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

Michael N. Routledge,3, Keith I. E. McLuckie,1, George D. D. Jones,2, Peter B. Farmer,1 and Elizabeth A. Martin,1

Department of Biological Sciences, De Montfort University,
1 MRC Toxicology Unit and
2 Department of Oncology, Hodgkin Building, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 104 transformants) was higher than BPDE alone (12 in 104 transformants) or UVB alone (446 in 104 transformants), and the mutation frequency for BPDE + UVC (197 in 104 transformants) was higher than BPDE alone or UVC alone (26 in 104 transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC->AT as the most common mutation) and BPDE signature mutations (GC->TA and GC->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.

Abbreviations: BPDE, benzo[a]pyrene diol epoxide; PAH, polycyclic aromatic hydrocarbon; HRP, horseradish peroxidase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutagenic DNA lesions can arise as the result of the interaction of DNA with a myriad of agents that are derived, in vivo, exogenously and/or endogenously. In many cases, exposure to mutagenic agents occurs in the form of combined exposures or complex mixtures of mutagens e.g. cigarette smoke or diesel exhaust emissions (1). In experimental studies it is often desirable to reduce exposure to the level of the individual agent, in order to elucidate the processes involved. However, it is possible that the induction of damage and mutations by a specific agent may be altered if the exposure occurs in combination with another agent or as a mixture of agents. For example, it has been demonstrated that some carcinogenic compounds that normally require metabolic activation to reactive species before binding to DNA occurs can be photoactivated to direct acting mutagens by UVA irradiation (25). As well as interactions between agents prior to DNA binding, which may act to modulate either the binding of genotoxic agents or the mutagenicity of the reactive species, it may be possible that the mutagenicity of bound adducts is modified by other DNA reactive agents, or by the presence of other lesions on the DNA molecule. As a first step in addressing this possibility we have combined UV irradiation with adduction by benzo[a]pyrene diol epoxide (BPDE) as the DNA damaging events. BPDE is the ultimate carcinogen formed by the metabolism of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, which is found in cigarette smoke, burnt food and smoke from the burning of fossil fuels. BPDE binds mainly to guanines (6) and is known to induce primarily GC->TA transversions (7). UV irradiation, a known mutagen and carcinogen, induces GC->AT transitions, mainly through the formation of cyclopyrimidine dimers and (6–4)photoproducts (811). Evidence that PAH and UV exposure (in sunlight) may have a combined toxic effect in vivo has come from studies on the phototoxicity of PAH in aquatic organisms (1214). In terms of human carcinogenesis, a combination effect of UV irradiation and PAH could be postulated in situations where occupational exposure to PAH is combined with regular exposure to high levels of sunlight, such as for road maintenance workers (15) or in medicinal therapies for psoriasis that combine coal tar application and UV irradiation (16).

Amongst the different types of DNA lesions that are induced by UV irradiation, DNA single strand breaks may be easily monitored using a plasmid strand break assay, as the introduction of a single strand break into supercoiled plasmid DNA results in the conversion of the supercoiled plasmid into relaxed or nicked plasmid, which migrates more slowly than supercoiled plasmid upon agarose gel electrophoresis (17). A versatile method for analysing induced mutations is the supF forward mutation assay (18), which has been applied to the study of a wide range of mutagens (10,1821). Using the supF mutation assay, information can be obtained as to the type of mutations induced by a compound and the distribution of these mutations within the target gene.

In this pilot study, we have investigated the effects of UV irradiation on DNA that has been adducted in vitro with BPDE, by measuring DNA single strand breaks in a plasmid strand break assay and the effect of the presence of BPDE adducts in the target DNA on UV induced mutations in the supF gene forward mutation assay (10).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of DNA damage
Plasmid DNA (pUC18, 20 µg in 20 µl water) was incubated with either acetone (20 µl) or BPDE dissolved in 20 µl acetone (approximately 10 µM) for 10 min at 37°C. Unreacted BPDE was removed by extraction into ethyl acetate. DNA was precipitated by addition of 0.1 vol 2.5 M sodium acetate, pH 5 and 2.5 vol ethanol, followed by storage at –20°C, overnight. Precipitated DNA was pelleted and washed twice with 70% (v/v) ethanol. BPDE adduction levels were determined by 32P-post-labelling assay, as described previously (22) except that visualization and quantification of adducts was performed using a PhosphorImager (Molecular Dynamics, Sevenoaks, Kent) with ImageQuant software, version 3.3. For UV irradiation, DNA was re-dissolved in water (0.2 µg/µl) and 50 µl DNA solution was pipetted onto the surface of a plastic Petri dish. The DNA was irradiated with a UV lamp at a dose rate of 0.2 mW/cm2 for UVB and 0.7 mW/cm2 for UVC. At set time points (0, 10, 20, 30, 40, 50 and 60 min for UVB and 0, 0.75, 1.5, 2, 3, 4 and 5 min for UVC), 5 µl aliquots were removed and added to 20 µl gel loading buffer (0.05% bromophenol blue, 0.05% xylene cyanol, 6% glycerol in water) and stored on ice. Upon completion of the dosing regime, samples were analysed by agarose gel electrophoresis for single strand breaks (see below).

For the supF mutagenesis experiments the same procedures as above were performed using the pSP189 plasmid, except that one UV exposure was used for each wavelength. For UVB irradiation, the plasmid samples were irradiated for 20 min (giving a dose of 2.4 kJ/m2), whilst for UVC irradiation, the plasmid samples were irradiated for 1.5 min (giving a dose of 0.63 kJ/m2). After irradiation plasmid samples were stored at 4°C until transfection into Ad293 cells, which occurred within 24 h of irradiation.

Single strand break assay
After UV irradiation DNA samples were loaded onto a 0.8% (w/v) agarose gel made up with 1x TBE (90 mM Tris–borate, 1 mM EDTA, pH 8.0), on which supercoiled and relaxed plasmid were separated by electrophoresis at 12 V, overnight, with 1x TBE as running buffer. Following electrophoresis the gel was stained in ethidium bromide (0.5 µg/ml) for 1 h followed by destaining in water for 1 h. DNA bands were visualized and quantified using a Biorad MultiImager (BioRad, Hercules, CA). The amount of DNA in the supercoiled and relaxed forms of the plasmid was estimated by integration of the intensity of the pixels comprising each band. As supercoiled DNA binds lower amounts of ethidium bromide compared with open circular DNA, the intensities of supercoiled bands were adjusted by a factor of 1.4 (17).

SupF mutation assay
Subconfluent Ad293 cells (an adenovirus transformed embryonic kidney cell line, which was a kind gift from Tony Dipple, NCI–FCRDC, Frederick, MD) were transfected with control or treated plasmid (10 µg per 9 cm culture plate) using the calcium phosphate precipitation technique (23). After 48 h, plasmid was recovered using a Qiagen plasmid purification kit (Qiagen, Crawley, West Sussex). Aliquots of recovered plasmid were used to transform electrocompetent MBM7070 Escherichia coli by electroporation using a Gene Pulser apparatus (BioRad). Transformants were plated onto LB agar plates containing 100 µg/ml ampicillin, 75 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactose (Xgal) and 25 µg/ml isopropyl-ß-D-thiogalactoside (IPTG).

Plasmid was extracted from white and pale blue mutant colonies and sequenced using the primer 5'-GGCGACACGGAAATGTTGAA-3' (Protein and Nucleic Acid Chemistry Laboratory, CMHT, University of Leicester). The shuttle vector pSP189 contains an eight base `signature sequence' giving 48 (65 536) possible unique sequences (note that this figure differs from that of 2x48 given in the original reference (10), as we do not believe that insertion of the sequence in either direction doubles the number of possible sequences). Any mutants with a duplicated `signature' were excluded from further analysis. Poisson distribution analysis was used to assess the randomness of spectra. Hotspots were assumed when >5% of total mutations occurred at a single point in the spectrum.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of DNA single strand breaks in plasmid DNA
Supercoiled plasmid (pUC18) that had been treated with BPDE in acetone, or acetone alone (control), was irradiated with up to 7.2 kJ/m2 UVB or up to 2.1 kJ/m2 UVC. The BPDE adduct level in the treated DNA was determined by 32P-post-labelling to be about 1 adduct in 103 nucleotides (data not shown). The UVB and UVC doses used were chosen from preliminary range-finding experiments with untreated plasmid (data not shown), as doses that gave measurable increases in the amount of DNA in the form II plasmid band (relaxed circular plasmid containing at least one strand break), without inducing significant levels of linear DNA. After determination of the relative amount of DNA in the form I and form II bands, the data was plotted as the negative natural logarithm of the fraction of form I DNA remaining at each dose, against time of exposure (Figure 1Go). Plotting the results in this way is equivalent to plotting the average number of single strand breaks per plasmid molecule versus UV dose. The UV dose at which there were on average, one single strand break per plasmid molecule (D0) can be read from these plots, or calculated from the reciprocal of the slope. From Figure 1Go it can be seen that D0 for BPDE + UVB and UVB control were 5.9 and 24.5 kJ/m2, respectively, whilst D0 for BPDE + UVC and UVC control were 1.9 and 10.9 kJ/m2, respectively. In other words, there was a greater number of UV-induced single strand breaks introduced into the plasmid DNA that had been adducted with BPDE, compared with the control plasmid, at any given dose of either UVB or UVC irradiation.



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Fig. 1. Induction of single strand breaks in plasmid DNA. Control pUC18 DNA and BPDE adducted pUC18 DNA was irradiated with (a) UVB, or (b) UVC. Supercoiled (form I) plasmid was separated from open circular (form II) plasmid by agarose gel electrophoresis. The reduction in the fraction of supercoiled DNA with increasing time of exposure was calculated and the negative natural logarithm of this fraction plotted against time of UV irradiation. The values on the y axis are equivalent to the average number of single strand breaks per plasmid molecule.

 
Mutation frequency in supF assay
Aliquots of six pSP189 samples, untreated control (control), BPDE treated (BPDE), plasmid irradiated with UVB (UVB), BPDE treated plasmid that was subsequently irradiated with UVB (BPDE + UVB), plasmid irradiated with UVC (UVC) and BPDE treated plasmid that was subsequently irradiated with UVC (BPDE + UVC), were transfected into Ad293 cells to allow for mutagenesis to occur within the human cell line. The UVC dose of 0.63 kJ/m2 was chosen based on results obtained in preliminary supF assay experiments in this laboratory (unpublished results), whilst the UVB dose (2.4 kJ/m2) was selected on the basis of this dose of UVB irradiation inducing a similar level of conversion of supercoiled to relaxed plasmid as was induced by the 0.63 kJ/m2 dose of UVC irradiation, as determined in the plasmid single strand break assay (Figure 1Go). Extracted plasmid that had been replicated in the Ad293 cells was screened for the presence of mutant supF gene by transformation into E.coli MBM7070. Table IGo shows the mutation frequency that was observed in these experiments. It can be seen from the control plasmid that the background mutation frequency was suitably low, at 3 mutants in 105 transformants. For all other treatments mutation frequency was significantly enhanced. The level of BPDE adducts in the BPDE treated pSP189 was determined by 32P-postlabelling to be about 2.2 adducts in 104 nucleotides, which is the equivalent of about 1 adduct per plasmid molecule. In this experiment, this has led to a mutation frequency of 12 mutants per 104 transformants (Table IGo). It can be noted as well, that the presence of BPDE damage in the plasmid led to an increase in mutation frequency upon UV irradiation with UVB or UVC that was greater than predicted from the mutation frequency of either treatment alone. For example, the mutation frequency for BPDE + UVB was 1167 in 104 transformants, which was ~2.5-fold greater than the addition of the BPDE mutation frequency (12 in 104 transformants) and the UVB mutation frequency (446 in 104 transformants). Similarly, the mutation frequency for BPDE + UVC (197 in 104 transformants) was ~5-fold greater than the additive mutation frequencies for BPDE (12 in 104 transformants) and UVC alone (26 in 104 transformants). When mutant plasmids were sequenced it was found that two classes of mutated plasmid were present: those containing a single or tandem mutation in the supF gene and those containing two or more non-adjacent (multiple) mutations. The frequency of multiple mutations was greatest in the combined treatments (Table IGo), with 20 out of 91 sequenced BPDE + UVB plasmids containing a total of 46 multiple mutations and 11 out of 64 sequenced BPDE + UVC plasmids containing a total of 27 multiple mutations. The only other protocol to yield multiple mutations was UVB treated plasmid, for which 3 out of 66 sequenced plasmids contained six multiple mutations. Interestingly, two of these plasmids contained identical multiple mutations, AT->GC at site 120 and GC->AT at site 124.


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Table I. Mutation frequency induced by the various treatments of pSP189
 
Mutation spectra in supF assay
Mutant colonies were collected from the different transformations and sequenced to determine the types and distribution of induced mutations in the supF gene. It can be seen from Table IIGo that in all treatments induced mutations were mainly at GC pairs. There were two classes of mutant plasmid, those that contained single or tandem mutations and those that contained multiple mutations. The types of mutations for these two classes of mutant plasmid (singles and multiples) are shown separately in Table IIGo, as it has been shown that multiple mutations arise by a separate mechanism to single or tandem mutations (24), a mechanism that involves the induction of error prone replication (25). For the singles class of mutants, the most common base substitution mutation induced by BPDE was the GC->TA transversion (65%), followed by GC->CG transversions. For all the treatments involving UV irradiation, the GC->AT mutation was the most common (84% for UVB and 79% for UVC). However, it can be seen that the percentage of both GC->TA and GC->CG mutations was higher in BPDE + UVB compared with UVB, and BPDE + UVC compared with UVC. It can also be seen from Table IIGo that the percentage of GC->AT mutations is lower in the multiple class mutants compared with the single class mutants for both BPDE + UVB and BPDE + UVC. For BPDE + UVB this is due to an increase in the percentage of GC->TA, GC->CG, AT->GC and AT->CG mutations, whereas for BPDE + UVC it is due to an increase in the percentage of AT->GC and AT->TA mutations.


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Table II. Percentage single base substitution mutationsa induced by the various treatments of pSP189b
 
Table IIIGo shows the types of single class base substitution mutations that were induced at GC base pairs, expressed in terms of the absolute frequency of these mutations. By expressing the results in this way it can be seen more clearly that the increase in mutation frequency of different types of GC base substitutions varies. Hence, the increase in frequency of GC->AT mutations is from 375 to 899 in 104 transformants in UVB compared with BPDE + UVB samples, whereas the increase in frequency of GC->TA mutations in the same samples is much greater, going from 31 to 163 in 104 transformants. This is an increase of 5.3-fold for GC->TA mutations, compared with an increase of 2.4-fold for GC->AT mutations in these samples. Similarly, for the UVC treated samples, the increase in GC->TA transversions (22-fold) and GC->CG transversions (10-fold) in BPDE + UVC versus UVC samples was greater than the increase in GC->AT transitions between the two samples (6-fold).


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Table III. Absolute mutation frequency (per 104 transformants) for mutations at GC pairs
 
The distribution of mutations (excluding multiple mutations) within the supF gene for the five treatments is shown in Figure 2Go. The distribution spectra of the multiple mutations have been shown separately (Figure 3Go). In each spectrum of mutations there are hotspots for mutation. Examination of the spectra in Figure 2Go reveals some differences in the distribution of these hotspots, particularly between the BPDE spectrum and the UV-induced spectra. For the BPDE treated spectrum there are clusters of mutations between sites 109–115 and sites 172–178. In particular hotspots of mutation are seen at sites 110, 115, 142 and 175. The UVB spectrum contains hotspots at 108, 122, 155, 156, 159 and 172, whilst the UVC spectrum contains hotspots at 104, 123, 156, 159 and 172. When comparing the BPDE + UVC spectrum to the BPDE and UVC spectra it can be seen that the BPDE + UVC spectrum contains aspects of both individual spectra. The BPDE-induced hotspot at site 110 is present, as is the UVC hotspot at 172. Interestingly, the strong GC->AT mutation hotspot at site 156 (a G in the transcribed strand of supF) in the UVC spectrum has switched to a strong GC->AT mutation hotspot at site 155 (a C in the transcribed strand) in the BPDE + UVC spectrum. Site 163, which was not a hotspot in either BPDE or UVC spectra, appears as a hotspot, with both UV consistent GC->AT mutations and BPDE consistent GC->TA and GC->CG mutations at this site in the BPDE + UVC spectrum.



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Fig. 2. Mutation spectra induced by the various treatments used. The 5' to 3' sequence of the transcribed strand of the wild-type supF gene is shown, with letters below the wild-type sequence indicating the position and type of point mutations induced by the various treatments.

 


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Fig. 3. A comparison of the spectra of single and tandem mutations with the spectra of multiple mutations (two or more non-adjacent mutations in a sequenced plasmid) for the BPDE + UVB and BPDE + UVC treatments.

 
Figure 3Go shows the spectra of multiple mutations observed for the BPDE + UVB and BPDE + UVC protocols. For ease of comparison, the single mutation spectra for these protocols are reproduced in this figure. For the BPDE + UVB multiple spectrum it can be seen that there were hotspots of mutation at sites 155, 156 and 163, which were also hotspots in the BPDE + UVB single mutation spectrum, and at site 124, which was not classified as a hotspot in the single mutation spectrum. A notable hotspot at site 159 in the BPDE + UVB single mutation spectrum was not present in the multiple spectrum. Comparing the BPDE + UVC single and multiple mutation spectra it can be seen that the only common hotspots occur at sites 115 and 172.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The majority of in vivo exposure to DNA damaging agents occurs as a result of interactions of complex mixtures of chemicals, such as those found in cigarette smoke or diesel exhaust emissions, with DNA (1). As well as complex mixtures, combined exposures to agents from different sources may also occur, for example bitumen and sunlight (15). When studying DNA damage and mutagenesis, it is often necessary to reduce this complex situation to a more simple single-agent exposure system, and the types of DNA damage and mutations induced by specific agents have been studied in a wide number of experimental systems. To start to address the question of what the effect of combined exposures may be on individual DNA damaging agents, we have investigated the combination of two mutagens that have been extensively studied: BPDE, a chemical carcinogen that forms well characterized bulky DNA adducts, and UV irradiation, a physical mutagen that induces cyclopyrimidine dimers and (6–4)photoproducts. The reason for choosing this combination was twofold. Firstly, as an experimental system the types of mutations induced by BPDE and UV irradiation are quite distinct, with the most common mutation induced by BPDE in a number of mutation assays being GC->TA transversions (7,26), whilst GC->AT mutations are the most common base substitutions induced by UV irradiation (9,27). These mutations arise as a result of damage to purines (chiefly guanine) in the case of BPDE (6,7) and pyrimidines in the case of UV (11,28). Hence, it would be possible to distinguish between the mutagenic contributions of the two agents in a sequential exposure protocol. (In this paper, GC->TA mutations are referred to as BPDE signature mutations, whilst GC->AT mutations are referred to as UV signature mutations as a short hand term to distinguish between the likely source of these mutations in these experiments). Secondly, there is evidence that in aquatic organisms, the toxicity of PAH pollutants is enhanced by UV irradiation (1214). There is, therefore, some rationale to thinking that DNA damaged by a PAH such as BPDE may be more susceptible to UV induced damage or mutations.

One type of damage induced in DNA by UV irradiation is the single strand break, and the induction of single strand breaks can be easily analysed using a plasmid strand break assay. In our system, the level of UV-induced single strand breaks was higher for any given UV dose when the plasmid was adducted with BPDE at a level of ~1 adduct in 103 nucleotides, than when it was not adducted. This increased susceptibility of BPDE adducted DNA to UV-induced strand breaks applied to both UVB and UVC irradiation. Whilst in cells there is evidence that some UVB-induced strand breaks may be mediated by repair enzymes (29), in the cell-free system studied here all strand breaks must be induced directly, or by production of reactive oxygen species in the irradiated DNA solution. It is possible that the BPDE adduct acts as a site of preferential induction of strand breaks, or that the presence of the BPDE adduct is involved in the UV-induced generation of reactive oxygen species from water molecules, and it is these reactive oxygen species that induce the strand breaks. It has previously been shown that laser pulse irradiation at 355 nm of site-specifically modified BPDE-guanine oligonucleotides induces strand cleavage at, and to a lesser extent near to, the adducted base, by a mechanism that involves either photo-induced electron transfer or the production of a local free radical that ultimately results in strand breaks (30,31).

DNA single strand breaks are formed by UVB and UVC at much lower levels than are mutagenic cyclopyrimidine dimers and 6–4 photoproducts (32). In order to investigate whether mutagenicity was increased when BPDE adducts were present on plasmid DNA prior to UV irradiation, we repeated the exposures using the pSP189 plasmid that carries the supF target gene as the substrate DNA and studied the induced mutations of the different treatments. Mutations induced by BPDE and UV irradiation in separate exposures have previously been reported in this system (7,10). Based on the quantification of BPDE adducts by the 32P-post-labelling assay, our BPDE-induced mutation frequency was ~5-fold higher at a similar adduct level (about one adduct per plasmid molecule, on average) than that reported by Maher et al. (7). The spectrum of mutation types was similar in the two studies with GC->TA, GC->AT and GC->CG mutations accounting for 63, 9 and 18%, respectively, in the earlier study (7) and for 65, 11 and 22%, respectively, in this study. The distribution of mutations, however, was quite different, with no common hotspots of mutation observed in the two spectra. The sites of hotspot for mutation in the current study, 110, 115, 142 and 175, were not hotspots in the previous study. Interestingly, the hotspots in the current study all correlate with hotspots for BPDE adduction in the supF gene, as determined by a polymerase stop assay in a previous study (7). Maher et al. (7) showed that hotspots of damage and hotspots of mutation did not correlate. However, as we did not perform the polymerase stop assay here, we cannot comment on whether our results disagree with the previous finding, but merely point this finding out as an interesting observation. Differences in the distribution of mutations between this study and Maher et al. (7) are probably due to the fact that some of the plasmid sequence in pSP189 differs from that of pZ189 used in the earlier study. Such differences in induced mutation spectra in pS189 (an earlier version of pSP189) and pZ189 have been reported for aflatoxin B1 (33) and it is known that alterations in DNA sequence some distance from the target gene can modulate mutational spectra (34,35).

In our experiments, the most notable effect on UV-induced mutagenicity when the DNA was already adducted with BPDE was to increase the mutation frequency of the combination treatment compared with UV alone, for both UVB and UVC. For UVB and UVC the mutation frequency was 2.6- and 7.6-fold higher, respectively, when the plasmid was already adducted with BPDE than when it was not. The effect of the combined treatment on mutation frequency was more than just additive, as the mutation frequency of the BPDE adducted plasmid was lower than the mutation frequency of either UV irradiation treatment. Hence, the observed increase in one type of measure of DNA damage, the single strand break, was reflected by an increase in mutation frequency when the target DNA was already modified with BPDE.

Analysis of the mutation spectra for the combined treatments shows an increase in the percentage of GC->TA and GC->CG mutations in the BPDE + UV treatments compared with UV alone, which is expected. However, when the absolute mutation frequency for each type of base substitution is considered (Table IIIGo), the increase in GC->TA and GC->CG mutations in the BPDE + UV samples compared with UV alone is higher than the increase in GC->AT mutations. For example, the mutation frequency for GC->AT mutations increases just over 2-fold for UVB and almost 6-fold for UVC when the BPDE adducts are present, whereas the increase in GC->TA mutations is over 5- and 22-fold, respectively, for UVB and UVC. It appears that the mutations induced by UV treatment of BPDE-adducted plasmid include a greater frequency of BPDE signature mutations (GC->TA, GC->CG) as well as a greater frequency of UV signature mutations (GC->AT), and that the level of increase in the BPDE signature mutations is higher than the level of increase in the UV signature mutations. This is important because the level of BPDE adducts is the same in both cases, but the subsequent exposure of BPDE plasmid to UV has led to a greater increase in a type of mutations associated with BPDE adducts, compared with the increase in the mutations associated with UV adducts. For the UVC treatment, the relative increase of apparent BPDE signature mutations is also reflected by the presence of a hotspot of GC->TA and GC->CG transversions at site 110 in the BPDE + UVC spectrum. There is a hotspot at this site in the BPDE spectrum, but not in the UVC spectrum. This implies that the hotspot is related to BPDE damage. Previous studies of PAH mutagenicity in the supF gene have shown that whilst the hotspots of damage and mutation do not correlate, mutations are always targeted to bases that are adducted (20).

It has previously been shown that compounds that normally require enzyme mediated activation to be mutagenic in bacterial mutagenicity assays, such as aflatoxin, dimethylbenzo[a] anthracene and N-nitrosodimethylamine, are direct acting mutagens when exposed to UVA radiation or sunlight (25). For N-nitrosodimethylamine, the formation of O6-methylguanine and N7-methylguanine, DNA lesions known to be formed by activated metabolites of N-nitrosodimethylamine, as well as 8-oxo-deoxyguanosine, an oxidative lesion formed as a result of UVA irradiation, were detected (5). In a study using N-nitrosopyrrolidine it was shown that the compound was a direct acting mutagen in M13mp2 phage in the presence of UVA radiation, with DNA lesions formed by N-nitroso-1-phosphonooxypyrrolidine, a photoactivation product of N-nitrosopyrrolidine (36). In these studies it should be noted that UVA irradiation, and in the case of activation by sunlight possibly UVB irradiation also, increases binding of the mutagen to DNA by activation of the parent compound to a reactive intermediate that binds to DNA, whereas in the experiments we describe, the BPDE adducts are already bound to the DNA at the time of UV irradiation. In terms of the induction of DNA strand breaks, it has previously been shown in experiments using site-specifically BPDE modified oligonucleotides that the presence of non-bound benzo[a]pyrene tetraols also led to photoinduced strand cleavage of the oligonucleotide, albeit at a 7-fold higher tetraol concentration for a response equivalent to the BPDE–DNA oligonucleotide (30).

The explanation for the increased mutagenicity in UV irradiated BPDE-adducted plasmid may be that this increased mutagenicity is the direct result of the increase in single strand breaks shown by the plasmid strand break experiments. Although the single strand breaks are not miscoding lesions, it has previously been reported that the introduction of single strand breaks into the plasmid pZ189 (to yield nicked plasmid) led to an increase in mutation frequency over background in a repair deficient cell line (25). Furthermore, the presence of multiple mutations within a single plasmid was found to be associated with the presence of nicked plasmid. Such multiple mutations also occurred when UV irradiated plasmid was transformed into repair proficient cells. Thus, the presence of nicks in transformed plasmid, or the repair of UV damaged plasmid (during which nicks may be formed), was associated with an error prone repair replication that accounted for some of the observed mutations (25). Although the use of different plasmid and cell types between the experiments reported here and the earlier experiments (in which spontaneous mutation frequency was higher) makes direct comparison of the results difficult, it seems likely that the increase in levels of UV-induced single strand breaks, as shown in the plasmid strand break assay experiments, has contributed to the increased mutation frequency. The presence of increased strand breaks certainly correlates with the higher levels of multiple mutations in the BPDE + UVB or BPDE + UVC treated samples compared with the other samples. Nevertheless, it is unlikely that this is the only explanation for the increase in mutation frequency. In our mutation experiments the doses of UVB and UVC used were selected because they gave similar levels of strand breaks in the plasmid strand break assay, not because they gave similar levels of mutation. Although this means that the UVB and UVC mutation frequency results cannot be directly compared, it also means that plasmid with different UV treatments but similar levels of single strand breaks gave very different mutation frequencies. This would not be predicted if the induction of single strand breaks was entirely responsible for the increase in mutation frequency.

We consider there to be four other possible explanations for the increased mutation frequency when BPDE adducts are present: (i) GC->TA mutations are induced by oxidative DNA damage, and the production of reactive oxygen species as a result of an interaction between the BPDE adduct and UV radiation could account for increased mutation frequency (a mechanism similar to that by which non-bound benzo[a]pyrene tetraol enhanced photoinduced cleavage of BPDE-modified oligonucleotides as discussed above). (ii) The presence of the BPDE adducts may cause the DNA to absorb more energy from the UV radiation, leading to enhanced UV damage, which could be targeted around the sites of BPDE adduction. (iii) UV irradiation of the BPDE adduct may produce a photoactivation product that is inherently more mutagenic than the original adduct. (iv) The presence of UV and BPDE adducts in the same DNA molecule alters the mutagenicity of one or the other.

Looking at the mutation spectra generated in this study, it would appear that in the BPDE + UV spectra, the BPDE adducts have contributed to the increased frequency of mutations in the combination treatment of BPDE + UV versus BPDE alone, even though the level of BPDE adducts has not altered. This evidence suggests that the BPDE adducts have themselves become more mutagenic, either due to photoactivation of the adduct, or to effects of UV adducts nearby (i.e. a change in DNA conformation caused by the presence of a UV adduct has led to a BPDE adduct becoming more mutagenic than before). The question of what factors may influence the formation of different mutagenic spectra by the same adduct forming agent, or even by the same specific adduct, under different circumstances has been addressed in elegant detail by Rodriguez and Loechler (34,37) and Seo et al. (38), with particular reference to adducts formed by BPDE. They have provided a strong case that the same adduct [specifically the major adduct of (+)-anti-BPDE formed by trans addition of N2-dG to C10 of (+)-anti-BPDE] can be locked into two conformations, one that leads to G->T mutation, and another that leads to G->A mutation. Perhaps the outcome of the UV irradiation of BPDE adducted DNA is to induce an analogous conformational change in the adducts?

Whilst our results may relate specifically to the interaction of UV irradiation with BPDE adducted DNA, this work was inspired by a general interest in the possible mutagenic effects of combined exposures that may result from the presence of more than one type of mutagenic lesion in DNA. By examination of data from an earlier study of the mutagenicity of the tamoxifen derivatives, {alpha}-acetoxytamoxifen and 4-hydroxy-tamoxifen (39), we note another potential example of such a situation. In order to activate 4-hydroxytamoxifen to 4-hydroxytamoxifen quinone methide, which reacts with DNA, the 4-hydroxytamoxifen was incubated with horseradish peroxidase (HRP) and H2O2. As this activation step generates mutagenic oxidative DNA lesions, the HRP/H2O2 treatment without 4-hydroxytamoxifen was used as a control. In a lacI gene bacterial mutagenicity assay, 1 µM activated 4-hydroxytamoxifen induced mutations at a frequency ~16-fold higher than untreated control, whilst the HRP/H2O2 treatment itself induced mutations at a frequency ~7-fold higher than control. It was found that the mutagenicity of the HRP/H2O2 activated 4-hydroxytamoxifen was two orders of magnitude higher than that of {alpha}-acetoxytamoxifen in this system. Whilst the authors' conclusion that the adducts formed by 4-hydroxytamoxifen quinone methide are more mutagenic than the adducts formed by {alpha}-acetoxytamoxifen is justified, we would suggest that if our combined BPDE + UV results are indicative of a general enhancement of mutagenicity of combined exposures, then there is a possibility that some proportion of the higher mutagenicity of HRP/H2O2 activated 4-hydroxytamoxifen is due to the effects of the oxidative lesions enhancing the mutagenicity of the tamoxifen adducts, or vice versa.

If the presence of UV lesions and BPDE adducts in DNA does influence the mutagenicity of one or more of these adducts, it follows that mutations induced by agents that cause a range of adducts in DNA might demonstrate a similar effect. This would have implications for the conclusions drawn from experiments in which the mutagenicity of single DNA adducts are assessed in site-specific assays.

For the results we present here, we have not ruled out the possibility that the increase in GC->TA mutations is due to oxidative DNA damage induced by reactive oxygen species generated through an interaction of the BPDE adduct and UV radiation. We aim to investigate this and other questions that arise from these results in future experiments. For example, we aim to repeat the treatments of pSP189 in the reverse order (i.e. UV irradiation prior to BPDE adduction). This is unlikely to lead to enhanced single strand breaks, but will test whether the presence of UV and BPDE adducts on the same plasmid interact to increase mutation frequency. The results presented here demonstrate that UV-induced DNA damage and mutagenicity is enhanced by the presence of one class of bulky aromatic DNA adducts. This observation may be directly relevant to the enhanced toxicity of PAH by UV that has been observed in aquatic organisms, and may also be indicative of a general effect of synergistic mutagenicity by combined exposures of genotoxic agents. This possibility warrants further investigation.

Finally, the supF assay has been applied to a wide range of mutagenic treatments and of the 255 possible base substitution mutations in the 85 base pair tRNA gene sequence, 245 have previously been reported (40). We note that in the experiments reported here we can add two more mutations to that list: a C->G transversion at site 142 (BPDE spectrum, Figure 2Go) and an A->C at site 157 (UVB spectrum, Figure 2Go).


    Notes
 
3 To whom correspondence should be addressed at present address: Molecular Epidemiology Unit, School of Medicine, University of Leeds, Leeds LS2 9JT, UKEmail: medmnr{at}leeds.ac.uk Back


    Acknowledgments
 
This work was partly funded by a Medical Research Council Grant (G95277655MA) awarded to G.D.D.J.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 12, 2001; revised March 19, 2001; accepted April 10, 2001.