Heavy‐ion‐induced mutations in the gpt delta transgenic mouse: Effect of p53 gene knockout

JOURNAL OF HEALTH SCIENCE

2009

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Transgenic rodents are valuable models for investigation of genotoxicity of chemicals in vivo. We have developed gpt delta transgenic mice (C57BL/6J background) and rats (Sprague-Dawley, SD), which have the ability to identify both point mutations by the gpt assay [6-thioguanine (6-TG) selection] and certain types of deletions by the Spi − (Spi, sensitive to P2 interference) assay. Recently, the gpt delta SD rat was backcrossed with the Fisher 344 (F344) rat to establish an gpt delta F344 rat. The average spontaneous gpt mutation frequencies (MFs) are about 4.5 × 10 −6 in both SD and F344 gpt delta rats as well as in gpt delta mice. The G:C to A:T transitions at 5 -CpG-3 sites and G:C to T:A transversions are the predominant spontaneous gpt mutations in rats and mice. However, there is one false mutation (e.g. A:T to T:A at position 299) in the rats. The base substitution may have arisen when the lambda EG10 transgene was introduced into the genome of the SD rat during transgenesis. In the Spi − assay, 1-bp deletions in repetitive sequences are predominantly observed in both mice and rats. Possible mechanisms underlying the spontaneous mutations in gpt delta rodents are discussed.Key words --gpt delta transgenic rodent, spontaneous mutation, mutation spectrum, gpt assay, Spi − assay OVERVIEW OF gpt DELTA TRANSGENIC RODENTSGene mutations play an important role in the etiology of many human diseases including cancer. Since humans are exposed to a variety of endogenous and exogenous mutagens, there has been considerable interest in the relationship between exposure, genotoxic effects, and cancer incidence. To assess the risk of mutagens to the human genome, genotoxicity tests have been developed, including in vivo mutation assays using experimental animals, which play a crucial role in risk assessment. To investigate in vivo genotoxicity, a number of transgenic rodent mutation assays have been developed by introducing reporter transgenes into the chromosome of every cell of the animal. 1, 2) Using these systems, mutagenic events induced in a rodent can * To whom correspondence should be addressed: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Tel.: +81-3-3700-1141 (Ext. 280); Fax: +81-3-3707-6950; E-mail: masumura@nihs.go.jp be detected by recovering the transgene and analyzing the phenotype of the reporter gene in a bacterial host. These models permit quantitation of mutations and identification at the sequence level in any tissue or organ in the body. lacZ, lacI or cII have been employed as reporter genes in transgenic rodents, such as the Muta TM mouse, and the Big Blue R mouse and rat. 3-7) Despite differences in size and sequence context, spontaneous mutation frequencies of these reporter genes are in the mid-10 −5 range and those are predominantly base substitutions in most tissues. This high background of base substitutions may make it difficult to detect rare mutations such as deletions induced by ionizing radiation. 8,9) To...

Section: Spi − Assay (Spi − Selection) For Deletionssupporting

confidence: 80%

Section: Overview Of Gpt Delta Transgenic Rodentsmentioning

confidence: 99%

Spontaneous Mutagenesis in Rodents: Spontaneous Gene Mutations Identified by Neutral Reporter Genes in gpt Delta Transgenic Mice and Rats

Masumura

JOURNAL OF HEALTH SCIENCE

2009

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“…3). IR induces DSBs through direct deposition of energy on DNA and through oxidation of DNA Yatagai et al, 2002]. We suggest that UVB and MMC induce lesions that block the progression of the replication fork, thereby inducing DSBs in DNA [Horiguchi et al, 2001;Takeiri et al, 2003].…”

Section: Molecular Nature Of Spi à Mutationsmentioning

confidence: 98%

“…The sequence alterations can be characterized at the molecular level. Here, we summarize the characteristics of deletions and base substitutions induced by ionizing radiation (IR) in liver and spleen Masumura et al, 2002;Yatagai et al, 2002], ultraviolet B (UVB) light in epidermis [Horiguchi et al, 2001], mitomycin C (MMC) in bone marrow [Takeiri et al, 2003], 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in colon, and aminophenylnorharman (APNH) in liver [Masumura et al, 1999a[Masumura et al, , 2003b and discuss the possible mechanisms of mutation induction.…”

Section: Introductionmentioning

confidence: 99%

Molecular nature of intrachromosomal deletions and base substitutions induced by environmental mutagens

Environ and Mol Mutagen

Masumura

2005

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Cellular DNA is exposed to a variety of exogenous and endogenous mutagens. A complete understanding of the importance of different types of DNA damage requires knowledge of the specific molecular alterations induced by different types of agents in specific target tissues in vivo. The gpt delta transgenic mouse model provides the opportunity to characterize tissue-specific DNA alterations because small and large deletions as well as base substitutions can be analyzed. Here, we summarize the characteristics of intrachromosomal deletions and base substitutions induced by ionizing radiation in liver and spleen, ultraviolet B (UVB) radiation in epidermis, mitomycin C (MMC) in bone marrow, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in colon, and aminophenylnorharman (APNH) in liver of gpt delta mice. Carbon-ion radiation, UVB, and MMC induced large deletions of more than 1 kb. About half of the large deletions occurred between short direct-repeat sequences and the remainder had flush ends, suggesting the involvement of nonhomologous end joining of double-stranded breaks (DSBs) in DNA. UV photoproducts and interstrand crosslinks by MMC may block DNA replication, thereby inducing DSBs. In contrast, PhIP and APNH mainly generated 1 bp deletions in runs of guanine bases. As for base substitutions, UVB and MMC induced G:C-->A:T transitions at dipyrimidine sites and tandem base substitutions at GG sites, respectively. PhIP and APNH induced G:C-->T:A transversions. Translesion DNA synthesis across the lesions, i.e., UV photoproducts, intrastrand crosslinks by MMC, and guanine adducts by the heterocyclic amines, may be involved in the induction of base substitutions. These results indicate the importance of sequence information to elucidate the mechanisms underlying deletions and base substitutions induced in vivo by environmental mutagens.

“…The tumor suppressor gene p53 is involved in various aspects of genome stability and is thus referred to as``the guardian of the genome'' (80). Indeed, the p53 defect enhances Spi -mutation frequency in the kidney of mice after g-ray irradiation (81). The enhancement was primarily due to an increase in complex deletions.…”

Section: Establishment Of Gpt Delta Transgenic Mice For In Vivo Genotmentioning

confidence: 99%

Novel DNA Polymerases and Novel Genotoxicity Assays

2007

Genes and Environment

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Human genome is continuously exposed to exogenous and endogenous genotoxic agents. The most hazardous and ubiquitous exogenous mutagen may be cigarette smoke, which contains more than 4,000 chemicals including about 60 known carcinogens. About 1% of oxygen metabolism leads to production of reactive oxygen species, a major source of endogenous mutagens. These genotoxic agents induce a variety of lesions in DNA, which results in mutations and chromosome aberrations upon replication. If such genetic alterations occurred in the genes involved in cell proliferations and/or maintenance of genome stability, the cells would proceed in multi-steps of carcinogenesis. The goal of environmental mutagenesis and genetic toxicology is to elucidate the mechanistic links between exposure to genotoxic agents and the health consequences, and to prevent the health hazard associated with DNA damage. To this end, we have investigated the mechanisms of mutagenesis induced by environmental chemicals and contributed to establish the paradigm that Y-family DNA polymerases play central roles in mutagenesis via translesion DNA synthesis across damaged bases in DNA. We also developed genotoxicity assays with bacteria and mice to evaluate the potential risk of environmental chemicals. Here, I review the roles of Y-family DNA polymerases in mutagenesis and introduce features of the novel bacterial and rodent genotoxicity assays. Future directions of environmental mutagenesis and carcinogenesis are also discussed.