• Environmental Analysis Health and Toxicology
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• v.14 no.1_2
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• pp.1-12
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• 1999

Recently, several new methods for the detection of genetic damages in vitro and in vivo based on molecular biological techniques were introduced according to the rapid progress in toxicology combined with cellular and molecular biology. Among these methods, mouse lymphoma thymidine kanase (tk) gene forward mutation assay, single cell gel electrophoresis (comet assay) and transgenic animal and cell line model as a target gene of lac I (Big Blue) and lac Z (Muta Mouse) gene mutation are newly introduced based on molecular toxicological approaches. The mouse lymphoma tk$\^$+/-/ gene assay (MOLY) using L5178Y tk$\^$+/-/ mouse lymphoma cell line is one of the mammalian forward mutation assays, and has many advantages and more sensitive than hprt assay. The target gene of MOLY is a heterozygous tk$\^$+/-/ gene located in 11 chromosome, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. The comet assay is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakages in mammalian cells, Also, transgenic animal and cell line models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease process, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Also in vivo acridine orange supravital staining micronucleus assay by using mouse peripheral reticulocytes was introduced as an alternative of bone marrow micronucleus assay. In this respect, there was an International workshop on genotoxicity procedure (IWGTP) supported by OECD and EMS (Environmental Mutagen Society) at Washington D. C. in March 25-26, 1999. The objective of IWGTP is to harmonize the testing procedures internationally, and to extend to finalization of OECD guideline, and to the agreement of new guidelines under the International Conference of Harmonization (ICH) for these methods mentioned above. Therefore, we introduce and review the principle, detailed procedure, and application of MOLY, comet assay, transgenic mutagenesis assay and supravital staining micronucleus assay.

• Environmental Mutagens and Carcinogens
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• v.18 no.1
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• pp.15-21
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• 1998

Transgenic animal and cell line models which are recently developed and used in toxicology fields combined with molecular biological technique, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Transgenic models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease processes. The lac I and lac Z gnee most widely used as a mutational target in transgenic systems. The assay is performed by treatment with putative mutagenic agents, isolation of genomic DNA from cells or tissues, exposure the isolated DNA to in vitro packaging extract, plating and sequencing. The results from these processes provide not only mutant frequency as quantitative evaluation but also mutational spectrum as qualitative evaluation of various agents. Therefore we introduce and review the principle, detailed procedure and application of transgenic mutagenesis assay system in toxicology fields especially in mutagenesis and carcinogenesis.

• Environmental Mutagens and Carcinogens
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• v.22 no.1
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• pp.1-11
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• 2002

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 1996.12a
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• pp.64-64
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• 1996

Transgenic animal and cell line models which are recently developed in toxicology field combined with molecular biological technique, are powerful tools for studying of mutation in vivo and in vitro, respectively. The Big Blue mutagenesis assay system is one of the most widely used transgenic systems. Especially, for the study of direct acting mutagens, Big Blue cell line is very useful and powerful to evaluate mutagenicity because the mutation frequency and mutationspectrlun showed no distinct differences between cell line and animal. The Big Blue cell lines carry stably integrated copies of lambda shuttle vector containing lac I gene as a mutational target. These lambda shuttle vectors are useful for various mutagenesis related studies in eukaryotic system due to their ability to be exposed mutagen and then transfer a suitable target DNA sequence to it convenient organism for analysis. We tried to assess the mutagenic effect of 4-NQO with Big Blue cell line. After the treatment of 4-NQO, genomic DNA was isolated and lambda shuttle vector was packaged by in Vitro packaging and then these were plated on bacterial host in the presence of X-gal to screen mutation in the lac I. We determined MF as a ratio of blue plaques versus colorless plaques and now undergoing the mutation spectrum of 4-NQO in lac J gene sequence.

• Proceedings of the Korean Society of Toxicology Conference
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• 2003.10b
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• pp.109-110
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• 2003

To validate a novel mouse model, gpt delta, for in vivo mutagenesis, the Mammalian Mutagenesis Society (MMS), a subgroup of the Environmental Mutagen Society of Japan (JEMS) (JEMS/MMS), performed a collaborative study as the third trial for transgenic animal assay. In this mouse model, point mutations and deletions re separately identified by gpt (6-thioguanine-resistant) and Spi- (sensitive to P2 interference) selections, respectively.(omitted)

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.71-71
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• 2002

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. ${\beta}$-casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of ${\beta}$-casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. ${\beta}$-casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of ${\beta}$-casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine ${\beta}$-casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2003.10a
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• pp.109-110
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• 2003

• Toxicological Research
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• v.34 no.4
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• pp.281-290
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• 2018

Exposure to chemical agents is an inevitable consequence of modern society; some of these agents are hazardous to human health. The effects of chemical carcinogens are of great concern in many countries, and international organizations, such as the World Health Organization, have established guidelines for the regulation of these chemicals. Carcinogens are currently categorized into two classes, genotoxic and non-genotoxic carcinogens, which are subject to different regulatory policies. Genotoxic carcinogens are chemicals that exert carcinogenicity via the induction of mutations. Owing to their DNA interaction properties, there is thought to be no safe exposure threshold or dose. Genotoxic carcinogens are regulated under the assumption that they pose a cancer risk for humans, even at very low doses. In contrast, non-genotoxic carcinogens, which induce cancer through mechanisms other than mutations, such as hormonal effects, cytotoxicity, cell proliferation, or epigenetic changes, are thought to have a safe exposure threshold or dose; thus, their use in society is permitted unless the exposure or intake level would exceed the threshold. Genotoxicity assays are an important method to distinguish the two classes of carcinogens. However, some carcinogens have negative results in in vitro bacterial mutation assays, but yield positive results in the in vivo transgenic rodent gene mutation assay. Non-DNA damage, such as spindle poison or topoisomerase inhibition, often leads to positive results in cytogenetic genotoxicity assays such as the chromosome aberration assay or the micronucleus assay. Therefore, mechanistic considerations of tumor induction, based on the results of the genotoxicity assays, are necessary to distinguish genotoxic and non-genotoxic carcinogens. In this review, the concept of threshold of toxicological concern is introduced and the potential risk from multiple exposures to low doses of genotoxic carcinogens is also discussed.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.10a
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• pp.14-14
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• 2010

Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed two different kinds of transgenic potato plants (Solanumtuberosum L. cv. Taedong Valley) overexpressing strawberry GalUR and mouse GLoase gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the these genes in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid(AsA) levels in transgenic tubers were determined by high-performance liquid chromatography(HPLC). The over-expression of these genes resulted in 2-4 folds increase in AsA intransgenic potato and the levels of AsA were positively correlated with increased geneactivity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen(MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of these gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. We have also investigated the mechanism of the abiotic stress tolerance upon enhancing the level of the ascorbate in transgenic potato. The transgenic potato plants overexpressing GalUR gene with enhanced accumulation of ascorbate were investigated to analyze the antioxidants activity of enzymes involved in the ascorbate-glutathione cycle and their tolerance mechanism against different abiotic stresses under invitro conditions. Transformed potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed significant increase in the activities of superoxide dismutase(SOD, EC 1.15.1.1), catalase, enzymes of ascorbate-glutathione cycle enzymes such as ascorbate peroxidase(APX, EC 1.11.1.11), dehydroascorbate reductase(DHAR, EC 1.8.5.1), and glutathione reductase(GR, EC 1.8.1.7) as well as the levels of ascorbate, GSH and proline when compared to the untransformed tubers. The increased enzyme activities correlated with their mRNA transcript accumulation in the stressed transgenic tubers. Pronounced differences in redox status were also observed in stressed transgenic potato tubers that showed more tolerance to abiotic stresses when compared to untransformed tubers. From the present study, it is evident that improved to lerance against abiotic stresses in transgenic tubers is due to the increased activity of enzymes involved in the antioxidant system together with enhanced ascorbate accumulated in transformed tubers when compared to untransformed tubers. At moment we also investigating the role of enhanced reduced glutathione level for the maintenance of the methylglyoxal level as it is evident that methylglyoxal is a potent cytotoxic compound produced under the abiotic stress and the maintenance of the methylglyoxal level is important to survive the plant under stress conditions.

• Toxicological Research
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• v.17
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• pp.293-297
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• 2001

The international pharmaceutical and regulatory communities had been recognizing the limited utility of conventional rodent carcinogenicity study particularly on the second species, mouse, after intense investigation of carcinogenicity data base worldwide, and a new scheme for carcinogenicity testing for pharmaceuticals was proposed at the Expert Working Group on Safety in the International Conference on Harmonization (ICH) in 1996. CB6F 1-Tg rasH2 mouse carrying human prototype c-Ha-ras gene with its own promoter/enhancer is one oj the new carcinogenicity assay model for human cancer risk assessment. Studies have been conducted since 1992 to validate the transgenic (Tg) mice for rapid carcinogenicity test-ing, short term (26 weeks) studies with genotoxic (by Salmonella), non-genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic non-carcinogens revealed relatively high concordance oj the response of the Tg mouse with classical bioassay across classes of carcinogenic agents. Mechanistic basis for carcinogensis in the model are being elucidated in terms of the role of overexpression and/or point mutation of the transgene. This report review the initial studies of validation of the model and preliminary results of on-going ILSI HESI ACT project will be presented.