• 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.

• 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.

• 한국균학회지
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• 제1권1호
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• pp.17-21
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• 1973

Fifty-eight strains of Aspergillus spp. isolated from various grains were examined for the screening of aflatoxins by the method of the Thin-Layer Chromatography (TLC), using the aflatoxin producing strain of Aspergillus flavus ATCC 15517 as a control. The results as follows: 1. Samples of Aspergillus spp. isolated from various grains were extracted with chloroform and chromatographied by the thin-layer chromatography method. 2. Three strains of Aspergillus spp. among the 58 strains can be found that the spots having the same Rf value as control with culture extract of Aspergillus flavus ATCC 15517. 3. The further prove studies of aflatoxins were proceed by the methods of in vivo and in vitro tests. And this methods considered to capable for the use of mass screening among the samples.

• 약학회지
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• 제49권1호
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• pp.68-73
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• 2005

Epi-xanthatin analogs containing hydrophilic substituents such as carboxylic acid, alcohol, morpholine, amino acid, and glucose derivatives were synthesized and their in vitro cytotoxicity and in vivo antitumor activity were evaluated. The target compounds were generally cytotoxic against tumor cell lines of human origin with $ED_{50}$ values of $0.1{\sim}30{\mu}g/ml$, except the highly hydrophilic analog 6 containing aspartic acid. Contrary to the potent cytotoxicity weakly hydrophilic analogs 2 and 8 were not active in vivo, or even toxic to the test animals. As a result, hydrophilic analog of epi-xanthatin did not show in vitro cytotoxicity and hydrophobic analogs did not show in vivo antitumor activity, thus it is presumed that amphiphilic analogs or those with medium hydrophilicity would exhibit the antitumor potency in vivo.

• Toxicological Research
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• 제17권2호
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• pp.139-142
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• 2001

Sexual behavior and reproductivity of male fIsh were studied as an in vivo screening method of endocrine disruptors. Male medaka (Oryzias latipes) were exposed to 17$\beta$-estradiol at nominal concentrations of 2 and 20 $\mu\textrm{g}$/l for 14 days. After exposure of the chemical, sexual behavior between male medaka and normal female which were injected with prostaglandin $F_{2\alpha}$ just before the test, was analysed by using video camera for one hour. Normal control male showed courtship dancing such as following, guarding, dancing and crossing while 17$\beta$-estradiol treated male did not show any type oj courtship dancing. Furthermore, fecundity and fertility were significantly decreased in the treated group. It was suggested that analysis of sexual behavior could be a useful endpoint for the screening of the endocrine disruptors.

• 생약학회지
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• 제28권4호
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• pp.286-292
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• 1997

We studied the screening condition for immune regulatory responsor. We focused on the T-lymphocytes leer this purpose. Mouse fetal thymic organ culture (FTOC) system and flow cytometric analysis were mainly used in this experiment. Even if FTOC is carried out in vivo condition, the pattern of thymic development in the condition of FTOC is similar to that of in vivo condition. In this regard, FTOC system might be very powerful tool to screen the immune regulator, especially concerning on T cells. To establish the optimum condition of FTOC to screen the Immune regulator, we focused on the optimum amount of dose and culture period. The cell number and surface antigens on T cells were also analysed by using hemacytometer and flow cytometer. To monitor the differentiation event, anti-CD3, anti-CD4 and anti-CD8 antibodies were used. Alkoxyglycerol and Phellodendri Cortex were used fur positive and negative control, respectively. Astragalus membranceus was used as test sample. From our analysis, we reached to conclusions that the best dose of extract is $50\;{\mu}g/ml$ of culture medium, the best culture period is for 9 days, and ethanol used as solvent has no toxicity to FTOC.

• 대한화장품학회지
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• 제22권2호
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• pp.201-210
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• 1996

Predictive tests for the identification of contact sensitizing chemicals have been developed. We measured the sensitization potential with three predictive tests, the in vitro and the in vivo Local Lymph Node Assay(LLNA), ELISA to detect interferon-gamma(IFN-${\gamma}$) from supernatant and flow cytometry to detect change of cell surface proteins, using draining lymph nodes of mice. BALB/c mice were exposed to various chemicals or vehicles on the ears daily for 3 consecutive days in all experiments. With some exceptions of propyl paraben, neomycin sulfate, the in vivo LLNA was able to detect the sensitizing capacity of test chemicals and was more sensitive than the in vitro LLNA for chemicals used in the present study. In another experiment, contact sensitivity was assessed by the ELISA to detect IFN-Υ from the supernatants of the cultured LNCs after sensitization with chemicals. There was a good correlation between the LLNA and the IFN-Υ production for test chemicals. We also examined the change of cell surface proteins on LNCs after sensitization by flow cytometry for some cell adhesion molecules(ICAM-1, E-cadherine, B7 molecule), T cell markers(CD3, CD4, CD8, T$\alpha$$\beta$,T${\gamma}$$\delta$) and B cell markers(LR1, CD45R, I-Ad). The number of ICAM-1 positive cells and B cells in LNCs were increased after sensitization with DNCB, TNCB, isoeugenol and 25%, 50% cinnamic aldehyde compared with that of vehicle as a control. In conclusion, the in vivo LLNA could provide more sensitive screening test for moderate to strong sensitizers and some weak sensitizers including cosmetic raw materials than the in vitro LLNA. The production of IFN-Υ by allergen-activated LNCs might be a values indicators without radioisotopes for the identification of contact allergens. Detection of allergens by testing the increase of ICAM-1 positive cells and B cells in LNCs by flow cytometry might be used as a test method to detect allergens.

• 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.

• Toxicological Research
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• 제16권1호
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• pp.33-37
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• 2000

Hershberger assay is known as one of the in vivo-short-term scrrning assays for endocrine disrupting chemicals (EDCs), but this method is not a validated test system. In the present study, the establishment of Hershberger assay to detect EDCs was tried using a model substance, di(n-butyl)phthalate (DBP), a plasticizer for plastics. Thirty-six immature male rats were randomly assigned to six groups: DBP 0, 40, 200, and 1000mg/kg, a positive control (flutamide 20 mg/kg), and a combination group(DBP 1000mg/kg and testosterone 50 ug/kg). DBP and flutamide were administered by gavage to male rats from day 21 to 40 post partum. Testosterone was subcutaneously injected during the same period. We evaluated body weigth gain, weights of ventral prostate, seminal vesicle, and levator ani and bulvocavernous muscle, and serum concentrations of testosterone and lutenizing hormone in male rats. The weights of seminal vesicle and levator ani and bulvocavernous muscle of males receiving 1000mg/kg of DBP was significantly lower than controls. There was no effect of DBP-treatment on body weight gain, prostate weight, and hormone concentrations. In the positive control group, the weights of seminal vesicle and levator ani and bulvocavernous muscle of males receiving 20mg/kg of flutamide were significantly lower than controls. In the combination group, there was no effect of co-treatment of DBP and testosterone on all parameters effect against DBP. This method was found to be a useful short-term screening assay system for EDCs.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2001년도 International Symposium on Signal transduction in Toxicology
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• pp.117-117
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• 2001

The aim of this study was to assess a possible alternative method as replacement for in vivo phototoxicity test. The 3T3 mouse fibroblast neutral red uptake phototoxicity assay (3T3 NRU PT assay) is a screening method for studying DNA or cellular damage. Photohemolysis assay is a mechanistic study for investigating oxygen-dependent membrane damage.(omitted)