In vivo mutagenesis induced by benzo[a]pyrene instilled into the lung of gpt delta transgenic mice

Benzo[a]pyrene (B[a]P) is a ubiquitous airborne pollutant whose mutagenicity has been evaluated previously by oral and intraperitoneal administration to experimental animals. In this study, mutagenesis in the lungs, the target organ of air pollutants, was examined after a single intratracheal instillation of 0-2 mg B[a]P into gpt delta transgenic mice. Intratracheal injection of B[a]P resulted in a statistically significant and dose-dependent increase in gpt mutant frequency as measured by 6-thioguanine selection. The mutant frequencies at B[a]P doses of 0.5, 1, and 2 mg were 2.8, 4.2, and 6.8 times higher than the frequency seen in nontreated mice (0.60 +/- 0.13 x 10(-5)). The most frequent mutations induced by B[a]P treatment were G:C-->T:A transversions, which are characteristic of B[a]P mutagenesis in other models, and single-base deletions of G:C base pairs. To characterize the hotspots of B[a]P-induced mutations in the gpt gene, we analyzed sequences adjacent to the mutated G:C base pairs. Guanine bases centered in the nucleotide sequences CGT, CGA, and CGG were the most frequent targets of B[a]P. Our results indicate that intratracheal instillation of B[a]P into gpt delta mice causes a dose-dependent increase in gpt mutant frequency in the lung, and that the predominant mutation induced is G:C-->T:A transversion.     

Mutations in the lungs of gpt delta transgenic mice following inhalation of diesel exhaust

Diesel exhaust (DE) is a major airborne pollutant of urban areas. It contains various polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs. In this study, gpt delta mice were treated with inhalation of 1 or 3 mg m(-3) DE, or a single intratracheal instillation of diesel exhaust particles (DEP) or DEP extract. In the lungs of mice treated with inhalation of 3 mg m(-3) DE for 12 weeks, the mutant frequency (MF) was 3.2-fold higher than that of the control group (1.90 x 10(-5) and 0.59 x 10(-5), respectively). An instillation of DEP and DEP extract resulted in a significant dose-dependent linear increase in MF. In mice treated with 0.5 mg DEP and 0.2 mg DEP extract, the MFs were 3.0- and 2.7-fold higher than that of the control group, respectively. The mutagenic potency (MF mg(-1)) of DEP extract (5.6 x 10(-5)) was double that of DEP (2.7 x 10(-5)), suggesting that the mutagenicity of the latter is derived primarily from compounds in the extract, which itself is responsible for ca. 50% of the weight of DEP. G:C-->A:T transitions were the predominant gpt mutation induced by all three treatments and G:C-->T:A transversions were induced by DEP and DEP extract. Guanine bases centered in nucleotide sequences such as GGA, TGA, CGG, and CGT were the major mutation targets of all three treatments. Thus, our results suggest that the mutagens contained in DEP such as PAH and nitrated PAHs induce mutations and may be responsible for carcinogenesis caused by inhalation of DE.     

Transmission of mutations in the lacI transgene to the offspring of ENU-treated Big Blue male mice

The purpose of the study was to determine: 1) if male germ cells of Big Blue mice carrying newly induced mutations in the lacI transgene were effective in fertilization; 2) if offspring arising from such mutant sperm had the mutation in germ cells and multiple somatic tissues; and 3) how the frequency of mutants induced in the lacI transgene compared to the frequency induced in endogenous genes traditionally employed to study germ cell mutagenesis in mice. Male B6C3F(1) mice hemizygous for the lambda/lacI transgene were treated weekly with 100 mg/kg body weight of the mammalian germ cell mutagen N-ethyl-N-nitrosourea (ENU). The cumulative dose for each treated animal was 300 mg ENU/kg body weight. Ten weeks later the treated mice were mated to T stock females and the resulting offspring were screened for specific-locus mutations at six loci affecting external appearance, as well as for mutations in the lacI transgene in multiple somatic tissues and germ cells. Five offspring carrying recessive specific-locus mutations were observed among 597 offspring screened (mutant frequency = 139.6 x 10(-5) per locus). Four offspring carrying lacI mutations were observed among 280 offspring screened (mutant frequency = 35.7 x 10(-5) per locus (assuming 40 target loci)). Each of the four lacI mutant offspring carried a different mutation. Three of the mutations were A:T-->G:C transitions and one a G:C-->A:T transition. Consistent with the expectation that a mutation induced in a parental germ cell and transmitted to a conceptus would exist in every cell of the offspring, each mutant mouse had identical mutations in all somatic tissues sampled, as well as in its germ cells. These data provide preliminary evidence for the biological validity of assessing induced, heritable mutations using transgenic mice, without the need for generating an F(1) generation.     

Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice.

We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al. [1996] Env. Mol. Mutagen. 28:465-470). In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations. Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several-fold the mutant frequency of gpt in bone marrow. Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU-treated and untreated transgenic mice. In the gpt mutants rescued from ENU-treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions. On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions. Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants). Three of these occurred at 5'-CpG-3' sites. Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU-treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU-treated and untreated other transgenic mice or cultured mammalian cells. We also report the establishment of homozygous transgenic mice that have transgene lambdaEG10 DNA in both chromosome 17 of C57BL/6J mouse.     

Comparative analysis of the mutagenic activity of oxaliplatin and cisplatin in the Hprt gene of CHO cells

Oxaliplatin is a platinum-derived antitumor drug that is active against cisplatin-resistant tumors and has lower overall toxicity than does cisplatin. DNA adduct formation is believed to mediate the cytotoxic activity of both compounds; however, the adducts may also be responsible for mutagenic and secondary tumorigenic activities. In this study, we have compared the mutagenicity of oxaliplatin and cisplatin in the Hprt gene of CHO-K1 cells. Both drugs produced dose-related increases in mutant frequency. For 1-hr treatments, oxaliplatin was less mutagenic than cisplatin at equimolar doses, while similar mutant frequencies were induced at equitoxic doses. Sequencing of mutant Hprt genes indicated that the mutation spectra of both oxaliplatin and cisplatin were significantly different from the spontaneous mutation spectrum (P = 0.014 and P = 0.008, respectively). A significant difference was also observed between the spectra of oxaliplatin- and cisplatin-induced mutations (P = 0.033). Although G:C-->T:A transversion was the most common mutation produced by both compounds, oxaliplatin produced higher frequencies of A:T-->T:A transversion than did cisplatin, most commonly at nucleotide 307, and higher frequencies of small deletions/insertions. Also, cisplatin induced tandem base-pair substitutions, mainly at positions 135/136, and a higher frequency of G:C-->A:T transition than did oxaliplatin. These results provide the first evidence that oxaliplatin is mutagenic and that the profiles of cisplatin- and oxaliplatin-induced mutations display not only similarities but also distinctive features relating to the type and sequence-context preference for mutation. Environ.     

Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadiene-exposed male B6C3F1 mice and F344 rats

A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C-->C:G and A:T-->T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5' partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats.     

Effect of nucleotide excision repair on ENU-induced mutation in female germ cells of Drosophila melanogaster

The role of nucleotide excision repair (NER) in the repair of alkylation damage in the germ cells of higher eukaryotes has been studied mainly by treating postmeiotic male germ cells. Little is known about repair in actively repairing female germ cells. In this study, we treated NER-deficient (ner(-)) mus201(D1) Drosophila females with N-ethyl-N-nitrosourea (ENU) and determined both the mutant frequencies in the multiple locus recessive lethal (RL) test and in the single locus vermilion gene and determined the ENU mutation spectrum in the vermilion gene. The results show that ENU is mutagenic in all cell stages and that the induced frequencies increase with cell maturation, from oogonia to mature oocytes. In addition, the induced spectrum consists mainly of A:T-->T:A transversions (43.8%), A:T-->G:C transitions (21.9%), and A:T-->C:G transversions (15.6%). G:C-->A:T (3.1%) transitions, other transversions (9.4%), frameshifts (3.1%), and deletions (3.1%) were also found. Comparison of these results with those previously obtained for repair-proficient (ner(+)) female germ cells reveal: 1) Differences in the RL and vermilion mutation frequencies for ner(+) and ner(-) germ cells, indicating that NER is involved in the repair of ENU-induced damage to these cells. 2) At least 15.6% of mutations in ner(-) cells may be the consequence of N-ethylation damage and mutations of this type were not detected in ner(+) cells. 3) Although differences were found in transition frequencies between ENU-treated ner(+) and ner(-) germ cells (52.2% vs. 25%), suggesting that a functional NER is involved in processing O-ethylated damage, the role of NER in repairing O-ethylated adducts is uncertain.     

Mutagenic activity of cisplatin in the lacZ plasmid-based transgenic mouse model

Cis-diamminedichloroplatinum (II) (cisplatin) is a well characterized antitumor drug used for the treatment of a variety of human cancers. The cytotoxicity of cisplatin is mainly mediated through the formation of DNA adducts, which are also believed to be responsible for the secondary malignancies produced by the drug. The aim of this study was to determine the in vivo mutagenic activity of cisplatin in the lacZ plasmid-based transgenic mouse model. The mutant frequency (MF) and the spectrum of mutations induced by cisplatin in the mouse liver were analyzed and compared to controls. The mean MF in the lacZ gene was increased 2-fold in mice treated with a single 6 mg/kg body weight dose of cisplatin and sacrificed after 17 and 28 days (P = 0.001 and P < 0.0001). Restriction analysis and sequencing of mutant DNA showed that cisplatin was able to induce both large deletions and point mutations. A specific profile of base substitution and frameshift mutations was identified in treated mice, consisting primarily of G:C-->A:T transitions at GpG and ApG sites, the preferential DNA binding sites of cisplatin, and single basepair deletions/insertions. The present results provide the first evidence that cisplatin has mutagenic activity in vivo and induces a characteristic pattern of mutations in the mouse liver. This mutagenicity may be responsible for its tumorigenic activity.     

17 Beta-estradiol and not genistein modulates lacI mutant frequency and types of mutation induced in the heart of ovariectomized big blue rats treated with 7, 12-dimethylbenz[a]anthracene

In industrialized countries, heart disease rates are higher among women after menopause. Recent studies indicate that consumption of phytoestorogens, e.g., isoflavones such as genistein (GE), may have potential cardiovascular health benefits; however, no studies have evaluated the effect of these agents on toxicant-induced damage in the heart. Since estrogen receptors are found in the heart, and GE mimics estrogenic effects, we have examined whether or not dietary GE or 17 beta-estradiol (E2) modulates the lacI mutant frequency (MF) in the heart of ovariectomized (OVX) Big Blue rats exposed to the model carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Groups of female rats were administered 80 mg/kg DMBA or vehicle by gavage and were chronically fed with diets containing 0, 250, or 1,000 microg/g GE or 5 microg/g E2. Sixteen weeks after carcinogen treatment, the animals were sacrificed and the hearts were removed and processed for determining the frequency and types of mutations in the heart tissue. GE and E2 supplementation alone resulted in nonsignificant increases in MF. The DMBA-induced lacI MF in the heart was sevenfold higher than the control (119.8 +/- 18.7 x 10(-6) vs. 17.4 +/- 3.2 x 10(-6); P < 0.001). GE in the diet had no significant effect on DMBA mutagenicity, while feeding E2 to DMBA-treated rats caused a significant reduction in the MF (119.8+/- 18.7 x 10(-6) vs. 61.4 +/- 13.5 x 10(-6); P < 0.017). DNA sequence analysis revealed that the majority of DMBA-induced mutations in rats fed control diet were A:T-->T:A (42%) and G:C-->T:A (19%) transversions, followed by G:C-->A:T (13%) and A:T-->G:C (8%) transitions. Feeding E2 altered the DMBA-induced mutational spectra by decreasing A:T-->T:A (23%) and G:C-->T:A (13%) transversions and increasing G:C-->A:T (24%) and A:T-->G:C (21%) transitions. Taken together, the results suggest that DMBA can induce gene mutations in heart tissue of OVX rats, and while dietary GE had little or no effect on DMBA-induced mutation, dietary E2 reduced the mutagenicity of DMBA.     

Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice

Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073-9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.     

Spontaneous mutation of the lacI transgene in rodents: absence of species, strain, and insertion-site influence

Comparison of spontaneous mutation spectra derived from different transgenic constructs can provide valuable insights for interpreting the mechanisms of spontaneous mutation. In this study, spontaneous mutation frequencies and spectra of the lacI transgene are compared in the liver of C57BL/6, B6C3F1, and BC-1 mice and F344 rats. Before correction for clonal expansion, the mutant frequency varied from 2.6 +/- 0.45 to 5.0 +/- 2.4 x 10(-5). Correction for potential clonal expansion reduced the range in mutation frequency to between 2.3 +/- 0.45 and 3.5 +/- 2.0 x 10(-5). There is thus no statistical difference in spontaneous mutation frequency between the different strains and species. G:C --> A:T transitions and to a lesser extent, G:C --> T:A transversions dominate the mutational spectra in all of these animals. In three strains of mice, G:C --> A:T transitions account for 50.7-53.3% of mutation, 81.7-83.8% of which involve CpG sites, whereas G:C --> T:A transversions account for 17.8-32.9% of mutations with 43.2-50.0% found at CpG sites. In rats, G:C --> A:T transitions account for 38.0% of the spectra, 70.0% of which involve CpG sites, whereas G:C --> T:A transversions account for 23.0% of the spectra, 70.0% of which involve CpG sites. The distribution of other classes of mutations is also very similar. We conclude that, despite reports about species and strain differences in induced mutation, spontaneous mutations in the lacI transgene appear to be similar, regardless of genomic location, rodent strain, or species. In addition to insights into spontaneous mutation, this study also provides essential data for comparison with and interpretation of induced mutations.     

Characterization of the mutational specificity of DNA cross-linking mutagens by the Lac+ reversion assay with Escherichia coli.

The mutational specificities of DNA cross-linking compounds such as cisplatin, transplatin, carboplatin, mitomycin C, psoralen, and 8-methoxypsoralen were investigated in lacZ reversion assay systems of Escherichia coli. Tester strains were constructed by introducing the six kinds of F' plasmids (lacI-, lacZ461, and proAB+), each of which carries a different base-substitution mutation within the lacZ gene. Each of the six possible base-substitution mutations was assayed by Lac+ reversion. Cisplatin induced G.C-->A.T transitions and G.C-->T.A transversions, with the former predominating. Transplatin induced A.T-->G.C transitions in addition to G.C-->A.T transitions and G.C-->T.A. Carboplatin weakly induced G.C-->A.T transitions. On the other hand, mitomycin C induced only G.C-->T.A transversions, while psoralen and 8-methoxypsoralen reactivated with near-UV irradiation induced A.T-->G.C transitions preferentially. The Lac(+) reversion system was very convenient for rapidly determining mutational spectra.     

O-ethylthymidine adducts are the most relevant damages for mutation induced by N-ethyl-N-nitrosourea in female germ cells of Drosophila melanogaster

Responses to genotoxic agents vary not only among organisms, test systems, and cellular stages, but also between sexes; little, however, is known about the mutagenic consequences of chemical exposures to female germ cells. In this study, the mutagenicity of N-ethyl-N-nitrosourea (ENU) was analyzed in female germ cells of Drosophila melanogaster using the recessive-lethal test and the vermilion system, which simultaneously generates information on induced mutation frequency and mutation spectrum. ENU was mutagenic in all stages of oogenesis, although there were differences among the stages. In mature and immature oocytes, ENU-induced mutations in the vermilion locus were 43.5% A:T-->G:C transitions, 39.1% A:T-->T:A transversions, 8.7% G:C-->A:T transitions, and 8.7% A:T-->C:G transversions, indicating that the most important premutagenic lesions induced by this chemical are O(4)-ethylthymine and O(2)-ethylthymine. The low frequency of mutation involving O(6)-ethylguanine (i.e., G:C-->A:T transitions) could be a consequence of the repair of these lesions by O(6)-methylguanine DNA methyltransferase. Comparison of these results with those previously obtained in male germ cells stresses the importance of the repair activity of the analyzed cells, because the mutation spectrum in female germ cells was similar to the spectrum obtained with repair-proficient spermatogonial cells and different from repair-deficient postmeiotic cells. The results also indicate that studies with female germ cells could be an alternative to the use of premeiotic male germ cells, especially when the analysis of these cells is difficult or almost impossible and when studies of in vivo DNA repair in premeiotic germ cells are performed.     

Analysis of mutation spectra in UVB-exposed mouse skin epidermis and dermis: frequent occurrence of C-->T transition at methylated CpG-associated dipyrimidine sites

We recently reported the kinetics of mutation induction by UVB in the skin epidermis and dermis of transgenic Muta trade mark mice [Ikehata and Ono, Mutat Res 508:41-47, 2002]. In the present study we determined the complete DNA sequence of the lacZ transgene in 208 mutants isolated from the dermis and epidermis of UVB-irradiated and control mice. The resulting mutation patterns for the dermis and epidermis were similar, although two CC-->TT tandem substitutions, one of the signature mutations for UV insult, were detected only among the UVB-induced epidermal mutants. The spectra of the UVB-induced and control mutations were both dominated by C-->T transitions (83% and 62%); however, the C-->T transitions from irradiated mice occurred almost exclusively in dipyrimidine sites, while those from control mice preferred CpG sites. Thus, the mutation spectrum detected for the irradiated skin tissues was different from the background spectrum and UV-specific, confirming the utility of the transgenic system for UVB-induced mutation studies in vivo. An analysis of the bases adjacent to the mutated cytosines from irradiated mice revealed that the dipyrimidine sites preferred for UVB-induced mutation were 5'-TC-3' > 5'-CC-3' > 5'-CT-3'. Among mutants from irradiated mice, C-->T transitions were recovered frequently at dipyrimidine sites associated with CpG. We showed that CpG sites in the lacZ transgene of Muta trade mark mice were heavily methylated in both the epidermis and dermis. Thus, CpG methylation could contribute to the UVB-induced recurrent or hotspot mutations in the mammalian genome.     

Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichia coli

Using purified photolyases for pyrimidine (6-4) pyrimidone photoproducts [(6-4)PP] and cyclobutane pyrimidine dimers (CPD), the effects of photoreactivation on mutagenesis were examined in the supF gene on a plasmid transfected into repair-deficient SOS-induced Escherichia coli host cells. More than 95% of CPD and (6-4)PP were removed from plasmid DNA by treatment with CPD photolyase and (6-4)photolyase, respectively. In each photolyase treatment, base substitutions at dipyrimidine sequences were predominantly observed. Of the single base substitutions observed after CPD photoreactivation, 83% were A:T-->G:C transitions at 5'-TT-3' sites. After (6-4)photolyase treatment, 81% were G:C-->A:T transitions at 5'-CC-3' and 5'-TC-3' sequences. Thus, the major mutagenic photoproducts of single-base substitutions were CPD at 5'-CC-3' or 5'-TC-3' sites and (6-4)PP at 5'-TT-3' sites. Tandem double mutations occurred mainly at 5'-CC-3' sites and were CPD-photoreactivated, suggesting that CPD at 5'-CC-3' was responsible for tandem double mutations. After photoreactivation of both CPD and (6-4)PP, single-base substitutions were primarily G:C-->A:T transitions at 5'-CC-3' or 5'-TC-3' sites and A:T-->G:C transitions at 5'-TT-3' sites, and secondarily G:C-->T:A transversions at 5'-CC-3' sites, G:C-->C:G transversions at 5'-CC-3' sites and A:T-->T:A transversions at 5'-TT-3' sites, which were essentially the same as those observed after photoreactivation of CPD alone, (6-4)PP alone and without photoreactivation. Thus, these transversions were not derived from unknown UV adducts but from incompletely repaired CPD and (6-4)PP.     

Relatively high rates of G:C → A:T transitions at CpG sites were observed in certain epithelial tissues including pancreas and submaxillary gland of adult big blue® mice

With few exceptions, spontaneous mutation frequency and pattern are similar across tissue types and relatively constant in young to middle adulthood in wild type mice. Underrepresented in surveys of spontaneous mutations across murine tissues is the diversity of epithelial tissues. For the first time, spontaneous mutations were detected in pancreas and submaxillary gland and compared with kidney, lung, and male germ cells from five adult male Big Blue® mice. Mutation load was assessed quantitatively through measurement of mutant and mutation frequency and qualitatively through identification of mutations and characterization of recurrent mutations, multiple mutations, mutation pattern, and mutation spectrum. A total of 9.6 million plaque forming units were screened, 226 mutants were collected, and 196 independent mutations were identified. Four novel mutations were discovered. Spontaneous mutation frequency was low in pancreas and high in the submaxillary gland. The submaxillary gland had multiple recurrent mutations in each of the mice and one mutant had two independent mutations. Mutation patterns for epithelial tissues differed from that observed in male germ cells with a striking bias for G:C to A:T transitions at CpG sites. A comprehensive review of lacI spontaneous mutation patterns in young adult mice and rats identified additional examples of this mutational bias. An overarching observation about spontaneous mutation frequency in adult tissues of the mouse remains one of stability. A repeated observation in certain epithelial tissues is a higher rate of G:C to A:T transitions at CpG sites and the underlying mechanisms for this bias are not known. Environ. Mol. Mutagen. 55:51–63, 2014. © 2013 Wiley Periodicals, Inc.     

UVA induces C-->T transitions at methyl-CpG-associated dipyrimidine sites in mouse skin epidermis more frequently than UVB

We studied the kinetics of mutation induction in skin epidermis and dermis of UVA-irradiated transgenic Muta mice and analyzed the sequence changes in 80 lacZ transgene mutants from the irradiated epidermis. The mutant frequency increased linearly in both the epidermis and dermis up to 240 kJ/m2 UVA, twice as efficiently in the epidermis as in the dermis, without provoking any inflammatory reactions in the exposed skin. The 83 mutations detected in the UVA-exposed epidermis were dominated by C-->T transitions (88%), found almost exclusively at dipyrimidine sites, and specified by four occurrences of CC-->TT tandem substitutions, suggesting that UV-specific photoproducts induced in DNA have a major role in the genotoxicity. No T-->G transversions, which have been considered as a UVA signature mutation, and few mutations suggesting the relevance of oxidative damage were recovered in the present study. An analysis of the bases adjacent to the mutated cytosines revealed that the 3'-cytosine of dipyrimidine sites is the preferred target of UVA-induced C-->T transition. Moreover, C-->T transitions were induced at dipyrimidine sites associated with CpG much more frequently by UVA than by UVB, forming hotspots at several of these sites. These results suggest that UVA contributes more to the formation of recurrent or hotspot mutations at methylated CpG sites in the mammalian genome than UVB, since methylation of the CpG motif is observed entirely in the lacZ transgenes and is known to enhance the formation of cyclobutane pyrimidine dimers by longer wavelength UV.     

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.     

The tumor promoter TPA enhances benzo[a]pyrene and benzo[a]pyrene diolepoxide mutagenesis in Big Blue mouse skin

The Big Blue mouse was used to investigate the role of cell proliferation in mutation fixation in the mouse back skin model of carcinogenesis. Phorbol 12-myristate 13 acetate (TPA) was applied to the dorsum of Big Blue mice to manipulate cell proliferation, and benzo[a]pyrene (BaP) or BaP-diolepoxide (BPDE) was applied to produce premutagenic DNA damage. Mutations in the lacI transgene of skin DNA were measured. BaP and BPDE elevated mutant frequency, DNA adducts, and cell damage over untreated and acetone-treated mice. BPDE-DNA adducts peaked within 30 min of exposure and DNA adducts, formed after application of both BaP and BPDE, declined rapidly with time. As the dose of BaP increased (4 to 64 microg), DNA adducts, mutant frequency, and cell damage increased in a dose-dependent manner. TPA applied after BaP and BPDE further increased mutant frequency, DNA adducts, and cell damage, while variably affecting mitotic index and other measures of cell proliferation. TPA became less effective at increasing mitotic index as the dose of BaP increased, although all measures of cell proliferation, taken together, increased. The most effective production of DNA adducts and mutations occurred when the carcinogen was applied simultaneously with or within 1 hr of TPA. Mutations induced by BPDE were predominantly base substitutions: of these base substitutions, 35% were G:C --> A:T transitions, and 36% were G:C --> T:A and 29% G:C --> C:G transversions. Approximately 88% of all mutations and 100% of base substitutions were at G:C sites; 60% of all mutations and 70% of the base substitution mutations occurred at CpG sites. A:T --> G:C transitions were not found. All of the single-base deletions were at G:C base pairs.     

Tissue-specific mutant frequencies and mutational spectra in cyclophosphamide-treated lacI transgenic mice.

The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T-lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle-treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP-treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high-dose CP treatment, with a significant increase in the frequency of A. T --> T. A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle-treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G. C --> A. T transitions at CpG sites and G. C --> T. A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP-induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP-induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo.