Mutagenesis Advance Access published online on June 23, 2008
Mutagenesis, doi:10.1093/mutage/gen033
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Mutations induced by benzo[a]pyrene and dibenzo[a,l]pyrene in lacI transgenic B6C3F1 mouse lung result from stable DNA adducts
Environmental Carcinogenesis Division, National Health and Environmental Effects Research, US Environmental Protection Agency, MD B143-06, Research Triangle Park, NC 27711, USA
Dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P) are carcinogenic polycyclic aromatic hydrocarbons (PAHs) that are each capable of forming a variety of covalent adducts with DNA. Some of the DNA adducts formed by these PAHs have been demonstrated to spontaneously depurinate, producing apurinic (AP) sites. The significance of the formation of AP sites as a key event in the production of mutations and tumours by PAHs has been a subject of ongoing investigations. Because cells have efficient and accurate mechanisms for repairing background levels of AP sites, the contribution of PAH-induced AP site mutagenesis is expected to be maximal in conditions where those induced AP sites are produced in significant excess of the endogenous AP sites. In this study, we investigated the effect of two dosing regimens on the mutagenicity of DB[a,l]P and B[a]P in vivo using the Big Blue® transgenic mouse system. We compared administration of a single highly tumorigenic dose of each PAH with a fractionated delivery of the same total dose administered over 5 days, with the expectation that PAH-induced AP sites would be produced at a greater margin above background levels in animals receiving the high single dose than in the animals receiving the fractionated doses. Treatment with DB[a,l]P yielded a 2.5-fold (single dose) to 3-fold (fractionated dose) increase in mutant frequencies relative to controls. Both single-dose and fractionated dose treatment regimens with B[a]P produced about a 15-fold increase in mutant frequencies compared to controls. The mutations induced by B[a]P and DB[a,l]P correlated with the stable covalent DNA adducts produced by each. These mutation results are consistent with the previously identified stable covalent DNA adducts being the promutagenic lesions produced by these two PAHs and do not support a major role for depurinating adducts, contributing to PAH-induced mutagenesis in mouse lung in vivo.
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Polycyclic aromatic hydrocarbons (PAHs) comprise a large class of environmental pollutants. The tumorigenic potencies of PAHs vary widely, ranging from inactive to highly potent compounds (1
–3). It has been reported previously that dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P) are highly tumorigenic in strain A/J mouse lung following intra-peritoneal (i.p.) administration (4,5). It was found that DB[a,l]P was 
100-fold more potent in producing lung adenomas than B[a]P as a function of administered dose (5).
In vitro, and in mouse skin and rat mammary gland in vivo, both B[a]P and DB[a,l]P have been shown to be metabolically activated to reactive intermediates capable of forming both stable covalent DNA adducts as well as unstable covalent DNA adducts which spontaneously depurinate, leaving potentially promutagenic apurinic (AP) sites (1,6–11). Some studies have suggested a major role for these AP sites in carcinogenicity of these two PAHs (9,10,12–15), while other studies have demonstrated the importance of stable DNA adducts and lack of evidence for depurinating adducts in contributing to their biological activities (5,16,17). One of the reasons that AP sites have not been readily demonstrated in vivo following exposure to these two PAHs is that efficient mechanisms are in place to rapidly repair AP sites. Generation of AP sites in DNA is a routine consequence of cellular metabolism, and repair of this background level of AP damage is rapid and relatively error free. In order to contribute to the carcinogenicity of the PAH, the AP sites that are induced by PAH treatment must be produced at a level great enough to either allow some of the AP sites to remain unrepaired during replication or to be processed by error-prone repair mechanisms. Base excision repair is inducible and exhibits decreased fidelity at higher levels of AP sites. To evaluate the relative contributions of stable and unstable PAH–DNA adducts to mutagenic processes in vivo, we compared quantitative and qualitative aspects of mutation production for both B[a]P and DB[a,l]P under conditions where the total administered dose of each PAH was kept constant, but where peak levels of PAH-derived AP sites were expected to vary.
To investigate the mutagenicity of these two PAHs in vivo, we used the Big Blue® B6C3F1 transgenic mouse mutagenesis assay. The assay is based on detecting mutations produced in the lacI target gene which is stably integrated in multiple copies into the mouse genome. The lacI gene can be readily recovered from tissue DNA and assessed for mutations using a phenotypic assay. This assay offers the advantage of allowing for metabolic activation and detoxification of a chemical in a whole animal system (18–20). In this study, the mutagenicity of both DB[a,l]P and B[a]P was examined in the lungs of transgenic mice.
Our study design examined the production of mutations in lung DNA following either a single high dose of PAH or daily administration of fractionated doses of the PAH (20% of single high dose administered on each of five consecutive days). Our previous studies have shown that, within a range of doses encompassing the fractionated doses and the total doses used in the present study, formation of total stable covalent DNA adducts in lung is linear as a function of administered dose for both of these PAHs (5,17). Further, there is no background level of stable PAH adducts in untreated mice. In contrast, untreated mice exhibit a high steady-state level of AP sites which represent a balance between constant production and efficient repair of DNA base damage. AP sites produced by a large single dose of PAH would be expected to exceed the steady-state background level of AP sites by a greater margin than would AP sites induced by the lower fractionated doses spread over time because the fractionated doses will each induce lower peak values than the single dose and because some portion of the AP damage induced by each fractionated dose will be accurately repaired by the constitutive repair processes. Therefore, if AP sites were produced by the PAH in lung DNA, a greater contribution of the mutational sequelae of AP damage resulting from the high single dose would be expected compared to the fractionated dose treatment. This should be reflected in both the observed mutant frequencies and in the mutational spectra. Further, since DB[a,l]P has been proposed to produce proportionately more depurinating DNA adducts than B[a]P, a proportionately greater difference in the mutational outcomes between single and fractionated doses is predicted for the former.
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Treatment of animals
Male B6C3F1 (Big Blue®) mice [
LIZ: C57BL/6 (LIZ) female x C3H male] were obtained from Stratagene Cloning Systems (La Jolla, CA). All animals were 8- to 9-week old at time of dosing. Animals were housed one per cage in a facility accredited by the American Association for Accreditation of Laboratory Animal Care and used in accordance with protocols approved by the US Environmental Protection Agency Institutional Animal Care and Use Committee. B[a]P was purchased from Sigma Chemical Co. (CAS no. 50-32-8, St Louis, MO) and DB[a,l]P was obtained from Chemsyn Science Laboratories (CAS no. 191-30-0, Lenexa, KS). The PAHs were dissolved in tricaprylin (1,2,3-trioctanoylglycerol, CAS no. 538-23-8, Fluka Chemical Corp., Milwaukee, WI) to yield the desired dose in an injection volume of 0.2 ml. Treatment groups consisted of six animals which were randomly assigned to the group. Animals in the control group received one i.p. injection of 0.2 ml tricaprylin. Animals treated with DB[a,l]P were either treated with one i.p. injection of 6 mg/kg or daily injections of 1.2 mg/kg for 5 days. B[a]P-treated animals received either one i.p. injection of 200 mg/kg or daily injections of 40 mg/kg for 5 days. The total dose for animals treated with either a single injection or daily injections for 5 days was 6 mg/kg for DB[a,l]P and 200 mg/kg for B[a]P. All the animals survived until the end of the study, with no significant changes in weight observed between the control and PAH-treated groups.
For this study, the doses selected produce significant numbers of lung tumours in A/J mice and have been well tolerated in previous studies (5,17). The differences in dose levels for the two PAHs were dictated by the differences in their tumorigenic potencies and toxicites. At 6 mg/kg, B[a]P does not induce a significant increase in lung tumours in A/J mice, whereas DB[a,l]P is toxic at doses well below 200 mg/kg. No overt toxicity was observed in any of the experimental groups in the present study.
Isolation, packaging and sequence analysis of DNA
At 31 days after the final injection, animals were euthanized by asphyxiation with carbon dioxide and cervical dislocation. Lungs were removed, quickly frozen in liquid nitrogen and stored at –80°C. DNA isolation, packaging and plating and DNA sequence analysis were performed as previously described (21,22). Sectored plaques, which were very rarely observed, were not included in the analysis of mutant frequency, as those have been attributed to mutations arising during packaging and plating, and not reflective of mutations produced in vivo (23).
Statistical analysis
Analyses of the statistical significance of increases in mutant frequencies were performed using the generalized Cochran–Armitage test using the COCHARM program written by Troy D. Johnson (24,25). Comparison of mutation spectra was performed using the hypergeometric test as implemented in the program iMARS (26).
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Mutant frequencies
The mutant frequencies observed in the control and PAH-treated mice are summarized in Table I. Administration of B[a]P and DB[a,l]P i.p. to male B6C3F1 Big Blue® mice induced significant increases in phenotypically detectable lacI– mutants recovered from lung tissue DNA. A 2.4-fold increase in mutant frequency was observed in the lungs of mice given a single injection of 6 mg/kg DB[a,l]P. For mice receiving daily injections of 1.2 mg/kg DB[a,l]P for 5 days, a 2.8-fold increase in mutant frequency was observed. The difference in mutant frequency between the treatment regimens for DB[a,l]P was not statistically significant (P = 0.16). The mice receiving a single injection of 200 mg/kg B[a]P showed a 15.3-fold increase in mutant frequency relative to the control value. For the mice receiving daily injections of 40 mg/kg B[a]P for 5 days, a 16.1-fold increase in mutant frequency was observed. The difference in overall mutant frequencies between the treatment regimens for B[a]P was not significant (P = 0.71).
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Mutation spectra
Table II shows the distribution of mutations found in both control mice and mice treated with either DB[a,l]P or B[a]P. The majority of mutations recovered from control mice were GC to AT transitions. This class of mutations occurred in 73% of the independent mutants sequenced. Among this class of transitions, 40% were at CpG sites. The next most frequently observed class of mutation in the control mice were GC to TA transversions, constituting 13% of the total mutations detected.
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DB[a,l]P treatment produced a pattern of mutations different from the distribution of mutations recovered from the control animals. Both DB[a,l]P treatment regimens produced a similar percentage of GC to AT transitions,
25 to 30% of the total mutants sequenced. There was an increase in GC to TA and GC to CG transversions and in AT to TA and AT to GC transitions in both DB[a,l]P treatment groups compared to the control mice. There was a highly significant difference between the mutation spectra of the control group and the two DB[a,l]P treatment groups (P = 0.001 for single dose; P < 0.001 for fractionated dose).
For the B[a]P-treated mice, the distribution of mutations was different from the distributions of mutations in both the control and DB[a,l]P-treated animals. GC to AT transitions constituted 20% of the total mutations observed for both B[a]P treatment regimens. GC to TA transversions were the most frequently recovered mutation in the B[a]P-treated mice, occurring in 50% of the mutants. As seen in the DB[a,l]P-treated mice, there was also an increase in GC to CG transversions in the B[a]P-treated mice from both dosing regimens. Mutations at adenine sites were less frequent in the B[a]P-treated mice than in the DB[a,l]P-treated animals, constituting 1% of the total mutation spectrum.
The mutation spectra induced by single-dose administrations of B[a]P and DB[a,l]P were significantly different from each other (P = 0.001), as were the mutation spectra resulting from fractionated doses of B[a]P and DB[a,l]P (P < 0.001). In contrast, the observed mutation spectrum resulting from the single dose of B[a]P was not significantly different from that produced by the fractionated dose (P = 0.99). While generally quite similar, some differences were observed between the mutation spectrum induced by the single dose of DB[a,l]P and that induced by the fractionated dose (P = 0.80), with the single dose producing relatively more GC to TA transitions and the fractionated dose producing relatively more AT to TA transitions and complex mutations.
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Expected mutant frequencies
AP sites are produced in DNA as a consequence of normal cellular respiration and mechanisms exist for the efficient and accurate repair of such sites. One can generalize for the situation of DNA damage of any particular type where there exists a background internal steady-state level, DI, for which the cell has repair mechanisms in place capable of efficiently repairing that level of damage as well as some capacity for repairing slightly increased levels of DNA damage, DI +
D, without significantly increasing the probability of subsequently producing mutations. That is the repair processes are rapid compared to the time frame for mutation fixation, and the repair processes are not saturated in a cell under normal conditions. As D increases, DI + D approaches some level, DR, corresponding to the maximum repair capacity of the cell for the specific damage, these constitutive repair processes will become saturated and residual unrepaired damage will begin to contribute to the production of mutations. Mutation production by an increase in DNA damage from an external exposure that induces a level of damage, DE, is then proportional to DE – DR, the amount of damage that exceeds the constitutive repair capacity. If we consider the case of a fractionated dose yielding the same total level of damage divided into n independent doses, DE/n, then mutation production by each fractionated dose is proportional to (DE/n – DR), and the mutation production expected from the combination of the fractionated doses is n(DE/n – DR). The ratio, therefore, of the expected induction of mutations from the single dose compared to the fractionated dose is 
, which simplifies to 
. So, for all values of n > 1, the promutagenic damage induced by the single dose will always exceed that induced by the fractionated dose, provided that the time between fractionated doses is large compared to the time required for repair.
In the situation for induction of DNA damage for which there is no endogenous level in the cell, e.g. stable PAH–DNA adducts, no difference is predicted between the mutation induction by single versus fractionated doses, i.e. as 
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Observed mutant frequencies
In this study, both B[a]P and DB[a,l]P produced mutations in the lungs of B6C3F1 lacI transgenic mice. Both PAHs produced significant increases in mutant frequency as compared to the control animals, regardless of treatment regimen. However, there was not a significant difference in mutant frequency for either chemical as a function of the dosing regimen. Although the mutation spectra induced by each PAH treatment were statistically different from the mutation spectrum observed in control animals, our data indicate no significant difference in mutant frequency between single and fractionated doses for either PAH in the mouse lung. If unstable DNA adducts were produced by PAH treatment in mouse lung, then the transient peak levels of resultant AP sites should have been higher following the single high-dose administration compared to the lower fractionated doses administered over 5 days. If these AP sites were the primary promutagenic lesions in the DNA of the PAH-exposed animals, then the observed mutant frequency would be expected to be higher in the single-dose treatment groups. In fact, the single-dose treatment groups had a slightly lower observed mutant frequency than the fractionated dose treatment groups for both PAHs.
B[a]P mutation spectrum
The mutation spectra for single and fractionated doses of B[a]P were not significantly different, a result not consistent with a major role for depurinating adducts as promutagenic lesions induced by B[a]P in mouse lung. We also note a very large preponderance of mutations targeted to guanines, consistent with the spectrum of stable DNA adducts formed in lung DNA after B[a]P administration. Other investigators have reported on the in vivo mutagenicity of B[a]P, although the present study is the first to examine the lung. In a study using C57BL/6 Big Blue® mice, the authors noted that GC to TA and GC to CG transversions were the most common mutations recovered from the spleens of B[a]P-treated mice (18). Another study using the same strain reported that the mutation spectrum recovered from the livers of B[a]P-treated mice exhibited primarily GC to TA and GC to CG transversions (27). The percentage of GC to TA transversions was also found to be increased at the cII/cI loci in T cells of B[a]P-treated Big Blue® mice (28). These results are consistent with the mutation spectrum recovered from the lungs of B[a]P-treated Big Blue® B6C3F1 mice in the present study. These results are also consistent with our previous study of mutations in the Ki-ras oncogene in lung tumours induced by B[a]P, where GC to TA transversions at the first and second base of codon 12 and GC to AT transitions at the third base of codon 12 were the major mutations detected (4). These all involved mutations at guanines and are consistent with previous studies showing that stable covalent DNA adducts are produced by B[a]P primarily at guanines (8,17,29).
Chakravarti et al. (15) found that B[a]P induced GC to TA transversions in codon 13 and AT to TA transversions in codon 61 in the c-Ha-ras oncogene from mouse skin papillomas. The investigators postulated that these mutations were due to misreplication of unrepaired AP sites resulting from the formation of depurinating adducts. Although we also observed a significant increase in GC to TA transversions, we did not detect production of AT to TA transversions as a significant consequence of B[a]P exposure by either dosing regimen.
If AP sites were contributing to the production of mutations in the present study, we would expect to see a higher proportion of AP site-derived mutations in those animals that received a single high dose of PAH, where the transient levels of AP sites are expected to be higher relative to background levels than in the animals receiving the fractionated dose. However, for both DB[a,l]P and B[a]P, the observed mutant frequencies are slightly higher in the animals receiving the fractionated dose than in the animals receiving the single high dose of PAH.
DB[a,l]P mutation spectrum
The mutation spectra produced by single and fractionated doses of DB[a,l]P are qualitatively quite similar, but do show some differences. Analysis of these differences does not support a major role for unstable adducts as premutagenic lesions in this tissue. The reported distribution of depurinating adducts formed by DB[a,l]P in vivo in mouse skin is 80% adenine adducts and 20% guanine adducts (9). We would therefore expect to see a higher proportion of mutations targeted to adenines in the single-dose treatment group, where the contribution of AP sites should be maximized, than we would in the fractionated dose treatment group, if AP sites were being formed in similar proportions in the lung. However, we find a greater proportion of GC to TA transversions and a lower proportion of AT to TA transversions in the single-dose treatment group than in the fractionated dose group. This finding is the opposite of the predicted mutational outcome for depurinating DB[a,l]P–DNA adducts formed primarily at adenines.
The reason for the increased fraction of GC to TA transversions and decreased AT to TA transversions in the single-dose group relative to the fractionated dose group is unknown. One possibility is that a greater proportion of stable DNA adducts is formed at guanines than at adenines in the single-dose group. This could reflect a difference in cytochrome P450 activities being induced by multiple- versus single-dose treatments leading to a shift in the proportion of adducts at guanines and adenines. This could also reflect differential repair of the adducts formed, perhaps through differential induction or saturation of repair pathways. Regardless of the mechanism, the shift in mutation spectra is the opposite of what would be expected if AP sites comprised the promutagenic damage.
Several studies have examined Ki-ras proto-oncogene activation in mice exposed to DB[a,l]P to elucidate the mechanism of action of this PAH. We previously reported (5) that the Ki-ras mutation spectrum in DB[a,l]P-induced A/J mouse lung tumours was predominantly GC to TA transversions in the first base of codon 12, AT to GC transitions in the second base of codon 12 and AT to TA transversions in the second or third base of codon 61. This distribution of mutations in the lung tumours is very similar to the DB[a,l]P-induced mutant spectrum observed in the present study. Furthermore, the distribution of mutations targeted to adenines and guanines in both Ki-ras and lacI mirrors the distribution of stable DB[a,l]P–DNA adducts that are formed in approximately equal amounts at adenines and guanines in mouse lung DNA in vivo (5).
Mutations induced by the reactive metabolite (–)anti-11R,12S-dihydrodiol 13S,14R-epoxide of DB[a,l]P [(–)anti-DB[a,l]PDE] have been studied in the Hprt gene in V79 cells (30). This reactive metabolite induced a variety of mutations, with base substitutions being the predominant type detected. The most prevalent base substitutions were AT to TA transversions and AT to GC transitions. GC to TA transversions were noted at higher concentrations of the (–)anti-DB[a,l]PDE. Other mutations detected were frameshifts, deletions, insertions and tandem mutations. These results are quite similar to our results with DB[a,l]P in the lacI gene in vivo, including the increased production of GC to TA transversions at higher doses.
Quantitative relationships between DNA adducts, mutations and tumorigenesis
If AP sites were major contributors to PAH carcinogenesis, then the extent of production of AP damage should correlate with the extent of production of mutations and tumours. The relative amounts and identities of stable and depurinating adducts formed in mouse skin following topical administration of 200 nmol of DB[a,l]P have been described (9,10). It was reported that 99% of the total adducts formed are depurinating, with 80% of the total depurinating adducts formed at adenines and 20% formed at guanines. The remaining 1% of stable adducts formed are approximately equally divided between guanine and adenine adducts. Similarly, the relative amounts of stable and depurinating adducts formed in mouse skin following topical administration of B[a]P have also been reported (8). Unstable adducts accounted for 70% of the total adduction with 35% of depurinating adducts formed at adenines and 65% formed at guanines. Among the stable DNA adducts, 80% were formed at guanines with the remaining 20% unidentified. We have previously measured the stable DNA adducts in mouse lung following administration of 200 mg/kg B[a]P and 6 mg/kg DB[a,l]P (5,17). If the same proportions of depurinating adducts to stable adducts were to be formed in mouse lung as are reported in mouse skin for each of these PAHs, then it would be expected that 6 mg/kg DB[a,l]P would form 25-fold more depurinating DNA adducts than would 200 mg/kg B[a]P. However, we observed that 200 mg/kg B[a]P induces both a higher mutant frequency in lung DNA and more numerous pulmonary adenomas than does 6 mg/kg DB[a,l]P (5). Indeed, we have previously shown that the difference in observed tumorigenic potency for B[a]P and DB[a,l]P in mouse lung can be quantitatively accounted for solely by differences in the formation and persistence of stable covalent PAH–DNA adducts (5).
Implications for role of other types of DNA damage in PAH mutagenesis
Although this study has primarily focused on the roles of stable and depurinating PAH–DNA adducts in contributing to in vivo mutagenesis, the same considerations may apply to other types of DNA damage that might be produced by exposure to PAHs. In particular, oxidized DNA bases, including 8-oxo-deoxyguanosine, are a type of promutagenic endogenous DNA damage produced as a consequence of normal cellular respiration for which efficient and accurate repair mechanisms have evolved. PAHs have also been shown to induce the same type of oxidative DNA damage in vitro by redox cycling of PAH–quinones (11,31,32). If PAH-induced oxidized DNA bases were major contributors to in vivo mutagenesis in the present study, then the relative contribution of these lesions to the observed mutational outcome should also have been greater for the single-dose administration than for the fractionated dose administration since oxidative DNA base damage is also produced as a consequence of normal cellular respiration and is rapidly and accurately repaired by constitutive repair pathways. Our data do not provide any supporting evidence for a significant role of PAH-induced reactive oxygen species in mutagenesis in mouse lung in vivo.
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The data from the present study do not provide support for a significant role of unstable DNA adducts in PAH carcinogenesis in mouse lung. While our data do not directly address the question of whether AP sites are induced by these PAHs in vivo, comparison of the mutational consequences of exposure to either B[a]P or DB[a,l]P under conditions where the contribution of AP sites, if formed, are predicted to have been maximal shows no evidence of either increased mutant frequency or shift in mutational specificity consistent with abasic site damage. Rather, the mutant frequencies and spectra of mutations observed are readily explained by the stable covalent DNA adducts formed in mouse lung DNA after exposure to these PAHs. This interpretation is bolstered by the observation that the spectra of mutations observed in Ki-ras genes recovered from adenomas induced by these PAHs in strain A/J mouse lung are also consistent with the mutations produced in the lacI reporter gene in this study and with the stable covalent adducts produced by each PAH in lung DNA. Furthermore, the differences in potency of these two PAHs as lung tumorigens can be explained quantitatively by the differences in stable DNA adducts formed in vivo, but not by the predicted levels of abasic sites formed by putative unstable DNA adducts.
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Acknowledgments |
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The authors would like to thank Drs Stephen Nesnow, Gary Foureman and Julian Preston for their helpful comments on this manuscript. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory of the US Environmental Protection Agency and approved for publication. Approval does not signify that the contents reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +1 919 541 2974; Fax: +1 919 541 0694; Email: ross.jeffrey{at}epa.gov
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Received on March 13, 2008; revised on May 6, 2008; accepted on May 19, 2008.
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