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Toxicological studies on benzophenone-type UV filters : environmental exposure, toxicokinetic, genotoxic and toxicogenomic assessment

Other Titles
 벤조페논계 자외선차단제에 대한 독성학적 연구 : 환경노출, 독성동태, 유전독성 및 독성유전체학적 평가 
Authors
 전희경 
Issue Date
2007
Description
Dept. of Public Health/박사
Abstract
[한글]벤조페논계 자외선차단제는 주로 방향강화제나 살충제, 농약, 의약품 제조의 원료로 사용되어지며, 플라스틱, 잉크 등의 첨가제로서 사용되고 있다. 특히 2-하이드록시-4-메톡시벤조페논은 2,2‘-다이하이드록시-4-메톡시벤조페논과 함께 화장품, 자외선차단로션, 립스틱, 모발 염색제, 샴프 및 세제 등에서 자외선안정제의 기능성 원료로서 많이 사용되고 있다. 그러므로, 본 연구에서는 벤조페논계 자외선차단제의 환경 중 노출 수준과 대사작용, 동력학적 동태, 유전독성, 특이적으로 조절되는 지표유전자 도출에 이르기까지 전반적인 독성학적 접근법을 이용하여 연구를 수행하였고, 그 결과는 다음과 같다.첫째로, 환경시료에서 기체크로마토그래피-질량분석기를 이용하여 유도체화 과정을 거쳐 7종의 벤조페논계 자외선차단제의 동시분석법을 개발하였다. 분석된 벤조페논계 자외선차단제는 벤조페논, 벤조하이드롤, 4-하이드록시벤조페논, 2-하이드록시-4-메톡시벤조페논, 2,4-다이 하이드록시벤조페논, 2,2‘-다이하이드록시-4-메톡시벤조페논과 2,3,4-트리하이드록시벤조페논이다. 최적조건하에서 수질 (62-114 %)과 토양 (60-125 %)시료는 높은 회수율을 나타내었으며, 낮은 상대표준편차 값을 보여, 분석방법의 정확도와 정밀도를 검증하였다. 검출한계는 분석대상물질에서 5-100 ng/L (또는 kg)의 범위로 산출되었으며, 정량한계는 25-100 ng/L (또는 kg)로 계산되었다. 이와 같이 검증된 분석방법을 통하여 우리나라의 환경시료를 분석한 결과, 수질 (27-204 ppt)보다 토양 (500-18,380 ppt)에서 더 높은 농도로 검출되어, 1차적으로 수질오염 일어난 이후, 2차적으로 토양으로 축적 되어지는 것으로 사료된다. 또한, 벤조페논계 자외선차단제의 환경 오염원으로 사료되는 생활하수, 레저용수, 공단배수 중에서 공단배수가 주오염원으로 작용하는 것으로 보여진다.둘째로, 랫드(rat)의 혈액 중에서 기체크로마토그래피-질량분석기를 이용하여 벤조페존계 자외선차단제의 최적분석방법을 개발하였다. 벤조페논과 2-하이드록시-4-메톡시벤조페논 100 mg kg-1 bw을 랫드에 경구투여하여 시간별로 혈액시료를 채취하였고, 시료는 pH 9.5에서 에틸아세테이트로 추출하여 유도체화 후 분석하였다. 최적분석조건 하에서, 검량선은 r2 > 0.999이상의 좋은 직선성을 보였으며, 76 % 이상의 회수율을 나타내었고, 상대표준편차 9.87 % 이하의 일내 정밀성, 13.89 % 이하의 일간 정밀성과 상대표준오차 -14.59 % 값 이하의 정확성이 검증되었다. 본 분석방법을 적용한 결과, 랫드에 벤조페논과 2-하이드록시-4-메톡시벤조페논을 경구투여한 이후 30분부터 그 대사체인 벤조하이드롤, 4-하이드록시벤조페논, 2,4-다이하이드로시 벤조페논, 2,2‘-다이하이드록시-4-메톡시벤조페논과 2,3,4-트리 하이드록시벤조페논이 검출되었다. 독성동태학적 생체이용률 파라미터는 Win-Nonline 프로그램을 이용하여 산출되었으며, 벤조페논은 8시간 에서 2.06 &micro;g/ml의 최고농도를 보였고, 2-하이드록시-4-메톡시 벤조페논은 3시간에서 21 ㎍/ml로 검출되었다. 벤조페논과 2-하이드록시-4-메톡시벤조페논은 다이페닐케톤 구조로서 두개의 방향족 고리를 가지고 있으며, 이러한 구조는 소수성(지방친화성) 분자로 작용하여 상대적으로 빠른 흡수가 일어나는 것으로 사료되어진다.세번째 연구에서는, 세포수준에서 DNA손상을 확인하기 위하여 단세포전기영동법(comet assay)을 이용하여 벤조페논계 자외선차단제의 위해성을 평가하였다. 그 결과, 벤조페논과 그 대사체인 4-하이드록시 벤조페논은 통계적으로 유의한 DNA 손상을 보였으며 (P < 0.05), 벤조하이드롤은 대사활성존재 및 부재하 모두에서DNA 손상을 유발하지 않는것으로 나타났다. 2-하이드록시-4-메톡시벤조페논은 대사체인 2,4-다이하이드록시벤조페논과 2,3,4-트리하이드록시벤조페논에서 대사활성 존재 및 부재시 모두 DNA 손상을 유발하였으나, 2,2‘-다이하이드록시-4-메톡시 벤조페논은 DNA 손상을 유발하지 않았다. 따라서 본 실험을 통하여 유추한 결과, 벤조페논과 2-하이드록시-4-메톡시벤조페논의 대사체들 중에서 para-위치에 하이드록실기를 가진 대사체들에서 DNA 손상을 유발하는 것으로 사료되어진다.네번째로, 이들 벤조페논계 자외선차단제는 세계야생보호기금과 일본 국립의약품 식품위생연구소에서 내분비계장애물질로 분류되어지고, 다수의 문헌을 통하여 내분비계 호르몬 (특히 성호르몬)과 유사한 작용을 하는 것으로 보고되고 있다. 따라서 본 연구에서는 인간 유방암 세포주인 MCF 7에 벤조페논계 자외선차단제를 48시간 동안 처리한 후, cDNA 마이크로어레이 실험을 통해 유전자 발현 양상을 분석하였다. 벤조페논과 4-하이드록시벤조페논, 2-하이드록시-4-메톡시벤조페논에 의해 각각 32개, 38개, 30개 유전 유의한 발현 변화를 보였으며, 벤조페논계 자외선차단제에 대해 공통적으로 발현이 증가한 4개의 유전자와 6개의 감소한 유전자들을 선별할 수 있었다. 이들 유전자 중에서 발현이 증가한 3개 유전자와 감소한 3개의 유전자가 17β-에스트라다이올과 일치하는 결과를 보여 높은 상관관계를 나타내었다. 이들은 세포증식, 갑상선호르몬, 전사조절, 대사작용, 면역작용과 관련된 유전자들로 벤조페논계 자외선차단제의 새로운 지표 유전자로 도출하기 위하여 RT-PCR을 이용하여 확인시험을 수행하였으며, 추가적으로 다른 벤조페논계 자외선차단제들에 연구가 수반되어야 할 것으로 사료된다.요약하면, 본 연구에서는 벤조페논계 자외선차단제의 노출에 대하여 각기 다른 독성학적인 기법을 활용하여 다양한 각도에서 접근하고자 하였다. 이러한 연구를 통하여 벤조페논계 자외선차단제의 위해성 평가와 법률 제정에 유용한 기초자료로서 제공되어 질 것으로 사료된다.
[영문]UV filters of benzophenone (BP)-type are used primarily as photoinitiators, fragrance enhancers, as ultraviolet curing agents and occasionally, as flavor ingredients. They are also used in the manufacture of insecticides, agricultural chemicals and pharmaceuticals and as additives for plastics, coatings and adhesives. Especially, 2-hydroxy-4-methoxybenzophenone (HMB) is widely used as a UV stabilizer in skin moisturizing products and sunscreen lotions, usually in conjunction with 2,2’-dihydroxy-4-methoxybenzophenone (DHMB). Therefore, this study investigated environmental exposure levels, metabolism, kinetic behavior, genotoxicity and specific-regulated genes of BP-type UV filters using overall toxicological approaches.First, a novel method has been developed to determine and quantify seven organic UV filters simultaneously employing liquid (solid)-liquid extraction, derivatization with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and gas chromatography with mass spectrometry (GC-MS) in various environmental matrices. The UV filters determined were: BP, benzhydrol (BH), 4-hydroxybenzophenone (HBP), HMB, 2,4-dihydroxybenzophenone (DHB), DHMB and 2,3,4-trihydroxylbenzophenone (THB). Under optimal conditions, the high recovery (62-114 % and 60-125 % for water and soil, respectively) and the low relative standard deviation (RSD) values (less than 13.9% and 17.2% for water and soil, respectively) were indicated the high performance of this method. The detection limits of method were relatively low, ranging from 5 to 100 ng/L or kg and the quantification limits of method ranged between from 25 to 500 ng/L or kg for all test compounds. This validated method was applied to the analysis of seven BP-type UV filters in water and soil samples in Korea, collected between April and May 2003. The overall concentration of UV filters in the soil samples (500-18,380 ppt) was higher than in water samples (27-204 ppt). Therefore the established method in this study was successfully applied to monitor the residue measurement of the BP-type UV filters in environmental water and soil samples.Secondly, in rat blood, the simultaneous analysis of BP-type UV filters was optimized by GC-MS. The male Sprague-Dawley rats were used in this study and BP and HMB were administered orally at a dose of 100 mg kg-1 body weight. Plasma sample was adjusted to pH 9.5 and extracted with ethyl acetate for 20 mins. The organic solvent was reduced to dryness and the residues were derivatized with MSTFA and determined by GC-MS. Under optimal conditions, calibration curves showed a good linearity (r2 > 0.999) and the recovery after extraction and concentration was above 76%. Intra-day and inter-day RSD values were within 9.87 and 13.89%, respectively, indicating good precision. The accuracy of the method expressed as relative mean error (RME) was below - 14.59% which was shown to be satisfactory. BP was mainly converted to BH and HBP in rat. Also, HMB was enzymatically converted to at least three intermediates. DHB was a major intermediate formed by o-demethylation, which in turn was converted to THB and DHMB by aromatic hydroxylation. The toxicokinetic parameters were presented by the noncompartmental analysis. The Cmax value of BP was 2.06 ± 0.46 μg/ml and the Tmax was 3.83 hr. The t1/2 of BP was approximately 19.28 hr and the AUC0-∞ was 47.17 ㎍/ml&#8226;hr. The Cmax value of HMB was 21.21 ± 11.61 μg/ml and the Tmax was 2.71 hr. The t1/2 of HMB was approximately 4.58 hr and the AUC0-∞ was 104.89 ㎍/ml&#8226;hr. The established method was successfully applied to various biological sample (urine and bile, etc.) for the determination of BP-type UV filters.Thirdly, to evaluate the magnitude of DNA damage, the single cell gel electrophoresis assay was performed. The comet assay in alkaline condition is a rapid, sensitive, and reliable biochemical technique for evaluating single-stand DNA breakage in mammalian cells, as well as high throughput toxicity screening tool. From these results, BP and its metabolite, HBP, were observed statistically significant differences of tail moment values compared with negative control (P < 0.05). BH at tested all concentrations was not observed significant difference of tail moment. Also, HMB and its metabolite, DHB and THB, were revealed statistically significant differences of tail moment values compared with negative control (P < 0.05), except for DHMB. Therefore, we suggest that BP derivatives with hydroxyl group at the para-position such as HBP, DHB and THB possibly induce the single stranded DNA breakage in L5178Y cells.Finally, these BP-type UV filters are thought to mimic estrogens in their action, and are called endocrine disrupting chemicals. In the previous studies, they have been seen to exert an uterotrophic effect in vivo and to stimulate cell proliferation of MCF-7 breast cancer cells in vitro. Therefore, to identify genes elicited by three of BP-type UV filters, a human cDNA microarray analysis was carried out to MCF-7 cells, treated with BP, HBP and HMB using KISTCHIP-400 including 401 endocrine system related genes. Out of the analyzed genes, 32, 38 and 30 genes were identified showing significant changes (minimum 1.5-fold) in gene expression resulting from BP, HBP and HMB, respectively. Through the clustering analysis of gene expression profiles, 4 up-regulated and 6 down-regulated common genes by three UV filters were identified. The functions of these genes were found related to cell proliferation, thyroid hormone, regulation of transcription, metabolism and immune response. Among the genes, especially, 3 genes were induced and 3 genes were repressed by three BP-type UV filters as 17β-estradiol (E2). Therefore it is suggested that these genes may be associated with estrogenic effect of three UV filters. For the confirmation of the gene expression profiles identified from microarray analysis and the expression patterns of the other UV filters with estrogenic activity by dose- and time- dependent manner, real time RT-PCR will be performed. Taken together, this study provides evidence for previously unknown gene regulation by BP-type UV filters and E2, raising interesting possibilities regarding to the role of endocrine disruptors.In summary, this thesis represents significant effects for exposure of BP-type UV filters viewed from different angles using toxicological tools. Through further investigation in vitro and in vivo, it should be characterized completely the toxic mechanisms of BP-type UV filters and applied as basic information for risk assessment and regulation establishment of BP-type UV filters.
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https://ir.ymlib.yonsei.ac.kr/handle/22282913/137072
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