• 대한수의학회지
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• 제38권3호
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• pp.589-594
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• 1998

The in vitro screening tests against the in vitro cultivated $L_3$ of Ascaris suum (in vitro $L_3$), which were cultivated from the embryonated egg to third-stage larva on 7 days in culture(DIC) and the in vivo rat's lung-derived $L_3$ of Ascaris suum (in vivo $L_3$), which were recovered from the lungs of rat on 7 days after infection, carried out in order to compare the anthelmintic efficacy of in vitro $L_3$ and that of in vivo $L_3$ in RPMI medium 1640 with 5% bovine calf serum. And also a screening test of efficacy against adult worms of Trichuris suis performed. The efficacies of screening tests were as follows : 1. The screening efficacies of abamectin and ivermectin against the in vitro $L_3$ were all 100% at the 10ppm concentration in RPMI medium 1640 on 5 DIC. 2. The screening efficacies of abamectin and ivermectin against the in vivo $L_3$ were all 100% at the 20ppm on 5 DIC or at 40ppm on 3 DIC. 3. The screening efficacies of abamectin and ivermectin against the adult worms of Trichuris suis were all 100% at 20ppm on 4 DIC. And therefore, the in vitro cultivated $L_3$ of Ascaris suum were used in the screening test as well as the in vivo rat's lung-derived $L_3$ of Ascaris suum. And also the adult worms such as Trichuris suis and filaroids which is small size and difficult to cultivate to vitro, were used in the screening test in vitro.

• 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.

• Toxicological Research
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• 제35권4호
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• pp.343-351
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• 2019

Many in vitro developmental toxicity assays have been proposed over several decades. Since the late 1980s, we have made intermittent attempts to introduce in vitro assays as screening tests for developmental toxicity of inhouse candidate products. Two cell-based assays which were developed two decades apart were intensively studied. One was an assay of inhibitory effects on mouse ascites tumor cell attachment to a concanavalin A-coated plastic sheet surface (MOT assay), which we studied in the early days of assay development. The other was an assay of inhibitory effects on the differentiation of mouse embryonic stem cell to beating heart cells (EST assay), which we assessed more recently. We evaluated the suitability of the assays for screening in-house candidates. The concordance rates with in vivo developmental toxicity were at the 60% level. The EST assay classified chemicals that inhibited cell proliferation as embryo-toxic. Both assays had a significant false positive rate. The assays were generally considered unsuitable for screening the developmental toxicity of our candidate compounds. Recent test systems adopt advanced technologies. Despite such evolution of materials and methods, the concordance rates of the EST and MOT systems were similar. This may suggest that the fundamental predictivity of in vitro developmental toxicity assays has remained basically unchanged for decades. To improve their predictivity, in vitro developmental toxicity assays should be strictly based on elucidated pathogenetic mechanisms of developmental toxicity.

• 대한화장품학회지
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• 제30권1호
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• pp.129-133
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• 2004

In vitro method는 in vivo results를 예측하기 위해 사용되어지는 것이 가장 큰 목적이므로 지급까지 in vitro SPF test는 여러 formulations를 screen 하거나 self-tanners의 activity에 미치는 cosmetic ingredients의 영향을 연구하는 데에 이용되어져 왔다. In vitro SPF test는 신속하고 객관적이며 적은 비용으로 사람에게 in vivo test를 하기에 앞서 protective formulas를 pre-screen 하며, 따라서 in vitro test가 유용하게 원하는 역할을 하기 위해서는 in vitro SPF 평가법의 정확성이 무엇보다 중요하다. 본 연구에서는 건조시간을 15분으로 고정하면서 기존에 사용해온 substrate인 Transpore$^{(R)}$ tape을 이용, 도포 방법을 개선하기 위한 시도를 하였다. 우선 기존 시험법의 분석을 통한 현 수준을 파악하고, 사용되고 있는 Transpore$^{(R)}$ tape의 외측으로부터 일정 부위만 사용하도록 개선하였다. 또한 다양한 시도를 통해 광원의 scan 부위에만 국소적으로 도포하는 방법이 도포시 발생하는 오차를 줄일 수 있음을 확인하였으며, 개선된 시험법을 이용하여 반복성과 선형성이 뛰어난 시험 결과를 얻어낼 수 있었다. 통계 패키지 분석을 통한 시험법의 신뢰성 검토에서도 우수한 결과를 보여 이와 같은 시험법을 통해 in vivo와 in vitro SPF의 보다 정확한 예측 시스템 관계를 구축할 수 있을 것으로 기대한다.

• Toxicological Research
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• 제33권2호
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• pp.107-118
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• 2017

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

• Environmental Analysis Health and Toxicology
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• 제11권3_4호
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• pp.65-68
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• 1996

Selaginellae Herba has been used as folk medicine for antineoplastics, coagulants, antidotes and invigorants. To find an in vitro screening method for liver protective effect of Selaginellae Herba in benzo(a)pyrene intoxicated injury were examined in primary cultured rat hepatocytes. Using MTT assay, herba concentration showed dose dependently viability. The lowest concentration of benzo(a)pyrene giving cytotoxicity revealed around 50gM. The hepatoprotective effect of Selaginellae Herba in both water and chloroform extracts was also increased dose dependently.

• 대한화장품학회지
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• 제29권2호
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• pp.37-65
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• 2003

새로운 주름개선 효능물질을 개발하기 위하여 여러 가지 세포배양 모델을 이용한 in vitro 효능물질의 검색방법을 이용하여 후보물질을 도출하고 있다. 이처럼 In vitro에서 선별된 주름개선 후보물질은 사람피부에 직접 도포 하였을 때 in vitro에서 와 는 달리 주름개선 효능이 없는 경우가 많이 있다. 즉 in vitro 검색결과와 사람피부에서의 효능은 현실적으로 큰 차이를 보인다는 것이다. 그러나 모든 효능 후보물질을 사람 피부에 직접 검사할 수는 없는 현실적 어려움이 있다. 이러한 애로점을 해결하기 위하여, 동물모델이나, 인공피부배양모델 등을 이용하는 추세이다. 이번 강의에서, 본인은 동물모델과 in vitro에서 효능 측정방법을 상세하게 리뷰 하고 각 방법에 대해 토의할 것이며, 임상 전 시험 결과를 보고 할 것이다. 그 중 특히 동물 모델은 주름개선 효능물질의 효능을 평가하는데 좋은 대상이 될 것이다.

• 한국축산식품학회지
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• 제35권1호
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• pp.91-100
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• 2015

The present study was conducted to screen candidate probiotic strains for anti-inflammatory activity. Initially, a nitric oxide (NO) assay was used to test selected candidate probiotic strains for anti-inflammatory activity in cultures of the murine macrophage cell line, RAW 264.7. Then, the in vitro probiotic properties of the strains, including bile tolerance, acid resistance, and growth in skim milk media, were investigated. We also performed an in vitro hydrophobicity test and an intestinal adhesion assay using Caenorhabditis elegans as a surrogate in vivo model. From our screening, we obtained 4 probiotic candidate lactic acid bacteria (LAB) strains based on their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell cultures and the results of the in vitro and in vivo probiotic property assessments. Molecular characterization using 16S rDNA sequencing analysis identified the 4 LAB strains as Lactobacillus plantarum. The selected L. plantarum strains (CAU1054, CAU1055, CAU1064, and CAU1106) were found to possess desirable in vitro and in vivo probiotic properties, and these strains are good candidates for further investigations in animal models and human clinical studies to elucidate the mechanisms underlying their anti-inflammatory activities.

• 한국환경보건학회지
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• 제15권1호
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• pp.1-9
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• 1989

환경인자가 인체의 발암에 크게 기여하며, 대기오염도 흡연인자와 더불어 폐암발생의 주요인자로 주목되고 있다. 발암성 시험은 mouse, rat 등의 소동물을 이용하는 in vivo 시험법이 원칙이나, 시간과 경비가 많이 소요되므로, in vitro 생물시험법이 현재 발암물질의 screening 수법으로 많이 이용되고 있다. 특히 B.N.Ames에 의해 개발된 Salmonella균 변이주를 이용하는 방법은, 간편성, 신속성에 뛰어나고, 정성적 또는 정량적으로 발암성과 매우 높은 상관관계를 보이고 있어 최근 10여년간에 걸쳐, 수많은 화학물질 또는 대기오염물질과 같은 복합오염물질의 변이원성 및 발암성의 screening test로서 널리 응용되어 왔다.

• Journal of Pharmaceutical Investigation
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• 제30권3호
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• pp.191-199
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• 2000

Genomics is providing targets faster than we can validate them and combinatorial chemistry is providing new chemical entities faster than we can screen them. Historically, the drug discovery cascade has been established as a sequential process initiated with a potency screening against a selected biological target. In this sequential process, pharmacokinetics was often regarded as a low-throughput activity. Typically, limited pharmacokinetics studies would be conducted prior to acceptance of a compound for safety evaluation and, as a result, compounds often failed to reach a clinical testing due to unfavorable pharmacokinetic characteristics. A new paradigm in drug discovery has emerged in which the entire sample collection is rapidly screened using robotized high-throughput assays at the outset of the program. Higher-throughput pharmacokinetics (HTPK) is being achieved through introduction of new techniques, including automation for sample preparation and new experimental approaches. A number of in vitro and in vivo methods are being developed for the HTPK. In vitro studies, in which many cell lines are used to screen absorption and metabolism, are generally faster than in vivo screening, and, in this sense, in vitro screening is often considered as a real HTPK. Despite the elegance of the in vitro models, however, in vivo screenings are always essential for the final confirmation. Among these in vivo methods, cassette dosing technique, is believed the methods that is applicable in the screening of pharmacokinetics of many compounds at a time. The widespread use of liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) allowed the feasibility of the cassette dosing technique. Another approach to increase the throughput of in vivo screening of pharmacokinetics is to reduce the number of sample analysis. Two common approaches are used for this purpose. First, samples from identical study designs but that contain different drug candidate can be pooled to produce single set of samples, thus, reducing sample to be analyzed. Second, for a single test compound, serial plasma samples can be pooled to produce a single composite sample for analysis. In this review, we validated the issue whether the second method can be applied to practical screening of in vivo pharmacokinetics using data from seven of our previous bioequivalence studies. For a given drug, equally spaced serial plasma samples were pooled to achieve a 'Pooled Concentration' for the drug. An area under the plasma drug concentration-time curve (AUC) was then calculated theoretically using the pooled concentration and the predicted AUC value was statistically compared with the traditionally calculated AUC value. The comparison revealed that the sample pooling method generated reasonably accurate AUC values when compared with those obtained by the traditional approach. It is especially noteworthy that the accuracy was obtained by the analysis of only one sample instead of analyses of a number of samples that necessitates a significant man-power and time. Thus, we propose the sample pooling method as an alternative to in vivo pharmacokinetic approach in the selection potential lead(s) from combinatorial libraries.