Big BlueTM Research Articles

Commentary on “Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl) nitrosamine as the basis for urothelial cell carcinogenesis.” He Z, Kosinska W, Zhao ZL, Wu XR, Guttenplan JB, Department of Basic Science, New York University Dental College, NY, USA.: Mutat Res 2012;742(1–2):92–5 [Epub 2011 Dec 4

Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue™) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue™) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis.

Encephalitozoon intestinalis infection increases host cell mutation frequency

BackgroundMicrosporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels.FindingsAs oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big BlueTM transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores.ConclusionsThese data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which E. intestinalis infection increases host mutation frequency are warranted.

Abstract 2548: Quantitative changes in endogenous DNA adducts correlate with conazole mutagenicity and tumorigenicity in mouse liver

Abstract We have previously shown that the conazole fungicides triadimefon and propiconazole, which are tumorigenic in mouse liver, are in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses. The nontumorigenic conazole myclobutanil was not mutagenic under the same conditions. DNA sequencing of the mutants recovered from each treatment group and from untreated control animals revealed that the mutations induced by propiconazole and triadimefon do not result from clonal expansion of background mutations. We hypothesized that these mutations arise from the accumulation of reactive electrophilic metabolic byproducts within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies in order to determine if quantitative or qualitative differences in DNA adducts correlated with mutagenicity and tumorigenicity. We resolved and quantitated 16 individual adduct spots by 32P postlabeling and thin layer chromatography using 3 solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. We hypothesize that this treatment-related increase in endogenous DNA adducts may explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon. This abstract does not necessarily reflect EPA policy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2548. doi:1538-7445.AM2012-2548

Quantitative changes in endogenous DNA adducts correlate with conazole in vivo mutagenicity and tumorigenicity

The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo. We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by (32)P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups (P = 0.002), as were total endogenous DNA adduct levels (P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon.

Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis

Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue™) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2–6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue™) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis.

Mutagenesis by asbestos in the lung of λ-lacI transgenic rats

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic λ-lacI (Big Blue™) rats were intratracheally instilled with single doses of 1 or 2 mg, or with four weekly doses of 2 mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg ( P = 0.035) and of 4 × 2 mg ( P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.

No-effect level in the mutagenic activity of the drug cyproterone acetate in rat liver: part I. Single dose treatment

The anti-androgen and progestagen cyproterone acetate (CPA) is known to cause liver tumors in rats. The drug has been identified recently as a mutagen in the liver of female transgenic λlacI (Big Blue™) rats at high doses after an expression time of 6 weeks. A dose of 50 mg CPA/kg BW, however, did not increase the mutation frequency (MF) of controls indicating a no-effect level of mutagenicity [Carcinogenesis 19 (1998) 241]. The present study was performed to assess the existence of a no-effect level of mutagenicity. In order to figure out conditions of maximum response, the time course of the MF was determined after administration of a single dose of 100 mg CPA/kg BW to female Big Blue™ rats. The MF showed a strong initial rise to a maximum 2 weeks after CPA administration accompanied by a corresponding increase of cell proliferation and of DNA adduct levels. Thereafter, the MF decreased within further 2 weeks to one third of the maximum level which was maintained for another 4 weeks. The DNA adduct levels decreased only by 15% during this time period suggesting that mutated hepatocytes were eliminated predominantly. A dose dependence curve determined at a fixation time of 2 weeks revealed a no-effect level of 5 mg CPA/kg BW for mutagenicity. In conclusion, our findings indicate that the length of the observation period may be a critical determinant for the outcome of a mutagensis study in rat liver. Furthermore, the existence of a no-effect level for the mutagenicity of CPA in rat liver was confirmed. However, it has to be clarified whether the dose of 5 mg CPA/kg BW corresponding to the “transient” type of mutations or the previous dose of 50 mg CPA/kg BW related to a “permanent” type of mutations is more relevant for the assessment of the genotoxic risk.

No-effect level in the mutagenic activity of the drug cyproterone acetate in rat liver: Part II. Multiple dose treatment

In order to probe for the existence of a no-effect levels for mutations induced by multiple dose treatment with cyproterone acetate (CPA), female lacI-transgenic Big Blue™ rats were treated daily for 3 weeks with oral doses of 5.0, 1.0 or 0.2 mg/kg CPA b.w., respectively. The dose of 5 mg/kg CPA b.w. ineffective as a single dose (see part I) increased the mutation frequency by 2.5-fold. Daily treatment with 1.0 and with 0.2 mg/kg CPA b.w. for 3 weeks, however, was not effective indicating that the 1 mg dose represents a no-effect level for multiple dose treatment. The finding that a total dose of 21×5=105 mg/kg CPA b.w. caused 50% less DNA adducts, but a mutation frequency three-fold higher (data extrapolated from part I) than observed after daily treatment with 5 mg/kg CPA b.w. for 21 days points to a crucial role of cell proliferation in mutagenesis by CPA. Our present results, in combination with previous findings, offer a basis to estimate the risk to develop mutations in human liver following treatment with CPA. Previous studies revealed that CPA-DNA adducts are formed in human hepatocytes at lower levels than in those of female rats [Mutat. Res. 395 (1997) 179; Cancer Res. 56 (1996) 4391]. Moreover, an in vitro-study indicated that human hepatocytes in culture do not respond to the mitogenic effect of CPA, while rat hepatocytes did [Cancer Res. 51 (1991) 1143]. We conclude that the risk of humans to develop mutations under treatment with CPA is substantially lower than in the female rat.

Dietary low-dose sucrose modulation of IQ-induced genotoxicity in the colon and liver of Big Blue™ rats

Earlier studies have indicated that sucrose increases 2-amino-3-methylimidazo[4,5- f]quinoline (IQ)-induced aberrant crypt foci in the colon. In this study, we investigated the role of sucrose in IQ-induced genotoxicity of the colon mucosa and liver. Big Blue™ rats were fed with IQ (20 ppm in feed) and/or sucrose (3.45 or 6.85 wt.% in feed) for 3 weeks. IQ increased DNA strand breaks in the colon, whereas the mutation frequency was increased in the liver. The level of IQ-induced DNA adducts was elevated in both colon mucosa cells and liver. In the liver, high sucrose intake increased the level of DNA adducts above that of IQ and low sucrose intake. Oxidative DNA damage detected in terms of 7-hydro-8-oxo-2′-deoxyguanosine by HPLC-EC, or endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites were unaltered in the colon and liver. Expression of ERCC1 and OGG1 mRNA levels were unaffected by IQ or sucrose feeding. Biomarkers of oxidative stress, including Vitamin C, malondialdehyde and protein oxidations (γ-glutamyl semialdehyde and 2-amino adipic semialdehyde) were unaltered in plasma and in liver. In conclusion, sucrose feeding increases IQ-induced genotoxicity in liver but not in colon, suggesting different mechanisms for sucrose and IQ in colon mutagenesis.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis in cultured Big Blue rat mammary epithelial and fibroblast cells.

Epithelial cells are the primary site of carcinogenesis in most tissues, including the mammary gland. As an alternative to the study of mutation induction in whole tissues in vivo, we have established Big Blue transgenic rat cell lines from the mammary epithelium (BBR/ME) and the mammary stroma (BBR/MFib), to permit a comparison of their mutagenic responses to carcinogens. We previously demonstrated their responsiveness to the alkylating agent N-ethyl-N-nitrosourea (ENU) (McDiarmid H et al. [2001]: Mutat Res 497:39-47). Here, we examined the responses of cultured epithelial and stromal cells to the protein pyrolysis product and mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rat hepatic S9 was used as a source of bioactivation enzymes. Mutant induction (cII locus) and clonogenic survival were measured as a function of PhIP concentration. PhIP mutagenicity was observed in the fibroblast cells, but the greater toxicity of PhIP to the epithelial cells prevented a definitive evaluation of mutagenicity. Since PhIP may be detoxified by conjugation with glutathione, we measured glutathione levels and glutathione-S-transferase expression and activities in both cell lines. The epithelial cells had higher glutathione-S-transferase enzyme activity and protein expression than did the fibroblast cell line. Because the epithelial cells were more sensitive to toxicity, glutathione conjugation evidently plays only a minor role in PhIP toxicity and mutagenicity in our cell lines.

The male rat carcinogens limonene and sodium saccharin are not mutagenic to male Big Blue rats.

Limonene and sodium saccharin are male rat specific carcinogens giving rise to renal and bladder tumours, respectively. Both compounds give negative results in genetic toxicity assays suggesting a non-genotoxic mode of action for their carcinogenicity. The alpha 2U-globulin accumulation theory has been invoked to explain the renal carcinogenicity of limonene: the accumulation of micro masses of calcium phosphate in the bladder, coupled with a high pH environment in the male rat bladder, has been suggested to be responsible for the bladder carcinogenicity of sodium saccharin. The implication of these proposed mechanisms is that limonene and sodium saccharin will not be mutagenic to the rat kidney and bladder, respectively. This proposal has been evaluated by assessing the mutagenic potential of the two chemicals to male lacI transgenic (Big Blue) rats. Male Big Blue rats were exposed for 10 consecutive days to either limonene in diet, at a dose level in excess of that used in the original National Toxicology Program gavage carcinogenicity bioassay, or to sodium saccharin in diet at the dose known to induce bladder tumours. The multi-site rat carcinogen 4-aminobiphenyl was used as a positive control for the experiment. Limonene failed to increase the mutant frequency in the liver or kidney of the rats, and sodium saccharin failed to increase the mutant frequency in the liver or bladder of the rats. 4-Aminobiphenyl was mutagenic to all three of these tissues. These results add further support to a non-genotoxic mechanism of carcinogenic action for both limonene and sodium saccharin.

Extent of CpG methylation is not proportional to the in vivo spontaneous mutation frequency at transgenic loci in Big Blue™ rodents

The lacI transgene used in the Big Blue™ (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5×10 −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 (∼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.

On clonal expansion and its effects on mutant frequencies, mutation spectra and statistics for somatic mutations in vivo.

Jackpots of mutations are the bane of transgenic mutation assays, for they can produce large mutant frequencies even in the absence of a mutagen. A jackpot is a large number of mutants that arose from a single mutation. They arise when there is a clonal expansion of a mutated cell. Jackpots as large as 1000 mutants/100 000 plaques have been observed, a mutant frequency of 1%. Such a jackpot can swamp not only the spontaneous mutant frequency but even the mutant frequency induced by potent mutagens, which are rarely .0.2% with even the most potent mutagens and are usually much lower (Hoorn et al., 1993; Tao et al., 1993; Morrison and Ashby, 1994; Douglas et al., 1995; Mirsalis et al., 1995; Skopek et al., 1995; Cosentino and Heddle, 1999). Very large jackpots are not normally a problem, for they are statistical outliers and can be discarded legitimately by standard statistical tests. These large jackpots arise when a mutation occurs early in development in one of the progenitor cells for one or more tissues. The normal growth of the organism is by clonal expansion of the cells present during the preceding stage. Accordingly, mutations arising at an early stage of development produce a large number of mutant cells, i.e. a jackpot, when these are recovered together. Smaller jackpots, from mutations arising later in development, are harder to identify and are, thus, more problematic. Numerous proposals have been made to deal with this problem including complex statistical analysis and the sequencing of each and every mutant (de Boer et al., 1996; Knoll et al., 1996). There is a simple method of dealing with the data without such extraordinary efforts. In fact, current methods over-compensate for jackpots and thus distort both the mutant frequency and the mutation spectrum. The transgenes present in the MutaTM Mouse and the Big BlueTM Mouse are bacterial genes embedded in a recoverable λ vector (Gossen et al., 1989; Kohler et al., 1991). They lack mammalian promoters or polyadenylation sites and are, presumably, unexpressed and unimportant to the mouse. Theoretically and practically they are genetically neutral, providing neither benefit nor harm to the cell containing them (Tao et al., 1993; Tao and Heddle, 1994; Cosentino and Heddle, 1996). This being the case, a cell containing a mutated transgene is neither more nor less likely to expand than any cell without such a mutation. In a large number of animals, therefore, clonal expansion will have no effect on the average mutant frequency: expansions of the numerous non-mutants will each tend to decrease the mutant frequency somewhat while expansions of the rare mutants will each tend to increase the mutant frequency to a larger extent (Figure 1). Nevertheless,

The Big Blue® transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques

There are mutational artifacts in the Big Blue® 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® 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.

Effects ofO6-alkylguanine-DNA alkyltransferase deficiency inEscherichia coli as the host for the detection of mutations inlacI transgenic mice

Transgenic mice are widely used to detect gene mutations in vivo induced by a variety of chemicals. It is known, however, that no mutagenicity of methyl methanesulfonate (MMS) is detected in epididymal sperm in various transgenic mice assays, although MMS induces the dominant lethal and specific locus mutations in male mice. To investigate the issue of whether unrepaired lesions in DNA of mature sperm can be transformed into mutations during replication of the lambda phage in Escherichia coli cells, we developed an E. coli strain YG5152, which is a derivative of strain SCS-8 but is deficient in the genes encoding O (6)-alkylguanine-DNA alkyltransferases. When lambda LIZalpha phages were treated with MMS or N-ethyl-N-nitrosourea (ENU) in vitro and infected to the E. coli strains, the mutant frequencies of lacI were markedly higher in strain YG5152 than in strain SCS-8. When Big Blue(trade mark) mice were treated with MMS (160 mg/kg) or ENU (125 or 250 mg/kg) and the phages rescued from mature sperm were infected to the strains, the mutation frequency (MF) of phages from ENU-treated mice at a dose of 250 mg/kg in strain YG5152 was about two times higher than that in strain SCS-8. However, no increase in the MF was observed in the MMS-treated mice even in strain YG5152. These results suggest that, although strain YG5152 efficiently detects ex vivo mutations caused by mutagenic alkyl adducts formed by MMS in lambda phage DNA, no detectable levels of mutagenic methyl adducts are present in mature sperm of MMS-treated mice. Possible reasons for this lack of mutagenicity of MMS in mature sperm using transgenic mice assays are discussed.

Hepatic gene mutations induced in Big Blue™ rats by both the potent rat liver Azo‐carcinogen 6BT and its reported noncarcinogenic analogue 5BT

Hepatic gene mutations induced in Big Blue™ rats by both the potent rat liver Azo‐carcinogen 6BT and its reported noncarcinogenic analogue 5BT

A comparative study of in vivo mutation assays: analysis of hprt, lacI, and cII/cI as mutational targets for N-nitroso- N-methylurea and benzo[ a]pyrene in Big Blue™ mice

We have compared the response of the native hprt gene and the lacI, cII, and cI transgenes in Big Blue™ B6C3F1 mice following treatment with either N-nitroso- N-methylurea (MNU) or benzo[ a]pyrene (B aP). Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis. Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-β- d-galactopyranoside (X-gal) to score lacI mutations, or plated on a hflAB lawn to score cII and cI mutants. With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg). In comparison, the lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI transgenes did not show any increase. With 150 mg/kg B aP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and lacI, respectively, while only a 1.3-fold increase was observed at cII. DNA sequencing revealed an increase in GC→TA transversions among the cII mutants, suggesting that the increase was related to B aP exposure. No significant increase in cI mutant frequency was observed. Therefore, the order of mutation assay sensitivity was hprt> lacI> cII/cI with MNU, and hprt≈ lacI> cII/cI with B aP. While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted.

P1B53 - Studies on tamoxifen — induced mutations in the livers and uteri of big blue™ transgenic F344 rats

P1B53 - Studies on tamoxifen — induced mutations in the livers and uteri of big blue™ transgenic F344 rats

Effect of target gene CpG content on spontaneous mutation in transgenic mice

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene ( mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10 −5 range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.

4-Chloro- o-phenylenediamine: a 26-week oral (in feed) mutagenicity study in Big Blue™ mice

4-Chloro- o-phenylenediamine (4-C- o-PDA) is a liver carcinogen in mice and was found to be weakly mutagenic in the liver of female Big Blue™ mice after short term treatment. In the present study the test compound was given subchronically in the diet for 26 weeks at doses of 0, 5000 and 10,000 ppm. The corresponding average test substance intake was 2166 mg kg −1 day −1 (males: 1794 mg kg −1 day −1; females: 2539 mg kg −1 day −1) and 4610 mg kg −1 day −1 (males: 3926 mg kg −1 day −1; females 5925 mg kg −1 day −1) at the low and high dose, respectively. After sacrifice, tissues were flash frozen in liquid nitrogen. The lacI mutant frequency in the liver was determined from three male and three female mice per dose group. The genomically integrated transgene was recovered by packaging into λ phage using Transpack™ packaging extract (Stratagene, La Jolla, USA) followed by infection of Escherichia coli strain SCS-8. Blue mutant plaques were scored against a background of clear non-mutant plaques. Food consumption decreased initially at 10,000 ppm, while no treatment related effect on food intake was observed at 5000 ppm. Body weight gain was found to be decreased in all treated animals. Absolute and relative liver weight increased in a dose-related manner, but only the latter effect was statistically significant. A clear dose dependent increase in lacI mutant frequencies was observed in the liver of both sexes. The following mutant frequencies (×10 −5) were observed: 2.73±1.01 (males, untreated), 7.24±1.50 (females, untreated), 18.91±5.30 (5000 ppm, males), 24.91±7.58 (5000 ppm, females), 20.47±6.68 (10,000 ppm, males) and 36.17±14.98 (10,000 ppm, females). It is therefore concluded that 4-C- o-PDA is a strong mutagen in the liver of mice treated subchronically for 26 weeks.