Application of bioassay for endocrine disrupting chemicals( ) B.C.Lee, S.J.Kim, J.H.Yoon, Duong N.C., K.E.Jung, I.S. Choi, C.R.Cho, J.C.Cho, E.J.Kim,

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1 Application of bioassay for endocrine disrupting chemicals( ) 2009

2 Application of bioassay for endocrine disrupting chemicals( ) B.C.Lee, S.J.Kim, J.H.Yoon, Duong N.C., K.E.Jung, I.S. Choi, C.R.Cho, J.C.Cho, E.J.Kim, I.C.Eom, K.H.Choi, J.S. H an Risk Assessment Division Environmental Health Risk Research Department National Institute of Environmental Research 2009

3 1. ( ) : Estrogenic effect bioassay 2.. in vitro screening. in vitro bioassay screening cell line, ) screening, Bioassay 2) 3) agonist, antagonist effect

4 , bio monitoring 3. Bioassay (estrogen receptor, ER) Bioassay bioassay screening / / In vivo / vitro bioassay ( ) Bioassay Human/rat/medaka estrogen receptor cell Yeast two-hybrid assay MCF-7 BUS cell E-screen assay cell dose-response curve

5 bioassay, bioassay 4.. bioassay 1) β α

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10 4. / OECD OECD,

11 Abstract The main objective of this study is to investigate the effects of pollutants originated from manufactured as well as environmental sources using yeast-two hybrid system and human breast cancer cell (MCF-7) assays. Chemicals used in the experiments include 4 hormones, 4 pharmaceuticals, 9 phenols, 9 phthalates and 4 nanoparticles (NPs). Toxicity and estrogenic activity of target pollutants were estimated after the exposure on yeast (Saccaromycescervisiae Y190) carrying medaka and estrogen receptors (mer and her, respectively) as well as MCF-7 cell line. 17-β estradiol (E2, 10-6 M) was used as an estrogenic activity reference chemical. Dose-response curves were established using EC 50 values of corresponded chemicals. Environmental samples were collected in amber glass bottles and stored at 4 o C immediately in prior of further treatment. Different mixtures of solvent were used in extraction steps in order to maximize the efficiency of pollutants isolation. Fractions of eluted solvent after the extraction were kept in separated vials for the test. In yeast-two hybrid assays, yeast was exposed to the NPs for 4 hours and then the estrogenic activity was evaluated using chemiluminesence assay. The viability of yeast exposed to NPs was examined by agar plate and blue staining methods. The results showed that the NPs did not induce estrogenic activity in mer-yeast

12 system. Without sonication, low concentrations of silver NPs (< 0.1 mg/l for Ag-S NPs and < 10 mg/l for Ag-C NPs) and ZnO NPs (<10 mg/l) significantly increased estrogenic activity of E2 in mer-yeast. At concentration less than 10 mg/l, Al 2 O 3 NPs showed no significant effects to estrogenic activity of E2. However, the exposure of yeast system to Al 2 O 3 NPs at concentration of 10mg/L and greater in creased the estrogenicity of E2. With 30min of sonication before the exposure, Ag-S, Ag-C, ZnO and Al 2 O 3 NPs showed significant impacts in reducing estrogenic activity of E2 in mer-yeast at 0.5, 0.5, 1 and 5 mg/l, respectively. In cell viability assay results, at lower concentrations of silver NPs (<0.1 mg/l for Ag-S and < 10 mg/l for Ag-C), there was no significant difference of yeast viability between the control and the exposures. There was also high correlation (r 2 >0.9) between yeast viability (as % of control) and estrogenic activity of E2 in yeast (as % of control). The viability test also showed that cell counts were reduced significantly in the exposure of Al 2 O 3 and ZnO NPs with concentrations ranged from 50 to 500 mg/l. The results suggested that the toxicity level can be sorted as Ag-S NPs > Ag-C NPs > ZnO NPs > Al 2 O 3 NPs. After the exposure of other 26 chemicals, dose response curves were obtained and used to calculate the relative potency of each chemical to E2. Dose response curve of E2 was also used to calculate the EEQ concentration of estrogenic compounds in the environmental samples. The results showed that the sensitivity of

13 mer is higher than that of her in yeast system. EEQ concentrations detected in environmental samples of the 1 st sampling were ranged from less than 1 µg/l (surface water samples) to 18 µg/l (influents of wastewater treatment plant) when using mer yeast. These values in her system were lower than those of mer system. It showed the consistent results with pure chemical test. Similar results were obtained in the samples of the 2 nd sampling. Estrogenic compounds in the environmental samples were also monitored by chemical analysis method using LC/MS/MS. The concentration of target compounds in the environmental samples were relatively correlated with estrogenic activity by yeast two-hybrid assays (R 2 =0.68). Statistical analysis of estrogenic activity and SUVA index also showed the relatively high relationship with R 2 =0.57 and 0.65 in the samples of the 1 st and the 2 nd sampling, respectively. The dose-response curve of target compounds was also obtained by MCF-7 assays and applied to estimate the estrogenic activity in the environmental samples.

14 i Abstract ix xii xv xvii xviii E-screen EDCs 4 2. Yeast two-hybrid assay EDCs (QSAR) 10. In vivo assay 11

15 . In vitro assay E-screen assay Cell Seeding SRB assay E-screen Yeast two-hybrid assay Yeast two-hybrid assay 29. Yeast Yeast two-hybrid assay dose-response 36. bioassay 44. bioassay 47

17 1. E-screen 5 2. Yeast two-hybrid system 8 3. E-screen assay MCF-7 cell ( ) MCF-7 BUS Yeast two-hybrid assay her her her her mer mer mer mer mer Ag-C E2 45

18 18. mer Ag-B E mer Al 2 O 3 E mer ZnO E mer her mer her A ( ), ( ) 65

19 1. E-screen (MCF-7 cell ) 6 2. Yeast two-hybrid assay 9 3. Yeast two-hybrid assay (Relative activity) LC/MS/MS (unit : μg/l) GC/MS (unit : μg/l) A GC/MS A (unit : μg/l) 64

20 (Abbreviation Lists) AhR : Aryl hydrocarbon Receptor (Aryl hydrocarbon ) BPA : Bisphenol A ( A) DMEM : Dulbecco's modified eagles medium ( ) E1 : Estrone (, ) E2 : 17β-estradiol (, ) E3 : Estriol (, ) ECACC : European collection of cell culture ( ) EDCs : Endocrine Disrupting Chemicals ( ) EE2 : 17α-Ethynylestradiol (, ) EEQ : Estradiol Equivalent Concentration ( ) ELISA : Enzyme-Linked Immunosorbent Assay ( ) ER : Estrogen Receptor ( ) FBS : Fetal bovine serum ( ) GN : Genistein (, ) her : human Estrogen Receptor (human ) MCF-7 : Michigan Cancer Foundation 7 cell line ( ) mer : Japanese medaka Estrogen Receptor (Japanese medaka ) NP : Nonylphenol ( ) OP : Octylphenol ( ) PBS : Phosphate Buffer Saline ( )

21 QSAR : Quantitative Structure-Activity Relationship ( - ) REC10 : 10% relative effective concentration (10% ) rer : rat Estrogen Receptor (rat ) SRB assay : Sulforhodamin B assay ( ) TIF2 : Transcriptional mediators / Intermediary Factor 2 (co-activator) YES : Yeast Estrogen Screening ( )

23 μ

24 μ

27 β α

28 β

30 α α

한다. 그리고 cell culture flask를 사용할 경우 5 ml의 DMEM

39 풀어진 세포현탁액 중에서 10 μl를 뽑아내어 70% 알코올로 소독한 hemocytometer 격자위에 주입한다. 9) 현미경을 이용하여 세포를 관찰하고 세포수를 센다 (그림 5). 10) 각 cell culture flask마다 cell이 주입되도록 해야 한다. 그리고 cell culture flask를 사용할 경우 5 ml의 DMEM 배지를 주입하고, 계산된 셀 양만큼 주입한다. 11) CO2 incubator (5%, 37 )에서 3일간 배양한다. 12) 1)~11)과정을 3일에 한번씩 동일하게 반복한다. 8) ① DMEM, PBS 등 해동 ② DMEM 배지 제거 ③ DMEM 주입 후 세포 탈착 ④ 원심분리 그림 4. MCF-7 cell 계대배양 실험순서(①~⑩)

40 ⑤ 원심분리 후 세포 ⑥ 세포 풀기 ⑦ Homocytometer에 주입 ⑧ Cell counting ⑨ DMEM 주입후계산된양주입 ⑩ CO2 Incubator 배양 그림 4. MCF-7 cell 계대배양 실험순서(①~⑩)

42 μ

43 μ μ μ

44 μ μ

45 μ μ μ μ

46 μ

47 μ β β

48 β α

49 μ

52 μ

53 μ μ μ μ μ μ μ μ μ μ μ

54 μ μ

57 β α

64 Chemicals mer(medaka ER) her(human ER) REC10(M) R. Potency REC10(M) R. Potency E2(17β-Estradiol) 5.E-10 1.E+00 5.E-10 1.E+00 E1(Estrone) 2.E-08 3.E-02 1.E-08 3.E-02 E3(Estriol) 1.E-08 5.E-02 7.E-08 6.E-03 Genistein 2.E-08 3.E-02 2.E-05 2.E-05 EE2(17α-ethinylestradiol) 2.E-10 3.E+00 6.E-10 7.E-01 Ibuprofen 3.E-04 2.E-06 9.E-04 5.E-07 Acetaminophene 8.E-05 7.E-06 1.E-04 4.E-06 Lyncomycin 7.E-05 8.E-06 9.E-05 5.E-06 4-t-butylphenol 1.E-07 5.E-03 6.E-05 7.E-06 4-n-butylphenol 3.E-07 2.E-03 1.E-04 4.E-06 4-n-pentylphenol 5.E-06 1.E-04 2.E-03 3.E-07 4-n-hexylphenol 1.E-06 5.E-04 5.E-06 9.E-05 4-t-octylphenol 1.E-03 4.E-07 2.E-07 2.E-03 4-n-heptylphenol 1.E-06 3.E-04 2.E-05 3.E-05 4-n-nonylphenol 1.E-06 4.E-04 9.E-07 5.E-04 4-n-octylphenol 1.E-05 5.E-05 9.E-06 5.E-05 bisphenol-a 4.E-07 1.E-03 5.E-06 1.E-04 diethyl phthalate 1.E-03 3.E-07 4.E-04 1.E-06 dipropyl phthalate 7.E-04 7.E-07 5.E-04 9.E-07 di-n-butyl phthalate 1.E-06 5.E-04 7.E-05 7.E-06 dipentyl phthalate 1.E-05 4.E-05 3.E-05 2.E-05 di-n-hexyl phthalate 4.E-05 1.E-05 9.E-05 5.E-06 butyl benzyl phthalate 9.E-04 6.E-07 5.E-04 9.E-07 dicyclohexyl phthalate 7.E-05 8.E-06 2.E-04 2.E-06 di-2-ehylhexyl phthalate 7.E-06 7.E-05 5.E-06 9.E-05 bis(2-ethylhexyl) adipate 1.E-02 5.E-08 3.E-04 2.E-06

68 ( ) 3 ( ) 4 ( ) 5 ( ) ( ) ( )

69 μ μ

72 μ μ μ

74 μ μ

75 μ

76 μ

78 μ

79 μ μ μ

80 μ μ μ

81 μ

83 μ μ μ

84 μ μ 시료 : 수질 500ml 내부표준물질 (Bisphenol-A-d 16 20μl ) 첨가 HLB 카트리지 (20CC) 추출 메탄올 10ml 첨가 (2회반복 ) 트리메칠아민 50μl첨가농축 (Tem:40 ) 유도체화시약 (BSTFA 50μl ) 첨가오븐 (Tem:60, 30분 ) 측정용시료 50μl GC/MS 측정

85 μ

87 IV.

88 V.

한국성인에서초기황반변성질환과 연관된위험요인연구

한국성인에서초기황반변성질환과 연관된위험요인연구 한국성인에서초기황반변성질환과 연관된위험요인연구 - - i - - i - - ii - - iii - - iv - χ - v - - vi - - 1 - - 2 - - 3 - - 4 - 그림 1. 연구대상자선정도표 - 5 - - 6 - - 7 - - 8 - 그림 2. 연구의틀 χ - 9 - - 10 - - 11 -