• Toxicological Research
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• v.19 no.4
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• pp.285-291
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• 2003

The use of skin whitening agents has been recently increased in various kinds of cosmetic products, although there were some reports that whitening agents might cause allergic contact dermatitis. A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pigs for contact sensitization potential. This study was carried out to investigate the skin sensitization potential of three whitening agents, arbutin, azelaic acid, and kojic acid, by LLNA using a non-radiois-topic endpoint. Female Balb/c mice were exposed topically to a weak allergen, $\alpha$-hexylcinnamalde-hyde (HCA), and three whitening agents following LLNA protocol. Lymph node (LN) weight and cell proliferation in ears and auricular lymph node using bromodeoxyuridine (BrdU) immunohistochemistry were evaluated. LN weights were significantly increased at the HCA group compared to the vehicle control. A weak allergen, HCA elicited 3-fold or greater increase in cell proliferation of lymph nodes as well as increase in cell proliferation of ear as measured by BrdU immunohistochemistry. However, in the case of skin whitening agent groups, there were no significant changes in LN weight and cell proliferation in the ear and lymph node of mice treated with 5, 10 and 20% of three whitening agents compared to the vehicle control. These results show that these three skin whitening agents may not have contact sensitization potentials at tested concentrations in Balb/c mice by LLNA.

• Toxicological Research
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• v.19 no.2
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• pp.133-139
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• 2003

Allergic contact dermatitis may be caused by a wide variety of chemicals. A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for assessing the contact sensitization potential of chemical. However, there is a need to develop a nonradioisotopic endpoint for the LLNA, because of the radioisotopic method's requiring the use of special facilities. In this study, we investigated the development of a nonradioisotopic endpoint for LLNA using ELISA (enzyme-linked immunosorbent assay). Female Balb/c mice were treated by the topical application on the dorsum of both ears with four different strong sensitizers, 2,4-dinitrochlorobenzene (DNCB), oxazolone (OXZ), toluene diisocyanate (TDI), and trimellitic anhydride (TMA), and a strong irritant, sodium lauryl sulfate (SLS), once daily for three consecutive days. The proliferation of cells in the auricular Iymph node was analyzed by means of the labelling index (Ll) of bromodeoxyuridine (BrdU) incorporation into cells. The weights of the Iymph nodes in the mice treated with allergens, DNCB, OXZ, TDl and TMA were increased compared to the vehicle control. The stimulation index (Sl) of mice treated with DNCB, OXZ, TDl, and TMA was over three-fold increase compared to the vehicle control. However, the S1 of mice exposed to SLS was not significantly increased compared to the vehicle control, while the lymph node weight of SLS was significantly increased. These results suggest that the LLNA modified endpoint using ELISA based on BrdU incorporation could provide a useful method of screening for irritants and allergens.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.11b
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• pp.185-185
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• 2002

A murine local lymph node assay (LLNA) has been developed as an alternative test to guinea pig maximization test. The disadvantage of LLNA is the need for the use of radioactive material. In this study, we aimed to investigate the development of non-radio isotopic endpoint for local lymph node assay in Balb/c mice using Enzyme-linked immunosorbent assay (ELISA) based on Bromodeoxyuridine (BrdU) incorporation.(omitted)

• Journal of Oriental Neuropsychiatry
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• v.21 no.2
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• pp.29-44
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• 2010

Objectives : The purpose of this study was to investigate the effect of Gentianae Radix on neurogenesis and apoptosis in ethanol- induced newborn rats hippocampus dentate gyrus. Methods : In vivo, laboratory animals were divided into three groups; Normal group(N), Control group(C) and Treated group (TG)(n=7 for each group). N were treated saline daily for five days. C were treated 1.5 g/kg ethanol and saline daily for five days. TG were treated 1.5 g/kg ethanol and 300 mg/kg Gentianae Radix daily for five days. BrdU(5-bromo-2-deoxyuridine) assay was used to test neurogenesis in the dentate gyrus. And TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was used to test apoptosis in the dentate gyrus. Three groups were measured body weight, serum ethanol concentration, BrdU-positive cells and TUNEL-positive cells in the dentate gyrus. In vitro, MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to test viability in SK-N-MC cells. BrdU assay was used to test neurogenesis in SK-N-MC cells. DNA fragmentation and caspase-3 enzyme activity assay were used to test apoptosis in SK-N-MC cells. And treated ethanol and Gentianae Radix of all in vitro tests were made various concentration. Results : In vivo, Gentianae Radix modulated ethanol-induced neurogenesis and apoptosis in newborn rats hippocampus dentate gyrus. In vitro, TG 100 ${\mu}g/ml$ have significantly modulated ethanol-induced neurogenesis and apoptosis in SK-N-MC cells. And only TG 100 ${\mu}g/ml$ have significantly protected SK-N-MC cells from ethanol-induced cytotoxicity. Conclusions : Gentianae Radix may have the effect that modulated ethanol-induced neurogenesis and apoptosis in SK-N-MC cells.

• Journal of Veterinary Clinics
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• v.14 no.2
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• pp.230-234
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• 1997

In vitro effect of cortisol on the proliferation of canine peripheral blood mononuclear cells (MNC) was examined. The MNC was isolated from peripheral blood by a gradient centrifugation with Picoll-Hypaque. The cell proliferation assayed using a noneradioactive 5-Bromo-2'-deoxy-uridine (BrdU) kit. The MNC proliferated well in response to either phrtobeRagg$]$utinin-p (PHA-P) or culture supernatant from MNC stimulated with PHA-p. However, these proliferative responses of MNC were not affected by addition of coitisol of 1 to 1,OOfl ng/ml. The addition of cortisol in MNC culture with either PHA-P or corture supernatBnt from MNC stimulated with PHA-P far 4 days wag not also influenced on the viabilities of cultured MNC. In conclusions it was able to assay the cell proliferation with BrdU instead of radioactive isotope e.g. tritiated thymidine (3H-TdR). These results suggested that cortisol does not at least influence on MNC proliferation in vitro.

• Korean Journal of Acupuncture
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• v.19 no.1
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• pp.77-84
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• 2002

목적 : 소아의 정신적 및 신체적 발달지연에 사용되고 있는 신기환이 해마의 신경세포 생성에 미치는 영향을 관찰하고자 하였다. 방법 : 신기환이 세포생성에 미치는 영향을 알아보기 위하여 해마 세포인 HiB5에 신기환을 처리, 배양하여 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay를 통해서 세포생성을 관찰하였다. 또한 3주령의 Sprague-Dawley (S-D)계 흰쥐의 중완혈에 신기환 약침을 자침후 해마의 치상회에서 bromodeoxyuridine (BrdU) immunohistochemistry를 시행하여 세포생성을 관찰하였다. 결과 : HiB5 배양세포에서는 신기환을 처리시 배양시간과 농도별로 세포생성율이 증가하였다. 또한, 대조군의 치상회에서 BrdU 양성세포수는 $128.50{\pm}9.53$, 1 mg/kg 신기환 자침군은 $157.08{\pm}10.82$ 및 10 mg/kg 신기환 자침군은 $204.80{\pm}17.68$로 나타났다. 이러한 결과로 신기환은 어린 S-D계 흰쥐의 치상회에서 세포생성을 증가시키는 것으로 사려된다.

• The Journal of Internal Korean Medicine
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• v.29 no.2
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• pp.500-511
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• 2008

The aim of this study was to investigate the inhibitory effect of Saengangeonbitang-gasamchilgn(SGGBTGSCG) on collagen production in rat hepatic stellate cells(HSC) and on the TAA-induced chronic liver injury model in rats. Methods : 1) HSCs were treated with SGGBTGSCG extract powder(50% EtOH SGGBTGSCG, dw SGGBTGSCG). After the treatment, MTT assay, BrdU assay and procollagen assay were done. In addition, gene expressions of collagen type $1{\alpha}2$, ASMA, TIMP1, and TIMP2, all of which are known to be associated with liver fibrosis, were analyzed by RT-PCR. 2) Liver fibrosis was developed in rats by injection of TAA 3 times a week for 5 weeks. After the SGGBTGSCG-treatment, body weight, liver & spleen weight, liver function test, the complete blood cell count and the change of portal pressure were studied. Results : In MTT assay, SGGBTGSCG significantly decreased the viability of HSCs in a dose-dependent manner. In BrdU assay, SGGBTGSCG significantly inhibited the HSC proliferation in a dose-dependant manner. In procollagen assay, SGGBTGSCG decreased procollagen production by HSC. In the change of rats' liver and spleen weight, TAA+SGGBTGSCG groups showed little difference compared with TAA-only group. In the liver function test, SGGBTGSCG decreased the serum level of ALT, AST, and Alp elevated by TAA. In the complete blood cell count, SGGBTGSCG significantly decreased WBC elevated by TAA and increased RBC and Hct lowered by TAA. In the change of portal pressure, SGGBTGSCG decreased portal pressure elevated by TAA. Conclusions : These results suggest that SGGBTGSCG is beneficial in the treatment of cirrhotic patients as well as for patients with chronic hepatitis.

• Journal of Haehwa Medicine
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• v.28 no.2
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• pp.41-47
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• 2019

Objectives : Atopic dermatitis is an irritable skin disease accompanying rash and itching leading to impaired skin barrier. ATO-ALL is an ethanol extract of natural products comprising 12 herbs and effective on atopic dermatitis. In this study, we aimed to propose that the effect of ATO-ALL on skin regeneration in human keratinocyte cell line, HaCaT cells. Methods : To evaluate the skin regenerating effects of ATO-ALL, scratch wound healing assay, bromodeoxyuridine (BrdU) assay, and propidum iodide (PI) assay were performed using cultured HaCaT cell line. Result : Scratch wound healing assay showed that ATO-ALL was able to enhance the gap filling activity more than 2-fold at 7 ppm concentration compared with control group. BrdU assay demonstrated that ATO-ALL treatment increased the de novo cell proliferation in a dose-dependent manner. Finally, PI assay indicated that the cell cycle of HaCaT cells was modulated by ATO-ALL treatment. Conclusions : These results suggested that ATO-ALL may have skin regenerating effects by increasing cell proliferation via cell cycle regulation. Taken together, ATO-ALL is supposed to have a potential on regeneration of damaged skin or functional disease including atopic dermatitis.

• Journal of Veterinary Clinics
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• v.23 no.2
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• pp.153-157
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• 2006

The purpose of this study was to determine the effects of mitomycin C on conjunctival fibroblast proliferation and apoptosis. Rabbit conjunctival fibroblast mono layers were treated with 48 hours application of mitomycin-C. The viability of cells was evauated by MTT assay. To examine the effect of mitomycin-C on the cell proliferation, immunocytochemistry for BrdU staning was performed. Then, we investigated whether mitomycin-C induced cell's apoptosis by TUNEL staining. As a result of MTT assay, the viability of cells was gradually inhibited in a dose dependent manner by mitomycin-C. The BrdU staining showed that mitomycin-C significantly suppressed the proliferation of conjunctival fibroblast at concentrations of 0.02% or more. When TUNEL assays were performed, the number of apoptotic cells increased 5.6-, 18.5-, and 33.8-fold compared with the control at 0.01, 0.02, and 0.04%, respectively, of mitomycin-C at 48 hours of exposure. Therefore, mitomycin-C may be useful as a regulator in the treatment of corneal diseases that manifest with scar formation and tumor of fibroblast expansion.

• Proceedings of the Korean Society of Toxicology Conference
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• 2005.05a
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• pp.140-140
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• 2005