• 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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• 제2권2호
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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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• 제33권2호
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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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• 제18권4호
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• pp.255-260
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• 2003

현재는 재조합 Erythropoietin의 생물학적 활성을 mouse를 이용한 in vivo bioassay로 실시하고 있으나, 이 방법의 여러가서 불편함으로 인하여, 이를 대체하기 위해 Ba/F3 세포주를 이용한 in vitro assay 방법을 확립하였으나, 위에 상술한 바와 같이 in vitro assay는 erythropoietin의 당분포에 의한 차이를 보이지 않게 때문에, 이를 보완하기 위해서는 in vivo bio-activity와 정량적인 상관관계가 있는 당단백질의 isoform 분석법인 Capillary zone electrophoresis (CZE) 와 sialic acid 함량 결과를 동시에 분석해야 했다. 위의 결과 sialic acid 함량은 erythropoietin 원액에서 10 mol/mol,EPO 이상의 sialic acid 함량을 가져야 되며, CZE 결과는 재조합 erythropoietin이 isoform 분포가 isoform 3의 경우 5.594～0.593％, isoform 4의 경우 31.598～11.704％, isoform 5의 경우 37.033～29.301％, isoform 6의 경우 27.837～18.807％, isoform 7의 경우 17.085～7.824％, isoform 8의 경우 7.642～1.964％을 보여줌과 동시에 isoform 4의 면적은 isoform 5의 면적보다 항상 작아야한다. 이상 두 가지 시험결과와 in vitro assay 결과를 combine해서 in vivo assay를 대체할 수 있다는 좋은 실험적 data를 얻었으므로 이상 위의 3가지 분석법을 활용한 combined in vitro bioassay 법의 기초를 확립하였다.

• 한국식품영양과학회지
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• 제33권9호
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• pp.1486-1491
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• 2004

식품오염 관련 중금속들의 에스트로겐성을 in vitro 와 in vivo 분석방법을 병행하여 평가하였다. 분석방법은 1) estrogen receptor dependent transcriptional expression 분석법, 2) E-screen assay 그리고, 3) 마우스 자궁비대시험 (uterotropic assay)을 사용하였다. 시험에 사용한 물질로는 $17\beta$-estradiol, diethylstilbestrol(DES), arsenic oxide, bis (tri-n-butyltin), cadmium chloride, chromium chloride, lead acetate, mercuric chloride을 사용하였다. Estrogen receptor dependent transcriptional expression 분석 결과, bis(tri-nbutyltin) > cadmium chloride > chromium chloride 순으로 에스트로겐성이 크게 나타났으며, mercuric chloride, lead acetate, arsenic oxide는 거의 나타나지 않았다. E-screen test 결과, bis(tri-n-butyltin) > cadmium chloride > chromium chloride 순으로 에스트로겐성이 크게 나타났으며, mercuric chloride, lead acetate, arsenic oxide는 거의 나타나지 않았다. 자궁비대시험 결과도 마찬가지로 bis(tri-nbutyltin), cadmium chloride, chromium chloride은 자궁중량 비대를 크게 초래하였으며, 반면에 mercuric chloride, lead acetate, arsenic oxide는 그러한 효과가 미약하거나 없었다. 세 분석방법 결과 bis(tri-n-butyltin), cadmium chloride, chromium chloride 순으로 에스트로겐성이 크게 나타났다. 이러한 결과는 최근 bis(tri-n-butyltin)과 cadmium chloride이 에스트로겐성이 있다는 다른 연구결과들과 잘 일치하며, 또한 크롬화합물도 에스트로겐성이 있다는 것을 새롭게 제시하고 있다. 본 연구는 세 단계 수준(전사활성화단계, 세포증식작용, in vivo assay)의 분석을 병행함으로써 수많은 중금속의 에스트로겐성을 효과적으로 평가할 수 있다는 것을 제시해주고 있다.

• 생명과학회지
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• 제10권1호
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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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• 제16권2호
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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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• 제26권3호
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• pp.166-172
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• 2008

목 적: 암환자의 방사선 치료 전 방사선에 대한 감수성을 미리 측정할 수 있다면 임상적으로 많은 도움이 될 것이다. 본 연구는 전 임상 실험을 통하여 MTT assay가 세포집락 측정기법과 비교해서 방사선 감수성을 예측할 수 있고, 직장암 환자의 조직에 사용할 수 있는지 가능성을 확인하고자 하였다. 대상 및 방법: 대장암 세포 주인 HCT-8, LoVo, CT-26, WiDr을 이용하여 세포집락 측정기법을 통해 세포생존곡선 및 2 Gy에서의 세포생존확률(SF2)을 구하였다. 세포 주 자체를 대상으로 MTT assay를 시행하는 실험(in vitro) 및 환자의 암 조직과 같은 상태를 만들기 위하여, 누드 마우스에 세포 주를 주입하여 암 조직을 형성한 후 in vitro와 같은 방식으로 MTT assay를 시행(ex vivo)하였다. 이 두 실험에 대한 흡광도 값에 따른 저해율(inhibition rate, %)을 구하였다. 결 과: $SF_2$ 및 세포생존곡선에 따르면 CT-26 및 LoVo가 HCT-8, WiDr에 비해 방사선에 민감하였다(p<0.05). In vitro MTT assay 결과 WiDr, HCT-8, LoVo와 CT-26의 방사선 저해율이 각각 17.3%, 21%, 30%, 56.5%를 나타내었다. 또한 ex vivo MTT assay의 저해율은 HCT-8, WiDr, LoVo와 CT-26에서 각각 23.5%, 26%, 38%, 53%를 나타내었다. 통계적인 차이를 감안하였을 때 세포생존곡선을 통해 얻은 방사선 감수성의 결과와 동일한 순서를 가졌다. 결 론: 4개의 세포 주의 방사선의 감수성의 순서가 세포집락 측정기법 및 in vitro와 ex vivo MTT assay 결과에서 거의 일치함을 보였다. 이는 직장암 환자에서 MTT assay를 통해 방사선 감수성을 예측할 수 있는 가능성을 제시하였다.

• Toxicological Research
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• 제16권1호
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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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• 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.