Ken-ichi Masumura

Recent advances in the protocols of transgenic mouse mutation assays

Transgenic mutation assays were developed to detect gene mutations in multiple organs of mice or rats. The assays permit (1) quantitative measurements of mutation frequencies in all tissues/organs including germ cells and (2) molecular analysis of induced and spontaneous mutations by DNA sequencing analysis. The protocols of recently developed selections in the lambda phage-based transgenic mutation assays, i.e. cII, Spi(-) and 6-thioguanine selections, are described, and a data set of transgenic mutation assays, including those using Big Blue and Muta Mouse, is presented.

Enhanced Spontaneous and Benzo(a)pyrene-Induced Mutations in the Lung of Nrf2-Deficient gpt Delta Mice

The lung is an organ that is sensitive to mutations induced by chemicals in ambient air, and transgenic mice harboring guanine phosphoribosyltransferase (gpt) gene as a target gene are a well-established model system for assessing genotoxicity in vivo. Transcription factor Nrf2 mediates inducible and constitutive expression of cytoprotective enzymes against xenobiotics and mutagens. To address whether Nrf2 is also involved in DNA protection, we generated nrf2+/-::gpt and nrf2-/-::gpt mice. The spontaneous mutation frequency of the gpt gene in the lung was approximately three times higher in nrf2-null (nrf2-/-) mice than nrf2 heterozygous (nrf2+/-) and wild-type (nrf2+/+) mice, whereas in the liver, the mutation frequency was higher in nrf2-/- and nrf2+/- mice than in nrf2+/+ wild-type mice. By contrast, no difference in mutation frequency was observed in testis among the three genotypes. A single intratracheal instillation of benzo(a)pyrene (BaP) increased the lung mutation frequency 3.1- and 6.1-fold in nrf2+/- and nrf2-/- mice, respectively, compared with BaP-untreated nrf2+/- mice, showing that nrf2-/- mice are more susceptible to genotoxic carcinogens. Surprisingly, mutation profiles of the gpt gene in BaP-treated nrf2+/- mice was substantially different from that in BaP-untreated nrf2-/- mice. In nrf2-/- mice, spontaneous and BaP-induced mutation hotspots were observed at nucleotides 64 and 140 of gpt, respectively. These results thus show that Nrf2 aids in the prevention of mutations in vivo and suggest that Nrf2 protects genomic DNA against certain types of mutations.

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 λEG10 DNA in both chromosome 17 of C57BL/6J mouse. Environ. Mol. Mutagen. 34:1–8, 1999

Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1−/−) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1−/− and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1−/− mice was 1.6-fold higher than that in Parp-1+/+ mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1–5 bp insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1−/− than in Parp-1+/+ mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1−/− and Parp-1+/+ mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-1 is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements.

Spi− selection: An efficient method to detect γ‐ray‐induced deletions in transgenic mice

Despite the importance of genome rearrangement in the etiology of cancer and human genetic disease, deletion mutations are poorly detectable by transgenic rodent mutagenicity tests. To facilitate the detection and molecular analysis of deletion mutations in vivo, we established a transgenic mouse model harboring a λEG10 shuttle vector that includes the red and gam genes for Spi− (sensitive to P2 interference) selection [Nohmi et al. (1996] Environ. Mol. Mutagen. 28:465–470]. This selection has a great advantage over other genetic systems, because phage deletion mutants can be preferentially selected as Spi− plaques, which can then be subjected to molecular analysis. Here, we show nucleotide sequences of 41 junctions of deletion mutations induced by γ‐irradiation. Unlike spontaneous deletion mutants, more than half of the large deletions occurred between short homologous sequences from one to eight bp. The remaining junctions had no such homologous sequences. Intriguingly, two Spi− mutants had P (palindrome)‐like nucleotide additions at the breakpoints, which are frequently observed in the coding junctions of V(D)J recombination, suggesting that broken DNA molecules with hairpin structures can be intermediates in the repair of radiation‐induced double‐strand breaks. We conclude that Spi− selection is useful for the efficient detection of deletion mutations in vivo and that most rearrangements induced by γ‐rays in mice are mediated by illegitimate recombination through DNA end‐joining. Environ. Mol. Mutagen. 34:9–15, 1999

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gptΔ transgenic mouse

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

In vivo mutational analysis of liver DNA in gpt delta transgenic rats treated with the hepatocarcinogens N-nitrosopyrrolidine, 2-amino-3-methylimidazo[4,5-f]quinoline, and di(2-ethylhexyl)phthalate

In order to cast light on carcinogen‐specific molecular mechanisms underlying experimental hepatocarcinogenesis in rats, in vivo mutagenicity and mutation spectra of known genotoxic rat hepatocarcinogens N‐nitrosopyrrolidine (NPYR), and 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), as well as the nongenotoxic hepatocarcinogen di(2‐ethylhexyl)phthalate (DEHP) and the noncarcinogen acetaminophen (AAP), were investigated in guanine phosphoribosyltransferase (gpt) delta transgenic rats, a recently developed animal model for genotoxicity analysis. After 13‐wk treatment, glutathione S‐transferase placental form (GST‐P)‐positive liver cell foci were significantly increased in NPYR‐treated and IQ‐treated rats. In the DEHP‐treated rats, marked hepatomegaly with centrilobular hypertrophy of hepatocytes occurred, although GST‐P staining was consistently negative. Positive mutagenicity was detected in IQ‐ and NPYR‐treated rats. Mutant frequencies (MFs) in the liver DNA were 188.0 × 10−6 and 56.5 × 10−6, approximately 35‐fold and 10‐fold higher, respectively, than that of nontreatment control rats (5.5 × 10−6). There were no increases in MFs in the DEHP‐ or AAP‐treated rats as compared to the nontreatment control value. IQ induced mainly base substitutions leading to G:C to T:A transversions (56.9%) and deletions of G:C base pairs. In contrast, NPYR primarily caused specific A:T to G:C transitions (49.3%), which are very rare in the other groups. These data provided support for the conclusion that IQ and NPYR hepatocarcinogenesis depends on genotoxic processes and specific DNA adduct formation while DEHP exerts its influence via a nongenotoxic promotional pathway. Our data also indicate that analysis of specific in vivo mutational responses with transgenic animal models can provide crucial information for understanding the molecular mechanisms underlying chemical carcinogenesis.

Low dose genotoxicity of 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) in gpt delta transgenic mice

Although humans are chronically exposed to most environmental chemicals at low doses, genotoxicity assays with rodents are usually performed at high doses with short treatment period. To investigate the dose-response of genotoxicity at lower doses, gpt delta transgenic mice were fed a diet containing 300, 30 or 3 parts per million (ppm) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) for 12 weeks and the gpt mutations in the liver were analyzed. In addition, the mice were continuously fed a diet containing MeIQx at a dose of 300 ppm for 78 weeks to examine the effect of a long-term treatment. In the mice treated for 12 weeks, the gpt mutant frequencies (MFs) were 8.6-, 2.3- and 1.2-fold higher than the control level at the doses of 300, 30 and 3 ppm, respectively. G:C to T:A transversion was the most predominant type of mutations and the fold increases in the specific MF of G:C to T:A were 58.2, 4.4 and 1.7 above the control at the three doses, respectively. The increases in the whole gpt and specific MFs at 3 ppm were not statistically significant. In the mice treated with 300 ppm of MeIQx for 78 weeks, the gpt MF was about 20 times higher than that of the untreated mice fed a control diet for 78 weeks, which was about two times higher than that of the untreated mice at 12 weeks. These results suggest that no obvious genotoxic effects can be detectable at the dose of MeIQx at 3 ppm in the liver and a longer treatment substantially enhances the genotoxicity. Factors constituting the practical threshold dose are discussed.

UVB-induced gpt mutations in the skin of gpt delta transgenic mice.

Ultraviolet light B (UVB)‐induced mutagenesis was studied in gpt delta transgenic mice, which contain the λEG10 shuttle vector as a transgene. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m2. At 4 weeks after irradiation, the mutant frequencies (MF) of the gpt gene were determined in the epidermis and the dermis, and the gpt mutations in the epidermis were identified by DNA sequencing. The epidermis exhibited a higher sensitivity to UVB than the dermis at doses of 0.3 and 0.5 kJ/m2 UVB: the MF of the epidermis were more than nine times higher than those of the nonirradiated mice, whereas the MF of the dermis were only two to three times higher than the nonirradiated level at the doses used. The UVB‐induced mutation spectrum in the epidermis was dominated by G:C to A:T transitions at dipyrimidine sites, such as 5′‐TC‐3′, 5′‐CC‐3′, and 5′‐T/C‐CG‐3′. Tandem transitions such as CC to TT were also observed. Interestingly, a remarkable bias towards the template strand of the gpt gene was observed in the single transitions at 5′‐TC‐3′ and 5′‐CC‐3′ sites, but not at 5′‐T/C‐CG‐3′ site. In contrast, G:C to A:T transitions at CpG sites and deletions were observed in nonirradiated mice. Hot spots of transitions were observed at different sites in UVB‐irradiated and nonirradiated mice. These results indicate that gpt delta transgenic mouse is a suitable model for studying in vivo UVB‐induced mutations at the molecular level. Environ. Mol. Mutagen. 34:72–79, 1999

Integration of In Vivo Genotoxicity and Short-term Carcinogenicity Assays Using F344 gpt Delta Transgenic Rats: In Vivo Mutagenicity of 2,4-Diaminotoluene and 2,6-Diaminotoluene Structural Isomers

An important trend in current toxicology is the replacement, reduction, and refinement of the use of experimental animals (the 3R principle). We propose a model in which in vivo genotoxicity and short-term carcinogenicity assays are integrated with F344 gpt delta transgenic rats. Using this model, the genotoxicity of chemicals can be identified in target organs using a shuttle vector λ EG10 that carries reporter genes for mutations; short-term carcinogenicity is determined by the formation of glutathione S-transferase placenta form (GST-P) foci in the liver. To begin validating this system, we examined the genotoxicity and hepatotoxicity of structural isomers of 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Although both compounds are genotoxic in the Ames/Salmonella assay, only 2,4-DAT induces tumors in rat livers. Male F344 gpt delta rats were fed diet containing 2,4-DAT at doses of 125, 250, or 500 ppm for 13 weeks or 2,6-DAT at a dose of 500 ppm for the same period. The mutation frequencies of base substitutions, mainly at G:C base pairs, were significantly increased in the livers of 2,4-DAT–treated rats at all three doses. In contrast, virtually no induction of genotoxicity was identified in the kidneys of 2,4-DAT–treated rats or in the livers of 2,6-DAT–treated rats. GST-P–positive foci were detected in the livers of rats treated with 2,4-DAT at a dose of 500 ppm but not in those treated with 2,6-DAT. Integrated genotoxicity and short-term carcinogenicity assays may be useful for early identifying genotoxic and nongenotoxic carcinogens in a reduced number of experimental animals.