魚類を用いた内分泌かく乱化学物質評価法に関する研究
Contents
1. CD TD FD DeKoven 5 Oryzias latipes2. soya bean milk 88 FD FD
3. 90 0 ELISA CD
4. 9 29 A 2 TD CD OF 68 LC MS MS No 2 FD 3
5. OECD USEPA _ Pimephales promelas Danio rerio Oncorhynchus mykiss Oryzias latipes 67 O RNA UU Hk RNA
6. 1 FD TD CD 48 31 GSI HSI CD 78 01 48 18 ug ml TD 3 51 3 83 pg ml FD lt 39 ng ml 89 TD CD FD FD TD CD
7. FD TD 50 ugm E2
8. ANOVA FD 9 11 9 g TD 10 2 1 6 g CD 11441 7 g 31 WER BIL FD 9 041 9 g TD 8 9 1 5 g CD 10 2 1 5 g Fig 1 B 3 ANOVA 31 GSI HSI Fig 1 GSI 0 3 0 4 Fig 2 A T HSI 2 3 Fig 2 B GSI RU HSI ANOVA 31 Fig 3 CD CruZ vi amp E 78 01 48 18 ug ml TD SERE 3 5143 83 pg ml p lt 0 01 Scheffe s F post hoc FD 39 ng ml 79 Day 0 oDay 7 Day 14 ebay 21 E Day 31 HT LLL NNNNN HTT oe a ee se ee a ee THEE EET NN
9. PJ TD X UCD LC MS MS FD CD 390 800 ng g 416 800 ng g 1 324 8 ng g 3 FD 93 2 ng g 129 6 ng g 8 8 ng g 3 CD soya bean milk 4 milk A BRUC
10. GSI 1 57 1 99 HSI 1 8 2 2 Fig 5 B HSI ANOVA 31 Fig 6 gt CD FD TD Kruskal Wallis E2 100 ug l 83 ZREL FD FD TD CD p 0 05 Mann Whitney s U FD FD CD CD
11. 14 ng ml 13 5 ng ml 7 ng ml soya bean milk 5 5 ng ml Milk A BRUC soya bean milk 2 5 ng ml 77 Table 2 Concentrations of phytoestrogens in fish diets and commercial powdered infant milks using LS MS MS analysis Diet Genistein Daidzein Bquol Coumestrol FD 93 2 129 6 1 027 2 8 8 TD 47 680 0 41 120 0 117 2 226 4 CD 390 800 0 416 800 0 6 4 1 324 8 MilkA 475 0 194 5 14 0 N D Milk B N D N D 13 5 N D Milk C 148 0 47 5 7 0 N D Soya bean milk 211 600 0 91 200 0 5 5 2 5 The data of fish diets and commercial infant powdered milks were represent total ng g diet and total ng ml milk respectively N D Not Detected Data represents the mean n 3 78 3 3 3 GSI HSI FD 6 66 0 50 cm TD 6 98 0 43 cm CD 7 22 0 46 cm 31 FD 6 56 0 43 cm TD 6 30 0 34 cm CD 6 73 0 47 cm Fig 1 A 3
12. 1999 58 K L Thorpe T H Hutchinson M J Hetheridge J P Sumpter C R Tyler Development of an in vivo screening assay for estrogenic chemicals using juvenile rainbow trout Oncorhynchus mykiss Environ Toxicol Chem Vol 19 2812 2820 2000 59 D L DeKoven J M Nunez S M Lester D E Conklin G D Marty L M Parker D E Hinton A purified diet for medaka Oryzias latipes refining a fish model for toxicological research Lab Anim Sci Vol 42 180 189 1992 60 C Pelissero B Cuisset F LeMenn The influence of sex steroids in commercial meals and fish diets on plasma concentration of estrogens and vitellogenin in cultured Siberian sturgeon Acipense baeri Aquat Living Resour Vol 2 161 168 1989 97
13. two hybrid MCF 7 in vitro A 7 vivo Pelissero D Acipenser baeri in vivo
14. yo 69 3 2 3 2 1 3 TD CD No 2 FD Table 1 FD 2 3 9 C 3 2 2 3 2 2 1 FRA
15. 92 FD CD FD FD _93 3 5 36 T Colborn F S vom Saal A M Soto Developmental effects of endocrine disrupting chemicals in wildlife and humans Environ Health
16. 37 C ml 2 BPM ATT 250 ul 07 230 ul 1 ml Ultrafree MC 0 22 rm 3 000 rpm 10 5 wl LC MS MS 3 2 5 4 LC MS MS HP 1100 Series U Quattro UltimaTM LC MS MS LC il ODS column 4 6X250 mm 40 C 1 0 m min Buffer A acetic acid to purified water 1 999 by vol Buffer B methanol acetonitrile 2 1 by vol B in A by vol at 30 for 1 min from 30 to 100 in 20 min from 100 to 100 75 in 25 min from 100 to 30 in 26 min and from 30 to 30 in 35 min MS multiple reaction monitoring mode collision energy 30 eV 4
17. ER PC FL A EL A 91 F A 3 PC FD Pelissero E2 ED
18. GSI 100 HSI X100 3 2 4 ED HPLC BRET n7 Bis Bradford 2 ELISA ELISA 1 2
19. 15 eV cone voltage 80 V masses to charge ratio m z 269 133 253 208 241 121 267 266 3 2 6 Stat View 5 0 for Macintosh lt 0 05 GSI HSI one way ANOVA Scheffe s F post hoc Kruskal Wallis Bonferroni adjusement Mann Whitney U 76 3 3 3 3 1 FD TD CD
20. 7 Carassius auratus 17B E2 11 E2
21. 200ml AAA 200 ml 1 ml 200 pl B 2 100 U ml RU 1 050 U ml 0 5M pH 4 5 17 GTC BRISK 5 ml 2 200 pl 2 1 3 74 1 Ultrafree MC 0 22 pum 10 000 rpm 1 ul LC MS MS 3 2 5 3 10 g 30 ml SOC 0 ml B 2 100 U ml RU 1 050 U ml 0 5M pH 4 5 1
22. 9 3543 5 ng g 6 13 1 9 ng g Acipenser baeri E2 E1 E2 E1 two hybrid in vitro TD CD
23. Table 2 CD 390 800 ng g FD 93 2 ng g TD 47 680 ng g CD 416 800 ng g FD 129 6 ng g TD 41 120 ng g CD 1 324 8 ng g FD 8 8 ng g TD 226 4 ng g CD 64 ng g TD 117 2 ng g _ FD 1027 2 ng g 3 3 2 Table 2 Milk A milk C 475 ng ml 148 ng ml milk B soya bean milk 211 600 ng ml Milk A milk C 194 5 ng ml 47 5 ng ml milk B soya bean milk 91 200 ng ml MilkA B C
24. 3 2 2 2 21 6 4 13 4 g 3 251 23 26C 12 12 Lic TD CD FD 1 0 48 31 72 Kobayashi Stacey 0 02 MS 222 70 0 5 30 mg 2 20C 2 7 10 FD TD CD FD FD CD 1 0 48 31 13 3 30 5 g 17B
25. Kruskal Wallis RE 84 Day 0 gDay 7 lt 12 0 10 0 Day 14 Day 21 m Day 31 TE LT EL ELULT LL ELT BSS SS LELELEELLLEETELLULELL LET ELLELELLEELT SS SSS Se SS SSSSNSSSSSNSSNSNSSRSSSSSNSNSSSNSNSNSNNNO DEE SX RRR MET NNNNNNN NN NNNNNNNNNNNNNNNNNN HLELLELLELL LEE ET LT LLL LEE EL LEL ELT LLL IT pese eene soos sos AELEELTTEEEELEEETCELETEELECTLELTEELEEELTTT SS SSS SS SSS SAS SUSE SESS SESS 0 o o 8 0 w bual Apog 2 0 0 LUELLULULUUHLHLLLLILLIIUII NNNNNNNNNNNNNNNNN LILLULELELELLULLLELTL B LLL TEL TI SSS CC EE ULL NN a NNNNNNNNNNNNNNNNNNNNNNNNNNNNN E LLLLUEELEELELULELLLLLLULELULTT SNSSSNNSNSENSSSSNSSNSNSNNSSNNN EE CELL NN NN B o o e Te o 1 o N N T 6 1uBieM Apog 35 0 30 0 5 0 0 0 FD FD CD _ SO FD E2 CD OVX TD FD FM Fish diet Fig 4 Change in body length A and body weight B in the ovariectomized OVX sham operated SO and normal female FM goldfish fed FD TD or CD for 31 days Fish were fed 1 0 body weight volume of one of the three diets TD CD and FD every two days for 31 days Ovariectomized goldfish fed with the FD were also exposed to nominal concentration of 100 ug l estradiol 17B for 31 days Columns and bars represent the mean and standard d
26. o 73 ELISA 39 ng ml 3 2 5 3 2 5 1 R E B HPLC 3 2 5 2 3 g 40 ml 1M 9 1 30 ml 10 10 2 000 rpm 10
27. A O or O O dt OF NN Ff OF T uio ujbue Apog MEE EE ERU NH AAA nae se se Ba ooo aoe eee seseeoesnseos see sr er ee ea sa ses LLU ee CE at4806055250242a205469098060c6 9729 980225 5 6495 LLL TOATA TTNET ERSE O CO t N e 6 UPleA Apog Q O C r e LL h diet Fi length A and body weight B in the male goldfish fed FD TD Fig 1 Change in body body weight volume of one of the three diets or CD for 31 days Fish were fed 1 0 TD CD and FD every two days for 31 days Columns and bars represent the mean and standard deviation 80 0 7 A 4 B 3 se lI 0 FD TD CD Fish diet Fig 2 GSI A and HSI B in adult male goldfish Fish were fed 1 0 body weight volume of one of the three diets TD CD and FD every two days for 31 days Columns and bars represent the mean and standard deviation 81 140 120 100 80 60 Vitellogenin ug ml 40 20 FD TD CD Fish diet Fig 3 Plasma vitellogenin levels in adult male goldfish Fish were fed 1 0 body weight volume of one of the three diets TD CD and FD every two days for 31 days N D Not Detected less than 39 ng ml Columns and bars represent the mean and standard deviation Significant difference compared to TD fed fish p lt 0 01 ANOVA and Scheffe s F Post hoc test 82 3 3 4 G
28. E2 100 ug CREL FD MARE Le 71 Table 1 Ingredient and composition of the three diets Fish diets J Ingredients casein 80 96 wheat flour 18 96 vitamin mix 0 5 mineral FD mix 1 5 96 i fish meal 60 wheat flour 29 soya bean 4 rice bran 2 yeast and vegetable oil 5 vitamin mix mineral mix wheat flour soya bean meal fish meal alfalfa meal rice CD bran shrinp meal spirulina vitamin mix mineral mix methionin Composition which does not include vitamin mix and mineral mix Composition of the ingredients is not shown 72 3 2 3 25 G 0 1 aprotinin 10 000 KIU ml 0 1 PMSF 14 0 U ml 4 20 1 800Xg HEBEL Melt L5ml 30C 4 31
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30. NAOSITE Nagasaki University s Academic Output SITE Author s Citation 2003 03 31 Issue Date 2003 03 31 URL http hdl nandl e net 10069 7316 This document is downloaded at 2015 11 06T04 42 38Z http naosite lb nagasaki u ac jp 3 3 1 9 49 70 in vitro in vivo
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32. SI HSI FD 9 240 9 cm TD 8 5 0 9 cm CD 8 640 6 cm 31 FD 9 1 0 9 cm TD 8 540 8 cm CD 8 70 7 cm Fig 4 A 3 ANOVA FD 24 145 5 g TD 20 6 5 6 g CD 19 9 4 7 g 31 FD 24 6 7 2 g TD 20 9 5 4 g CD 20 4 4 7 g Fig 4 B 3 ANOVA EK ANOVA 31 GSI HSI Fig 4 GSI 0 02 0 14 Fig 5 A ANOVA
33. eviation 85 3 A m Ovariectomized ti Sham operated Normal GSI HSI FD TD CD FD E2 FD FD CD Fish diet Fig 5 GSI A and HSI B in adult ovariectomized sham operated and normal female goldfish which were fed to three diets for 31 days Fish were fed 1 0 body weight volume of one of the three diets TD CD and FD every two days for 31 days Ovariectomized goldfish fed with the FD were also exposed to nominal concentration of 100 ug l estradiol 17B for 31 days Columns and bars represent the mean and standard deviation 86 1000000 iOvariectomized Sham operated Normal 100000 10000 1000 100 Vitellogenin ug ml 10 FD TD CD FD E2 FD FD CD Fish diet Fig 6 Plasma vitellogenin levels in adult ovariectomized sham operated and normal female goldfish which were fed to three diets for 31 days Fish were fed 1 0 body weight volume of one of the three diets TD CD and FD every two days for 31 days Positive control ovariectomized goldfish fed with the FD were exposed to nominal concentration of 100 ug l estradiol 17B for 31 days Columns and bars represent the mean and standard deviation Significant difference compared to FD TD and CD fed ovariectomized fish p lt 0 05 Kruskal Wallis test and Mann Whitney s U test 87 3 4 ZZ
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