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The Correlation Between Deltamethrin Exposure and Urinary 3-PBA Concentrations in Rats
김아름누리 ( Areumnuri Kim ),박지현 ( Ji Hyun Park ),전경미 ( Kyongmi Chon ),박경훈 ( Kyung-hun Park ),문병철 ( Byeong-chul Moon ),백민경 ( Min Kyoung Paik ) 한국환경농학회 2016 한국환경농학회 학술대회집 Vol.2016 No.-
Pyrethroids (PYRs) are a widely used insecticide in agriculture and household area.PYRs caninduce neurotoxic effects in mammalsby disrupting voltage-sensitive sodium channelswhen exposed to overdose.In mammals, PYRs such as deltamethrinis metabolized to 3-phenoxybenzoic acid (3-PBA) in liver that is mainly excreted in urine.Urine is highly useful as a testing matrix because it is easily accessible and can be collected noninvasively. Little studies have reported on the PYRs-dosedependent effects on urinary 3-PBA.This study is designed to identify the correlation of deltamethrinamount exposured orally and dermally to rats and its metabolite(3-PBA) excreted in urine.This study is designed to exposure deltamethrin to rats in a dose-dependent manner and identify the correlation between deltamethrin exposure and its metabolite(3-PBA) in urine. Exposure levels consisted of three concentration of deltamethrin; control (0mg/kgbw), low (0.0705 mg/kgbw), medium (0.705 mg/kgbw) and high(7.05 mg/kgbw) dose. Low concentration of deltamethrin was calculated on the basis of Korea predictive operator exposure model (KoPOEM), indicating the field-realistic exposure to pesticides for farmer when mixing and applying pesticides. Single exposure of deltamethrin to Spraque-Dawley rats were conducted through oral and dermal pathway. Dermal exposure persisted for 6 h, and urine specimens were collected for 24h after exposure of both two pathway. The following procedures including hydrolysis, extraction and derivatization were conducted prior to the analysis of urinary 3-PBA. After acidic hydrolysis for the cleavage of conjugates, the analytes3-PBAwas extracted from urine matrix using n-hexane. Then, 3-PBA was derivatized to volatile estersusing N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection was carried out using GC/MS. 2-Phenoxybenzoic acid served as internal standard for the quantification of 3-PBA. The limit of quantificationfor all analyteswas 0.98ng/ml urine.In the low-, medium- and high-dose oral deltamethrin groups, the concentrations of urinary 3-PBA were 44±2, 104±37, 943±65 ng/ml urine, respectively, showing good correlation between oral deltamethrin exposure and urinary 3-PBA in rats. This result was similar with those for dermal administration of deltamethrin. Based on these results, therefore, 3-PBA that is a main metabolite of PYRs may be suitable for urinary biomarker of exposure to PYRs.This study can be used as an index that can be predictedto exposuredosage of deltamethrinin the human from detection of urinary 3-PBA excretion.
HaCaT cells에서 소핵시험을 이용한 positive control 시험
신혜림 ( Shin Hye Rim ),김아름누리 ( Areumnuri Kim ),전경미 ( Kyongmi Chon ),박경훈 ( Kyung-hun Park ),문병철 ( Byeong-chul Moon ),백민경 ( Min Kyoung Paik ) 한국환경농학회 2016 한국환경농학회 학술대회집 Vol.2016 No.-
It has been reported that human keratinocyte cell line (HaCaT cells) are a valuable tool for studying the metabolism of chemicals that penetrate the skin. However, little is known about the genotoxicity and carcinogenicityin HaCaT cellson skin cancer study of carcinogens, such as inorganic arsenic.Therefore, the present studyinvestigated genotoxicity in HaCaT cells by the in vitro micronucleus (vitMN) test. The vitMN test is a method that typically uses cultured human or rodent cells. It provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected. Mitomycin C (MMC; a DNA cross linking agent) and Colchicine (COL; an aneugen) were used as positive controls. One of the most important considerations in the performance of the vitMNtest is ensuring that the cells being scored have completed mitosis during the treatment or the post-treatment incubation period, if one is used. Therefore,following exposure to MMC and COL were harvested after an additional 1.5 to 2.0 times the normal cell cycles. This study was composed of two parts; with cytochalasinB(cyto B) and without cyto B.Positive controls should be used at concentrations expected to give reproducible and detectable increase over negative control which demonstrated the sensitivity of vitMN test. The present study investigated to define the appropriate test concentrations of two positive controlsat significantly increases number of MN in the range of 55±5% cytotoxicity of HaCaT cells. As a result, the appropriate MMC concentrations for short treatment with cyto B and without cyto B were 60 and 10μg/ml, respectively. But 30 μg/ml was selected at COL concentration for short treatment, regardless of cyto B. For continuous treatment, respective 4 and 0.25 μg/ml were as appropriate MMC concentration, and respective 0.2 and 0.1 μg/mlwere as appropriate COL concentrations with cyto B and without cyto B.To the best of our knowledge, this is the first study to establishfor genotoxicity onHaCaT cells in association with carcinogenesis. The present study will provide an important insight into the genotoxicity on HaCaT cells with respect to the genotoxic chemicals.
In vitro comet assay를 이용한 HaCaT 세포의피부 유전독성 예측
이선의 ( Seoneui Lee ),김아름누리 ( Areumnuri Kim ),신혜림 ( Hye Rim Shin ),전경미 ( Kyongmi Chon ),박경훈 ( Kyung-hun Park ),문병철 ( Byeong-chul Moon ),백민경 ( Min Kyoung Paik ) 한국환경농학회 2016 한국환경농학회 학술대회집 Vol.2016 No.-
Skin carcinogen such as inorganic arsenic has been shown to promote skin cancer by stimulating DNA damaging, tumorigenicity, carcinogenicity and cell apoptosis due to play a role in cell cycle control. Methyl methanesulfonate (MMS) is a genotoxic chemical and breaks DNA double-strand, and therefore it is used for a reference substances to induce DNA damage induction. HaCaT cells are immortal keratinocyte cell line from human skin.Genotoxicity in HaCaTcells provides some clues of genotoxic and carcinogeniceffects in skin tissue. The alkaline single cell gel electrophoresis (comet) assay is a sensitive and simple test for the detection of DNA damage at the individual cell level. Thus, in the present study, the comet assay was used to evaluate DNA damage and genotoxicity in HaCaT cells. DNA damage in HaCaT cells was elicited by treatment with MMS as positive control for 3 hrs.Theseresults ofDNA damage in HaCaT cells might reveal the possibility ofpredictingthe potential skin carcinogenicity.It is impossible of genotoxicity tests to detect perfectly with a single test, due to the fact that it is designed to detect on particular characteristic.Other genotoxicity tests such as in vitro micronucleus, Ames and lymphoma assay arerecommended, based on the standard test battery for genotoxicity.
경피를 통한 Deltamethrin의 반복 노출과 흰쥐의 뇨중 3-PBA 배설량과의 상관성 연구
박지현 ( Jihyun Park ),김아름누리 ( Areumnuri Kim ),전경미 ( Kyongmi Chon ),박경훈 ( Kyung-hun Park ),문병철 ( Byeong-chul Moon ),백민경 ( Min Kyoung Paik ) 한국환경농학회 2016 한국환경농학회 학술대회집 Vol.2016 No.-
3-PBA는 대부분의 pyrethroid계 농약에서 공통적으로 대사되기 때문에 pyrethroid계 농약의 생물학적 모니터링 지표로 많이 사용되고 있다. 본 실험에서는 랫드에 deltamethrin을 반복적으로 노출하여 뇨중 대사체인 3-PBA 배설량을 확인하고, deltamethrin과 뇨중 3-PBA의 상관관계를 확인하고자 하였다. Deltamethrin의 노출농도는 실제 농작업자가 농업현장에서 농약의 조제 및 살포과정에서 노출되는 실제 노출수준으로 시험하기 위하여 deltamethrin 1% 유제사용에 따른 KoPOEM 산출량을 저농도로 하여 중농도와 고농도를 설정하였다. 실험은 8주령의 Spraque-Dawley 수컷 흰쥐에 농작업자의 주농약노출경로인 피부노출로 deltamethrin을 처리하였다. 반복노출조건은 deltamethrin 1% 유제의 안전사용지침에 근거하여 1주일 간격으로 3회 노출하였으며, 반복노출에 따른 뇨중 3-PBA 분석을 위해 deltamethrin 노출직후와 최종 노출 1주일후 뇨시료를 수거하였다. 뇨중 3-PBA 분석을 위해 HCl가 수분해를 거쳐 N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA)를 이용한 유도체화 과정을 거친 후 GC/MS로 분석하였다. 시험결과, deltamethrin의 반복노출시 처리농도 증가에 따라 뇨중 3-PBA 배설량은 높은 상관관계를 보이며 증가하는 것으로 나타났다(R²=0.99). 또한 deltamethrin의 반복노출 횟수에 따라 뇨중 3-PBA 배설량은 증가하나 최종 노출 1주일후의 뇨에서는 유의적으로 감소하였다. 본 연구결과는 농약에 반복적으로 노출되는 농작업자의 만성적인 농약노출에 따른 배설량 추이를 살펴보기 위한 자료로 활용될 것이다.
Chinese Hamster Lung Cell을 이용한 in vitro 소핵시험의 세포질 최적화 연구
백민경 ( Min Kyoung Paik ),김아름누리 ( Areumnuri Kim ),신혜림 ( Hye Rim Shin ),전경미 ( Kyongmi Chon ),박경훈 ( Kyung-hun Park ),류지혁 ( Byeong Chul Moon ),문병철 한국환경농학회 2018 한국환경농학회지 Vol.37 No.3
In vitro 소핵시험(vitMNT)은 유전독성의 유망한 대체시험법 중 하나로, OECD에서 TG로 채택되어 화학물질의 등록에 사용되고 있다. 본 시험에서는 CHL cell을 사용한 vitMN test에서 소핵을 판별하기 위한 최적화된 세포질 조건을 찾고자 하였으며, 양성대조물질로 MMC와 Col을 사용하고 세포 염색을 위해 giemza 용액을 사용하였다. 시험결과, 세포현탁을 위해 사용되는 고정액의 acetic acid의 농도는 1%로 하는 것이 band의 두께와 세포질의 퍼짐성 측면에서 적당하였다. 또한 세포의 깨짐을 최소화하는 최종 고정액의 적하시간은 현탁 후 1~4시간이었다. 이러한 결과는 vitMNT에서 소핵관찰의 신속성, 용이성 및 정확성을 확보하는데 도움이 될 것이다. BACKGROUND: in vitro micronucleus test (vitMNT) is one of the promising alternative testing methods in genotoxicity test and was adopted as OECD test guideline for chemical registration. This study was conducted to optimize the cytoplasm conditions in vitMNT using Chinese hamster lung (CHL) cell. METHODS AND RESULTS: In this study cytokinesisblock micronucleus test was conducted. Mitomycin C and colchicine were used as positive control chemicals and were treated for three hours (short time) or twenty-four hours (long time). Giemsa solution was used for cell staining. For optimization of vitMNT, the final fixative was prepared as five concentrations (0%, 1%, 3%, 5%, and 25%) of acetic acid in methanol, and treatment times of the final fixative were varied under four conditions (immediately, one hour, four hours, and one day). CONCLUSION: Acetic acid at 1% in methanol as the final fixative was most adequate to preserve the cytoplasm around the nucleus in the interphase cells. Also, fixative treatment time of cell suspension for one to four hours may minimize the cell rupture. These results can be helpful for getting an accurate result promptly due to clear visual distinction to score micronucleus in vitMNT using giemsa solution.