Development of bioassay system for evaluation of materials for personal protective equipment (PPE) against toxic effects of ionizing radiations.

Health effects caused by ionizing radiations raise considerable concern among general public and radiation workers. To estimate ability of personal protective equipment (PPE) materials that reduce toxic effects of ionizing radiations, we developed an experimental bioassay system using Chinese Hamster V79 cells. The system developed here distinguished the biological effectiveness of X-ray that was significantly affected by elements composed of shielding materials. Survival of the cells exposed to sub-lethal dose of X-ray was enhanced more than 2 times when the X-ray was filtrated by a lead plate. Also filtration of the X-ray with a tungsten plate enhanced the cell survival more than three times. These results suggested the colony assay system developed here was useful for examination of the biological effectiveness of X-ray and the ability of PPE materials reducing the toxic effects caused by ionizing radiations.

Triphala, a formulation of traditional Ayurvedic medicine, shows protective effect against X-radiation in HeLa cells.

Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations in Ayurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblica officinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigated its activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin, both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygen species (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actions against X-radiation and bleomycin because both agents damage DNA through the generation of ROS. These observations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cells cultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of the molecular basis for the beneficial effects of Triphala.

Transgenic medaka fish which mimic the endogenous expression of neuronal kinesin, KIF5A.

Intracellular transport is spatiotemporally controlled by microtubule-dependent motor proteins, including kinesins. In order to elucidate the mechanisms controlling kinesin expression, it is important to analyze their genomic regulatory regions. In this study, we cloned the neuronal tissue-specific kinesin in medaka fish and generated transgenic fish which mimic endogenous neuronal kinesin expression in order to elucidate the mechanisms which regulate kinesin expression. Searches for medaka neuronal orthologues by RT-PCR identified a candidate gene expressed only in neuronal tissues. Using BAC clones, we determined the cDNA sequence and the gene structure of the candidate neuronal kinesin. Evolutionary analysis indicated that the candidate gene encoded medaka KIF5Aa. The endogenous medaka orthologue was found to be expressed only in the nervous system, including the brain and spinal cord, while expression of KIF5Ab was not exclusive to neuronal tissues. Transgenic (Tg) medaka that expressed EGFP under the control of the 6.9 kbp 5' and 1.9kbp 3' flanking regions of the KIF5Aa gene showed characteristic expression throughout the nervous system, including the brain, spinal cord, olfactory pit, eye and cranial nerve. Immunohistological analysis showed that EGFP expression in Tg fish co-localized with expression of HuC/D, a neuronal marker. These results demonstrate that the 6.9 kbp 5' and 1.9 kbp 3' flanking regions of medaka KIF5Aa have neuronal-specific promoter activity mimicking endogenous expression of medaka KIF5Ab. This transgenic fish strain will be useful for further functional analysis of the effects of these regulatory regions on gene expression.

Identification of mouse mutant cells exhibiting the plastic mutant phenotype.

The initial processes involved in radiation carcinogenesis have not been clearly elucidated. We isolated mouse mutant cells exhibiting plasticity in their mutation phenotypes. These mutant cells were originally isolated from an irradiated cell population as 6-thioguanine resistant (6TGR) mutants that were deficient in hypoxanthine phosphoribosyl transferase (Hprt, E.C.2.4.2.8) activity at the frequency of approximately 6.2 x 10(-5). Approximately 10% of 6TGR cells showed plasticity in their mutant phenotypes and reverted to HAT-resistant (HATR), which is Hprt-proficient, wild type phenotype. Eventually we identified the plastic mutants in the un-irradiated wild type cell population as well and found that ionizing irradiation enhanced the frequency of the plastic mutation approximately 24 times. Treatment with 5-aza-cytidine did not affect the plasticity of mutant phenotypes identified in this study, suggesting that DNA methylation was not involved in the plastic changes of the mutant phenotypes. The plastic mutant phenotype identified in our study is a new type of genomic instability induced by ionizing irradiation, and it is likely to be involved in one of the primary changes that occur in the process of radiation carcinogenesis, and may explain one element of carcinogenesis, which is composed of multi-stages.

Identification of mouse mutant cells exhibiting plastic mutant phenotype II; ionizing radiation-induced mutant phenotype plasticity is not dependent on DNA methylation of the hypoxanthine phosphoribosyl transferase gene in mouse FM3A cells.

As we previously reported, we isolated and examined mouse mutant cells exhibiting phenotypic plasticity. Approximately 10% of 6-thioguanine resistant (6TG(R)) cells derived from the irradiated cell population exhibited phenotypic plasticity and reverted to wild type HAT resistance (HAT(R)). Similar mutant cells were also identified in an un-irradiated wild type cell population, but at a lower frequency. Ionizing irradiation enhanced the frequency of the plastic mutation approximately 24 times in our experiments. Treatment with 5-aza-cytidine did not affect phenotypic plasticity. In this study, we further performed detailed molecular analysis of the promoter region of the hypoxanthine phosphoribosyl transferase (Hprt) gene. The analysis revealed that most cytidine residues were not methylated, even in 6TG(R) mutant cells, in which Hprt activity must be down-regulated. These results suggested that DNA methylation was not involved in mutant phenotype plasticity, a new type of genomic instability induced by ionizing radiation. Plasticity in gene regulation may play an important role in radiation carcinogenesis, which is a multiple-stage process.

Loss-of-heterozygosity analysis of 6-thioguanine-resistant mutants induced by radon exposure in mouse FM3A cells.

Radon is an inert gas that can migrate from soils and rocks and accumulate in enclosed areas such as buildings and underground mines. The ubiquitous occurrence of radon in the environment is the primary cause of harmful radiation exposure to the public. To investigate the mutagenic effect of radon, mouse FM3A cells growing on soft agarose plates were exposed to alpha particles disintegrated from radon-222 and daughter elements. Mutation induction at the hypoxanthine phosphoribosyl transferase (Hprt) allele was examined at radon concentrations of 10, 230, 1,100, 6,500, 200,000, 1,000,000, and 10,000,000 Bq/m(3) for an exposure period of 1 week. A typical inverse dose-rate effect was observed in the frequencies of 6-thioguanine-resistant (6TG(R)) mutations, and lower mutation frequencies were exhibited at 230, 1,100, 6,500, and 200,000 Bq/m(3) than at 10, 1,000,000, and 10,000,000 Bq/m(3). Loss-of-heterozygosity (LOH) analysis at the Hprt locus revealed that deletion mutations were dominant at radon concentrations of 230, 1,100, 6,500, and 10,000,000 Bq/m(3), but not at 10, 200,000, and 1,000,000 Bq/m(3). These results suggested that alpha particles released from radon in the normal atmosphere did not exhibit the measured mutagenic effect in mouse FM3A cells, but that increased concentrations of radon led to a significant increase in the mutagenic effect of radon. At 6,500 Bq/m(3), radon exposure induced the least number of 6TG(R) mutants but all had LOH deletion mutations, which is the typically observed type of mutation in radiation carcinogenesis. Our results suggest that certain concentrations of environmental radon may have specific carcinogenic potential, and it should be avoided by proper ventilation wherever possible.

A transgene and its expression profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method.

A particle gun is used in a potential method for introducing foreign genes into fish. In this paper, we report on the stable transmission of a transgene and its expression profile of the F4 generation in the transgenic medaka (Oryzias latipes). We established four transgenic strains, which contained a green fluorescent protein (GFP) gene controlled by a medaka beta-actin promoter, using a particle gun. One more transgenic strain was also generated by microinjection for comparison. In all five strains, the founder was discovered to be mosaic for the transgene. However, from the F1 to F4 generations, transgenes and their expression profiles were stably inherited in the Mendelian manner. The expression profile was common among the five strains regardless of the method for gene transfer: GFP fluorescence became detectable at an early neurula stage. In this stage, the fluorescence was observed ubiquitously in most tissues. As somite developed, GFP fluorescence became intense only in the skeletal muscle and lens but it decreased in other tissues. In adult fish, an intense fluorescence was restricted in the skeletal muscle and lens, while a considerably weak fluorescence was observed in the brain, gill, heart, kidney, spleen, and ovary. From these results, it was concluded that the transgene and its expression profile were stably transmitted to offspring, and thus the particle gun is an effective method for transgenesis in spite of its easiness.

Effect of SCID mutation on the occurrence of mouse Pc-1 (Ms6-hm) germline mutations.

Mouse Pc-1 (Ms6-hm) is a hypervariable minisatellite locus that is unstable during intergenerational transmission. This hyper-instability of Pc-1 is useful for detecting germline mutation using a small number of experimental animals, although its molecular mechanism has not yet been elucidated. We examined the effect of severe combined immune deficiency (SCID) mutation on the spontaneous germline mutation at the Pc-1 locus using the CB17 mouse strain. Our results showed that the frequency of spontaneous germline mutation at Pc-1 in the offspring of wild-type parents was 9.7%. In F1 between SCID male and wild-type female, however, the frequency of germline mutation was drastically increased to 42.3%. When SCID female mice were mated with wild-type male, the frequency of germline mutation in F1 was slightly increased to 13.6%. These results suggest that DNA protein kinase catalytic subunit (DNA-PKcs), deficiency of which causes SCID mutation, plays an important role in the stable transmission of a genome containing hypervariable tandem repeats to progeny in male germ cells.