1 Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803, USA,
2 Centre for Environmental Health, University of Victoria, Victoria, BC V8W 2Y2, Canada,
3 Molecular Mutagenesis Group, Laboratory of Environmental Carcinogenesis and Mutagenesis and
4 Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-diol-9,10-epoxide; PAH, polycyclic aromatic hydrocarbon
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Introduction |
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More recently, another pathway for the metabolism of B[a]P to reactive intermediates has been proposed (1113). This involves one-electron oxidation to produce radical cation metabolites that bind covalently to DNA (1416). Unlike the diol epoxides, however, these radical cations do not form stable DNA adducts. Instead, binding of these radicals to a base results in rapid cleavage of the glycosyl bond, thereby generating an abasic site (13). Abasic sites produced by PAH radical cations are chemically identical to those produced by other cellular processes, including spontaneous depurination and the removal of modified DNA bases by glycosylases. Abasic sites are efficiently repaired by base excision repair (17), which is relatively error free. However, unrepaired abasic sites are mutagenic in bacteria (18,19) and in mammalian cells (2024). Thus, an alternative view by which B[a]P may induce cancer involves the production of abasic sites leading to the generation of mutations in critical oncogenes or tumor suppressor genes.
To better understand the mechanisms by which B[a]P causes mutations in animals, we have examined a collection of lacI mutants arising in the liver of untreated control and B[a]P-treated Big Blue® mice. Previous studies using Big Blue® mice showed that B[a]P is mutagenic in the liver (25), spleen (26) and in splenic T-cells (27). In other systems, B[a]P has been shown to be mutagenic in the small intestine (28), lung (29) and skin (3032). However, DNA sequence data following in vivo exposure to B[a]P are extremely limited (26,27). The mutation spectra presented here represent an extensive analysis of mutations isolated from liver tissue following in vivo B[a]P exposure. These data show that B[a]P is mutagenic both before and after partial hepatectomy and provide insights regarding the relative roles of BPDE and radical cations of B[a]P in B[a]P-induced mutagenesis in vivo.
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Materials and methods |
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PCR of the lacI gene
A 5 µl aliquot of phage stock was used as the template in a PCR containing 1x PCR buffer (15 mM TrisHCl pH 8.8, 1.5 mM MgCl2, 60 mM KCl), 200 µM dNTPs (Pharmacia Biotech), 2.5 U of Taq polymerase (Perkin Elmer) and 20 pmol each of forward [position 53 to 37, (5'-CCCGACACCATCGAATG-3')] and reverse [position 12011185 (5'-ACCATTCCACACAACATAC-3')] primers. Samples were subjected to 30 PCR cycles at 95°C for 36 s, 51°C for 36 s and 72°C for 90 s in a Perkin Elmer 9600 thermocycler. The PCR products were purified using Wizard PCR Prep DNA purification columns (Promega, Madison, WI), as specified by the supplier. The DNA concentration was determined on a Hoefer fluorometer.
Sequencing strategy
The methods used for sequencing the lacI gene have been described (34). Sequencing was initiated at the 5' end of the lacI gene using a primer that hybridizes at positions 41 to 21 (5'-GAATGGTGCAAAACCTTTCGCG-3'). Typically, the first 400 bases were resolved. If no mutation was observed, then a reverse primer was used that hybridizes at positions 11511128 (5'-TGCCTAATGAGTGAGCTAACTCAC-3'). Depending on the sequence coverage and ambiguities noted, further reactions were performed until a mutation was clearly defined. Other primers used for DNA sequence analysis of lacI can be found on the Big Blue® web site at (http://darwin.ceh.uvic.ca/bigblue/bigblue.htm). If a phenotypically silent mutation was identified, then sequencing continued until a mutation known to yield a phenotypic change was found. In this study, mutations could not be identified in 6% of the isolated mutants.
Statistical analyses
The data describing the spontaneous mutant frequency data from de Boer et al. (33) and the B[a]P-induced mutant frequency data from Shane et al. (25) that have been recalculated following DNA sequence analysis to correct for possible clonal mutations. The mutant frequency is the fraction of mutant (i.e. blue) plaques among all plaques scored. A corrected mutant frequency is possible only following sequencing since mutants that arise from clonal expansion (see below) can be eliminated. Mutation frequency is the frequency of each type of mutation observed. Mutation frequencies were calculated for each of the nine mutational classes listed in Table II. For the B[a]P-treated animals, the average mutant frequency observed following B[a]P-treatment was multiplied by the fraction of each type of mutation observed in each animal following DNA sequence analysis to yield a mutation frequency. An average mutant frequency of 4.2x105 was used to calculate the mutation frequencies of the spectrum of mutations previously reported for untreated liver tissue in 23 Big Blue® mice (33). This average spontaneous mutant frequency is similar to the frequency of 4.7x105 observed in the control animals in this study (25). Pairwise comparisons of these mutation frequencies were carried out using the MannWhitney U-test (35). Two-sided tests were used to compare the frequency of each type of mutation in the B[a]P-treated mice at hepatectomy and at sacrifice with one another and with the frequency of the same type of mutation in the untreated control group. A P < 0.05 level of significance was used in these analyses.
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Results |
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Mutations observed in lacI following B[a]P treatment
Table I lists the base pair substitution and single-base frameshift mutations observed in the liver tissue of B[a]P-treated Big Blue® mice at hepatectomy and at time of sacrifice. The sites of mutations observed within the lacI structural gene are indicated. All mutations are reported with respect to a change in the sense strand of lacI using the numbering system of Farabaugh (36). Mutations were identified in 147/156 (94%) of the mutants sequenced from B[a]P-treated mice. In order to more accurately determine both mutant and mutation frequencies, identical mutations recovered from the same tissue of the same animal were considered to represent the clonal expansion of a single mutant and only one of these mutations was scored. This conservative approach to scoring mutations guarantees that all mutations reported were of independent origin. A limitation to this approach is that mutational hotspots may be underestimated. Correcting for clonality resulted in 138 independent mutations. Of these, 125/138 (91%) were base pair substitutions and 115/138 (83%) occurred at a GC base pair. There were six single-base deletions and one single-base insertion. There were three deletions of two or more bases; and there were five double mutations, all of which were dinucleotide changes observed at adjacent nucleotides (Table I
). The occurrence of the single-base deletions or insertions can be explained by the slippage model first proposed by Streisinger et al. (37) and subsequently expanded by Schaaper et al. (38). Frameshift deletions of GC base pairs have been reported in bacteria treated with B[a]P (39) or with (±)-anti-BPDE (40) and to a lesser extent in B[a]P-treated mammalian cells (41,42). The conventional slippage model, however, cannot readily explain the single-base deletion observed at base pair 377.
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Hot spots
Mutation hot spots are specific sites (i.e. base pairs) within a gene where the number of independent mutations is observed at a frequency significantly greater than might be expected by a Poisson distribution. Although the number of independent mutants is low, six sites in lacI (bp 56, 86, 92, 93, 95 and 180) are putative hot spots for B[a]P-induced mutations (Table I). It is worth noting that multiple independent mutations at bp 92, 93 and 95 were also observed in the skin of Big Blue® mice treated topically with DMBA (43). Likewise, hotspots at several of these same sites, including bp 56, 93, 95 and 180, were noted in the spontaneous spectrum (33).
A closer examination of the 11 sites at which more than one type of mutation was observed revealed that with the exception of position 80, all involved 5'-CpG-3' sites. In the case of positions 56, 86, 92, 93, 95 and 180, mutations were recorded in two or three mice at one or both time points. Sites 92, 93 and 95 appear to be hot spots as three different types of mutations were recorded in multiple mice at these sites; in fact six and 12 mutations were recorded at positions 92 and 93, respectively. These data are consistent with the observation of mutation hotspots which have been observed in most genes where mutations have been studied (for example see refs 4446).
Analysis of mutation frequencies
Mutation spectra observed in the liver tissue of untreated control (33) and of B[a]P-treated mice at hepatectomy and at sacrifice are presented in Table II. For comparative analyses, the relative abundance (i.e. percentage) of each type of mutant is reported along with a calculated mutation frequency (see Materials and methods). While the distribution of mutations within a spectrum can be compared by looking at the relative percentage of each class of mutation, the frequency of each class provides the best measure of comparison between different spectra. There was evidence of animal-to-animal variability in the Big Blue® mutation assays from which these spectra were derived (25), indicating that the individual animal should be considered the experimental unit for statistical analysis. Similar variability has been described by others (48). Rather than assuming a specific underlying distribution, we used non-parametric (distribution-free) tests in the statistical analysis. Thus, the data in Table II
were analyzed by MannWhitney U-tests (35) applied to the individual animal responses.
The mutation frequencies of the nine classes of mutation were compared between the untreated control group and between the two B[a]P-treatment groups (at hepatectomy and at sacrifice; Table II). The mutations listed in Table II
were the six types of base pair substitutions, single-base additions and deletions as well as any `other' mutations (i.e. multi-base deletions and complex mutations) observed. In addition, the fold-increase in mutation frequency of a specific type of mutation in the treated group compared with the frequency of the same type of mutation in the spontaneous spectrum is reported. Comparisons were made between the mutation frequency of a given class of mutations between either of the B[a]P-treatment groups and the spontaneous group. In addition, mutation frequencies at hepatectomy and at sacrifice were compared with each other. There were no statistically significant differences for any of the nine classes of mutation presented in Table II
when comparing the two B[a]P-treatment groups. However, significant differences were observed between both of the B[a]P-derived spectra and the spontaneous spectrum.
Transitions:
With respect to transition mutations both of the B[a]P-derived spectra are similar to the spectrum of transitions observed in control animals (Table II). While the percentage of GC
AT transitions within either of the B[a]P-induced spectra is lower than the percentage observed in the untreated control, the mutation frequencies of GC
AT transitions between the B[a]P-treated and the untreated control spectra are comparable. The mutation frequencies of GC
AT transitions were 1.1- and 1.7-fold higher than the control for the B[a]P at hepatectomy and B[a]P at sacrifice, respectively. Likewise, AT
GC transitions, which are rarely observed in untreated tissues (33,49), were only observed once in the B[a]P-treatment spectrum at sacrifice. The mutation frequency (1.3x106) was lower than the frequency observed in the control spectrum (2.5x106) (Table II
). In the B[a]P-treatment spectrum generated at hepatectomy, no AT
GC transitions were observed; however, an upper limit for the frequency of this class of mutations was calculated to be <1.1x106(i.e. less than one mutant/8.47x105 p.f.u. scored). These data suggest that B[a]P treatment did not result in the production of either GC
AT or AT
GC transitions at the lacI locus in the liver of Big Blue® mice. Moreover, these data illustrate the importance of comparing mutation frequencies rather than relative percentages of different mutational classes to assess the mutagenic effects of mutagen treatment.
Other mutations:
The frequencies of frameshift mutations (single-base additions or deletions), and other mutations (including multi-base deletions, complex and double mutations) were not affected by B[a]P-treatment; however, very few of these types of mutations were observed (Tables I and II). Although the calculated mutation frequencies of these classes of mutants in the B[a]P-treatment spectra ranged from 1.3- to 1.7-fold over the mutation frequencies of the same type of mutation in the control spectrum, such differences were the result of calculating a frequency based on the presence of one to four mutants in a given class (Table II
). The fold difference in mutation frequencies must be considered in the context of the absolute number of mutations observed. Thus, upon statistical analysis, none of the differences in mutation frequency observed among frameshift, deletion, complex or double mutations in either of the B[a]P-induced mutant spectra was significantly different than the frequency of the same mutations in the untreated control (Table II
). These data are consistent with the view that B[a]P treatment in vivo does not result in a substantial induction of frameshift or complex mutations when selecting forward mutations at lacI.
Transversions:
GCTA was the most frequent transversion observed in B[a]P-treated animals (Table II
). However, GC
TA transversions were also frequent events in the spontaneous spectrum. Nevertheless, the mutation frequencies of GC
TA transversions in B[a]P-treated animals were elevated at hepatectomy (3.1-fold) and at sacrifice (4.3-fold) as compared with the frequency observed in the control spectrum. The frequencies of GC
TA transversions in B[a]P-treated animals at hepatectomy and at sacrifice are not different from each other but both are significantly different from the control frequency (P < 0.05).
GCCG transversions in the B[a]P treatment spectra comprise the most significant difference from the control. Compared with the spontaneous spectrum, the frequencies of these transversions were 10.5- and 11.9-fold greater at hepatectomy and at sacrifice, respectively. Again, the frequency of GC
TA transversions in B[a]P-treated animals at hepatectomy and at sacrifice are significantly different from the control (P < 0.05) but not from each other. The substantial increase in the frequency of GC
TA and GC
CG transversions following B[a]P treatment as compared with the frequency observed in control mice is consistent with their induction by B[a]P. Therefore, we conclude that B[a]P primarily induces both of these transversions in the liver of Big Blue® mice under the treatment conditions used in this study.
Interestingly, the frequency of ATTA transversions observed following B[a]P treatment at hepatectomy is 4.7-fold higher and significantly different from the control value (P < 0.05); however, the frequency of AT
TA transversions observed in the B[a]P-treatment spectrum at sacrifice is not statistically different from the control frequency. In addition, the frequency of AT
TA transversions at hepatectomy is not significantly different from the frequency at sacrifice. AT
TA transversions are intriguing since these mutations have been observed following B[a]P or BPDE treatment in vitro and in bacterial cells (50,51) and are thought to result from depurination following N3-modification of adenine. The fact that the mutation frequency of AT
TA transversions is not significantly different from the control in the B[a]P-treated mice at sacrifice may reflect the limited number of mutants in these spectra or may be the result of lesion instability and/or DNA repair during liver regeneration. Since the number of AT
TA transversions observed in all three spectra is limited and because the difference in these transversions is not observed in the B[a]P mutation spectrum at sacrifice, we consider the 4.7-fold increase in AT
TA transversions in the B[a]P mutation spectrum at hepatectomy to be equivocal, but worthy of further study.
Finally, ATCG transversions were infrequent in all three of the spectra shown in Table II
. There were no significant differences in the observed frequency of AT
CG transversions when either of the B[a]P treatment groups was compared with the control.
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Discussion |
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In this study, we used the Big Blue® system: (i) to determine if the mutation spectrum at lacI in B[a]P-treated mice was different from the spectrum observed in untreated mice; (ii) to determine if B[a]P-induced effects were apparent prior to and/or following cell proliferation in the liver induced by partial hepatectomy; and (iii) to evaluate whether the types of mutations observed in B[a]P-treated animals suggest BPDE or B[a]P-radical cation induced DNA damage.
B[a]P-induced effects
B[a]P is mutagenic in the liver of Big Blue® mice as indicated in Table II. Only the mutation frequencies of GC
TA and GC
CG transversions were significantly increased (P < 0.05) in the B[a]P-treatment spectra at hepatectomy and at sacrifice. In addition, AT
TA transversions are also significantly elevated in the B[a]P treatment spectrum at hepatectomy; however, as discussed above, we consider this observation to be equivocal and these mutations are not considered further.
While mutant frequencies were only significantly different from the control at sacrifice (25), such data represent a quantitative comparison of all classes of mutation with the control. Following DNA sequence analysis, it is clear that most types of mutations were not increased in the B[a]P treatment spectra. Thus, the sensitivity of detecting induced mutant frequencies may be limited by the number of different types (or classes) of mutations produced and by the quantitative yield of each mutational class. This is especially true when a given class of mutations rarely occurs in the spontaneous spectrum. For example, in Table II the frequency of GC
CG transversions in the spontaneous spectrum was 1.9x106. Following treatment with B[a]P, these transversions were significantly increased to frequencies of 19.9x106 at hepatectomy and 22.6x106 at sacrifice. Although these represent a 10.5- and 11.9-fold respective increase in GC
CG transversions relative to the spontaneous mutation frequency, both of the B[a]P-treatment frequencies for these transversions are well below the spontaneous mutant frequency of 42x106. Therefore, a substantial increase in the mutation frequency of a specific type of mutation may not significantly change the mutant frequency. These results provide an obvious example as to how DNA sequencing can complement a mutagenesis study.
Interestingly, the results presented here suggest that some of the types of mutations observed following B[a]P or BPDE treatment in vitro, in Escherichia coli or in mammalian cells in culture, may not be produced at significant levels in vivo. That is, in addition to the GCTA and GC
CG transversions seen in this study, a variety of in vitro bacterial and mammalian cell mutagenesis studies using B[a]P or DNA modified by specific enantiomers of BPDE have also reported ±1 base frameshift mutations and GC
AT transitions, as well as both AT
TA and AT
CG transversions (7,42,5462). The resulting mutation observed can vary with the nature of the assay system, the specific enantiomer of BPDE used, the sequence specificity of the target gene and the polymerase used to replicate past a defined adduct in vitro. For example, in Salmonella typhimurium strain, TA98, which is designed to detect frameshift reversion mutations, B[a]P efficiently induces frameshift mutations (63,64). Similarly, treatment of excision-repair deficient (uvr) E.coli with (±)-anti-BPDE, resulted predominantly in the induction of frameshift mutations in the lacI gene and a reduction in base pair substitutions as compared with the untreated control (40). In contrast, in Salmonella typhimurium strain TA100, which is designed to detect base pair substitution reversion mutations, 70% of the induced mutations elicited by B[a]P were GC
TA transversions while the remaining 30% were GC
AT transitions at a known hot spot for reversion (39).
It is of interest that there are differences in the quantitative yield and the types of mutations observed when treating with either (+)-anti or ()-anti-BPDE or a mixture of the tautomers i.e. (±)-anti-BPDE. This may be important as the (+)-anti and ()-anti tautomers bind with different affinities to the same guanine molecule (65,66) and can elicit different mutations at the same site (67,68). It has been reported that the (+)-anti-BPDE is formed at about 30 times the concentration of the ()-anti-BPDE tautomer when B[a]P is metabolized in vitro (69). Both the (+) and () isomer bind preferentially to 5'-GG-3' doublets (70), but the binding specificity for each isomer depends on the base 5' to the first guanine of the doublet. Thus, binding of (+)-anti-BPDE is favored if the base 5' to the doublet is a C or A, while binding of ()-anti-BPDE is increased if the flanking 5' base is a T. Mutations elicited by (+)-anti-BPDE were studied in supF following transfection into E.coli, while studies in mammalian cells mainly used (±)-anti-BPDE (6,7,71).
In mammalian tissue culture cells, the predominant mutations at the hprt gene following treatment with B[a]P or BPDE are base pair substitutions at guanine residues (7274). GCTA transversions are the most prevalent mutation followed by GC
AT transitions and GC
CG transversions. A similar spectrum has been observed in the supF gene in a shuttle vector treated with BPDE and transfected into mammalian cells (75). At the hprt locus in Chinese hamster V79 cells, GC
TA transversions predominated following treatment with BPDE, although at lower doses mutations were observed at AT base pairs as well (72). No differences were noted in the spectra of mutations induced by B[a]P and BPDE at the aprt locus in Chinese hamster ovary (CHO) cells (76). In this study, the predominant mutational change was GC
TA transversions.
It is worth noting that in the spleen of Big Blue® mice, Kohler et al. (26) observed GCTA, GC
CG and AT
TA transversions as well as one- and two-base deletions following B[a]P treatment. More recently, Monroe et al. (27) showed that the percentage of GC
TA transversions were also increased at the cII/cI loci in T-cells of B[a]P-treated Big Blue® mice.
Thus, the data presented here are consistent with the vast majority of studies reporting the production of GCTA and GC
CG transversions following treatment with B[a]P or BPDE. However, two classes of mutations often thought to be induced by B[a]P or BPDE, specifically GC
AT transitions and ±1 base frameshift mutations, are not observed to be significantly elevated in the liver of Big Blue® mice under the B[a]P treatment conditions described (Table II
).
Strand bias
Transcription-coupled repair appears to be an important pathway by which B[a]P-induced damage can be removed from DNA in mammalian cells. In diploid human fibroblasts, (±)-anti-BPDE-induced lesions were preferentially repaired on the transcribed strand (77). In addition, the lacI mutant frequency was substantially higher than the frequency of Hprt mutations in splenic T-cells following treatment of Big Blue® mice (5). Since Hprt is expressed in these cells and lacI is not, the implication is that a substantial number of B[a]P-induced premutagenic DNA lesions were removed by transcription-coupled repair leading to a reduction in the observed mutant frequency at Hprt. The data presented here are consistent with the findings of Skopek et al. (5). B[a]P-induced mutations occurred equally on both strands of lacI in the Big Blue® transgenic mouse (Table I). In fact, there were almost an equivalent number of mutations on both strands of the lacI gene when all base pair substitutions or only those at guanines were considered. Apparently, premutagenic damage that occurred on either adenine or guanine bases in either strand resulted in mutations with similar efficiency.
The effect of cell proliferation on mutant frequency and mutation spectra
One method of inducing cell proliferation in vivo is by partial hepatectomy. In the study presented here, the mutant frequency in the liver of the same animals was examined twice, once before and once after stimulation of cell proliferation. Following DNA sequencing of isolated mutants, mutation frequencies were calculated in these same tissue samples and were compared with spontaneous mutation frequencies (Table II). While the mutation frequencies of both GC
TA and GC
CG transversions were significantly increased at both time points relative to the control, they were not different from each other. Thus, there appears to be no affect of partial hepatectomy on the frequency of mutations observed following B[a]P treatment using the experimental design employed here. This lack of an effect of partial hepatectomy on B[a]P mutagenesis in mouse liver was unexpected. Stable B[a]PDNA adducts can be observed in the liver tissue of mice weeks after exposure (78). Therefore, we expected partial hepatectomy to force the replication of cells, thereby fixing mutations induced by B[a]P adducts. However, the mutation frequency data support the conclusion that the premutagenic lesions induced by B[a]P were mostly fixed as mutations in liver cells that had completed replication prior to partial hepatectomy.
How does B[a]P initiate mutations in exposed animals?
The literature reporting the mutagenic activity of B[a]P is extensive. However, most studies present the view that B[a]P causes mutations by forming stable, covalent, premutagenic adducts at guanine and adenine. Such studies emphasize the metabolism of B[a]P to BPDE as the pivotal metabolic activation pathway. However, an alternative activation pathway has been proposed, in which metabolism of B[a]P results in the production of radical cations which, in turn, lead to the formation of unstable purine adducts. Rapid depurination is then suggested to leave abasic sites in DNA and to produce mutations if these sites are not repaired (1113).
Although in this study there were no differences between the lacI mutation spectra in B[a]P-treated animals at sacrifice or at hepatectomy, both spectra differed significantly with respect to the frequency of GCTA and GC
CG transversions in the spectrum of spontaneous liver mutations (Table II
). This observation allows one to infer the role of different DNA lesions in B[a]P-induced mutagenesis in the liver of these mice. That is, whether the mutations observed are consistent with B[a]P metabolic activation by cytochrome P450 1A1 and epoxide hydrolase to yield mutagenic intermediates, including BPDE, or whether the production of PAH radical cations is the predominant pathway whereby B[a]P mutations are induced.
The radical cation hypothesis (1113) is more easily examined because an abasic site is thought to be the only mutagenic DNA lesion produced. Modification of nucleosides with PAH radical cations results in adduct formation with deoxyguanosine and deoxyadenosine, but not with deoxycytosine or thymidine (13,79). Therefore, unstable B[a]P adducts will result in the formation of abasic sites at purine (i.e. G and A) residues. Thus, the reactive specificity of PAH radical cations provides a basis for assuming that either a modified guanine or adenine produced a mutational change.
DNA replication opposite an abasic site in E.coli often leads to insertion of adenine (18,19,80). Subsequent replication fixes the mutation as a GT or A
T transversion, depending upon whether the depurinated base was guanine or adenine, respectively. This phenomenon of preferential incorporation of adenine opposite abasic sites in E.coli is commonly referred to as the `A-rule' (81), which has been evoked as evidence for the radical cation hypothesis of tumor initiation by PAHs (82). Indeed, an evaluation of c-Ha-ras mutations in PAH-induced tumors showed that the majority of mutations were consistent with depurination and insertion of adenine opposite an apurinic site. Thus, these data would seem to be consistent with the hypothesis that abasic sites generated by PAH radical cations may be responsible for tumor initiation by PAHs.
While abasic sites are mutagenic in mammalian cells, the types of mutations induced by these sites in mammalian cells differ from those observed in E.coli. In several studies using mammalian cells, there was no apparent preference for the incorporation of adenine opposite an abasic site (20,21,23,24,83). In fact, there is evidence that guanine and cytidine are more commonly inserted opposite abasic sites, which would give rise to ATCG, GC
CG transversions and AT
GC transitions (23,24). One can conclude from these data that the A-rule does not apply in mammalian cells. Furthermore, all the types of mutations that should result from PAH radical cation induced abasic sites are not observed in the spectrum reported here. Only two types of base substitutions (GC
TA and GC
CG transversions) are substantially increased by treatment of mice with B[a]P (Table II
). One can infer that a significant role for abasic sites in B[a]P-mediated mutagenesis would be reflected by increases in AT
CG, GC
CG transversions and AT
GC transitions. Since abasic sites are not expected to yield GC
TA transversions, as observed in this study, and since abasic sites have been shown to produce AT
GC transitions which were not observed here, we conclude that the single-electron oxidation pathway for B[a]P activation does not contribute substantially to mutagenesis of the lacI gene in the liver of Big Blue® mice. Of course, the GC
CG transversions observed in this study could be induced either by BPDE adducts or depurination of guanine following B[a]P-radical cation modification. We conclude that abasic sites are not responsible for the bulk of the detectable mutagenic events observed in the liver of B[a]P-exposed Big Blue® mice in these studies. Thus, these data suggest that the radical cation mechanism of mutation induction is less significant than BPDE-mediated mutagenesis in the liver of mice. Additional studies are needed to establish mutation spectra in other tissues and in other species in order to more fully evaluate which pathways of B[a]P metabolic activation are important for both mutagenesis and carcinogenesis.
The importance of sequencing mutants in the Big Blue® mutagenesis assay
Exposure of Big Blue® B6C3F1 mice to B[a]P caused significant changes in the hepatic mutation frequency in the lacI transgene (Table II). The differences in the mutation spectra were apparent both at the time of hepatectomy and at sacrifice, as revealed by sequencing (Tables I and II
). In contrast, the difference in mutant frequency between vehicle- and B[a]P-treated mice was only significant (P < 0.05) at sacrifice. In spite of the >2-fold induction in mutant frequency in B[a]P-treated animals at the time of partial hepatectomy, the increase relative to the control was not statistically significant. These data clearly demonstrate the sensitivity of the Big Blue® mutagenesis assay, especially when performed in combination with DNA sequencing. Similar observations have been made previously in Big Blue® studies with the mutagenic flame retardant, Tris(2,3-dibromopropyl)phosphate (84).
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Conclusions |
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Acknowledgments |
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Notes |
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6 To whom correspondence should be addressed Email: tindall{at}niehs.nih.gov
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References |
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