• Molecular & Cellular Toxicology
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• v.3 no.3
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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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• v.2 no.2
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• pp.106-113
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• 2006

Phthalate analogues are a plasticizer and solvent used in industry. Phthalates were classified in the category of "suspected" endocrine disruptors. The purpose of our study was to screen and elucidate the endocrine disrupting activity of seven phthalate analogues. E-screen assay was performed in MCF-7 human breast cancer cells with seven phthalate analogues. In this cell proliferation assay, benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) showed high estrogenic activity. Their relative proliferation efficiencies (RPE) were 109 and 106%, respectively. In vitro estrogen receptor (ER) binding assay, BBP, di-n-octyl phthalate (DOP) and dinonyl phthalate (DNP) showed weak relative binding affinity (RBA: 0.02%) compared to $17{\beta}-estradiol\;(E2)$ (RBA: 100%). In uterotrophic assay, E2 produced a significant increase, whereas four tested phthalate analogues had potential estrogenic effects in vitro did not increased in uterus weight in immature rats. From these results, we demonstrated that phthalate analogues exhibit weak estrogenic activity in vitro assays at high concentrations. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce a uterus weight increase in vivo. From these, we may suggest that these phthalate analogues are easily metabolized to inactive forms in vivo. Further investigation in other in vitro and in vivo experimental systems might be required.

• Ocean and Polar Research
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• v.33 no.2
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• pp.127-133
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• 2011

The comet assay, also called single-cell electrophoresis (SCGE) assay, is a potential sensitive monitoring tool for DNA damage in cells. The primary objective of this study was to use comet assay to ascertain if the blood cells of flounder (Pleuronichthys olivaceus) and muscle cells of clam (Saxidomus purpurata) are suitable for genotoxicity screening. This was achieved by initially exposing blood and muscle cells under in vitro conditions to the reference genotoxin hydrogen peroxide ($H_2O_2$); strong correlation between $H_2O_2$ concentration and comet values were found. Subsequently, the identification of DNA damage in isolated cells from flounder and clam was performed under in vivo exposure to benzo(a)pyrene (BaP) and tributyltin (TBT). Flounder and clam were exposed to different concentrations (1, 10, 50, 100 ${\mu}g/L$) of BaP or TBT for 4 days. Regardless of treated chemicals, blood cells of flounder were more prone to DNA breakage compared to muscle cells of clam. In conclusion, in vivo genotoxicity of BaP and TBT can be biomonitored using the comet assay. This study suggests that flounder and clam do show potential as mediums for monitoring genotoxic damage by comet assay.

• KSBB Journal
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• v.18 no.4
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• pp.255-260
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• 2003

In vivo bioassays for biological medicines have been considered final resort to unequivocally assess the biological activities for them because there are some cases in which the biological activities obtained from in vivo bioassay and in vitro bioassay quite differ each other. The in vivo biological activity of EPO depends on its sialic acid contents which confer microheterogeneity-isoforms to this protein. We have devise a method which consists of a in vitro bioassay using BaF3 cell line and a capillary zone electrophoresis (CZE) for the measurement of the EPO isoform distribution. The biological activity of EPO obtained using in vitro bioassay with BaF3 cell line showed good correlation (C.V.(％) 7.34, 5.85, 8,16, 8.08, 8.8) to EPO content measured either spectrophotometric assay (A280 0.1 ％ ＝0.743) or radio immunoassay. The assay validation results of in vitro bioassay with 3 lot of in house EPO showed good results to EPO content measured either in vivo assay or radio immunoassay. and also showed good results the robustness of our method in terms of precision, accuracy, repeatability. The isoform distribution for EPO-BRP (1 : 1 mixture of epoetin-${\alpha}$ and epoetin-${\beta}$, European Pharmacopoeia) by CZE method resulted in isoform 2 through isoform 8. The major peaks in electrophoregram were composed of isoform 3 through 7. Our recombinant EPO (epoetin-${\alpha}$) having equivalent in vivo biological activity showed the isoform distribution of isoform 3 through 9. The major peaks consisted of isoform 4 through 8. The peak area of isoform 4 was always smaller than that of isoform 5. The preparations of recombinant epoetin-${\alpha}$ with lower in vivo biological activity than EPO-BRP showed the isoform 2 through 8 in their electrophoregrams whose major peaks consisted of the isoform 3 through 7. The peak area of isoform 4 was larger than that of isoform 5.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.9
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• pp.1486-1491
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• 2004

The estrogenicities of six heavy metal compounds, which contaminate frequently in foods, were assayed using a combination of in vitro and in vivo assays. The assays were 1) estrogen receptor dependent transcriptional expression assay, 2) E-screen assay and, 3) the uterotropic assay in mice. The chemicals studied were 17$\beta$ -estradiol, diethylstilbestrol (DES), arsenic oxide, bis(tri-n-butyltin), cadmium chloride, chromium chloride, lead acetate, and mercuric chloride. Using the estrogen receptor dependent transcriptional expression assay, the following estrogenicity ranking was measured: bis(tri-n-butyltin) > cadmium chloride > chromium chloride >> mercuric chloride >lead acetate = arsenic oxide. Using E-screen test, the following estrogenicity ranking was measured: bis(tri-n-butyltin) > cadmium chloride > chromium chloride >> mercuric chloride > lead acetate = arsenic oxide. Results from the uterotropic assay showed that bis(tri-n-butyltin), cadmium chloride, chromium chloride caused an increase in uterine wet weight, while lead acetate, mercuric chloride, and arsenic oxide failed to do so. Bis(tri-n-butyltin), cadmium chloride and chromium chloride showed the highest estrogenicity in three assay systems. Recent studies suggesting that bis(tri-n-butyltin), cadmium chloride have estrogenicities are compatible with the present finding. Furthermore, our study is suggesting that chromium chloride may be estrogenic. The results demonstrate that this three level-assay combination (transcriptional activation, cell proliferation, and an in vivo effect in an estrogen-responsive tissue) could serve as a useful method to assess the estrogenicity of heavy metals.

• Journal of Life Science
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• v.10 no.1
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• pp.52-60
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• 2000

Up to now, there have been done much efforts in regard to nitrate reductase activity (NRA) of dicotyledonous herbs and important crop monocotyledons, but few to wild plants having canopy structure such as Carex. The objective of the present study are to determine: a) the optimum in vivo NR assay conditions for leaf samples of Carex species, b) changes of NRA according to section within leaf and leaf ages, c) diurnal variations. Optimized assay media of each Carex species were determined. NRA of C. rostrata adapted to oligotrophic habitats is readily saturated at lower substrate concentration than those of C. distans and C. gracilis, adapted to meso- and eutrophic habitats, respectively. All Carex species investigated have higher NRA in leaves than in roots. NRA of all species showed maximal values at the middle section of each leaf and in the youngest fully expanded leaves. Compared to C. gracilis, NR in leaves of C. distans was adapted readily to the light period. On the whole, Carex showed rather delayed diurnal variation. Even if the in vivo nitrate reductase assay based on nitrite estimation does not give an accurate estimation of total nitrate reduced, it still serves as a useful tool to find out relative differences in varying environmental conditions. Additionally, in vivo RNA measurements are helpful to understand nitrate reduction and basic nitrogen metabolism of Carex species having different canopy structure.

• Toxicological Research
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• v.16 no.2
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• pp.109-116
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• 2000

The purposes of our study were to optimize the conditions of the screening and testing methods for endocrine disruptors, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the sensitivity between these assays currently undergoing validation. Two in vitro test systems, MCF-7 cells proliferation (E-screen assay) and competitive binding to estrogen receptors (ER) were selected to evaluate the estrogenic effects. 17$\beta$-Estradiol (E2) and diethylstilbestrol (DES) were used as a positive control in vitro test. Also, E2 and ethinyl estradiol (EE) were used as a positive control in vivo uterotrophic assay. In in vitro test, E2 and DES showed a strong estrogenic response at concentration of 1.0 nM. In uterotrophic assay, E2 (0.3 $\mu\textrm{g}$/kg) and EE (0.3 $\mu\textrm{g}$/kg) produced a significant increase in uterus and vagina weight in both immature and ovariectomized rats. Although we did not com-pared the specificity between in vivo and in vitro assays, these assay systems may serve as a good tool for endocrine disruptors screening methods. Our data indicate that these assay systems exhibit some difference in their sensitivity to the same estrogenic compounds. Therefore, as a first rapid screening assay for estrogenic activity qf unknown chemicals, at least two assay systems should probably be carried out with a view of high sensitivity and standardization conditions. Also, a careful validation tests are necessary to obtain a reasonable degree of reproducibility.

• Radiation Oncology Journal
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• v.26 no.3
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• pp.166-172
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• 2008

Purpose: The measurement of radiosensitivity of individuals is useful in radiation therapy. Unfortunately, the measurement of radiation survival using a clonogenic assay, which is the established standard, can be difficult and time consuming. The aim of this study is to compare radiosensitivity results obtained from the MTT and clonogenic assays, and to evaluate whether the MTT assay can be used on clinical specimens. Materials and Methods: HCT-8, LoVo, CT-26, and WiDr were the colon cancer cell lines used for this study. The clonogenic assay was performed to obtain the cell survival curves and surviving fractions at a dose of 2 Gy ($SF_2$) as the standard technique for radiosensitivity. Also, the MTT assay was performed for each of the cell lines (in vitro). To simulate clinical specimens, the cell lines were inoculated into nude mice, removed when the tumors reached 1 cm in diameter, and chopped. Next, the tumors were subjected to the same process involved with the MTT assay in vitro. The inhibition rates (IR) of 10 Gy or 20 Gy of irradiation for in vitro and ex vivo were calculated based on the optical density of the MTT assay, respectively. Results: According to $SF_2$ and the cell survival curve, the HCT-8 and WiDr cell lines were more resistant to radiation than LoVo and CT-26 (p<0.05). The IR was measured by in vitro. The MTT assay IR was 17.3%, 21%, 30% and 56.5% for the WiDr, HCT-8, LoVo and CT-26 cell lines, respectively. In addition, the IR measured ex vivo by the MTT assay was 23.5%, 26%, 38% and 53% in the HCT-8, WiDr, LoVo and CT-26 tumors, respectively. Conclusion: The radiosensitivity measured by the MTT assay was correlated with the measures obtained from the clonogenic assay. This result highlights the possibility that the MTT assay could be used in clinical specimens for individual radiosensitivity assays.

• Toxicological Research
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• v.16 no.1
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• pp.77-82
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• 2000

This study was conducted to assess a possible alternative method as replacements for in vivo test. The human fibroblasts were exposed to several photoxic chemicals (promethazine, neutral red, chlortetracyclone, amiodatone, bithional, 8-methyooxypsorale) and non-phototoxi substance, ammonium laureth sulfate and irradiatied with 5 J/$cm^2$ of UVA (3320~420nm). The cell viability was measured by NRU (neutral red uptake) assay. The photoxic potential of test chemicals in the NRU PT (phototoxicity test) was assessed by determining the PIF (photoirritancy Factor) by using a cut-off value of 5. The NRU PT responses of most chemicals showed a close agreement with in vivo response except bithinol. There was a relatively good agreement between in vitro NRU assay and in vivo data. These results suggest that NRU assay using fibroblast could be used to predict the phototoxicity.

• 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.