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.

흰쥐의 뇨 중 3,5,6-Trichloro-2-Pyridinol 분석을 통한 Chloropyrifos 노출량 예측

김아름누리(Areumnuri Kim),백민경(Min-Kyoung Paik),이선영(Sun-Young Lee),홍수명(Su-Myeong Hong),정미혜(Mihye Jeong),오진아(Jin-A Oh) 한국농약과학회 2019 농약과학회지 Vol.23 No.4

Chlorpyrifos (CPF) is one of the organophosphates (OPs) pesticides, and has been widely studied for pesticide exposure by agricultural operator due to its high chemical penetration rate. However, studies to predict the exposure of acute CPF through the analysis of its metabolites using the biomonitoring for agricultural operators are insufficient. In this study, we examined the urinary excretion of 3,5,6-Trichloro2-Pyridinol (TCP) following the repeated exposure of CPF through the skin of rats and confirmed the correlation between CPF exposure and urinary TCP excretion, and finally predicted the CPF exposure by urinary TCP excretion in rats. TCP excretion was analyzed in urine samples of rats exposed through the skin for three times at low (1.01 ㎎/㎏), medium (10.05 ㎎/㎏) and high concentrations (100.50 ㎎/㎏) every 7 days. As the number of exposures increased, urinary TCP excretion decreased significantly, and the decrease in TCP excretion in urine increased with increasing exposure concentration. The correlation between dermal exposure of CPF and their excretion of TCP in urine samples were high, ranging from 0.67 to 0.86. In addition, the absorbed CPF ratio was 4.20~18.81% in rats of low-CPF exposed levels, which was selected as the realistic pesticide exposure level for the agricultural field in Korea. It would be considered to be a basic data for predicting acute pesticide exposure of agricultural operators through TCP analysis using biological samples for farmers in agricultural field.

Deltamethrin의 경피반복노출에 따른 흰쥐의 3-PBA 배설과의 상관관계

김아름누리(Areumnuri Kim),전경미(Kyongmi Chon),박병준(Byung-Jun Park),문병철(Byeong-Chul Moon),김병석(Byung-Seok Kim),백민경(Min-Kyoung Paik) 한국농약과학회 2018 농약과학회지 Vol.22 No.4

Deltamethrin, one of the Pyrethroids (PYRs) pesticides, has been widely used in agriculture and industry. The study on human metabolism and excretion of deltamethrin is needed since low level of pesticide is exposed repeatedly in agricultural fields. This study is aimed to analyse urinary 3-Phenoxybenzoic acid (3-PBA) in male rats following three times of dermal exposure to deltamethrin with interval of 7 days; low (0.0705 mg/kg bw), medium (0.7050 mg/kg bw) and high (7.0500 mg/kg) level of exposure, and then investigate the time courses of urinary 3-PBA and dermal deltamethrin exposure. As a result, the urinary 3-PBA excretion in both concentration and amount were eliminated with increasing dermal exposures in all levels of deltamethrin exposure groups. The low level of deltamethrin groups showed especially significant decrease in urinary 3-PBA. Likely, the more exposure level of deltamethrin, the more urinary 3-PBA excretion showed high correlation between the repeated dermal exposure to deltamethrin and urinary 3-PBA excretion. This result is similar with the results when the number of repetition increases. Base on the results, accumulation of deltamethrin in agricultural workers who generally exposure to low levels of chronic pesticide may be negligible in body. We suggested that urinary 3-PBA excretion may be acute biomarker available to evaluate dermal exposure to deltamethrin.

뒤영벌(Bombus spp.)에 대한 OECD 급성 접촉 및 섭식독성시험법의 국내 확립

김보선(Boseon Kim),김아름누리(Areumnuri Kim),전경미(Kyongmi Chon),이환(Hwan Lee),박연기(Yeon-Ki Park),유아선(Are-Sun You),윤형주(Hyung Joo Yoon) 한국농약과학회 2020 농약과학회지 Vol.24 No.1

Bumblebees are buzz pollinators and have been increasingly used in greenhouse tomato crops. Risk assessment for bees in EU includes bumble bees and solitary bees in addition to honeybees and new test methodologies for bees have been developed. Recently, OECD has published guidelines for bumblebee acute contact (TG 246) and oral toxicity (TG 247). The objective of this study was to establish and validate bumblebee acute toxicity tests using dimethoate for the reference chemical. The mortality of dimethoate 4 μg/ bee treatment was greater than 90% after 24 hr oral exposure. A droplet of 2 μL and 5 μL solution containing dimethoate for each treatment (10, 12, and 15 μg /bumblebee) was applied to the throax of each bumblebee. The mortality of each treatment group with a droplet of 2 μL and 5 μL was greater than 70% after 48 h exposure. There were no difference in mortality between 2 μL and 5 μL of droplets. The acute contact and oral toxicity tests were validated since the mortality of toxic reference group was greater than 50% at the end of the test period. In the near future, this test method could be used to assess the effect of various pesticides on bumblebees

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.

유세포분석기를 이용한 농약의 in vitro 소핵생성 평가

문지홍(Ji-Hong Moon),김아름누리(Areumnuri Kim),전경미(Kyongmi Chon),박경훈(Kyung-Hun Park),문병철(Byeong-Chul Moon),김병석(Byung-Seok Kim),백민경(Min-Kyoung Paik) 한국농약과학회 2019 농약과학회지 Vol.23 No.4

Pesticides have been recognized as one of major concern to human and environmental health. This study is aimed to evaluate whether flow cytometry analysis is a suitable method for micronuclei test, one of genotoxicity test of pesticides on human. We evaluated the number of micronuclei in the positive control (mitomycin C, colchicine) treated groups in Chinese hamster lung cells using flow cytometry. As a results, micronucleus rate in positive control were 2.4~4.2 much higher than in negative control group. These results indicate that flow cytometry method is a quicker and more efficient method for obtaining similar results to the conventional microscopic observation method. Also, pendimethalin (PM) and mancozeb (MZ), which are reported as pesticides causing genotoxicity, were treated with CHL cells, and the occurrence of micronuclei was confirmed by flow cytometry analysis. The occurrence of micronuclei in PM and MZ treated group was significantly increased with a dose-dependent manner, compared with negative control group. These results suggest that micronuclei assay using flow-cytometry analysis could be efficient high-throughput method for quantifying the formation of micronuclei.

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.

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.

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

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.