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[Cancer Research 63, 8708-8716, December 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Enhancement of the Mutagenicity of Benzo(a)pyrene Diol Epoxide by a Nonmutagenic Dose of Ultraviolet A Radiation

Ahmad Besaratinia and Gerd P. Pfeifer

Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the effects of single and combined exposures to two ubiquitous environmental carcinogens, polycyclic aromatic hydrocarbons and UVA radiation, in Big Blue mouse embryonic fibroblasts. We quantified the cytotoxicity, DNA adduct formation, and induction of mutations in the cII transgene in cells treated with a single agent or combinations of agents in both direct and reverse order. Mapping of DNA adducts by terminal transferase-dependent PCR showed the preferential formation of bulky adducts at identical nucleotide positions along the cII gene after treatment with the prototype polycyclic aromatic hydrocarbon, benzo(a)pyrene diol epoxide [B(a)PDE], or B(a)PDE plus UVA radiation treatments but not after UVA irradiation alone. The cII mutant frequency determined by a {lambda} phage-based mutation detection system was not increased significantly by UVA irradiation (1.7-fold over background; P < 0.3); however, B(a)PDE alone or in combinations with UVA radiation significantly increased the cII mutant frequency (P < 0.001). The highest cII mutant frequency was induced by the treatment with B(a)PDE followed by UVA irradiation, which was more than the added mutant frequencies of the two agents individually (>12.2-fold versus <7.6-fold over background; P < 0.01). In support of these findings, DNA sequencing analyses showed that the mutational spectra induced by B(a)PDE alone or combined with UVA radiation were significantly different from those derived spontaneously (P < 0.0001) or by UVA irradiation (P < 0.0005). The signature of mutations produced by B(a)PDE i.e., "G->T + C->A" transversions, was significantly enhanced when the B(a)PDE treatment was followed by UVA irradiation (47% versus 65%; P < 0.01). Also, the methylated CpG dinucleotide-targeted overall mutations specifically induced by B(a)PDE were increased after the subsequent UVA irradiation (43% versus 51%, respectively). Such enhancements in the mutational signature of B(a)PDE were most pronounced within the preferential DNA adduction sites along the cII gene after the treatment with B(a)PDE plus UVA radiation, which suggests that the primary B(a)PDE adducts are converted to more mutagenic species on UVA irradiation. We conclude that UVA radiation at a nonmutagenic dose has an enhancing effect on the mechanism by which B(a)PDE induces mutations.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Humans are continuously exposed to a myriad of environmental toxins throughout their lives. Generally, exposure profiles and biological consequences of the vast majority of these toxins are highly complicated. This is because most exposures occur while agents are in a mixture of or in combinations with various other agents, thereby imposing additive or multiplicative effects (1, 2, 3) . For instance, (in)voluntary inhalation of tobacco smoke or diesel engine exhaust in humans typifies a mixed exposure to multiple classes of chemicals. Also, photochemotherapy of a variety of human diseases in which a drug is administered before UV irradiation best exemplifies a combined exposure situation (4) . In experimental settings, however, single agent exposure is mostly in practice due to the limitations of the existing methodologies (2) .

Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental toxins present in air, water, and food (5) . Being formed as a result of incomplete pyrolysis of organic matter, PAHs find their way into our ambient air as a pollutant, e.g., emitted from natural sources such as forest fires or industrial sources such as power plants, as well as into our food chain as a contaminant e.g., in leafy vegetables or as a by-product of food processing e.g., in charbroiled meat (5 , 6) . More importantly, PAHs are found in substantial quantities in tobacco smoke (7) . Not only are many compounds of PAHs proven carcinogens in various test systems, but also the ever-occurring dietary/occupational/medicinal/recreational exposures to PAHs in humans are associated with different types of cancer (8) . Like most chemical carcinogens, PAHs undergo biotransformation to exert their biological effects (activation pathway) or otherwise to be eliminated (detoxification pathway; Refs. 9 , 10 ). The activated PAHs are mainly electrophilic reactants capable of covalently binding to subcellular targets, e.g., DNA, forming complex "adducts" (9 , 10) . The adducts refractory to repair have the potential to interfere with normal cellular functions, e.g., by causing miscoding during DNA replication and, as a result, induce mutations (11) . Of significance for cancer etiology are the DNA adducts that trigger mutations in critical oncogenes, e.g., ras or tumor suppressor genes, e.g., p53 (11) . Mechanistically, DNA binding of a prototype PAH reactant, benzo(a)pyrene diol epoxide [B(a)PDE], at known mutational hotspots of human lung cancer in both the k-ras and p53 genes, has provided putative links between PAH-DNA adducts and carcinogenesis (12, 13, 14) .

UV radiation in sunlight is a well-established physical environmental carcinogen and has long been implicated in human skin cancer (15 , 16) . The solar UV consists of UVC (<280 nm wavelength), UVB (280–320 nm wavelength), and UVA (>320–400 nm wavelength) radiations. Molecular oxygen (O2) generated during photosynthesis in plants and released into the atmosphere absorbs the entire UVC fraction of the sunlight and decomposes in the process. It then recombines to form ozone (O3), which, in turn, absorbs most of the solar UVB radiation before it can reach the surface of the earth. The remaining UV component to which living organisms on earth are exposed is mainly (~95%) comprised of UVA (17) . Unlike UVB radiation that induces promutagenic cis-syn cyclobutane pyrimidine-dimers, pyrimidine (6–4) pyrimidone photoproducts, and Dewar valence photoisomers, the carcinogenicity of UVA radiation is widely believed to involve endogenous photosensitizers and radical-mediated induction of mutagenesis (15 , 18) . UVA radiation per se as well as in combination with other agents has been identified as a risk factor for various benign or malignant human diseases. For example, combined exposure to tobacco smoke and sunlight is associated with dysplastic and malignant lip lesions and squamous cell carcinoma of the skin (19 , 20) . Also, in aquatic organisms the toxicity of PAH increases considerably on UV irradiation (21, 22, 23) . In real life situations, road maintenance workers handling asphalt (highly rich in PAHs) under the sun exemplify a typical combined exposure to PAH and UV radiation.

In the present study, we investigated the single and combined effects of B(a)PDE and UVA radiation on different pathways impacting on mutagenesis in embryonic fibroblasts of the Big Blue mouse. In this in vitro transgenic model system, genomic DNA containing an easily recoverable {lambda} shuttle vector, which carries the mutational cII target gene, can be screened for mutation induction, as well as assayed for the detection of DNA adducts at the nucleotide level. Here, we determined cytotoxicity, DNA adduct formation, and induction of mutations in the cII transgene after a single treatment with each agent and after combined treatments in direct and reverse orders with both agents.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Treatments.
Early passage Big Blue mouse embryonic fibroblasts (prepared from 13.5-day-old embryos) were grown to approximately 50–60% monolayer confluence in DMEM supplemented with 10% fetal bovine serum. The confluent cells were kept in phenol red and serum free medium, Opti-MEM I (Invitrogen Corporation, Carlsbad, CA), the night before treatments. The cells were treated with 1 µM benzo(a)pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (±; anti; Midwest Research Institute, Kansas City, MO) or control solvent, DMSO, for 30 min in the dark, or alternatively irradiated with a dose of 1.05 J/cm2 UVA. The UVA source consisted of two black-light lamps, F15T8.BLB, 15 W (each), 365 nm (General Electric), being filtered through a 3-mm window glass, yielding an average fluence rate of 1.75 mW/cm2 determined by a UVX radiometer (UV Products, Upland, CA). For homogeneous irradiation of the cells, the culture Petri dishes were placed on the filter glass and were rotated every 2–3 min during the course of irradiation. In case of combination treatments, irradiation preceded the chemical treatment or vice versa. The cells were washed thoroughly with PBS (pH 7.5) after the chemical treatment and/or the UVA irradiation. To determine cell survival and DNA adduct formation, two subsets of cells were harvested by trypsinization immediately after treatments and checked for viability using the trypan blue dye exclusion technique, or subjected to DNA isolation and subsequent terminal transferase-dependent PCR (TD-PCR), respectively. For mutation analyses, other subsets of the treated cells were cultured in complete growth medium for an additional 8 days, and afterward were used for determining mutant frequency and mutational spectrum of the cII transgene. The 8-day growing period is essential for the fixation of all of the induced/spontaneous mutations. All of the experiments were run in triplicate settings two to three times.

Genomic DNA Isolation.
Genomic DNA was isolated using a standard phenol-chloroform extraction and ethanol precipitation protocol (24) . The DNA was dissolved in TE buffer [10 mM Tris-HCl and 1 mM EDTA (pH 7.5)] and kept at -80°C until additional analysis.

TD-PCR for Mapping of DNA Adducts.
The entire length of the cII gene was subjected to TD-PCR as described earlier (25) . Briefly, genomic DNA (~1 µg) was used as a template, and single-stranded products were made by repeated primer extensions. The extension protocol consisted of primer U1: 5'-AATCGAGAGTGCGTTGCTT-3', Tm = 49.9°C in a mixture of Vent (exo-) DNA polymerase (New England Biolabs Inc., Beverly, MA) in a thermocycler setting of 2 min at 95°C, 2 min at 53°C, and 3 min at 72°C, nine cycles (in which one cycle consisted of 45 s at 95°C, 2 min at 53°C, and 3 min at 72°C), 45 s at 95°C, 2 min at 53°C, and 10 min at 72°C. The resulting product was precipitated with ethanol together with a salt solution containing 10 M ammonium acetate, 0.5 M EDTA (pH 8.0) and 20 mg/ml glycogen, and afterward subjected to the homopolymeric ribotailing and adapter ligation. The ligated fragments were PCR-amplified using primer U2: 5'-GCGTTGCTTAACAAAATCGCAATGCT-3', Tm = 63.1°C in a thermocycler for 2 min at 95°C, 2 min at 62°C, and 3 min at 72°C, 18 cycles (in which 1 cycle consisted of 45 s at 95°C, 2 min at 62°C, and 3 min at 72°C), 45 s at 95°C, 2 min at 62°C, and 10 min at 72°C. Final primer extension and labeling of the PCR-products were done using a fluorescence IR dye labeled primer (IRD-700; LI-COR Inc., Lincoln, NE) U3: 5'-GCAATGCTTGGAACTGAGAAGACAGC-3', Tm = 61.4°C. The thermocycler setting was 2 min at 95°C, 2 min at 65°C, and 3 min at 72°C, six cycles (in which one cycle consisted of 45 s at 95°C, 2 min at 65°C, and 3 min at 72°C), 45 s at 95°C, 2 min at 65°C, and 10 min at 72°C. The labeled reaction products were loaded onto a 5% acrylamide/urea gel for electrophoresis and simultaneous quantification by an IR2 Long Ranger 4200 system (LI-COR Inc.). The sites of DNA adduct formation were identified as the locations in which the presence of the lesions stopped the DNA polymerase from progressing, resulting in an intense dark band (dependent on the lesion frequency) in the sequencing gel.

cII Mutant Frequency Analysis.
The cII mutant frequency was quantified by the {lambda} select-cII mutation detection system for Big Blue rodents (Stratagene). The assay system is based on the ability of the {lambda} phage to multiply either lytically or lysogenically in Escherchia coli host cells (26) . The commitment of the {lambda} phage to lysis or lysogeny on infection of the host is dependent on a chain of events, of which cII transcription is most crucial. The cII protein activates the transcription of cI repressor and {lambda} integrase, both of which obligate the phage to undergo lysogenization (27) . Thus, only {lambda} phages carrying a mutated cII can enter the lytic pathway and as a result form visible plaques on an E. coli lawn (26) . The {lambda} LIZ vector, however, harbors a cI857 temperature sensitive (ts) mutation, which makes the cI(ts) protein labile at temperatures >32°C. Therefore, all of the {lambda} LIZ phages regardless of their cII mutant/nonmutant status multiply lytically in the host E. coli at incubating temperatures exceeding 32°C (nonselective condition; Ref. 26 ).

Briefly, the {lambda} LIZ shuttle vectors were recovered from the genomic DNA (~5 µg) and packaged into viable phage particles by Transpack packaging extract according to the manufacturer’s instructions (Stratagene). The phages were preadsorbed to G1250 E. coli, and the bacterial culture was plated on TB1 agar plates. The plates were incubated for 48 h at 24°C or overnight at 37°C (regarded as selective and nonselective conditions, respectively). The cII mutant frequency was expressed as the ratio of the number of plaques formed on the selective plates to that formed on the nonselective plates. As recommended by the manufacturer (Stratagene), a minimum of 3 x 105 rescued phages was screened for each experimental condition. For quality assurance, control phage solutions containing a mixture of {lambda} cII(+) and l cII(-) with known mutant frequencies (Stratagene) were assayed in all runs.

cII Mutational Spectrum Analysis.
All of the putative mutant cII plaques were verified after being replated under the selective conditions on a second TB1 agar plate. The verified plaques were subsequently amplified in a PCR using the {lambda} select-cII sequencing primers, according to the manufacturer’s recommended protocol (Stratagene). The PCR products were purified with QIA quick PCR purification kit (Qiagen GmbH, Hilden, Germany) and sequenced by a Big Dye terminator cycle sequencing kit on an ABI-377 DNA Sequencer (ABI Prism; PE Applied BioSystems, Foster City, CA).

Statistical Analysis.
Results are expressed as medians ± SE. Mutant frequencies in multiple groups and between two groups were compared using the Kruskall-Wallis one-way ANOVA and Mann-Whitney U tests, respectively. Mutational spectra were analyzed with the hypergeometric test of Adams and Skopek (28) and {chi}2 test where appropriate. All of the other variables in two different groups were compared by the Wilcoxon signed rank test or {chi}2 test where appropriate. Values of P < 0.05 were considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytotoxicity Examination.
The UVA irradiation and B(a)PDE treatment both individually and in combinations were cytotoxic in Big Blue mouse embryonic fibroblasts showing deteriorating effects on cell viability determined by the trypan blue dye exclusion technique (Fig. 1)Citation . The severity of the cytotoxicity was lowest for B(a)PDE treatment alone, and highest for UVA irradiation followed by B(a)PDE treatment. Although both combinations of the UVA irradiation and B(a)PDE treatment were significantly more cytotoxic than the individual agent treatment (P < 0.01), the cytotoxic effects of the combined B(a)PDE and UVA radiation in direct and reverse orders were not significantly different from one another (Fig. 1)Citation .



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  		Fig. 1. Cytotoxic effects of the single and combined exposures to benzo(a)pyrene diol epoxide [B(a)PDE] and UVA radiation on Big Blue mouse embryonic fibroblasts. Confluent cell cultures were treated with 1 µM of B(a)PDE for 45 min in the dark, or irradiated with 1.05 J/cm2 UVA or treated with a combination of the two agents in both direct and reverse order. Cell viability was determined by the trypan blue dye exclusion assay immediately after treatments. Viability is expressed as a percentage of total cell number. Results are expressed as medians of two independent experiments, with each experiment run in triplicate; bars, ±SE. *, as compared with nontreated control, P < 0.003; {dagger}, as compared with nontreated control, P < 0.01; {ddagger}, as compared with nontreated control, P < 0.001; §, as compared with nontreated control, P < 0.002.

 
DNA Adduct Formation.
Mapping of DNA adducts by TD-PCR revealed the preferential formation of bulky adducts at specific locations along the cII gene in cells treated with B(a)PDE alone or in combinations with UVA irradiation (Fig. 2)Citation . The UVA-alone irradiated cells, however, did not show appreciable DNA adducts relative to nontreated control (Fig. 2)Citation . Interestingly, the sites of DNA adduct formation in cells treated with B(a)PDE alone and those treated with both combinations of B(a)PDE and UVA radiation were identical. Of the eight preferential DNA adduction sites, six (75%) including nucleotide positions 47–50, 89–93, 103–107, 211–215, 226–231, and 256–261 contained methylated CpG dinucleotides (mCpGs; Fig. 2Citation ).



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  		Fig. 2. Mapping of DNA adducts along the lower strand of the cII gene in Big Blue mouse embryonic fibroblasts treated with 1 µM benzo(a)pyrene diol epoxide [B(a)PDE] for 45 min in the dark, or irradiated with 1.05 J/cm2 UVA or treated with a combination of the two agents in both direct and reverse order. Genomic DNA was extracted and subsequently subjected to the terminal transferase-dependent PCR for the mapping of bulky DNA adducts along the cII gene (28) . Numbers indicate the nucleotide positions. M, sizing standard; C, DMSO-treated control; nt, nucleotide.

 
cII Mutant Frequency and Mutational Spectrum.
The {lambda} phage-based mutation detection (Stratagene) analyses showed a significant increase in the cII mutant frequencies in cells treated with B(a)PDE alone or in combinations with UVA radiation relative to the spontaneous cII mutant frequency in nontreated controls (Fig. 3)Citation . The cells treated with B(a)PDE followed by UVA irradiation had the highest cII mutant frequency (>12.2-fold over background). This was significantly higher than the cII mutant frequency induced by a single treatment with B(a)PDE, or by an irradiation with UVA followed by a B(a)PDE treatment (P < 0.001 and P = 0.01, respectively). This induced mutant frequency was also higher than the added mutant frequencies induced by UVA irradiation alone (<1.7-fold over background) and B(a)PDE treatment alone (<5.9-fold over background; Fig. 3Citation ).



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  		Fig. 3. Mutant frequency of the cII transgene in Big Blue mouse embryonic fibroblasts treated with 1 µM benzo(a)pyrene diol epoxide [B(a)PDE] for 45 min in the dark or irradiated with 1.05 J/cm2 UVA or treated with a combination of the two agents in both direct and reverse order. Mutations were determined 8 days after treatments with the {lambda} select-cII mutation detection system for Big Blue rodents (Stratagene), a phage-based assay that permits detection of mutations within the transgene on the basis of plaque formation (nonmutant cII, no plaque formation; mutant cII, plaque formation). Mutant frequency was determined from a minimum of 3 x 105 plaques. Results are expressed as medians of three independent experiments in triplicate settings; bars, ±SE. *As compared with BPDE + UVA, P < 0.001; {dagger}, as compared with BPDE + UVA, P < 0.01; {ddagger}, as compared with BPDE plus UVA + BPDE, P < 0.01.

 
To examine the specificity of B(a)PDE and UVA radiation for affecting the mutagenicity of one another when combined, we double-treated a set of samples with UVA radiation as well as with B(a)PDE at various doses. We observed no increase in the cII mutant frequency after a double UVA irradiation relative to a single dose irradiation (UVA1.05 J/cm2 + UVA1.05 J/cm2: Mutant Frequency: 10.7 x 10-5). When doubling the second radiation dose, there was still no significant change in the cII mutant frequency (UVA1.05 J/cm2 + UVA2.1 J/cm2: Mutant Frequency: 9.1 x 10-5; Table 1Citation ). Instead, the cytotoxicity of UVA radiation in the double-dose regimens was increased, which could explain the resulting mutant frequencies (UVA1.05 J/cm2 + UVA1.05 J/cm2: Viability: 40%; UVA1.05 J/cm2 + UVA2.1 J/cm2: Viability: 28%). For B(a)PDE, we first established the nature of dose-response curve, and subsequently combined a comparable mutagenic dose of B(a)PDE relative to UVA, with a second dose of this chemical (Table 1)Citation . The treatment with B(a)PDE at increasing concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM yielded the following cII mutant frequencies: 6.1 x 10-5, 5.8 x 10-5, 13.9 x 10-5, 38.1 x 10-5, and 40.6 x 10-5, respectively. Consecutive treatments with double doses of B(a)PDE did not induce the cII mutant frequencies more than additively (Table 1)Citation . Like the UVA double irradiation, however, double doses of B(a)PDE were more cytotoxic than the single dose of this chemical (data not shown).


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  		Table 1 Dose-dependency of cII mutant frequency by UVA radiation and B(a)PDE

 
To compare the induced- and spontaneous-mutational spectra, sequencing analysis was performed on the DNA isolated from the verified mutant cII plaques of the cells treated with UVA radiation, B(a)PDE, UVA radiation plus B(a)PDE, and B(a)PDE plus UVA radiation, as well as from the spontaneously derived mutant cII plaques of the nontreated control cells (number of sequenced plaques = 120, 113, 186, 159, and 173, respectively). Overall, the DNA sequencing confirmed a mutated cII gene in 93–96% of the analyzed plaques. Of these, the vast majority was single base substitutions (78–87%), and less frequently, insertions, deletions, and multiple base substitutions, respectively (Fig. 4, A–E)Citation .



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  		Fig. 4. Detailed mutational spectra of the cII transgene from Big Blue mouse embryonic fibroblasts treated with 1 µM benzo(a)pyrene diol epoxide [B(a)PDE] for 45 min in the dark, or irradiated with 1.05 J/cm2 UVA or treated with a combination of the two agents in both direct and reverse order. A, control nontreatment (total number of sequenced plaques = 173); B, UVA radiation (total number of sequenced plaques = 120); C, B(a)PDE (total number of sequenced plaques = 113); D, UVA radiation plus B(a)PDE (total number of sequenced plaques = 186); E, B(a)PDE plus UVA radiation (total number of sequenced plaques = 159). Substituted bases are in bold. Deleted bases are underlined. Inserted bases are shown with an arrow. Numbers below the bases are the nucleotide positions.

 
In both the induced- and spontaneous-mutational spectra, there were four "jackpot" mutations at nucleotide positions 179–184 (G insertion/deletion), 196 (G->A transition), 211 (G->C transversion), and 221 (T->G transversion; Fig. 4, A–ECitation ). These jackpot mutations at specific locations in the cII gene, reported previously by us (25 , 29) and by others (30 , 31) , are thought to occur in the early development of the transgenic rodent and to undergo clonal expansion such that many cells from a single tissue harbor the same type of mutations. Alternatively, they might represent genuine hotspots of spontaneous mutagenesis (32) . Methodologically, therefore, it seems appropriate to exclude such jackpot mutations from the comparative spectra analysis. Excluding these jackpot mutations, except for the mutational spectrum induced by UVA irradiation alone, all of the other treatments including B(a)PDE, UVA radiation plus B(a)PDE, and B(a)PDE plus UVA radiation produced significantly different mutational spectra from that derived spontaneously (P < 0.0001). Also, the spectra of mutations induced by both combinations of B(a)PDE and UVA radiation differed significantly from that induced by UVA radiation alone (P < 0.0005). However, the mutational spectra induced by either combinations of the B(a)PDE and UVA radiation were not different from that induced by B(a)PDE alone (P = 0.12).

Because the cII gene is almost certainly not transcribed after being integrated into the genome of the animal (33) , the transgene is not biased for the strand preferences of DNA adduct formation/removal and mutagenesis associated with transcription-coupled DNA repair of mammalian endogenous genes (34, 35, 36) . Accordingly, it is appropriate to combine the strand mirror counterparts of all transitions (e.g., G->A + C->T) and transversions (e.g., G->T + C->A and G->C + C->G) occurring in the cII transgene when comparing different mutational spectra. As shown in Fig. 5Citation , both the B(a)PDE treatment alone and in combinations with UVA irradiation gave rise to specific base substitutions relative to control nontreatment as well as to UVA irradiation alone. The B(a)PDE treatment alone significantly increased the frequency of G->T + C->A transversions relative to both control and UVA irradiation-alone treatments (47% versus 12% and 19%, respectively; P < 0.0001; Fig. 5Citation ). This specific increase in the frequency of G->T + C->A transversions was most pronounced when the B(a)PDE treatment was combined with UVA irradiation. In fact, the B(a)PDE treatment followed by UVA irradiation induced more frequently the G->T + C->A transversions as compared with B(a)PDE treatment alone (65% versus 47%; P < 0.01). The frequency of the G->T + C->A transversions induced by B(a)PDE plus UVA radiation treatment was, however, nonsignificantly higher than that induced by UVA plus B(a)PDE treatment (65% versus 56%; P = 0.2; Fig. 5Citation ). These specifically induced G->T + C->A transversions as well as the overall induced mutations were targeted to the mCpGs along the cII gene in cells treated with B(a)PDE alone or in combinations with UVA irradiation (Fig. 6)Citation . The incidence of these mCpG-targeted mutations was the highest after the combined treatment of B(a)PDE followed by UVA irradiation (Fig. 6)Citation . The combination of B(a)PDE treatment preceded by UVA irradiation, however, showed less preference for targeting mCpGs for both the overall induced mutations and the G->T + C->A transversions relative to B(a)PDE treatment alone or combined with a subsequent UVA irradiation (Fig. 6)Citation .



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  		Fig. 5. Mutational spectra of the cII transgene in Big Blue mouse embryonic fibroblasts treated with 1 µM benzo(a)pyrene diol epoxide [B(a)PDE] for 45 min in the dark, or irradiated with 1.05 J/cm2 UVA or treated with a combination of the two agents in both direct and reverse order. The strand mirror counterparts of all transitions (e.g., G->A + C->T) and transversions (e.g., G->T + C->A and G->C + C->G) were combined and used to make the comparative analyses between different treatments. Ins, insertion; Del, deletion. *, BPDE versus nontreated control or UVA, P < 0.0001; *, BPDE + UVA versus BPDE, P < 0.01.

 


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  		Fig. 6. Overall mutations and specific G->T + C->A transversions at methylated CpGs dinucleotides in the cII transgene in Big Blue mouse embryonic fibroblasts treated with 1 µM benzo(a)pyrene diol epoxide [B(a)PDE] for 45 min in the dark, or irradiated with 1.05 J/cm2 UVA or treated with a combination of the two agents in both direct and reverse order. *, overall mutations: as compared with nontreated control, P < 0.04; *, G->T + C->A: as compared with nontreated control, P < 0.002; *, G->T + C->A: as compared with UVA, P < 0.0005; {dagger}, overall mutations: as compared with BPDE + UVA, P < 0.03; {dagger}, G->T + C->A: as compared with nontreated control, P < 0.002; {dagger}, G->T + C->A: as compared with UVA, P < 0.0007; {ddagger}, overall mutations: as compared with nontreated control, P < 0.003; {ddagger}, G->T + C->A: as compared with nontreated control, P < 0.0001; {ddagger}, G->T + C->A: as compared with UVA, P < 0.0001.

 
Altogether, the mutational spectra produced by B(a)PDE treatment alone or in combinations with UVA irradiation partially corresponded to the pattern of DNA adduct formation in the cII gene. In fact, some but not all of the frequently mutated sites induced by the treatments colocalized with the preferential DNA adduction sites. Also, within the preferential DNA adduct formation sites, the overall mutations and the specific G->T + C->A transversions induced by the B(a)PDE treatment alone or in combinations with UVA irradiation were significantly higher than their respective spontaneous mutations in the control nontreatment (P = 0.01). Of significance within the preferential DNA adduction sites was the increased frequency of the mCpG-targeted G->T + C->A transversions induced by B(a)PDE plus UVA radiation relative to UVA radiation plus B(a)PDE or B(a)PDE-alone treatments (15.4%, 10.9%, and 12.5%, respectively).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In real life, frequent exposures to a mixture or combination of cancer-causing agents occur; however, in experimental settings, single agent exposures are mostly investigated. This is mainly due to the limitations of the available methodologies to specifically explore the effects of an individual agent mixed or combined with other agents (1, 2, 3) . In the present study, we assessed the single and combined exposures to two ubiquitous environmental carcinogens, PAH and UVA radiation, in Big Blue mouse embryonic fibroblasts. This in vitro system offers a chromosomally integrated transgene, cII, which can be screened for the occurrence of all relevant events for mutagenesis and carcinogenesis (26) . Here, we quantified the cytotoxicity, DNA adduct formation, and induction of mutations in cells treated with a prototype PAH, B(a)PDE, or UVA radiation, or the combinations of B(a)PDE and UVA radiation in both direct and reverse order.

In the cytotoxicity examinations, all of the single-agent and combined-agent treatments appeared to be cytotoxic. The combinations of B(a)PDE treatment and UVA irradiation were significantly more cytotoxic than the individual agent treatment, with UVA irradiation followed by B(a)PDE treatment being the most severely cytotoxic (Fig. 1)Citation . In the mapping of DNA adducts, we found bulky adducts at identical locations along the cII gene induced by B(a)PDE treatment alone and in combinations with UVA irradiation (Fig. 2)Citation . To rule out the potential contribution of the most prevalent UV-associated photoadduct (37) , we analyzed the UVA-irradiated or UVA plus B(a)PDE-treated cells (both combinations) with a T4-endonuclease V enzymatic digestion assay for the detection of cyclobutane pyrimidine-dimers. No detectable photoproducts could be quantified in the cells treated with UVA radiation alone or in combinations with B(a)PDE (data not shown). In light of the observation that UVA irradiation per se did not induce appreciably bulky DNA adducts (Fig. 2)Citation , it is apparent that in the present study the B(a)PDE treatment alone or combined with UVA irradiation gave rise to similar types of bulky DNA adducts at almost equal levels. It is worth mentioning that the mapping data of B(a)PDE-DNA adducts by TD-PCR well-correlated with those data obtained by UvrABC-coupled ligation-mediated PCR in our previous work (38) . Methodologically, the latter technique has been criticized for not being able to locate DNA adducts directly but rather the sites of nicking by UvrABC, which can be sequence-dependent (11) . The correlation herein established between DNA adduct mapping by UvrABC-dependent and -independent methods argues against this criticism.

Mutational analyses of the cells irradiated with UVA showed that this treatment per se was not mutagenic, because it only slightly increased the cII mutant frequency (1.7-fold over background; P = 0.3) without producing a significantly different mutational spectrum relative to control (Fig. 4B)Citation . However, the B(a)PDE treatment individually and in combinations with UVA irradiation significantly increased the cII mutant frequency over the background (Fig. 3)Citation . Of most significance was the comutagenic effect of B(a)PDE and UVA radiation in inducing the cII mutations as the combined treatment of the B(a)PDE plus UVA irradiation induced more than additively the cII mutant frequency (>12.2-fold versus <5.9 + 1.7-fold over background; P < 0.01). Conformingly, the cII mutational spectra in cells treated with B(a)PDE alone or combined with UVA irradiation were significantly different from that derived spontaneously (Fig. 4, A–E)Citation . Although the overall cII mutational spectrum induced by B(a)PDE alone did not differ significantly from those induced by B(a)PDE plus UVA radiation, the signature of mutations produced by B(a)PDE was substantially enhanced when the B(a)PDE treatment was combined with UVA irradiation (Fig. 5)Citation . In fact, the specifically induced G->T + C->A transversions by B(a)PDE were statistically significantly more frequent when the B(a)PDE treatment was followed by the UVA irradiation (47% versus 65%; P < 0.01; Fig. 5Citation ). Also, the mCpG-targeted overall mutations and the specific G->T + C->A transversion characteristics for B(a)PDE treatment were increasingly pronounced after the combined treatment of B(a)PDE plus UVA radiation (Fig. 6)Citation . In agreement with the DNA mapping data, these findings support the notion that the enhanced mutagenicity of the combined B(a)PDE plus UVA radiation arises from a boost in the mechanism by which B(a)PDE induces mutations. Mechanistically, we have shown previously the hypermutability of the mCpG-targeted B(a)PDE-DNA adducts in the cII gene (38) as well as in the p53 tumor suppressor gene (12 , 13) . Given the similar induction of DNA adducts by B(a)PDE alone or in combination with UVA radiation, the observed augmented mutagenicity of the combined treatments suggests that B(a)PDE-DNA adducts become more mutagenic when they are irradiated with UVA. In fact, molecular modeling and computational chemistry data are supportive of this idea. The major DNA adduct of B(a)PDE, (+)-trans-anti-B(a)PDE-N2-dG, is known to adopt various conformations in the DNA duplex, of which some retain their base pairing potential, whereas others e.g., in the intercalative or base-displaced mode, have impaired base paring potential (39, 40, 41, 42, 43) . For example, the base-displaced conformation of (+)-trans-anti-B(a)PDE-N2-dG in which the B(a)P is stacked in the helix DNA and the guanine moiety is displaced in the major or minor grooves can induce G->T or G->A, mutations, respectively (39, 40, 41, 42, 43) . Theoretically, the UVA radiation possesses the physical property to change the chemistry or conformation of a given DNA adduct. This theory gains support from our demonstration that the increased G->T + C->A transversions induced by B(a)PDE plus UVA radiation relative to B(a)PDE alone were concurrent with a decrease in G->A + C->T transitions (Fig. 5)Citation . Also, consistent with this theory is the unchanged proportion of all base substitutions occurring at the guanine residues induced by B(a)PDE alone or combined with UVA radiation (~80% versus 81%). The B(a)P ring may act as a chromophore for absorption of UVA photons, thereby producing different promutagenic or nonmutagenic DNA adducts. It has been shown previously that 355 nm laser pulse irradiation of site-specifically modified B(a)PDE-guanine oligonucleotides induces strand cleavage at, and to a lesser extent near to, the adducted base on the same strand, by a mechanism that involves either photoinduced electron transfer or the production of a local free radical that ultimately results in strand breaks (44 , 45) . In addition, UVA irradiation of B(a)PDE-guanine adducts is known to produce a variety of oxidative guanine adducts.1 ,2 Altogether, if proven, this theory can only partially explain the observed mutagenicity of B(a)PDE and UVA radiation, because the B(a)PDE treatment preceded by UVA irradiation also showed more than added mutagenicity albeit to a lesser extent.

Alternatively, there are two other plausible scenarios that may explain the different mutagenic potencies of B(a)PDE alone and combined with UVA radiation. The first considers the possibility of DNA lesion-tolerant polymerases being differently affected after treatments with B(a)PDE alone or combined with UVA radiation. Pol {kappa} is a low-fidelity and moderate processivity polymerase (46, 47, 48) , which is shown to be overexpressed in tumorous lung tissues of cancer patients (49) . Also, the promoter region of the mouse Pol {kappa} gene contains two copies of the aryl hydrocarbon receptor binding sites, and the expression of the Pol {kappa} enzyme is induced by treatment of the animal with a PAH, 3-methylcholanthrene (50) . In vitro, Pol {kappa}, is capable of bypassing all stereoisomers of anti-B(a)PDE-N2-dG in both error-free and error-prone fashions (48) with dCMP insertion opposite the lesion at least three times more frequent than other incorporations (51) . Furthermore, mouse embryonic stem cells deficient in Pol {kappa} are hypersensitive to the lethal and mutagenic effects of B(a)P (52) . Of relevance for UV-associated DNA lesions is Pol {eta}, an intrinsically low fidelity and processivity DNA polymerase (53) . Pol {eta} efficiently and accurately bypasses UV-induced thymine-thymine dimers by inserting two dAMPs opposite the lesions. However, the enzyme is incapable of bypassing (6–4) photoproducts (54 , 55) . The extreme predisposition of the xeroderma pigmentosum patients to sunlight-associated skin cancer is now accounted for by their lack of a functional Pol {eta} gene (56 , 57) . Pol {eta} is also known to erroneously bypass the trans-anti-B(a)PDE-N2-dG with dAMP, dGMP, and dTMP being the most frequently misincorporated bases, respectively, opposite the lesion (58 , 59) . It was shown recently that UV irradiation of human host cells with wild-type or null Pol {eta} genotypes before their transfection with undamaged shuttle vectors significantly changed their mutant frequencies and mutational spectra independently of the Pol {eta} status. However, the increase in the induction of mutations in cells transfected with damaged shuttle vectors were dependent on the Pol {eta} status. The authors proposed that the pretreatment of the host cells with UV radiation, which causes the stalling of the replication machinery at sites of DNA damage in the genome, could disturb the balance between error-free and error-prone polymerases available for replicating the extrachromosomal plasmid. Accordingly, the induced untargeted mutagenesis in the undamaged and damaged plasmid might have originated from the activation of error prone polymerases other than Pol {eta}, and from the reduced fidelity of the classical replication machinery, respectively (60) . Altogether, it is conceivable that in the present study, the DNA polymerase machinery of the cells treated with B(a)PDE alone or combined with UVA irradiation might have been compromised differently. For instance, UVA irradiation could have down-regulated the fidelity and/or processivity of Pol {kappa}, thereby providing the opportunity for other more error-prone polymerases to bypass the B(a)PDE-DNA adducts. Currently, work in our laboratory is under way to explore the effects of B(a)PDE and UVA radiation on several relevant polymerases including the Pol {kappa} and Pol {eta}.

The second scenario takes into account the modulation of DNA repair systems, mainly the nucleotide excision repair, involved in the removal of both PAH-DNA adducts and most UV-associated photoproducts (61 , 62) . Thus far, both B(a)PDE and UV radiation have been shown to impact on nucleotide excision repair in various model systems as well as in human cells (61, 62, 63, 64, 65, 66, 67) . Of importance for the present study is our previous findings that early stage Big Blue mouse embryonic fibroblasts possess the nucleotide excision repair capacity to remove bulky DNA adducts such as 4-aminobiphenyl- and B(a)PDE-DNA adducts, as well as cyclobutane pyrimidine-dimers and pyrimidine (6–4) pyrimidone photoproducts 24–48 h after mutagen-treatments (68 , 69) .2 It is possible that the consecutive treatments with B(a)PDE and UVA radiation more significantly reduce the efficacy of the nucleotide excision repair process to remove the promutagenic DNA adducts than the B(a)PDE treatment does per se. If true, the presumably more persistent DNA adducts induced by B(a)PDE plus UVA radiation will have higher mutagenic potential as compared with those adducts induced by B(a)PDE alone.

In summary, we have demonstrated a unique comutagenicity of B(a)PDE and UVA radiation as representatives of the most prevalent environmental carcinogens. An unresolved issue is whether the combined mutagenicity of UVA and B(a)PDE represents a special case, perhaps due to the UVA-absorbing properties of the PAH ring system. This is the interpretation that we currently favor. Alternatively, combined exposures may commonly produce a different mutagenicity and mutation spectrum compared with single exposures. The mutational signature of mixed or combined mutagens may not always be recognizable due to the synergistic and/or multiplicative effects of the mutagenic agents. To single out an individual agent in a mixture or combination of mutagens, and to specifically determine its mutagenicity are highly complicated issues, which should be dealt with cautiously. These complications are well reflected in the exemplary case of smoking-associated lung cancer. The observation that the spectrum of mutations induced by UVA and B(a)PDE together differs from that produced by either alone makes the identification of a mutagen from its spectrum, for example p53 mutations in the lung cancers of smokers, less straightforward than was recognized previously. Evidently, not all preferential DNA adduct formation sites by known and/or suspected tobacco-related carcinogens are necessarily the sites of frequent mutations (hotspots) in the p53 tumor suppressor gene or ras oncogenes of smoking lung cancer patients. Our results may explain the lack of complete identity between the B(a)P-induced mutation spectrum and p53 mutations of smokers in one of several ways: it may be that B(a)P is indeed the cause of the p53 mutations but that other mutagens have distorted its mutation spectrum, it may be that the mutation spectrum of another component of cigarette smoke has been distorted to resemble that of B(a)P, or the mutation spectrum in lung tumors represents the combined output from several completely independent mutagens. Notwithstanding these theoretical considerations, it has been shown that the mutation spectrum of a mixture of mutagens, for example cigarette smoke condensate, is dominated and can be mimicked by a single potent mutagen found in the mixture, e.g., B(a)P (3) . One may argue that the herein reported results confirm our previous findings because they not only reaffirm the established B(a)PDE mutagenicity, but they also prove that even combined with another mutagen, B(a)PDE still leaves its specific signature of mutations on a chromosomal gene. Additional research is required to resolve these issues.


   ACKNOWLEDGMENTS
 
We thank Steven Bates for assistance in cell culturing.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Grant support: National Cancer Institute (CA84469) and National Institute of Environmental Health Sciences (ES06070; G. P. P.).

Requests for reprints: Ahmad Besaratinia, Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010-3000. Phone: (626) 359-8111; extension, 65918; Fax: (626) 358-7703; E-mail: ania{at}coh.org

1 N. Geacintov, personal communication. Back

2 A. Besaratinia and G. P. Pfeifer, unpublished observations. Back

Received 6/16/03. Revised 9/30/03. Accepted 10/ 2/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wilcosky T. C., Griffith J. D. Application of biological markers Hulka B. S. Wilcosky T. C. Griffith J. D. eds. . Biological Markers in Epidemiology, Vol. 1: 16-27, Oxford University Press New York 1990.
  2. Hulka B. S. Methodological issues in molecular epidemiology Hulka B. S. Wilcosky T. Griffith J. eds. . Biological Markers in Epidemiology, 214-226, Oxford University Press New York 1990.
  3. DeMarini D. M. Mutation spectra of complex mixtures. Mutat. Res., 411: 11-18, 1998.[Medline]
  4. Dolmans D. E., Fukumura D., Jain R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer, 3: 380-387, 2003.[Medline]
  5. Harvey R. G. Environmental occurrence and cancer Harvey R. G. eds. . Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity, Ed. 1 11-25, Cambridge University Press New York 1991.
  6. Phillips D. H. Polycyclic aromatic hydrocarbons in the diet. Mutat. Res., 443: 139-147, 1999.[Medline]
  7. Besaratinia A., Kleinjans J. C., Van Schooten F. J. Biomonitoring of tobacco smoke carcinogenicity by dosimetry of DNA adducts and genotyping and phenotyping of biotransformational enzymes: a review on polycyclic aromatic hydrocarbons. Biomarkers, 7: 209-229, 2002.[Medline]
  8. Phillips D. H. Smoking-related DNA and protein adducts in human tissues. Carcinogenesis (Lond.), 23: 1979-2004, 2002.[Abstract/Free Full Text]
  9. Harvey R. G. Metabolic activation, DNA binding, and mechanisms of carcinogenesis Harvey R. G. eds. . Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity, 50-95, Cambridge University Press New York 1991.
  10. Hall M., Grover P. L. Polycyclic aromatic hydrocarbons: metabolism, activation and tumour initiation Cooper C. S. Grover P. L. eds. . Chemical Carcinogenesis and Mutagenesis I, Vol. 94/I: 327-372, Springer-Verlag Berlin-Heidelberg 1990.
  11. Besaratinia, A. and Pfeifer, G. P. DNA Damage and Mutagenesis Induced by PAHs. In: A. Luch (ed.), The Carcinogenic Effects of Polycyclic Aromatic Hydrocarbons: World Scientific Publishing Company, Singapore, Singapore, in press, 2003.
  12. Denissenko M. F., Pao A., Tang M., Pfeifer G. P. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in. P53. Science (Wash. DC), 274: 430-432, 1996.[Abstract/Free Full Text]
  13. Smith L. E., Denissenko M. F., Bennett W. P., Li H., Amin S., Tang M., Pfeifer G. P. Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons. J. Natl. Cancer Inst., 92: 803-811, 2000.[Abstract/Free Full Text]
  14. Feng Z., Hu W., Chen J. X., Pao A., Li H., Rom W., Hung M. C., Tang M. S. Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer. J. Natl. Cancer Inst., 94: 1527-1536, 2002.[Abstract/Free Full Text]
  15. de Gruijl F. R. Skin cancer and solar UV radiation. Eur. J. Cancer, 35: 2003-2009, 1999.
  16. Woodhead A. D., Setlow R. B., Tanaka M. Environmental factors in nonmelanoma and melanoma skin cancer. J. Epidemiol., 9: S102-114, 1999.[Medline]
  17. de Gruijl F. R. Photocarcinogenesis: UVA vs. UVB radiation. Skin Pharmacol. Appl. Skin Physiol., 15: 316-320, 2002.[Medline]
  18. Cadet J., Berger M., Douki T., Morin B., Raoul S., Ravanat J. L., Spinelli S. Effects of UV and visible radiation on DNA-final base damage. Biol. Chem., 378: 1275-1286, 1997.[Medline]
  19. King G. N., Healy C. M., Glover M. T., Kwan J. T., Williams D. M., Leigh I. M., Worthington H. V., Thornhill M. H. Increased prevalence of dysplastic and malignant lip lesions in renal-transplant recipients. N. Engl. J. Med., 332: 1052-1057, 1995.[Abstract/Free Full Text]
  20. Vander Straten M., Carrasco D., Paterson M. S., McCrary M. L., Meyer D. J., Tyring S. K. Tobacco use and skin disease. South Med. J., 94: 621-634, 2001.[Medline]
  21. Nikkila A., Penttinen S., Kukkonen J. V. UV-B-Induced acute toxicity of pyrene to the waterflea Daphnia magna in natural freshwaters. Ecotoxicol. Environ. Saf., 44: 271-279, 1999.[Medline]
  22. Boese B. L., Ozretich R. J., Lamberson J. O., Cole F. A., Swartz R. C., Ferraro S. P. Phototoxic evaluation of marine sediments collected from a PAH-contaminated site. Arch. Environ. Contam. Toxicol., 38: 274-282, 2000.[Medline]
  23. Laycock N. L., Schirmer K., Bols N. C., Sivak J. G. Optical properties of rainbow trout lenses after in vitro exposure to polycyclic aromatic hydrocarbons in the presence or absence of ultraviolet radiation. Exp. Eye Res., 70: 205-214, 2000.[Medline]
  24. Pfeifer G. P., Chen H. H., Komura J., Riggs A. D. Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction. Methods Enzymol., 304: 548-571, 1999.[Medline]
  25. Besaratinia A., Pfeifer G. P. Weak yet distinct mutagenicity of acrylamide in mammalian cells. J. Natl. Cancer Inst., 95: 855-861, 2003.
  26. Jakubczak J. L., Merlino G., French J. E., Muller W. J., Paul B., Adhya S., Garges S. Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target. Proc. Natl. Acad. Sci. USA, 93: 9073-9078, 1996.[Abstract/Free Full Text]
  27. Herskowitz I., Hagen D. The lysis-lysogeny decision of phage lambda: explicit programming and responsiveness. Annu. Rev. Genet., 14: 399-445, 1980.[Medline]
  28. Adams W. T., Skopek T. R. Statistical test for the comparison of samples from mutational spectra. J. Mol. Biol., 194: 391-396, 1987.[Medline]
  29. Besaratinia A., Bates S. E., Pfeifer G. P. Mutational signature of the proximate bladder carcinogen N-hydroxy-4-acetylaminobiphenyl: inconsistency with the p53 mutational spectrum in bladder cancer. Cancer Res., 62: 4331-4338, 2002.[Abstract/Free Full Text]
  30. Harbach P. R., Zimmer D. M., Filipunas A. L., Mattes W. B., Aaron C. S. Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice. Environ. Mol. Mutagen., 33: 132-143, 1999.[Medline]
  31. Davies R., Gant T. W., Smith L. L., Styles J. A. Tamoxifen induces G:C–>T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene. Carcinogenesis (Lond.), 20: 1351-1356, 1999.[Abstract/Free Full Text]
  32. de Boer J. G., Erfle H., Walsh D., Holcroft J., Provost J. S., Rogers B., Tindall K. R., Glickman B. W. Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice. Environ. Mol. Mutagen., 30: 273-286, 1997.[Medline]
  33. Swiger R. R., Cosentino L., Shima N., Bielas J. H., Cruz-Munoz W., Heddle J. A. The cII locus in the MutaMouse system. Environ. Mol. Mutagen., 34: 201-207, 1999.[Medline]
  34. Mellon I., Spivak G., Hanawalt P. C. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell, 51: 241-249, 1987.[Medline]
  35. Hanawalt P. C. Transcription-coupled repair and human disease. Science (Wash. DC), 266: 1957-1958, 1994.[Free Full Text]
  36. Denissenko M. F., Pao A., Pfeifer G. P., Tang M. Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers. Oncogene, 16: 1241-1247, 1998.[Medline]
  37. Rochette P. J., Therrien J. P., Drouin R., Perdiz D., Bastien N., Drobetsky E. A., Sage E. UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells. Nucleic Acids Res., 31: 2786-2794, 2003.[Abstract/Free Full Text]
  38. Yoon J. H., Smith L. E., Feng Z., Tang M., Lee C. S., Pfeifer G. P. Methylated CpG dinucleotides are the preferential targets for G-to-T transversion mutations induced by benzo[a]pyrene diol epoxide in mammalian cells: similarities with the p53 mutation spectrum in smoking-associated lung cancers. Cancer Res., 61: 7110-7117, 2001.[Abstract/Free Full Text]
  39. Kozack R. E., Loechler E. L. Molecular modeling of the conformational complexity of (+)-anti-B[a]PDE-adducted DNA using simulated annealing. Carcinogenesis (Lond.), 18: 1585-1593, 1997.[Abstract/Free Full Text]
  40. Shukla R., Liu T., Geacintov N. E., Loechler E. L. The major. N2-dG adduct of (+)-anti-B[a]PDE shows a dramatically different mutagenic specificity (predominantly, G -> A) in a 5'-CGT-3' sequence context. Biochemistry, 36: 10256-10261, 1997.[Medline]
  41. Kozack R. E., Loechler E. L. Molecular modeling of the major adduct of (+)-anti-B[a]PDE (N2-dG) in the eight conformations and the five DNA sequences most relevant to base substitution mutagenesis. Carcinogenesis (Lond.), 20: 85-94, 1999.[Abstract/Free Full Text]
  42. Kozack R. E., Shukla R., Loechler E. L. A hypothesis for what conformation of the major adduct of (+)-anti-B[a]PDE (N2-dG) causes G–>T versus G–>A mutations based upon a correlation between mutagenesis and molecular modeling results. Carcinogenesis (Lond.), 20: 95-102, 1999.[Abstract/Free Full Text]
  43. Seo K. Y., Jelinsky S. A., Loechler E. L. Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens. Mutat. Res., 463: 215-246, 2000.[Medline]
  44. Li B., Mao B., Liu T. M., Xu J., Dourandin A., Amin S., Geacintov N. E. Laser pulse-induced photochemical strand cleavage of site-specifically and covalently modified (+)-anti-benzo[a]pyrene diol epoxide-oligonucleotide adducts. Chem. Res. Toxicol., 8: 396-402, 1995.[Medline]
  45. Routledge M. N., McLuckie K. I., Jones G. D., Farmer P. B., Martin E. A. Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations. Carcinogenesis (Lond.), 22: 1231-1238, 2001.[Abstract/Free Full Text]
  46. Ohashi E., Bebenek K., Matsuda T., Feaver W. J., Gerlach V. L., Friedberg E. C., Ohmori H., Kunkel T. A. Fidelity and processivity of DNA synthesis by DNA polymerase kappa, the product of the human DINB1 gene. J. Biol. Chem., 275: 39678-39684, 2000.[Abstract/Free Full Text]
  47. Ohashi E., Ogi T., Kusumoto R., Iwai S., Masutani C., Hanaoka F., Ohmori H. Error-prone bypass of certain DNA lesions by the human DNA polymerase kappa. Genes Dev., 14: 1589-1594, 2000.[Abstract/Free Full Text]
  48. Zhang Y., Yuan F., Xin H., Wu X., Rajpal D. K., Yang D., Wang Z. Human DNA polymerase kappa synthesizes DNA with extraordinarily low fidelity. Nucleic Acids Res., 28: 4147-4156, 2000.[Abstract/Free Full Text]
  49. O-Wang J., Kawamura K., Tada Y., Ohmori H., Kimura H., Sakiyama S., Tagawa M. DNA polymerase kappa, implicated in spontaneous and DNA damage-induced mutagenesis, is overexpressed in lung cancer. Cancer Res., 61: 5366-5369, 2001.[Abstract/Free Full Text]
  50. Ogi T., Mimura J., Hikida M., Fujimoto H., Fujii-Kuriyama Y., Ohmori H. Expression of human and mouse genes encoding polkappa: testis-specific developmental regulation and AhR-dependent inducible transcription. Genes Cells, 6: 943-953, 2001.[Abstract]
  51. Suzuki N., Ohashi E., Kolbanovskiy A., Geacintov N. E., Grollman A. P., Ohmori H., Shibutani S. Translesion synthesis by human DNA polymerase kappa on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N(2)-BPDE (7, 8-dihydroxy-anti-9, 10-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene). Biochemistry, 41: 6100-6106, 2002.[Medline]
  52. Ogi T., Shinkai Y., Tanaka K., Ohmori H. Polkappa protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene. Proc. Natl. Acad. Sci. USA, 99: 15548-15553, 2002.[Abstract/Free Full Text]
  53. Matsuda T., Bebenek K., Masutani C., Hanaoka F., Kunkel T. A. Low fidelity DNA synthesis by human DNA polymerase-eta. Nature (Lond.), 404: 1011-1013, 2000.[Medline]
  54. Masutani C., Araki M., Yamada A., Kusumoto R., Nogimori T., Maekawa T., Iwai S., Hanaoka F. Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity. EMBO J., 18: 3491-3501, 1999.[Medline]
  55. Johnson R. E., Washington M. T., Prakash S., Prakash L. Fidelity of human DNA polymerase eta. J. Biol. Chem., 275: 7447-7450, 2000.[Abstract/Free Full Text]
  56. Masutani C., Kusumoto R., Yamada A., Dohmae N., Yokoi M., Yuasa M., Araki M., Iwai S., Takio K., Hanaoka F. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature (Lond.), 399: 700-704, 1999.[Medline]
  57. Johnson R. E., Kondratick C. M., Prakash S., Prakash L. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science (Wash. DC), 285: 263-265, 1999.[Abstract/Free Full Text]
  58. Chiapperino D., Kroth H., Kramarczuk I. H., Sayer J. M., Masutani C., Hanaoka F., Jerina D. M., Cheh A. M. Preferential misincorporation of purine nucleotides by human DNA polymerase eta opposite benzo[a]pyrene 7, 8-diol 9, 10-epoxide deoxyguanosine adducts. J. Biol. Chem., 277: 11765-11771, 2002.[Abstract/Free Full Text]
  59. Zhang Y., Wu X., Guo D., Rechkoblit O., Wang Z. 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. DNA Repair (Amst.), 1: 559-569, 2002.
  60. Stary A., Kannouche P., Lehmann A. R., Sarasin A. Role of DNA polymerase eta in the UV mutation spectrum in human cells. J. Biol. Chem., 278: 18767-18775, 2003.[Abstract/Free Full Text]
  61. Friedberg E. C. How nucleotide excision repair protects against cancer. Nat. Rev. Cancer, 1: 22-33, 2001.[Medline]
  62. Batty D. P., Wood R. D. Damage recognition in nucleotide excision repair of DNA. Gene, 241: 193-204, 2000.[Medline]
  63. van Steeg H., de Vries A., van Oostrom C., van Benthem J., Beems R. B., van Kreijl C. F. DNA repair-deficient Xpa and Xpa/p53+/- knock-out mice: nature of the models. Toxicol. Pathol., 29(Suppl): 109-116, 2001.
  64. Geacintov N. E., Broyde S., Buterin T., Naegeli H., Wu M., Yan S., Patel D. J. Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery. Biopolymers, 65: 202-210, 2002.[Medline]
  65. Sarasin A. The molecular pathways of ultraviolet-induced carcinogenesis. Mutat. Res., 428: 5-10, 1999.[Medline]
  66. Conney A. H., Chang R. L., Cui X. X., Schiltz M., Yagi H., Jerina D. M., Wei S. J. Dose-dependent differences in the profile of mutations induced by carcinogenic (R, S, S, R) bay- and fjord-region diol epoxides of polycyclic aromatic hydrocarbons. Adv. Exp. Med. Biol., 500: 697-707, 2001.[Medline]
  67. Shyong E. Q., Lu Y., Goldstein A., Lebwohl M., Wei H. Synergistic enhancement of H2O2 production in human epidermoid carcinoma cells by Benzo[a]pyrene and ultraviolet A radiation. Toxicol. Appl. Pharmacol., 188: 104-109, 2003.[Medline]
  68. You Y. H., Lee D. H., Yoon J. H., Nakajima S., Yasui A., Pfeifer G. P. Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells. J. Biol. Chem., 276: 44688-44694, 2001.[Abstract/Free Full Text]
  69. You Y. H., Li C., Pfeifer G. P. Involvement of 5-methylcytosine in sunlight-induced mutagenesis. J. Mol. Biol., 293: 493-503, 1999.[Medline]



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