• Toxicological Research
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• v.16 no.1
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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.

• Toxicological Research
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• v.33 no.2
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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.

• Korean Journal of Food Science and Technology
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• v.32 no.4
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• pp.964-969
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• 2000

Di-(2-ethylhexyl) adipate(DEHA) has been used extensively as a plasticizer in the manufacture of plastic products such as PVC films. Though, phthalate esters plasticizers have been known to induce endocrine system-mediated responses, few studies have been conducted for the screening of estrogenic activity of DEHA, an adipate plasticizer. This study was initiated to evaluate the estrogenic activity of DEHA by in vitro E-screen assay and in vivo uterotrophic assay. MCF-7 human breast cancer cells were treated with $DEHA(5{\times}10^{-9}{\sim}5{\times}10^{-4}\;M)$, for 144 hr, and cell proliferation was determined by sulforhodamine B(SRB) assay. DEHA dissolved in corn oil was administered subcutaneously to ovariectomized(OVX) female Sprague-Dawley rats at dosage levels of 0, 2, 20 and 200 mg/kg/day for three consecutive days. Rats were sacrificed 24 hr after final treatment and vagina and uterus(wet and blotted) weights were obtained. E-screen assayed DEHA did not generate cell proliferation at treated concentrations$(5{\times}10^{-9}{\sim}5{\times}10^{-4}\;M)$, whereas 17 ${\beta}-estradiol$(E2), the positive control, induced cell proliferation at low concentrations$(5{\times}10^{-14}{\sim}5{\times}10^{-9}\;M)$. In the uterotrophic assay, DEHA did not change vagina and uterus(wet and blotted) weights at dosage levels up to 200 mg/kg/day treatment. These results demonstrated that DEHA did not exhibit the estrogenic activity as determined by in vitro E-screen assay and in vivo uterotrophic assay.

• Weed & Turfgrass Science
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• v.4 no.2
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• pp.118-123
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• 2015

Metagenomics is a powerful tool to isolate novel biocatalyst and biomolecules directly from the environmental DNA libraries. Since the metagenomics approach bypasses cultivation of microorganisms, un-cultured microorganisms that are majority of exists can be the richest reservoir for natural products discovery. To discover novel herbicidal substances from soil metagenome, we established three easy, simple and effective high throughput screening methods such as cucumber cotyledon leaf disc assay, microalgae assay and seed germination assay. Employing the methods, we isolated two active single clones (9-G1 and 9-G12) expressing herbicidal activity which whitened leaf discs, inhibited growth of microalgae and inhibited root growth of germinated Arabidopsis seeds. Spraying butanol fraction of the isolated active clones' culture broth led to growth retardation or desiccation of Digitalia sanguinalis (L) Scop. in vivo. These results represent that the screening methods established in this study are useful to screen herbicidal substances from metagenome libraries. Further identifying molecular structure of the herbicidal active substances and analyzing gene clusters encoding synthesis systems for the active substances are in progress.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.05a
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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)

• Food Science of Animal Resources
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• v.35 no.1
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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.

• Journal of Acupuncture Research
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• v.24 no.5
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• pp.23-32
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• 2007

Objectives : Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, and angiogenic factors and matrix protein interactions modulate this process. The aim of this study was to determine whether herbs medicine(KHBJs) could induce angiogenic activity in human umbilical vein endothelial cells(HUVECs). Methods : The angiogenic activity of KHBJs were evaluated by proliferation using BrdU assay, chemotactic migration assay, tube formation assay, and measurement of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor(VEGF) in HUVECs. Also, In order to identify enhance angiogenic activity by activity guided fractionation, the angiogenic activity of fractions of KHBJs such as KHBJB or KHBJR were evaluated in vitro and in vivo Matrigel plug angiogenesis asaay. Results : About 9 KHBJs significantly increased HUVECs proliferation in a dose-dependent manner. In addition, 9 herbs medicine(KHBJs) increased migration and tube-like formation in HUVECs. Interestingly the expression of bFGF and VEGF, an angiogenesis-inducing growth factor, were dose-dependently increased by KHBJs. However, angiogenic activity of fractionated KHBJs(KHBJB or KHBJR) not enhanced more than KHBJs in HUVECs and Matrigel plug in vivo angiogenesis assay. Conclusions : 9 KHBJs significantly induces angiogenesis in in vitro and in vivo. These results suggest that 9 KHBJs potent angiogenic agents and promising drug for the induction of neovascularization.

• Toxicological Research
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• v.20 no.3
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• pp.225-232
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• 2004

The suitability of the single cell gel electrophoresis (SCGE) assay as a test for the monitoring of genotoxicity of aquatic environment was evaluated. The SCGE assay was employed to detect DNA damage induced in clam (Spisula sachalinensis) exposed to a direct mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or an indirect mutagen, benzo[a]pyrene (B[a]P). The cells of gill and digestive glands were isolated from clam by homogenization, which was the optimized cell dissociation method, and the level of DNA damage was assessed and expressed as mean tail length. In the gill cells, significant dose- and time-dependent increase was observed in the mean tail length at the concentration from 0.01 to 0.5 ppm MNNG for 96 h. The linear correlation between relative dam-age index (RDI) values was suggested to provide criteria of genotoxicity monitoring for direct acting mutagen. The dose- and time-dependent responses of the digestive glands cells were less sensitive than those of the gill cells. In contrast, the genotoxic response resulting from the exposure of 0.01～1.0 ppm B[a]P to clam revealed a higher sensitivity in the digestive glands cells than the gill cells. The comparison between the time profiles of genotoxic responses in clam and carp, the latter had been obtained in our previous study, indicated that the metabolism of genotoxic compounds in the two aquatic organisms were quite different each other. We conclude that the SCGE assay has the potential as a screening test for routine genotoxicity monitoring of aquatic organisms because of its higher sensitivity and simplicity.

• Food Science of Animal Resources
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• v.33 no.4
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• pp.522-530
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• 2013

The ability of probiotics to adhere to the intestinal epithelium likely plays an important role in their colonization of the gastrointestinal tract. Here, we performed high-throughput screening (HTS) for suitable characteristics of potential probiotic bacteria using attachment and colonization ability through a C. elegans surrogate in vivo model. A total of 100 strains of lactic acid bacteria (LAB) isolated from infant feces were subjected to the colonization assay using C. elegans intestine. Based on colonization ability, we showed that nine isolates have a high attachment ability during whole experimental periods (up to 168 h), compared to Lactobacillus rhamnosus strain GG as a control. Also, through the use of an in vitro cell attachment model, nine isolates revealed highly binding activity to the mucus layer. Next, the selected 9 isolates were assayed for their survival ability when exposed to acidic and bile conditions as well as cholesterol reduction and the utilization of prebiotic substrates. As a result, the isolated nine strains were determined to be highly resistant to acid and bile conditions. In addition, they have significant activity for the reduction of cholesterol and utilization of several prebiotic substrates as a carbon source. Finally, the selected nine strains were identified by either L. rhamnosus or L. plantarum (4 strains for L. rhamnosus and 5 strains for L. plantarum, respectively). Taken together, we propose that the direct colonization of probiotics using C. elegans may be applicable to the rapid screening of valuable probiotic strains in vivo.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2001.05a
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• pp.126-126
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• 2001

Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wild range of environmental and industrial chemicals. It is clear that in vivo methods will be required to identify adverse effects produced by these chemicals. (omitted)