• 한국환경성돌연변이발암원학회지
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• 제19권1호
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• pp.7-13
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• 1999

The mouse lymphoma thymidine kinase (tk+/-) gene assay (MOLY) using L5178Y tk+/- mouse lymphoma cell line is one of the mammalian forward mutation assays. It is well known that MOLY has many advantages and more sensitive than the other mammalian forward mutation assays such as x-linked hyposanthine phosphoribosyltransferase (hprt) gene assay. The target gene of MOLY is a heterozygous tk+/- gene located in 11 chromosome of L5178Y tk+/- cell, 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. MOLY has relatively short expression time (2-3 days) compared to 1 week of hprt gene assay. MOLY can also induce relatively high mutant frequency so a large number of events can be recorded. The bimodal distribution of colony size which may indicate gene mutation and chromosome breakage potential of chemicals according to mutation scale such as large normal-growing mutants and small slow-growing mutants can be observed in this assay. The statistical analysis of data can be performed using the MUTANT program developed by York Electronic Research in association with Hazelton as recommended by the UKEMS (United Kingdom Environmental Mutagen Society) guidelines. This report reviewed MOLY using the microtiter cloning technique (microwell assay).

• Environmental Analysis Health and Toxicology
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• 제14권1_2호
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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.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 1996년도 제19회정기학술대회(The 19th Symposium of the Korean Society of Environmental Toxicology)
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• pp.66-66
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• 1996

• Molecular & Cellular Toxicology
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• 제3권3호
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• pp.172-176
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• 2007

Butylated hydroxytoluene (BHT) is widely used antioxidant food additives. It has been extensively studied for potential toxicities. BHT appears adverse effects in liver and thyroid. In this study, we evaluated the genetic toxicity of BHT with more advanced methods, in vitro mouse lymphoma assay $tk^{+/-}$ gene assay (MLA) and in vivo mouse supravital micronucleus (MN) assay. BHT did not appear the significantly results in the absence and presence of metabolic activation system with MLA. Also, in vivo testing of BHT yielded negative results with supravital MN assay. These results suggest that BHT itself was not generally considered genotoxic.

• Molecular & Cellular Toxicology
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• 제3권2호
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• pp.113-118
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• 2007

Chlorobenzenes due to their acute toxicity and the capability of bioaccumulating are of great health and environmental concern. Especially, 1, 2-dichlorobenzene (CAS No. 95-50-1) is used for organic synthesis, dye manufacture, as a solvent and for other applications in chemical industry. Adverse effects of 1, 2-dichlorobenzene includes increases in liver and kidney weights and hepatotoxicity. In this study, we evaluated the genetic toxicity of 1, 2-dichlorobenzene with more advanced methods, in vitro mouse lymphoma assay $tk^{+/-}$ gene assay (MLA) and in vitro mouse supravital micronucleus (MN) assay. 1, 2-Dichlorobenzene appeared the significantly positive results and the induction of large mutant colonies only in the presence of metabolic activation system with MLA. But in vitro testing of 1, 2-dichlorobenzene yielded negative results with supravital MN assay. These results suggest that 1, 2-dichlorobenzene may play a mutagen rather than clastogen in vitro mammalian system.

• Molecular & Cellular Toxicology
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• 제2권3호
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• pp.176-184
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• 2006

The controversy on genotoxicity of molinate, an herbicide, has been reported in bacterial system, and in vitro and in vivo mammalian systems. To clarify the genotoxicity of molinate, we performed bacterial gene mutation test, in vitro chromosome aberration and mouse lymphoma $tk^{+/-}$ gene assay, and in vivo micronucleus assay using bone marrow cells and peripheral reticulocytes of mice. In bacterial gene mutation assay, no mutagenicity of molinate ($12-185{\mu}g/plate$) was observed in Salmonella typhimurium TA 98, 100, 1535 and 1537 both in the absence and in the presence of S-9 metabolic activation system. The clastogenicity of molinate was observed in the presence ($102.1-408.2\;{\mu}g/mL$) of metabolic activation system in mammalian cell system using Chinese hamster lung fibroblast. However, no clastogenicity was observed in the absence ($13.6-54.3\;{\mu}g/mL$) of metabolic activation system. It is suggested that the genotoxicity of molinate was derived some metabolites by metabolic activation. Molinate was also subjected to mouse lymphoma L5178Y $tk^{+/-}$ cells using microtiter cloning technique. In the absence of S-9 mixture, mutation frequencies (MFs) were revealed $1.4-1.9{\times}10^{-4}$ with no statistical significance. However, MFs in the presence of metabolic activation system revealed $3.2-3.4{\times}10^{-4}$ with statistical significance (p<0.05). In vivo micronucleus (MN) assay using mouse bone marrow cells, molinate revealed genotoxic potential in the dose ranges of 100-398 mg/kg of molinate when administered orally. Molinate also subjected to acridine orange MN assay with mouse peripheral reticulocytes. The frequency of micronucleated reticulocytes (MNRETs) induced 48 hr after i.p. injection at a single dose of 91, 182 and 363 mg/kg of molinate was dose-dependently increased as $10.2{\pm}4.7,\;14.6{\pm}3.9\;and\;28.6{\pm}6.3\;(mean{\pm}SD\;of\;MNRETs/2,000\;reticulocytes)$ with statistical significance (p<0.05), respectively. Consequently, genotoxic potential of molinate was observed in in vitro mammalian mutagenicity systems only in the presence of metabolic activation system and in vivo MN assay using both bone marrow cells and peripheral reticulocytes in the dose ranges used in this experiment. These results suggest that metabolic activation plays a critical role to express the genotoxicity of molinate in in vitro and in vivo mammalian system.

• 약학회지
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• 제43권5호
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• pp.614-622
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• 1999

The standard operation procedure of mouse lymphoma L5178Y $tk^{+/-}-3.7.2C$ gene mutation assay (MOLY) has been regarded as a sensitive in vitro mammalian cell gene mutation assay that is capable of detecting clastogens as well as mutagens. Using MOLY, one of natural sweetner, stevioside (5mg/ml) and its aglycon, steviol ($340{\;}\mu\textrm{g}/ml$) were evaluated the mutagenicity. Stevioside and steviol did not induce mutagenicity in MOLY. On the other hand, stevioside (250mg/kg, B.W.) and steviol (200mg/kg, B.W.) were also evaluated their ability to induce micronuclei in regenerating hepatocytes and bone marrow cells of ddY mice. From these results, stevioside and steviol did not induce any mutagenic effect both MOLY and in vivo micronucleus test.

• Molecular & Cellular Toxicology
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• 제3권1호
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• pp.53-59
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• 2007

This study was conducted to evaluate the mutagenic potential of dimethyl isophthalate (DMIP) using Ames bacterial reverse mutation test, chromosomal aberration test and mouse lymphoma $tk^{+/-}$ gene assay. As results, in Ames bacterial reversion assay, DMIP was tested up to the concentration of 5,000 ${\mu}g$/plate and did not induce mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and Escherichia coli WP2uvrA with or without metabolic activation (S9 mix). Using cytotoxicity test, the maximal doses of DMIP for chromosomal aberration assay were determined at 1,250 ${\mu}g/mL$, which was a minimum precipitation concentration ($IC_{50}>1,940\;{\mu}g/mL$ or 10 mM) and at 155 ${\mu}g/mL$ ($IC_{50}:155\;{\mu}g/mL$) in the presence and the absence, respectively, of S9 mix. DMIP in the presence of S9 mix induced statistically significant (P<0.001) increases in the number of cells with chromosome aberrations at the dose levels of over 250 ${\mu}g/mL$, when compared with the negative control. However, DMIP in the absence of S9 mix did not caused significant induction in chromosomal aberrant cells. In MLA, DMIP at the dose range of 242.5-1,940 ${\mu}g/mL$ in the presence of S9 mix induced statistically significant increases in mutation frequencies related to small colony growth, whereas any significant mutation frequency was not observed in absence of S9 mix. From these results, it is conclusively suggested that dimethyl isophthalate may be a clastogen rather than a point mutagen.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2004년도 춘계학술대회
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• pp.82-82
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• 2004

• Molecular & Cellular Toxicology
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• 제3권1호
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• pp.23-30
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• 2007

The effects of high energy short wave solar radiation on human skin have received much publicity as the major cause of accelerated skin ageing and of skin cancers. To meet public demand, the cosmetic industry has developed sun protection factor products, which contain a variety of so-called "UV filters", among others benzophenone (BP) and its metabolites are the widely used UV filters. UV filters are also used to prevent UV light from damaging scents and colors in a variety of cosmetics products and to protect of plastic products against light-induced degradation. There are many variants of BP in use. In this respect, to regulate and to evaluate the hazardous effect of BP-type UV filters will be important to environment and human health. The genotoxicity of 7 BP-type UV filters was evaluated in L5178Y $(tk^{+/-})$ mouse lymphoma cells in vitro. BP, benzhydrol, 4-hydroxybenzophenone 2-hydroxy-4-methoxybenzophenone and 2, 4-dihydroxybenzophenone did not induce significant mutation frequencies both in the presence and absence of metabolic activation system. 2, 2'-Dihydroxy-4-methoxybenzophenone appeared the positive results at the highest dose, i.e. 120.4 ${\mu}g/mL$ only in the absence of metabolic activation system. And also, 2, 3, 4-trihydroxybenzophenone revealed a significant increase of mutation frequencies in the range of 138.1-207.2 ${\mu}g/mL$ in the absence of metabolic activation system and 118.3-354.8 ${\mu}g/mL$ in the presence of metabolic activation system. Through the results of MLA with 7 BP-type UV filters in L5178Y cells in vitro, we may provide the important clues on the genotoxic potentials of these BP-type UV filters.