LacI mutation spectra following benzo[a]pyrene treatment of Big Blue® mice

Barbara S. Shane1, Johan de Boer2, David E. Watson3,5, Joseph K. Haseman4, Barry W. Glickman2 and Kenneth R. Tindall3,6

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
The mutation spectrum of the lacI gene from the liver of C57Bl6 Big Blue® transgenic mice treated with benzo[a]pyrene (B[a]P) has been compared with the spectrum of spontaneous mutations observed in the liver of untreated Big Blue® mice. Mice were treated with B[a]P for 3 days followed by a partial hepatectomy one day after the last injection. Liver tissue was removed for analysis at hepatectomy and, again, 3 days later at the time of sacrifice. Earlier, we reported that the lacI mutant frequency in these B[a]P-treated mice was elevated in the liver both at the time of hepatectomy and at sacrifice; however, a statistically significant increase in the mutant frequency was observed only at sacrifice. In this study, the DNA sequence spectra of lacI mutations observed in the liver of B[a]P-treated Big Blue® mice at hepatectomy and at time of sacrifice were compared with each other and with the spectrum of spontaneous liver mutations. No differences were observed between the two B[a]P-treatment spectra. However, mutation frequencies of both GC->TA and GC->CG at the time of hepatectomy and at sacrifice were significantly elevated compared with the spontaneous frequency of these same transversions. Also, the frequency of AT->TA transversions was significantly higher than the spontaneous frequency at the time of hepatectomy but not at sacrifice. The frequency of all other classes of mutations scored was not significantly different from the frequency of these same events in the spontaneous spectra. These data support the view that B[a]P treatment results in the induction of GC->TA and GC->CG transversions within 1 day of the last injection and they provide insights regarding the relative roles of benzo[a]pyrene-7,8-diol-9,10-epoxide and radical cations of B[a]P in B[a]P-induced mutagenesis in vivo. Finally, these data provide evidence for B[a]P-induced mutagenesis under conditions where no statistical increase in mutant frequency could be shown.

Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-diol-9,10-epoxide; PAH, polycyclic aromatic hydrocarbon


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Benzo[a]pyrene (B[a]P) is a ubiquitous mutagenic carcinogen that undergoes biotransformation to produce intermediates that are both mutagenic and carcinogenic. However, the precise mechanism(s) by which B[a]P and other polyaromatic hydrocarbons (PAHs) induce mutational changes that lead to cancer remain unclear. The traditional view of B[a]P mutagenesis involves metabolic activation by cytochrome P450 1A1 and epoxide hydrolase to yield a variety of reactive intermediates. The most important of these appears to be the bay-region diol epoxide of B[a]P, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), for which there are four possible optical enantiomers (1). BPDEs are reactive metabolites that are known to form stable DNA adducts, predominantly at the exocyclic amino groups of guanine and adenine (24). These adducts are thought to be responsible for the mutations observed in bacteria and in mammalian cells following B[a]P or BPDE exposure (58). In addition, B[a]P was shown to be carcinogenic in the 2 year rodent cancer bioassay, causing mostly lung tumors (9), and all of the B[a]P diol epoxide enantiomers are carcinogenic in mammals (10). Thus, these data are consistent with the view that diol epoxide formation is a major metabolic pathway by which B[a]P initiates carcinogenesis.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Isolation of mutants
B[a]P treatment and mutant quantification have been described previously (25). Briefly, mutants were collected from three 6-week old male C57Bl6 Big Blue® transgenic mice following three i.p. injections of 40 mg/kg B[a]P (days 1–3) and a partial hepatectomy on day 4. All B[a]P-treated animals were sacrificed for mutant analysis on day 7. All lacI mutants arising in the liver tissue (both at the time of hepatectomy and at time of sacrifice) of the B[a]P-exposed mice were sequenced. The mutation spectrum for the untreated controls was from a separate study of spontaneous mutations in liver tissue of 23 6–8-week old male B6C3F1 and C57Bl6 mice (33). Mutant plaques were recovered from the genome as described previously (25). In short, recovered phage were plated at 12–15 000 p.f.u. per 25x25 cm plate in the presence of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal). Mutant (blue) plaques were collected, re-plated for purification and a single mutant (blue) plaque was isolated for DNA sequence analysis. All mutant phage were stored at 4°C in 500 µl SM buffer. Pinpoint or sectored blue plaques were not isolated for sequence analysis.

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 Tris–HCl 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 1201–1185 (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 1151–1128 (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 IIGo. 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 4.2x10–5 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 4.7x10–5 observed in the control animals in this study (25). Pairwise comparisons of these mutation frequencies were carried out using the Mann–Whitney 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.


View this table:
[in this window]
[in a new window]
 
Table II. Mutation spectra at lacI in the liver of untreated control and B[a]P-treated Big Blue® mice
 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Mutant frequency and mutational analysis
The mutant frequencies in the lacI gene of the liver of three C57Bl/6 mice treated for 3 days with 40 mg/kg B[a]P i.p. followed by a partial hepatectomy on the fourth day have been reported (25). Following DNA sequence analysis of isolated mutants, the previously reported mutant frequencies of the B[a]P-treatment groups have been corrected to exclude possible clonal mutations. The corrected mutant frequencies for the B[a]P treatment groups are 8.6x10–5 (at hepatectomy) and 10.6x10–5 (at sacrifice). The average spontaneous mutant frequency observed at lacI in the Big Blue® mouse liver is 4.2x10–5 (33).

Mutations observed in lacI following B[a]P treatment
Table IGo 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 IGo). 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.


View this table:
[in this window]
[in a new window]
 
Table I. Base pair substitution and single-base frameshift mutations observed in liver tissue of B[a]P-treated Big Blue® mice
 
Finally, there were three complex mutations (data not shown). In one, two adenine bases at position 304 replaced a cytosine. In the second, TT replaced the GCG sequence at position 222–224, and in the third, a GCG sequence was replaced by TGC at position 790–792. Sequence context and surrounding bases of these three complex changes did not readily offer a mechanistic explanation for the origin of these mutations. Similar to the earlier findings in untreated mice, a substantial portion, 57/138 (41%), of the mutations observed in B[a]P-treated mice were observed in the first 177 bp of the gene which codes for the DNA-binding domain of the lac repressor (33).

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 IGo). 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 44–46).

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 IIGo. 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 IIGo were analyzed by Mann–Whitney 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 IIGo). The mutations listed in Table IIGo 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 IIGo 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 IIGo). 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 (1.3x10–6) was lower than the frequency observed in the control spectrum (2.5x10–6) (Table IIGo). 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 <1.1x10–6(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 IIGoGo). 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 IIGo). 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 IIGo). 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:
GC->TA was the most frequent transversion observed in B[a]P-treated animals (Table IIGo). 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).

GC->CG 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 AT->TA 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, AT->CG transversions were infrequent in all three of the spectra shown in Table IIGo. 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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Transgenic mutation assays provide a means by which genomic alterations can be studied in vivo. The Big Blue® system conveniently allows for the assessment of both spontaneous and mutagen/carcinogen induced mutant frequencies at lacI, and more recently at cII (27,52,53). Additional information can be gained from sequencing mutants arising in different treatment groups to yield a spectrum of the types (or classes) of mutations present following treatment. In turn, these data can be used to quantify these mutational classes in both control and treatment groups. Where differences are observed, one can infer mutagenic activity of a given treatment.

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 IIGo. 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 IIGo the frequency of GC->CG transversions in the spontaneous spectrum was 1.9x10–6. Following treatment with B[a]P, these transversions were significantly increased to frequencies of 19.9x10–6 at hepatectomy and 22.6x10–6 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 42x10–6. 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 GC->TA 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). GC->TA 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 GC->TA, 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 GC->TA 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 IIGo).

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 IGo). 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 IIGo). 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]P–DNA 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 GC->TA and GC->CG transversions in the spectrum of spontaneous liver mutations (Table IIGo). 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 G->T 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 AT->CG, 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 IIGo). 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 IIGo). The differences in the mutation spectra were apparent both at the time of hepatectomy and at sacrifice, as revealed by sequencing (Tables I and IIGoGo). 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).


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Significant differences between the mutation spectra from untreated and B[a]P-treated mice were used to make inferences about the mechanism of B[a]P mutagenesis in the liver of exposed mice. These mutation spectra strongly suggest that mutagenic lesions are produced as a result of B[a]P activation by cytochrome P-450 and epoxide hydrolase, presumably to produce BPDEs. This study does not support the hypothesis that abasic sites are important in B[a]P-mediated mutagenesis in the liver of Big Blue® mice. Further investigations of the mutagenic properties of B[a]P and its metabolites in different tissues and in different species are required to provide a better understanding of the mechanism(s) of B[a]P mutagenesis in vivo. Partial hepatectomy of mice exposed to B[a]P had no significant effect on mutant frequency or on mutation spectra. It appears that under the treatment conditions described, B[a]P-induced mutations were fixed shortly after exposure. Finally, it was demonstrated that mutation spectra could provide a sensitive measure of quantitative increases in specific types of mutations in cases where mutant frequency is not substantially affected.


    Acknowledgments
 
We would like to acknowledge the assistance of Heather Erfle, David Walsh and James Holcroft in the sequencing of these mutants. This work was supported in part by funding to the Molecular Mutagenesis Group, NIEHS (K.R.T.) and by grants from the National Cancer Institute of Canada (to B.W.G.), the National Institute of Environmental Health Sciences (to B.W.G.) and RR 10230-01 from the National Institutes of Health (to B.S.S.).


    Notes
 
5 Present address: Lilly Research Laboratories, GL45, Greenfield, IN 46140, USA Back

6 To whom correspondence should be addressed Email: tindall{at}niehs.nih.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 

  1. Conney,A.H., Chang,R.L., Jerina,D.M. and Wei,S.J. (1994) Studies on the metabolism of benzo[a]pyrene and dose-dependent differences in the mutagenic profile of its ultimate carcinogenic metabolite. Drug Metab. Rev., 26, 125–163.[ISI][Medline]
  2. Kadlubar,F.F. (1980) A transversion mutation hypothesis for chemical carcinogenesis by N2-substitution of guanine in DNA. Chem. Biol. Interact., 31, 255–263.[ISI][Medline]
  3. Cosman,M., de los Santos,C., Fiala,R., Hingerty,B.E., Singh,S.B., Ibanez,V., Margulis,L.A., Live,D., Geacintov,N.E., Broyde,S. et al. (1992) Solution conformation of the major adduct between the carcinogen (+)-anti-benzo[a]pyrene diol epoxide and DNA. Proc. Natl Acad. Sci. USA, 89, 1914–1918.[Abstract]
  4. Bartsch,H. (1996) DNA adducts in human carcinogenesis: etiological relevance and structure–activity relationship. Mutat. Res., 340, 67–79.[ISI][Medline]
  5. Skopek,T.R., Kort,K.L., Marino,D.R., Mittal,L.V., Umbenhauer,D.R., Laws,G.M. and Adams,S.P. (1996) Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice. Environ. Mol. Mutagen., 28, 376–384.[ISI][Medline]
  6. Jelinsky,S.A., Liu,T., Geacintov,N.E. and Loechler,E.L. (1995) The major, N2–Gua adduct of the (+)-anti-benzo[a]pyrene diol epoxide is capable of inducing G->A and G->C, in addition to G->T, mutations. Biochemistry, 34, 13545–13553.[ISI][Medline]
  7. Loechler,E.L. (1995) How are potent bulky carcinogens able to induce such a diverse array of mutations? Mol. Carcinog., 13, 213–219.[ISI][Medline]
  8. Burgess,J.A., Stevens,C.W. and Fahl,W.E. (1985) Mutation at separate gene loci in Salmonella typhimurium TA100 related to DNA nucleotide modification by stereoisomeric benzo(a)pyrene 7,8-diol-9,10-epoxides. Cancer Res., 45, 4257–4262.[Abstract]
  9. Hicks,R.M. and Ashby,J. (1988) Review of the rodent carcinogenicity data for four test chemicals. In Ashby,J.J., de Serres,F.J., Shelby,M.D., Margolin,B.H., Ishidate,M. and Becking,G.C. (eds) Evaluation of Short Term Tests for Carcinogens. Vol. 2. Report of the International Programme on Chemical Safety's Collaborative Study on in vivo Assays. Cambridge University Press, Cambridge, UK, pp. 351–365.
  10. Buening,M.K., Wislocki,P.G., Levin,W., Yagi,H., Thakker,D.R., Akagi,H., Koreeda,M., Jerina,D.M. and Conney,A.H. (1978) Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Proc. Natl Acad. Sci. USA, 75, 5358–5361.[Abstract]
  11. Cavalieri,E.L. and Rogan,E.G. (1990) Radical cations in aromatic hydrocarbon carcinogenesis. Free Radic. Res. Commun., 11, 77–87.[ISI][Medline]
  12. Cavalieri,E.L. and Rogan,E.G. (1992) The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther., 55, 183–199.[ISI][Medline]
  13. Cavalieri,E. and Rogan,E. (1998) Mechanisms of tumor Initiation by polycyclic aromatic hydrocarbons in mammals. In Neilson,A.H. (ed.) The Handbook of Environmental Chemistry. Vol. 3, Part J. Springer-Verlag, Berlin/Heidelberg, Germany, pp. 81–117.
  14. Todorovic,R., Ariese,F., Devanesan,P., Jankowiak,R., Small,G.J., Rogan,E. and Cavalieri,E. (1997) Determination of benzo[a]pyrene– and 7,12-dimethylbenz[a]anthracene–DNA adducts formed in rat mammary glands. Chem. Res. Toxicol., 10, 941–947.[ISI][Medline]
  15. Devanesan,P.D., Higginbotham,S., Ariese,F., Jankowiak,R., Suh,M., Small,G.J., Cavalieri,E.L. and Rogan,E.G. (1996) Depurinating and stable benzo[a]pyrene–DNA adducts formed in isolated rat liver nuclei. Chem. Res. Toxicol., 9, 1113–1116.[ISI][Medline]
  16. Chen,J. and Thilly,W.G. (1996) Mutational spectra vary with exposure conditions: benzo[a]pyrene in human cells. Mutat. Res., 357, 209–217.[ISI][Medline]
  17. Sobol,R. W., Horton,J.K., Kuhn,R., Gu,H., Singhal,R.K., Prasad,R., Rajewsky,K. and Wilson,S.H. (1996) Requirement of mammalian DNA polymerase-beta in base-excision repair [published errata appear in Nature (1996) 379, 848 and Nature (1996) 383, 457]. Nature, 379, 183–186.[ISI][Medline]
  18. Schaaper,R.M., Kunkel,T.A. and Loeb,L.A. (1983) Infidelity of DNA synthesis associated with bypass of apurinic sites. Proc. Natl Acad. Sci. USA, 80, 487–491.[Abstract]
  19. Kunkel,T.A., Schaaper,R.M. and Loeb,L.A. (1983) Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro. Biochemistry, 22, 2378–2384.[ISI][Medline]
  20. Gentil,A., Cabral-Neto,J.B., Mariage-Samson,R., Margot,A., Imbach,J.L., Rayner,B. and Sarasin,A. (1992) Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells. J. Mol. Biol., 227, 981–984.[ISI][Medline]
  21. Gentil,A., Renault,G., Madzak,C., Margot,A., Cabral-Neto,J.B., Vasseur,J.J., Rayner,B., Imbach,J.L. and Sarasin,A. (1990) Mutagenic properties of a unique abasic site in mammalian cells. Biochem. Biophys. Res. Commun., 173, 704–710.[ISI][Medline]
  22. Kamiya,H., Suzuki,M. and Ohtsuka,E. (1993) Mutation-spectrum of a true abasic site in codon 12 of a c-Ha-ras gene in mammalian cells. FEBS Lett., 328, 125–129.[ISI][Medline]
  23. Klinedinst,D.K. and Drinkwater,N.R. (1992) Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells. Mol. Carcinog., 6, 32–42.[ISI][Medline]
  24. Cabral Neto,J.B., Cabral,R.E., Margot,A., Le Page,F., Sarasin,A. and Gentil,A. (1994) Coding properties of a unique apurinic/apyrimidinic site replicated in mammalian cells. J. Mol. Biol., 240, 416–420.[ISI][Medline]
  25. Shane,B.S., Lockhart,A.M., Winston,G.W. and Tindall,K.R. (1997) Mutant frequency of lacI in transgenic mice following benzo[a]pyrene treatment and partial hepatectomy. Mutat. Res., 377, 1–11.[ISI][Medline]
  26. Kohler,S.W., Provost,G.S., Fieck,A., Kretz,P.L., Bullock,W.O., Sorge,J.A., Putman,D.L. and Short,J.M. (1991) Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc. Natl Acad. Sci. USA, 88, 7958–7962.[Abstract]
  27. Monroe,J.J., Kort,K.L., Miller,J.E., Marino,D.R. and Skopek,T.R. (1998) A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and as mutational targets for N-nitroso-N-methylurea and benzo[a]pyrene in Big Blue mice. Mutat. Res., 421, 121–136.[ISI][Medline]
  28. Brooks,R.A., Gooderham,N.J., Edwards,R.J., Boobis,A.R. and Winton,D.J. (1999) The mutagenicity of benzo[a]pyrene in mouse small intestine. Carcinogenesis, 20, 109–114.[Abstract/Free Full Text]
  29. Muto,S., Yokoi,T., Gondo,Y., Katsuki,M., Shioyama,Y., Fujita,K. and Kamataki,T. (1999) Inhibition of benzo[a]pyrene-induced mutagenesis by (–)-epigallocatechin gallate in the lung of rpsL transgenic mice. Carcinogenesis, 20, 421–424.[Abstract/Free Full Text]
  30. Colapietro,A.M., Goodell,A.L. and Smart,R.C. (1993) Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels. Carcinogenesis, 14, 2289–2295.[Abstract]
  31. Ruggeri,B., DiRado,M., Zhang,S.Y., Bauer,B., Goodrow,T. and Klein-Szanto,A.J. (1993) Benzo[a]pyrene-induced murine skin tumors exhibit frequent and characteristic G to T mutations in the p53 gene. Proc. Natl Acad. Sci. USA, 90, 1013–1017.[Abstract]
  32. Cavalieri,E.L., Higginbotham,S., RamaKrishna,N.V., Devanesan,P.D., Todorovic,R., Rogan,E.G. and Salmasi,S. (1991) Comparative dose–response tumorigenicity studies of dibenzo[alpha,l]pyrene versus 7,12-dimethylbenz{alpha}anthracene, benzo{alpha}pyrene and two dibenzo[alpha,l]pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis, 12, 1939–1944.[Abstract]
  33. de Boer,J.G., Erfle,H., Walsh,D., Holcroft,J., Provost,J.S., Rogers,B., Tindall,K.R. and Glickman,B.W. (1997) Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice. Environ. Mol. Mutagen., 30, 273–286.[ISI][Medline]
  34. Erfle,H.L., Walsh,D.F., Holcroft,J., Hague,N., de Boer,J.G. and Glickman,B.W. (1996) An efficient laboratory protocol for the sequencing of large numbers of lacI mutants recovered from Big Blue transgenic animals. Environ. Mol. Mutagen., 28, 393–396.[ISI][Medline]
  35. Seigel,S. (1956) Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Company, New York, NY.
  36. Farabaugh,P.J. (1978) Sequence of the lacI gene. Nature, 274, 765–769.[ISI][Medline]
  37. Streisinger,G., Okada,Y., Emrich,J., Newton,J., Tsugita,A., Terzaghi,E. and Inouye,M. (1966) Frameshift mutations and the genetic code. Cold Spring Harbor Symp. Quant. Biol., 31, 77–84.[ISI][Medline]
  38. Schaaper,R.M., Dunn,R.L. and Glickman,B.W. (1987) Mechanisms of ultraviolet-induced mutations: mutational spectra in Escherichia coli lacI gene for a wild-type and excision-deficient strain. J. Mol. Biol., 198, 187–102.[ISI][Medline]
  39. DeMarini,D.M., Shelton,M.L. and Bell,D.A. (1994) Mutation spectra in Salmonella of complex mixtures: comparison of urban air to benzo[a]pyrene. Environ. Mol. Mutagen., 24, 262–275.[ISI][Medline]
  40. Bernelot-Moens,C., Glickman,B.W. and Gordon,A.J. (1990) Induction of specific frameshift and base substitution events by benzo[a]pyrene diol epoxide in excision-repair-deficient Escherichia coli. Carcinogenesis, 11, 781–785.[Abstract]
  41. Yang,J.L., Maher,V.M. and McCormick,J.J. (1987) Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells. Mol. Cell. Biol., 7, 1267–1270.[ISI][Medline]
  42. Carothers,A.M. and Grunberger,D. (1990) DNA base changes in benzo[a]pyrene diol epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells. Carcinogenesis, 11, 189–192.[Abstract]
  43. Gorelick,N.J., Andrews,J.L., Gu,M. and Glickman,B.W. (1995) Mutational spectra in the lacl gene in skin from 7,12-dimethylbenz[a]anthracene-treated and untreated transgenic mice. Mol. Carcinog., 14, 53–62.[ISI][Medline]
  44. Holmquist,G.P. and Gao,S. (1997) Somatic mutation theory, DNA repair rates and the molecular epidemiology of p53 mutations. Mutat. Res., 386, 69–101.[ISI][Medline]
  45. Kunala,S. and Brash,D.E. (1992) Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity. Proc. Natl Acad. Sci. USA, 89, 11031–11035.[Abstract]
  46. Farabaugh,P.J., Schmeissner,U., Hofer,M. and Miller,J.H. (1978) Genetic studies of the lac repressor. VII On the molecular nature of spontaneous hotspots in the lacI gene of E.coli. J. Mol. Biol., 126, 847–857.[ISI][Medline]
  47. Carr,G.J. and Gorelick,N.J. (1995) Statistical design and analysis of mutation studies in transgenic mice. Environ. Mol. Mutagen., 25, 246–255.[ISI][Medline]
  48. Piegorsch,W.W., Lockhart,A.M., Margolin,B.H., Tindall,K.R., Gorelick,N.J., Short,J.M., Carr,G.J., Thompson,E.D. and Shelby,M.D. (1994) Sources of variability in data from a lacI transgenic mouse mutation assay. Environ. Mol. Mutagen., 23, 17–31.[ISI][Medline]
  49. de Boer,J.G., Provost,S., Gorelick,N., Tindall,K. and Glickman,B.W. (1998) Spontaneous mutation in lacI transgenic mice: a comparison of tissues. Mutagenesis, 13, 109–114.[Abstract]
  50. Page,J.E., Pilcher,A.S., Yagi,H., Sayer,J.M., Jerina,D.M. and Dipple,A. (1999) Mutational consequences of replication of M13mp7L2 constructs containing cis-opened benzo[a]pyrene 7,8-diol 9,10-epoxide–deoxyadenosine adducts. Chem. Res. Toxicol., 12, 258–263.[ISI][Medline]
  51. Christner,D.F., Lakshman,M.K., Sayer,J.M., Jerina,D.M. and Dipple,A. (1994) Primer extension by various polymerases using oligonucleotide templates containing stereoisomeric benzo[a]pyrene-deoxyadenosine adducts. Biochemistry, 33, 14297–14305.[ISI][Medline]
  52. Watson,D.E., Cunningham,M.L. and Tindall,K.R. (1998) Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts. Mutagenesis, 13, 487–497.[Abstract]
  53. Harbach,P.R., Zimmer,D.M., Filipunas,A.L., Mattes,W.B. and Aaron,C.S. (1999) Spontaneous mutational spectrum at the lambda cII locus in liver, lung and spleen tissue of Big Blue transgenic mice. Environ. Mol. Mutagen., 33, 132–143.[ISI][Medline]
  54. Fernandes,A., Liu,T., Amin,S., Geacintov,N.E., Grollman,A.P. and Moriya,M. (1998) Mutagenic potential of stereoisomeric bay region (+)- and (–)-cis-anti-benzo[a]pyrene diol epoxide-N2–2'-deoxyguanosine adducts in Escherichia coli and simian kidney cells. Biochemistry, 37, 10164–10172.[ISI][Medline]
  55. Zhan,D.J., Heflich,R.H. and Fu,P.P. (1996) Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide and 3-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide. Environ. Mol. Mutagen., 27, 19–29.[ISI][Medline]
  56. Chary,P., Latham,G.J., Robberson,D.L., Kim,S.J., Han,S., Harris,C.M., Harris,T.M. and Lloyd,R.S. (1995) In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61. J. Biol. Chem., 270, 4990–5000.[Abstract/Free Full Text]
  57. Quan,T., Reiners,J.J.Jr, Culp,S.J., Richter,P. and States,J.C. (1995) Differential mutagenicity and cytotoxicity of (+/–)-benzo[a]pyrene-trans-7,8-dihydrodiol and (+/–)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide in genetically engineered human fibroblasts. Mol. Carcinog., 12, 91–102.[ISI][Medline]
  58. Suh,M., Jankowiak,R., Ariese,F., Mao,B., Geacintov,N.E. and Small,G.J. (1994) Flanking base effects on the structural conformation of the (+)-trans-anti-benzo[a]pyrene diolepoxide adduct to N2-dG in sequence-defined oligonucleotides. Carcinogenesis, 15, 2891–2898.[Abstract]
  59. Wei,S.J., Chang,R.L., Bhachech,N., Cui,X.X., Merkler,K.A., Wong,C.Q., Hennig,E., Yagi,H., Jerina,D.M. and Conney,A.H. (1993) Dose-dependent differences in the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster V-79 cells. Cancer Res., 53, 3294–3301.[Abstract]
  60. Mazur,M. and Glickman,B.W. (1988) Sequence specificity of mutations induced by benzo[a]pyrene-7,8-diol-9,10-epoxide at endogenous aprt gene in CHO cells. Somat. Cell, Mol. Genet., 14, 393–400.[ISI][Medline]
  61. Yang,J.L., Maher,V.M. and McCormick,J.J. (1987) Kinds of mutations formed when a shuttle vector containing adducts of (+/–)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene replicates in human cells. Proc. Natl Acad. Sci. USA, 84, 3787–3791.[Abstract]
  62. Mizusawa,H., Lee,C.H., Kakefuda,T., McKenney,K., Shimatake,H. and Rosenberg,M. (1981) Base insertion and deletion mutations induced in an Escherichia coli plasmid by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide. Proc. Natl Acad. Sci. USA, 78, 6817–6820.[Abstract]
  63. DeMarini,D.M., Shelton,M.L. and Levine,J.G. (1995) Mutational spectrum of cigarette smoke condensate in Salmonella: comparison with mutations in smoking-associated tumors. Carcinogenesis, 16, 2535–2542.[Abstract]
  64. Fahl,W.E., Scarpelli,D.G. and Gill,K. (1981) Relationship between benzo(a)pyrene-induced DNA base modification and frequency of reverse mutations in mutant strains of Salmonella typhimurium. Cancer Res., 41, 3400–3406.[Abstract]
  65. Pearl,L.H. and Neidle,S. (1986) Origins of stereospecificity in DNA damage by anti-benzo[a]pyrene diol-epoxides. A molecular modelling study. FEBS Lett., 209, 269–276.[ISI][Medline]
  66. Rao,S.N., Lybrand,T., Michaud,D., Jerina,D.M. and Kollman,P.A. (1989) Molecular mechanics simulations on covalent complexes between polycyclic carcinogens and B-DNA. Carcinogenesis, 10, 27–38.[Abstract]
  67. Moriya,M., Spiegel,S., Fernandes,A., Amin,S., Liu,T., Geacintov,N. and Grollman,A.P. (1996) Fidelity of translesional synthesis past benzo[a]pyrene diol epoxide-2'-deoxyguanosine DNA adducts: marked effects of host cell, sequence context and chirality. Biochemistry, 35, 16646–16651.[ISI][Medline]
  68. Shibutani,S., Margulis,L.A., Geacintov,N.E. and Grollman,A.P. (1993) Translesional synthesis on a DNA template containing a single stereoisomer of dG-(+)- or dG-(–)-anti-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). Biochemistry, 32, 7531–7541.[ISI][Medline]
  69. Cheng,S.C., Hilton,B.D., Roman,J.M. and Dipple,A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol., 2, 334–340.[ISI][Medline]
  70. Rill,R.L. and Marsch,G.A. (1990) Sequence preferences of covalent DNA binding by anti-(+)- and anti-(–)-benzo[a]pyrene diol epoxides. Biochemistry, 29, 6050–6058.[ISI][Medline]
  71. Loechler,E.L. (1996) The role of adduct site-specific mutagenesis in understanding how carcinogen–DNA adducts cause mutations: perspective, prospects and problems. Carcinogenesis, 17, 895–902.[Abstract]
  72. Wei,S.J., Chang,R.L., Wong,C.Q., Bhachech,N., Cui,X.X., Hennig,E., Yagi,H., Sayer,J.M., Jerina,D.M., Preston,B.D. et al. (1991) Dose-dependent differences in the profile of mutations induced by an ultimate carcinogen from benzo[a]pyrene. Proc. Natl Acad. Sci. USA, 88, 11227–11230.[Abstract]
  73. Wei,S.J., Chang,R.L., Hennig,E., Cui,X.X., Merkler,K.A., Wong,C.Q., Yagi,H., Jerina,D.M. and Conney,A.H. (1994) Mutagenic selectivity at the HPRT locus in V-79 cells: comparison of mutations caused by bay-region benzo[a]pyrene 7,8-diol-9,10-epoxide enantiomers with high and low carcinogenic activity. Carcinogenesis, 15, 1729–1735.[Abstract]
  74. Lambert,B., Andersson,B., Bastlova,T., Hou,S.M., Hellgren,D. and Kolman,A. (1994) Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide and ethylene oxide. Environ. Health Perspect., 102(Suppl. 4), 135–138.
  75. Rodriguez,H. and Loechler,E.L. (1993) Mutagenesis by the (+)-anti-diol epoxide of benzo[a]pyrene: what controls mutagenic specificity? Biochemistry, 32, 1759–1769.[ISI][Medline]
  76. Yang,H., Mazur-Melnyk,M., de Boer,J.G. and Glickman,B.W. (1999) A comparison of mutational specificity of mutations induced by S9-activated B[a]P and benzo[a]pyrene-7,8-diol-9,10-epoxide at the endogenous aprt gene in CHO cells. Mutat. Res., 423, 23–32.[ISI][Medline]
  77. Chen,R.H., Maher,V.M. and McCormick,J.J. (1990) Effect of excision repair by diploid human fibroblasts on the kinds and locations of mutations induced by (+/–)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the HPRT gene. Proc. Natl Acad. Sci. USA, 87, 8680–8684.[Abstract]
  78. Stowers,S.J. and Anderson,M.W. (1985) Formation and persistence of benzo(a)pyrene metabolite–DNA adducts. Environ. Health Perspect., 62, 31–39.[ISI][Medline]
  79. Hanson,A.A., Rogan,E.G. and Cavalieri,E.L. (1998) Synthesis of adducts formed by iodine oxidation of aromatic hydrocarbons in the presence of deoxyribonucleosides and nucleobases. Chem. Res. Toxicol., 11, 1201–1208.[ISI][Medline]
  80. Schaaper,R.M., Glickman,B.W. and Loeb,L.A. (1982) Role of depurination in mutagenesis by chemical carcinogens. Cancer Res., 42, 3480–3485.[Abstract]
  81. Strauss,B.S. (1991) The `A rule' of mutagen specificity: a consequence of DNA polymerase bypass of non-instructional lesions? Bioessays, 13, 79–84.[ISI][Medline]
  82. Chakravarti,D., Pelling,J.C., Cavalieri,E.L. and Rogan,E.G. (1995) Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites. Proc. Natl Acad. Sci. USA, 92, 10422–10426.[Abstract]
  83. Kamiya,H., Suzuki,M., Komatsu,Y., Miura,H., Kikuchi,K., Sakaguchi,T., Murata,N., Masutani,C., Hanaoka,F. and Ohtsuka,E. (1992) An abasic site analogue activates a c-Ha-ras gene by a point mutation at modified and adjacent positions [published erratum appears in Nucleic Acids Res. (1992) 20, 4964]. Nucleic Acids Res., 20, 4409–4415.[Abstract]
  84. de Boer,J.G., Mirsalis,J.C., Provost,G.S., Tindall,K.R. and Glickman,B.W. (1996) Spectrum of mutations in kidney, stomach and liver from lacI transgenic mice recovered after treatment with tris(2,3-dibromopropyl)phosphate. Environ. Mol. Mutagen., 28, 418–423.[ISI][Medline]
Received July 14, 1999; revised December 22, 1999; accepted December 28, 1999.