Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line.

This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide (4-NQO) in the lacI target gene of the transgenic Big Blue Rat2 cell line. There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied. Big Blue Rat2 cells were treated with 0.03125, 0.0625 or 0.125 microg/ml of 4-NQO, the highest concentration giving 85% survival. Our results indicated that the mutant frequency (MF) induced by 4-NQO was dose-dependent with increases from three- to seven-fold. The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations. In spontaneous mutants, transition (60%) mutations, especially G:C-->A:T transition (45%), were most frequent. However, the major type of base substitution after treatment of 4-NQO was transversions (68.8%), especially G:C-->T:A (43.8%), while only 25% of mutants were transitions. These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved.     

The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques

There are mutational artifacts in the Big Blue(R) assay and it is important to characterize the source and nature of these mutations. Differences were reported in the mutation patterns of a small sample of 23 sectored and 91 circular mutant plaques derived from skin using the Big Blue(R) transgenic mouse mutation detection system [G. R. Stuart, N.J. Gorelick, J.L. Andrews, J.G. de Boer, B.W. Glickman, The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice, Environ. Mol. Mutagen 28 (1996) 385-392.]. We have extended these observations by analyzing 46 sectored and 224 circular mutant plaques derived from seven tissues. The frequency of sectored mutant plaques is estimated to be 16% with no significant variation with tissue type. However, the patterns of mutation for sectored mutants and mouse-derived mutations differed significantly (p=0.04). Base substitutions in sectored mutant plaques do not show the asymmetries found in circular mutants consistent with integration of a GC rich transgene into the AT rich mammalian genome. Sectored mutants have mutation patterns consistent with a mixture of mouse, in vitro and Escherichia coli-derived mutations. Data on the relative frequencies of different mutant plaque morphologies suggests that overlapped plaques are substantially contaminated by sectored plaques at recommended plating densities.     

A more efficient Big Blue protocol improves transgene rescue and accuracy in a adduct and mutation measurement

Transgenic mutational systems have provided researchers with an invaluable tool, allowing the measurement of both spontaneous and induced mutations. The Big Blue transgenic rodent mutagenesis system developed by Stratagene (La Jolla, CA) uses a lambda shuttle vector carrying lacI as the mutational target gene. A common criticism of the Big Blue system is that it relies on visual screening to detect mutants rather than positive selection, which is employed in more recently developed systems. The lack of positive selection, however, has provided the Big Blue system with a unique advantage, as it allows for the dynamic quantification of mutation fixation, repair, and adduct stability, since both pre-mutagenic DNA adducts and mutations can readily be quantified [Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 11391]. Improvements to the standard Big Blue assay protocol are required for the visualization of mutant plaques resulting from pre-mutagenic damage, as these can appear much lighter in color than the lightest color control mutant (CM0). This increase in detection has been achieved by the development of a protocol that now permits the effective measurement of repair and mutation fixation utilizing the Big Blue system. This new protocol has also addressed efficiency, allowing for a two-fold increase in the number of plaques produced per packaging reaction and a decrease in both phage migration and plaque size, permitting a greater than three-fold increase in plating density. The implementation of this protocol will make the Big Blue assay more economical and less demanding than before, while providing researchers with an efficient means to measure both repair and mutation in this system.     

Genotoxic effects of bitumen fumes in Big Blue transgenic rat lung

Road paving workers are exposed to bitumen fumes (CAS No. 8052-42-4), a complex mixture of volatile compounds and particles containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning their toxicity. In order to gain better insights on their genotoxic potential, we used an experimental design able to generate bitumen fumes at road paving temperature (temperature: 170 degrees C, total particulate matter: 100mg/m3) and perform a nose-only exposure of Big Blue transgenic rodents 6h/day for five consecutive days. The mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the rodent genome. We previously observed in mouse lung, that bitumen fumes did not induce an increase of cII mutants, a modification of the mutation spectrum, nor the formation of DNA adducts. Since DNA adducts were found in the lungs of rats exposed to asphalt fumes in similar conditions, we decided to carry out an analogous experiment with Big Blue rats. A DNA adduct was detected 3 and 30 days after the end of treatment suggesting that these genetic alterations were quite steady. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rats. In addition, a slight but not significant modification of the mutation spectrum associated with an increase of G:C to T:A and A:T to C:G transversions was noticeable in the treated animals. Then, these data failed to demonstrate a pulmonary mutagenic potential for bitumen fumes generated at road paving temperature in our experimental conditions despite the presence of a DNA adduct. These results may provide information concerning the pulmonary mechanism of action of this aerosol and may contribute to the occupational health hazard assessment.     

Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice

The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations. Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed. Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment. We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice. A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups. While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not. The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007). The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI. The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C相似文献   

Subchronic administration of phenobarbital alters the mutation spectrum of lacI in the livers of Big Blue transgenic mice

Phenobarbital (PHE) is a liver carcinogen in B6C3F1 mice and a weak mutagen that does not appear to form DNA adducts. To investigate PHE mutagenicity in vivo, B6C3F1 Big Blue(R) male transgenic mice harboring the lambdaLIZ shuttle vector containing the lacI target gene were fed PHE at 2500 ppm for 180 days. A modest increase in the mutant frequency (MF) from 5.02+/-2.4x10(-5) in the control group to 6.88+/-0.754x10(-5) in the PHE-treated group, which was marginally different (p<0.05), was obtained. To better assess the relevance of this increase in MF, a random collection of mutants from each PHE-exposed mouse was sequenced. After correcting for clonal expansion, which is the most conservative approach, the MF in the PHE-treated mice decreased to 6.39+/-1.02x10(-5), an insignificant difference (p=0.10) from that in control group. Despite this modest increase in MF, the mutation spectrum obtained from the PHE-exposed group was significantly different (pA:T transitions remained the same in the two spectra. It is postulated that the increase in transversions at G:C base pairs found in the PHE-derived spectrum is likely due to oxidative damage as a result of induction of CYP2B isozymes by the chronic administration of PHE. Results from this study demonstrate that PHE alters the spectrum of mutations, rather than inducing a significant global increase in the MF. The PHE-derived spectrum of lacI mutants from the liver of Big Blue(R) B6C3F1 male mice was remarkably similar (p=0.8) to that generated by oxazepam (OX), a compound which also induces CYP2B isozymes following chronic administration of the drug.     

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.     

Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.     

Genotoxicity of malachite green and leucomalachite green in female Big Blue B6C3F1 mice

Malachite green, a triphenylmethane dye used in aquaculture as an antifungal agent, is rapidly reduced in vivo to leucomalachite green. Previous studies in which female B6C3F1 mice were fed malachite green produced relatively high levels of liver DNA adducts after 28 days, but no significant induction of liver tumors was detected in a 2-year feeding study. Comparable experiments conducted with leucomalachite green resulted in relatively low levels of liver DNA adducts but a dose-responsive induction of liver tumors. In the present study, we fed transgenic female Big Blue B6C3F1 mice with 450 ppm malachite green and 204 and 408 ppm leucomalachite green (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither malachite green nor leucomalachite green increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm leucomalachite green did increase the liver cII mutant frequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with malachite green or in female Big Blue rats treated with a comparable dose of leucomalachite green for 16 weeks in a previous study [Mutat. Res. 547 (2004) 5]. These results indicate that leucomalachite green is an in vivo mutagen in transgenic female mouse liver and that the mutagenicities of malachite green and leucomalachite green correlate with their tumorigenicities in mice and rats. The lack of increased micronucleus frequencies and lymphocyte Hprt mutants in female mice treated with leucomalachite green suggests that its genotoxicity is targeted to the tissue at risk for tumor induction.     

Mutagenicity of ultraviolet A radiation in the lacI transgene in Big Blue mouse embryonic fibroblasts

Sunlight ultraviolet A (UVA) irradiation has been implicated in the etiology of human skin cancer. A genotoxic mode of action for UVA radiation has been suggested that involves photosensitization reactions giving rise to promutagenic DNA lesions. We investigated the mutagenicity of UVA in the lacI transgene in Big Blue mouse embryonic fibroblasts. UVA irradiation of these cells at a physiologically relevant dose of 18J/cm(2) caused a 2.8-fold increase in the lacI mutant frequency relative to control, i.e., 12.12+/-1.84 versus 4.39+/-1.99 x 10(-5) (mean+/-S.D.). DNA sequencing analysis showed that of 100 UVA-induced mutant plaques and 54 spontaneously arisen control plaques, 97 and 51, respectively, contained a minimum of one mutation along the lacI transgene. The vast majority of both induced- and spontaneous mutations were single base substitutions, although less frequently, there were also single and multiple base deletions and insertions, and tandem base substitutions. Detailed mutation spectrometry analysis revealed that G:C-->T:A transversions, the signature mutations of oxidative DNA damage, were significantly induced by UVA irradiation (P<0.003). The absolute frequency of this type of mutations was 7.4-fold increased consequent to UVA irradiation as compared to control (3.38 versus 0.454 x 10(-5); P<0.00001). These findings are in complete agreement with those previously observed in the cII transgene of the same model system, and reaffirm the notion that intracellular photosensitization reactions causing promutagenic oxidative DNA damage are involved in UVA genotoxicity.     

High plating density improves Big Blue system efficiency without loss of sensitivity

To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.     

In vivo genotoxicity of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline in lacI transgenic (Big Blue) mice

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a heterocyclic amine found in cooked meat, is a strong mutagen in the Salmonella/microsome assay and was proven to be a hepatocarcinogen in rodents. We used the lacI transgenic (Big Blue(R)) mouse to investigate MeIQx genotoxicity in vivo. lacI mutant frequencies were examined in liver and colon after single intragastric administration of MeIQx (males) or 12 weeks of feeding in the diet (males and females). Micronucleus induction was monitored in the peripheral blood and cell proliferating activity was monitored by proliferating cell nuclear antigen (PCNA) immunostaining, but only after the intragastric administration. Intragastric treatment with MeIQx (100 mg/kg) did not increase mutant frequency (MF) in liver or colon but it did induce a slight but statistically significant increase in the incidence of micronucleated reticulocytes 48 h after the treatment. No apparent increase in PCNA-positive foci was observed in any of tissues analyzed 14 days after the treatment. Administration of MeIQx (300 ppm) in diet for 12 weeks, however, caused MF increases in liver and colon in male and female mice, with greater increases in the females. An increase was also obvious after 4 weeks, but only in females. The sex difference in MF is consistent with the fact that female mice are more susceptible to MeIQx carcinogenesis. These results demonstrated that in the transgenic mouse mutation assay, long-term feeding of MeIQx was more effective than single gastric exposures in revealing the compound's mutagenicity in the target organs of carcinogenicity and that sex differences in susceptibility can also be observed.     

Mutagenicity in lung of big Blue((R)) mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO(2) reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC-->TA, 29% GC-->AT, 2% GC-->CG, and 10% multiple mutations - primarily GG-->TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO(2)(+) resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG-->TT transversions. Nose-only exposure of Big Blue((R)) mice to PAN at 78ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency +/-S.E. of 6.16+/-0.58/10(5) for controls versus 8.24+/-0.30/10(5) for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with 3H-PAN showed that 24h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG-->TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.     

Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice

Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lacI transgenic mice aged 1.5-25 months. Mutations accumulated with age in liver and accumulated more rapidly in bladder. In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice. To investigate the origin of mutations, the mutational spectra for each tissue and age were determined. DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age. The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication. The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair. Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues.     

Spontaneous mutations in the Big Blue® transgenic system are primarily mouse derived

The Big Blue® transgenic mouse mutation detection system provides a powerful approach for measuring spontaneous and induced mutations in vivo. The observed mutations may contain a fraction of ex vivo or prokaryotic mutational events. Indeed, a modified, selectable form of the Big Blue® assay seem to generate artifactual mutants under certain circumstances. Herein we review the evidence that circular mutants (i.e., the plaque circumference is at least 50% blue) collected in the standard Big Blue® assay are derived primarily from the mouse. The most direct evidence is the similarity in the types of mutations found in jackpot and nonjackpot mutations. In addition, about half of the spontaneous mutations in the lacI transgene are transitions and transversions at CpG dinucleotides, a mammalian-specific feature. The mutation pattern observed at lacI is consistent with AT mutation pressure operating in a GC rich DNA and approaches that reported for observed germline human factor IX mutations. Furthermore, the spontaneous mutation pattern of circular Big Blue® mutants differs significantly from that of an endogenous lacI gene in E. coli. Pinpoint mutants (a dot of blue color peripherally located in a wild type plaque), which a priori were not expected to be mouse-derived, have a mutation pattern consistent with the mutation pattern of an endogenous E. coli lacI gene. Analysis of induced mutagenesis studies reveals mutation frequencies and patterns for the Big Blue® circular mutants which are comparable to endogenous genes. In reconstruction experiments, blue plaques derived from a superinfection with wild type and mutant phage produced approximately 50% blue and 50% clear plaques on replating. This phenomenon has not been seen when plaques derived from mouse were replated in the Big Blue® assay. Collectively, the evidence strongly supports a murine origin for circular mutants recovered in the standard Big Blue® assay. Validation of current assays is an essential step in determining the frequency and pattern of spontaneous murine-specific mutations. Defining this benchmark will be helpful in evaluating the next generation of transgenic mutation detection systems.     

iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.     

Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue rats

The cII assay provides an alternative choice to the lacI transgene for mutational studies involving Big Blue(R) transgenic mice and rats, or permits the evaluation of mutational responses in both genes. Here, we compare the mutational response of the cII gene from colon of Big Blue(R) F344 rats treated with a dietary mutagen and animal carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to those previously determined in the lacI transgene from colon of the same group of animals. A cursory inspection of PhIP-induced mutational spectra (MS) in cII and lacI suggests that the two transgenes respond differently to PhIP-induced mutation. However, a more thorough analysis of the MS in the two transgenes, including consideration of the number of mutational target sequences in each gene and nearest neighbor analyses of mutated nucleotides, indicates that PhIP-induced mutational specificity is similar in both genes. The evaluation of PhIP-induced mutational responses in these two transgenes serves as a model for intergenic mutational analyses.     

Age-dependent sensitivity of Big Blue transgenic mice to the mutagenicity of N-ethyl-N-nitrosourea (ENU) in liver

The incidence of childhood cancer is increasing and recent evidence suggests an association between childhood cancer and environmental exposure to genotoxins. In the present study, the Big Blue transgenic mouse model was used to determine whether specific periods in early life represent windows of vulnerability to mutation induction by genotoxins in mouse liver. Groups of mice were treated with single doses of 120 mg N-ethyl-N-nitrosourea (ENU)/kg body weight or the vehicle either transplacentally to the 18-day-old fetus or at postnatal days (PNDs) 1, 8, 15, 42 or 126; the animals were sacrificed 6 weeks after their treatment. The cII mutation assay was performed to determine the mutant frequencies (MFs) in the livers of the mice. Liver cII MFs for both sexes were dependent on the age at which the animals were treated. Perinatal treatment with ENU (either transplacental treatment to the 18-day-old fetus or i.p. injection at PND 1) induced relatively high MFs. However, ENU treatment at PNDs 8 and 15 resulted in the highest mutation induction. The lowest mutation induction occurred in those animals treated as adults (PND 126). For instance, the cII MF for the PND 8 female group was 646 x 10(-6) while the MF for female adults was only 145 x 10(-6), a more than 4-fold difference. Molecular analysis of the mutants found that A:T-->T:A transversions and A:T-->G:C transitions characterized the pattern of mutations induced by ENU in both the neonate and adult mice, while the predominate type of mutation in the controls was G:C-->A:T. The results indicate that mouse liver is most sensitive to ENU-induced mutation during infancy. This period correlates well with the age-dependent sensitivity to carcinogenicity in mouse liver, suggesting that mutation is an important rate-limiting factor for age-related carcinogenesis.     

Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.     

Mutation spectral changes in spermatogenic cells obtained from old mice

Male reproductive health is compromised with increased paternal age, due at least in part, to an increased frequency of de novo germline mutations. Because of technical and sample limitations, there is a dearth of empirical information on the mechanism(s) that mediate this age-related increase in mutant frequency. To study this phenomenon, investigators have used as a model system a transgenic mouse strain that carries a lacI mutagenesis reporter transgene. This transgene displays a paternal age effect and overcomes many of the technical difficulties that have inhibited experimental analyses of age-related changes in the male germline. In this study, approximately 300 mutant lacI transgenes were recovered from defined spermatogenic cell types obtained from various aged lacI transgenic mice and sequenced. The spectrum representing mutations from spermatogenic cells of old mice revealed an increased prevalence of transversions compared to spectra for young and middle-aged mice. Five mutation hotspots were identified in spectra for spermatogenic cells from young and middle-aged mice, but no hotspots were identified in the spectrum for spermatogenic cells from old mice. These results suggest that the challenges to germline DNA change as the animal ages and that the increased mutant frequency observed with increased paternal age is not simply a greater accumulation of mutagenic events characteristic of spermatogenic cells from the young animal.