pyrene, aflatoxin B1, 2-acetylaminofluorene, and 2-naphthylamine. The direct-acting compounds (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, N-acetoxy-2-acetylaminofluorene, and N-OH-2-naphthylamine were also studied. For all compounds except 2-naphthylamine and its active metabolite, the repair-deficient cells were significantly more sensitive to killing than the normal CHO cells. Mutation induction at both loci was also more efficient in UV5 cells in each instance where enhanced cytotoxicity was observed. By using tritium-labeled N-acetoxy-2-acetylaminofluorene, normal and mutant cells were shown to bind mutagen to their nuclear DNA with similar efficiency, and a greater amount of adduct removal occurred in the normal cells. From this study it is concluded that the use of excision repair-deficient CHO cells provides enhanced sensitivity for detecting mutagenesis and that a positive differential cytotoxicity response gives an indication of repairable, potentially lethal genetic damage.     

Comparative mutagenic efficiencies of the DNA adducts from the cooked-food-related mutagens Trp-P-2 and IQ in CHO cells

The relationship between DNA-adduct formation and mutagenicity of two heterocyclic aromatic amines associated with cooked foods was determined in a CHO cell strain lacking nucleotide excision repair. Cells were exposed to tritiated IQ (2-amino-3-methylimidazo[4,5-f]quinoline) or Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) supplemented with hamster S9 microsomal fraction for metabolic activation. DNA from nuclei was isolated by DNAase-mediated elution from polycarbonate filters after RNAase and proteinase treatment. The presumed metabolites of both compounds bound to DNA in a dose-dependent fashion. Although the dose required to produce 50% cell killing was 15 times higher for IQ than Trp-P-2, the amount of radioactive material bound to DNA at that dose was about 10-fold lower with IQ. When mutations at the hprt and aprt loci were compared with the estimated levels of adducts, the calculated mutagenic efficiency of the adducts was about 4 mutations per 1000 adducts for both compounds, assuming a target sequence of 1000 base pairs for either locus. We conclude that IQ is acting as a weak mutagen in this system because its extracellular metabolites either do not reach or do not react efficiently with the DNA of the CHO cells.     

Formation of DNA adducts in HL-60 cells treated with the toluene metabolite p-cresol: a potential biomarker for toluene exposure

We have examined DNA adduct formation in myeloperoxidase containing HL-60 cells treated with the toluene metabolite p-cresol. Treatment of HL-60 cells with the combination of p-cresol and H(2)O(2) produced four DNA adducts 1: (75.0%), 2: (9.1%), 3: (7.0%) and 4: (8.8%) and adduct levels ranging from 0.3 to 33.6 x 10(-7). The levels of DNA adducts formed by p-cresol were dependent on concentrations of p-cresol, H(2)O(2) and treatment time. In vitro incubation of p-cresol with myeloperoxidase and H(2)O(2) produced three DNA adducts 1: (40.5%), 2: (28.4%) and 3: (29.7%) with a relative adduct level of 0.7x10(-7). The quinone methide derivative of p-cresol (PCQM) was prepared by Ag(I)O oxidation. Reaction of calf thymus DNA with PCQM produced four adducts 1: (18.5%), 2: (36.4%), 3: (29.0%) and 5: (16.0%) with a relative adduct level 1.6x10(-7). Rechromatography analyses indicates that DNA adducts 1-3 formed in HL-60 cells treated with p-cresol and after myeloperoxidase activation of p-cresol were similar to those formed by reaction of DNA with PCQM. This observation suggests that p-cresol is activated to a quinone methide intermediate in each of these activation systems. Taken together, these results suggest PCQM is the reactive intermediate leading to the formation of DNA adducts in HL-60 cells treated with p-cresol. Furthermore, the DNA adducts formed by PCQM may provide a biomarker to assess occupational exposure to toluene.     

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.     

Benzo[a]pyrene diol-epoxides: different mutagenic efficiency in human and bacterial cells

Monolayer cultures of diploid human fibroblasts and suspensions of S. typhimurium TA100 cells were treated with [3H]-labelled enantiomeric forms of benzo[a]pyrene anti and syn 7,8-dihydrodiol 9,10-epoxides. In both cell types, all of the enantiomers induced the formation of mutant 6-thioguanine (human) or 8-azaguanine-(bacterial)resistant cells. Diol-epoxide-modified nucleosides from human and from bacterial DNA hydrolysates were characterized by HPLC and showed essentially the same adduct species for human and bacterial cells treated with the same enantiomers. There were substantial differences, however, in the efficiency with which structurally-different adduct species were converted to mutant genotypes. In human cells, the mutagenic efficiency (mutation frequency/unit modified DNA) of the respective adduct species (+ anti much greater than -anti = +/- syn) at the hprt locus was exactly the opposite of that seen at a similar gene locus (gpt) in TA100 (-anti = +/- syn greater than + anti). The results suggest that the structural configuration of adducts in genomic DNA is important in determining whether a mutant genotype will result, and likewise, that there are differences in specificity between the human and bacterial systems which process these adduct lesions.     

Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products.

We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of activated oxygen species in benzo[a]pyrene:DNA adduct formation in vitro.

The role of several activated oxygen species in the oxidation and binding of B[a]P to calf thymus DNA in vitro was investigated. B[a]P was reacted with calf thymus DNA in the presence and absence of scavengers of active oxygen species. Reactions were performed in the dark at 37 degrees C for 30 min in a buffered aqueous solution with 250 micrograms of calf thymus DNA. The levels of B[a]P:DNA adducts formed were determined using the 32P-postlabeling assay. B[a]P:DNA adduct levels ranged from 1.5-2.6 and 0.25 pmol adducts/mg DNA in reactions with 120 or 12 nmol of B[a]P, respectively. The addition of scavengers of reactive oxygen species to reaction mixtures resulted in a considerable decrease in the levels of DNA adducts formed in comparison to control reactions. Reactions performed with 500 units catalase or 100 units superoxide dismutase significantly inhibited DNA adduct formation. In these reactions adduct levels were 32 and 48% of control levels, respectively. The addition of both catalase and superoxide dismutase to reactions inhibited adduct formation by 95% relative to control reactions. A decrease in adduct levels was also observed when reactions were performed with citrate-Fe3+ chelate, a scavenger of superoxide. In reactions with 50 mM mannitol and 50 mM sodium benzoate, both of which are hydroxyl radical scavengers, adduct formation was significantly inhibited with adduct levels being 30 and 51% of control values, respectively. Adduct levels were decreased to 26% of control values in reactions with 10 mM 2,5-dimethylfuran, a scavenger of singlet oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory effects of different doses of alpha-tocopherol on benzo(a)pyrene-DNA adduct formation in the pulmonary tissue of cigarette smoke inhaling mice

Cigarette smoke (CS) has been established as one of the major risk factors for many pathologies including lung cancer in humans and experimental animals. In view of the discrepancy about the role of alpha-tocopherol (AT) in carcinogenesis, the present study was designed to investigate the effects of different doses of AT on benzo(a)pyrene-DNA [B(a)P-DNA] adduct formation in lungs of CS inhaling mice. Extent of carcinogen-DNA adduct formation has been considered as an index for carcinogenesis. Feeding of 35 IU AT/kg body weight increased B(a)P-DNA adducts formation significantly whereas feeding of 5 IU AT/kg body weight did not altered much the B(a)P-DNA adduct levels when both were compared to the control counterparts. With CS inhalation, the B(a)P-DNA adducts formation increased in all the groups when compared to their respective sham counterparts. Interestingly, in CS exposed groups, there was least increase in B(a)P-DNA adducts formation in 5 IU AT/kg fed animals followed by the control and 35 IU AT/kg body weight fed groups respectively. The results suggest that higher doses of AT accentuate DNA adduct formation in CS inhaling mice.     

Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.     

Mutation spectra induced by alpha-acetoxytamoxifen-DNA adducts in human DNA repair proficient and deficient (xeroderma pigmentosum complementation group A) cells

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.     

Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: role of quinone methide in DNA adduct formation.

We have investigated the activation of p-cresol to form DNA adducts using horseradish peroxidase, rat liver microsomes and MnO(2). In vitro activation of p-cresol with horseradish peroxidase produced six DNA adducts with a relative adduct level of 8.03+/-0.43 x 10(-7). The formation of DNA adducts by oxidation of p-cresol with horseradish peroxidase was inhibited 65 and 95% by the addition of either 250 or 500 microM ascorbic acid to the incubation. Activation of p-cresol with phenobarbital-induced rat liver microsomes with NADPH as the cofactor; resulted in the formation of a single DNA adduct with a relative adduct level of 0.28+/-0.08 x 10(-7). Similar incubations of p-cresol with microsomes and cumene hydroperoxide yielded three DNA adducts with a relative adduct level of 0.35+/-0.03 x 10(-7). p-Cresol was oxidized with MnO(2) to a quinone methide. Reaction of p-cresol (QM) with DNA produced five major adducts and a relative adduct level of 20.38+/-1.16 x 10(-7). DNA adducts 1,2 and 3 formed by activation of p-cresol with either horseradish peroxidase or microsomes, are the same as that produced by p-cresol (QM). This observation suggests that p-cresol is activated to a quinone methide intermediate by these activation systems. Incubation of deoxyguanosine-3'-phosphate with p-cresol (QM) resulted in a adduct pattern similar to that observed with DNA; suggesting that guanine is the principal site for modification. Taken together these results demonstrate that oxidation of p-cresol to the quinone methide intermediate results in the formation of DNA adducts. We propose that the DNA adducts formed by p-cresol may be used as molecular biomarkers of occupational exposure to toluene.     

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)     

Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner. Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct. Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion. Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis. Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis. These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells.

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360-1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2',7'-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 microM of cadmium acetate for 4h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2 x 10(-6)), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC-->AT transitions and decreased the frequencies of TA-->AT and TA-->GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC-->AT transitions upon cadmium exposure.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo . S -BMO adducts were the main product and represented 77 % ( n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo. S -BMO adducts were the main product and represented 77 % (n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.