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. 3 OECD _ _ 10 100 ug l NP2. OD RNA P DANZO RNA O Carassius auratus
3. yo 3 4 NP FD2 TD2 CD2 3 CD2 gt TD2 gt FD2 8
4. NP FD2 CD2 NP BESERE L 38V C RAVA a TS 11 KT CD2 NP RBHO MP TS 11 KT i FD2 NP RBH CD2 TS 11 KT GSI FD2 CD2 NP BRITT BEV aD E2 Villeneuve
5. CD2 10 ug NP MRER 7 21 28 NP 7 21 Panter ZM 189 154 RRA y bhay Ki CCR L Oryzias latipes 4 CHET in vivo NP SR NP
6. 141 E2 25 000 TS 25 000 11 KT 100 000 E2 25 000 TS 50 000 11 KT 50 000 10 30 3 0 05 M H3BOs 0 5 0 01 IgG IgG 00779 CAPPEL U S A
7. 1 Acipenser baeri DE E2 CD2 172
8. ANOVA FD2 CD2 NP 28 ARR GST Fig 9 GST FD2 CD2 NP ANOVA CD2 FD2 e eO Enzyme activity pmol mg min o o w Rh ad e M O o e CO Enzyme activity pmol mg min o o ho P e uh Control 0 1 1 10 100 E2 Nominal concentration of NP ug Fig 8 Levels of hepatic EROD and MROD activities in the FD2 A or CD2 B fed group exposed to the nominal concentrations of 0 1 1 0 10 and 100 pg l of NP for 28 days Columns and bars represent the mean and standard deviation 165 20 e FD2 CD2 15 S 2 o a 10 5 2 5 0 Control 0 1 1 10 100 E2 Nominal concentration of NP ug Fig 9 Levels of hepatic GST activity in the FD2 4 or CD2 fed group L1 exposed to the nominal concentrations of 0 1 1 0 10 and 100 ug l of NP for 28 days Columns and bars represent the mean and standard deviation 166 5 4
9. 174 175 5 5 1 T Colborn F S vom Saal A M Soto va effects of endocrine disrupting chemicals in wildlife and humans Environ Health Perspect Vol 101 378 384 1993 2 T Colborn D Dumanoshi J P Myers Our Stolen Future Dutton NY USA 1996 3 S Jobling M Nolan C R Tyler G Brighty J P Sumpter Widespread sexual disruption in wild fish Environ Toxicol Chem Vol 19 2812 2820 1998 4 Organization for Economic Cooperation and Development Final report of the fish expert consultation meeting London UK October 28 29 Environmental Health and Safety Division Paris France 1999 5 U S Environmental Protection Agency Endocrine Disruptor Screening Program Proposed Statement of Policy Fed Reg Vol 248 71542 71568 1998 6 J P Sumpter S Jobling Vitellogenesis as a b
10. 139 Table 1 Ingredients and compositions of fish diets Fish diets Formulated diet FD2 No 5 Trout diet TD2 Crumble for trout Carp diet CD2 Floating pellet for carp 1 5 15 Ingredients defatted rice bran 55 casein 40 vitamin mix 3 0 96 mineral mix 2 0 fish meal 65 wheat flour 21 defatted rice bran 6 0 soy bean meal 2 0 corn gluten meal 3 0 96 vitamin mix 2 0 mineral mix 1 0 fish meal 38 wheat flour 25 96 defatted rice bran 18 96 soy bean meal 15 96 corn gluten meal 1 0 vitamin mix 1 0 96 mineral mix 2 0 Composition which does not include vitamin mix and mineral mix 140 5 2 5 2 ELISA 96 100 pg E2 28
11. x B NP a AY 1 200 o 1 1 000 NP NP CD2 100 ug LNP L TIREE 28 CD2 5 XT Xr SEO fL FD2 100 Pelissero
12. 295 26 ng g 758 54 ng g Fig 1 D B TD2 CD2 hER B hER c 10 CD2 gt TD2 gt FD2 o 147 f CLN intensity T B ate 100 C D Rate of CLN intensity T B oi o 0 39 78 156 313 625 1250 2500 0 39 78 156 313 625 1250 2500 Concentration ug ml Concentration ug ml Fig 1 Dose response curves of agonist activity for FD2 O and TD2 A and A and CD2 LJ and W using the yeast antagonist assay system for human estrogen receptor a A and B and f C and D Fish diets with close circle or without open circle the hydrolysis were showed The values of antagonist activity were represented as the rate 96 of CLN intensity T B The all data represent the mean n 4 148 Table 3 Estrogenic activities of fish diet for human estrogen receptor C and f using yeast two hybrid assay Estradiol 17Q equivalent ng g Diet hER a hER B Without With Without With Formulated diet FD2 N D N D N D N D Trout diet TD2 N D 26 51 n d 295 26 Carp diet CD2 N D 95 91 39 91 758 54 Fifty micrograms of sample was
13. in vivo 115 2001 35 MERA in vivo TPT TBT 1 2002 36 A Arukwe F Lars G de Xenobiotic and steroid biotransformation enzymes in Atlantic salmon Salmo Salar liver treated with an estrogenic compound 4 nonylphenol Environ Toxicol Chem Vol 16 2576 2583 1997 37 P C Lee S C Patra C T Stelloh W Lee M Struve Interaction of nonylphenol and hepatic CYP 1A in rats Biochem Pharmacol Vol 52 885 889 1996 38 P C Lee S C Patra M Struve Modulation of rat hepatic CYP 3A by nonylphenol Xenobiotica Vol 26 831 838 1996 180
14. FD2 TD2 CD2 3 LC MS MS 4 two hybrid hER c B 5 2 3 WRA LC RER 5 2 3 1 Carassius auratus 30 1 6 3 63 2001 3 FD2 TD2 CD2 137 1 0 1 1 1 7 14 28 42
15. 10 13 ng ml Fig 7 A CD2 ANOVA ZED SURRBRO TS p lt 0 05 Fisher PLSD Post hoc NP 100 ug l IRERE CI 0 5 ng ml Fig 7 B 11 KT FD2 NP 0 1 ug l lt ANOVA GRA MBE Chor Fig 7 C CD2 fGBHR LCdSVCHEBR BEER LL 4907 D AREND ON ANOVA NP 100 ug l E2 1 ug l IRIRE IIIR p 0 05 Fisher PLSD Post hoc NP 100 ug l IRERE CIEI 0 3 ng ml Fig 7 D E2 FD2 CD2 NP MREEREAE CITT KERB 250 1000 pg ml Fig 7 E and F ANOVA 162 30 A initial 25 f mDay 28 20 Testosterone ng ml
16. ANOVA CD2 NP 28 HAJRIE L7 D GSI HSI Fig 5 B CD2 HSI 3 4 NP RRR LS ANOVA GSI NP REREH CREK E2 WESEREO LO RRE amp He TREA p lt 0 05 Dunnett s post hoc HSI FD2 CD2 GSI CD2 FD2 154 Table 4 1 Change in body length in the FD2 fed group exposed to NP concentrations of 0 1 1 0 10 and 100 ug l for 7 14 21 and 28 days NP treatment Body length cm ug Day 0 Day 7 Day 14 Day 21 Day 28 Initial 9 95 0 71 Control 10 08 0 47 10 10 0 27 10 20 0 39 10 34 0 38 0 1 10 00 0 32 9 90 0 33 10 14 0 32 10 16 0 29 1 0 9 82 0 78 9 74 0 78 9 92 0 76 10 00 0 76 10 10 02 0 78 9 90 0 87 10 14 0 79 10 12 0 85 100 9 96 0 84 9 8640 82 10 06 0 79 10 12 0 79 E2 1 0 9 82 1 23 9 74 1 27 9 92 1 19 9 921123 Data represent the mean and standard deviation The control fish were exposed to the solvent carrier only DMSO 0 01 ml l and the positive control fish were exposed to the nominal concentra
17. NP CYP 1A T amp CYP 3A NP CYP1A CYP3A Lee NP CYP 1A CYP 3A 39 NP NP CYP NNP CYP 1A CYA 3A GST 100 ugl NP X BEX FD2
18. 3 uq NP 4 4 t OP H E2 NP 4 rOP a BE2 1710 1 ua E2 1 2 000 1 3 000 171 4 17100 4 1 500 1 B E2 NP 1 110 30
19. p 0 05 Dunnett s post hoc FD2 CD2 AHIR 10 100 160 1000000 Day 0 100000 Day 7 E Day 21 1000 RS N X Do NNI N 100 Vitellogenin ug ml 10 A ees o1 L 1000000 100000 10000 1000 100 Vitellogenin ug ml 10 0 1 Initial Cont 0 1 10 0 Nominal concentration of NP ug m LN 2 10 oak Fig 6 Levels of plasma vitellogenin synthesis in the FD2 A or CD2 B fed group exposed to the nominal concentrations of 0 1 1 0 10 and 100 ug l of NP for 7 21 and 28 days Columns and bars represent the mean and standard deviation Significant difference compared to the control fish p 0 05 Dunnett s Post hoc test 161 5 3 6 FD2 CD2 1 0 1 1 10 100 ug l NP 28 TS 11 KT E2 Fig 7 FD2 NP 0 1 10 ug l RBC Sif TS p 0 05 Dunnett s post hoc NP
20. HER a B E2 B TD2 CD2 hBR B hER w 10 4 CD2 gt TD2 gt FD2 FD2 TD2 CD2 1 63 CD2 TD2 14 28 EFD2 7 14 28 42 63 167 3 CD2 gt TD2 gt FD2 FD2 TD2 CD2 CD2 gt TD2 gt FD2 3
21. 9 50 ug vitro tyo hybrid VS In vivo 134 8 4
22. FD 1 0 1 1 10 100 ug l NP 28 Fig 6 A 4 FD2 NP REN COREE 7 21 28 NP 0 1 10 ug l RRA C bI EI 10 ug ml FD2 NP 100 ug l E2 1 ug l RREI WRR L Heat L CARE 7 p 0 05 Dunnett s post hoc RE 21 28 p lt 0 05 Dunnett s post hoc RE CD2 1 0 1 1 10 100 ug l NP 28 Fig 6 B CD2 NP 0 1 100 ug l RRR OIRE 7 100 pg ml ANOVA NP 100 ug l BREE 28
23. E2 CD2 CD2 NP WRSEREO GSI X FD2 NP RIER e beg CD2 GSI 168 GnRH GTH GTH 11 KT
24. Cyprinus carpio 4 169 Giesy 2 Pimophales promelas 0 05 3 4 ug l NP 42 B REEL RE OWE hth E2 100 pg l NP E2 Villeneuve NP SH
25. NP NPEO NPEO NPEO APEO APEO OF VEVEOR C b RRE 9 NPEO 80 8 NPEO NP2bO NP1EO NP2EC NPIEC NP NP 4 17 7 ug UF OBRERIEE IC 33V C XFH Oryzias latipes
26. NP FD2 CD2 NP CML CMA LX 11 17B P430 1A CYP 1A 135 S GST 136 5 2 5 2 1 NP E2 DMSO No FD2 TD2 CD2 TD2 CD2 EP 1 5 Table 1 5 2 2
27. 9 gi 9 133 NP 3
28. 1 63 AME Fig 4 CD2 TD2 14 28 p lt 0 05 Mann Whitney s U test CD2 FD2 7 14 28 42 63 p lt 0 05 Mann Whitney s U test CD2 gt TD2 gt FD2 150 A FD2 gETD2 ee tn T NNN 7 NN NN Initial wo uibual Apog Feeding period days Fig 2 Change in body weight A and body length B in the male goldfish fed FD2 TD2 or CD2 for 63 days Fish were fed 1 0 body weight volume of one of the three diets TD2 CD2 and FD2 every day for 63 days Columns and bars represent the mean and standard deviation 151 GSI 1 e Oi HSI 96 FD2 TD2 CD2 Fish diet Fig 3 GSI A and HSI B in adult male goldfish Fish were fed 1 0 body weight volume of one of the three diets TD2 CD2 and FD2 every day for 63 days Columns and bars represent the mean and standard deviation 152 100000 initial 10000 Da
29. CD2 100 pg l NP 28 EROD MROD 3 FD2 NP cm 173 GST FD2 CD2 Arukwe 4 Salmo Salar 1 kg 1 5 25 125 mg 4 progesterone 6 16 17 hydroxylase BROD UDP glucuronosyltransferase 100 ug l NP CYP 1A GST Arukwe
30. HPLC 9 Bradford 2 1 0 78 ng 30 ng ABE 39 ng ml 5 2 6 17B E2 TS 11 11 KT Asahina 7 ELISA HRP E2 6 oxo E2 6 CMO HRP TS Testosterone 3 CMO HRP 11 KT 11 oxo testosterone 3 CMO HRP E2 anti 6 oxo E2 6 CMO BSA TS anti Testosterone 3 CMO BSA 11 KT anti 11 oxo testosterone 3 CMO BSA Cosmo Bio Co Ltd o OPD
31. NP CURE LICH a OM AT n4 _FD2 100 ug I NP LTR 7 21 28 Thorpe Tyler 2 4 A NP SOREL WEKT 14 ABICBO SHEE LOEC 16 ug l Jobling 514 4 t NP 21 ERRAL LOEC 20 3 pg 100 ug l NP 170 NP _
32. FD2 CD2 NP 28 A BREE LZ CYP 1A GST FD2 0 1 1 10 100 ug l NP 28 B RIRE NP BR EREL 33V C HSI GSI GSI CD2 NP CORE Lic Bee a ICT HSI GSI 100 ugl NP HSI GSI 1 ug l E2 MES SEO GSI Jobling 4 fESIO R72 5 7 wv 7 x 7 v b 8 o tnn Oncorhynchus mykiss GSI
33. Initial Cont 0 1 1 10 100 E2 Initial Cont 0 1 1 10 100 E2 O initial C aDay 28 NY N GO co a O O 11 Ketotestosterone ng ml o Oo co initial Cont 0 1 1 10 100 E2 o initial E F 2500 u Day 28 2000 1500 1000 Estraddiol 178 pg ml 500 Initial Cont 0 1 1 10 100 E2 Initial Cont 0 1 Nominal concentration of NP ug L Fig 7 Levels of plasma testosterone A and B 11 ketotestosterone C and D and estradiol 17B E and F synthesis in the FD2 left sides or CD2 right sides fed group exposed to the nominal concentrations of 0 1 1 0 10 and 100 pg l of NP for 28 days Columns and bars represent the mean and standard deviation Significant difference compared to the control fish p 0 05 Dunnett s Post hoc test and significant difference compared to the initial control fish p 0 05 Fisher PLSD Post hoc test 163 5 3 7 EROD MROD GST FD2 CD2 NP 28 HIRE LEE Fa OFFS 7 BY EROD MROD Fig 8 EROD U MROD FD ANOVA 0 1 pmol mg min Fig 8 A CD2 NP 100 pg MBRERYELSRER CEY 0 1 pmol mg min Fig 8 B NP 100 ug l MESERELOS REE P FERE LC 3
34. 63 63 80C GSI 9e HSI 5 2 3 2 NP ERB 60 BBA 18 5 46 6 12 WHE 0 1 1 10 100 g l NP IC THEA 211C 28 A BREE Lic 2001 5 6 A DMSO 0 01 m i 1 ug l E2 AROR CRE LE RENAR 251 12 12 pH 7 2 7 6 NP RRR 24 10 FD2 CD2
35. HRP 50 ul 4 18 0 85 NaCl KEAT 7 0 2M 0 01 H202 0 05 OPD 150 ul 20C 40 6 N 50 wl ELx808 Ultra Microplate Reader BIO TEK 490 nm ASSAY ZAP BIOSOFT U K 5 2 7 GST CYP1A 2 EROD MROD 142 HPLC 62 5 mM 6 25 mM MgCl 0 625 mM NADP G 6 P DHase 2 5 uM 320 ul 40 ul 5 nM G 6 P 40 pl 22 20 90C 400 yl 0 45
36. uM PTFT HPLC Waters SYMMETRY C 18 0 001 M Bf pH 5 7 60 40 1 ml min Ex 550 nm Em 585 nm GST GST Tag assay kit Lowry 5 2 8 Stat View 5 0 for Macintosh p lt 0 05 GSI HSI one way ANOVA Dunnett s post hoc Fisher PLSD post h
37. used for the assay Diets with or without the hydrolysis were also showed N D Not Detected n d not determined The all data represent the mean n 4 149 5 3 3 GSI HSI FD2 TD2 CD2 H 1 63 Fig 2 7 14 28 42 63 3 ANOVA FD2 TD2 CD2 1 63 Lit GSI HSI Fig 3 FD2 TD2 RERO GSI 1 27 1 21 CD2 0 97 Fig 3 A 3 GSI ANOVA FD2 HSI 2 38 TD2 CD2 3 39 ARU 3 19 WE Fig 3 B 3 HMO HSI ANOVA FD2 TD2 CD2
38. 1 1 28 NP RBA CORE 7 21 28 AIC BHR 5 28 80C GSI HSI 138 5 2 4 25 G 0 1 aprotinin 10 000 KIU ml 0 1 phenylmethylsufonyl fluoride heparin 14 0 U ml 4 20 1 800 Xg XE LC ifi SELL BE L7 WIE 1 5 ml 30 4C 4 0 25 M Arizono 9 Ariyoshi 5 Lowry
39. 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 06T09 40 54Z http naosite lb nagasaki u ac jp 455 P450 1A S 5 1 17B 11
40. ard deviation The control fish were exposed to the solvent carrier only DMSO 0 01 ml l and the positive control fish were exposed to the nominal concentration of 1 0 ug l of E2 157 Table 5 2 Change in body weight in the CD2 fed group exposed to NP concentrations of 0 1 1 0 10 and 100 ug l for 7 14 21 and 28 days NP treatment Body weight g ug l Day 0 Day 7 Day 14 Day 21 Day 28 Initial 30 8 5 7 Control 31 845 8 32 745 7 336456 34 7463 0 1 31 8460 328464 33 7466 34 7 7 1 1 0 32 545 7 33 6467 348 78 35 3 7 5 10 32 148 1 33 6479 34 6 8 2 35 2 8 5 100 30 0443 304 42 31 5445 324 438 E2 1 0 29 9463 29 3466 29 8 6 5 30 5 6 6 Data represent the mean and standard deviation The control fish were exposed to the solvent carrier only DMSO 0 01 ml l and the positive control fish were exposed to the nominal concentration of 1 0 ug l of E2 158 GSI and HSI GSI and HSI Control 0 1 1 10 100 E2 Nominal concentration of NP ug Fig 5 Levels of gonadosomatic index GSI and hepatosomatic index HSI D in the FD2 A or CD2 B fed group exposed to the nominal concentrations of 0 1 1 0 10 and 100 ug l of NP for 28 days The GSI and HSI was calculated as a ratio of gonad and hepatopancreas weight to body weight Columns and bars represent the mean and standard deviation Significant difference compared to the control fish p 0 05 Dunnett s Post hoc test 159 5 3 5
41. as Environ Toxicol Chem Vol 19 1368 1377 2000 28 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 29 C R Tyler R Aerle M V Nilsen R Blackwell S Maddix B M Nilsen K Berg T M Hutchinson A Goks yr Monoclonal antibody enzyme linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout Oncorhynchus mykiss Environ Toxicol Chem Vol 21 47 54 2002 30 OECD Fish acute basicity test Organization for Economic Cooperation and Development Guide line for icing and chemicals Method 203 OECD Paris 31 2001 179 32 C Pelissero B Bennetau P Babin F Le Menn J Dunogues The estrogenic activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri J Steroid Biochem Mol Biol Vol 38 293 299 1991 33 G H Panter T H Hutchinson H Lange C M Lye J P Sumpter M Zerulla C R Tyler Utility of a juvenile fathead minnow screening assay for detecting anti estrogenic substances Environ Toxicol Chem Vol 21 319 326 2002 34
42. ding lignans and phytoestrogens on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin SHBG J Steroid Biochem Vol 27 1135 1144 1987 15 M Kobayashi H Ishibashi T Moriwaki T Koshiishi S Ogawa T Matsumoto K Arizono S Watabe Development of low estrogen fish diet for in vivo endocrine disrupter test in preparation 2002 16 K Arizono T Ito M Yamaguchi T Ariyoshi Induction of zinc metallothionein in the liver of rats by lead Eisei Kagaku Vol 28 94 98 1982 17 T Ariyoshi E Takabatake H Remmer Drug metabolism in ethanol induced fatty liver Life sci Vol 9 361 369 1970 18 O H Lowry N J Rosebrough A L Farr R J Randall Protein measurement with the Folin phenol reagent J Biol Chem Vol 193 265 275 1951 177 19 S Yamanaka K Arizono Y Matsuda K Soyano H Urushitani T Iguchi R Sakakibara Beds and application of an effective detection method for fish plasma vitellogenin induced by NA estrogens Biosci Biotechnol Biochem Vol 62 1196 1200 1998 20 M M Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Anal Biochem Vol 72 248 254 1976 21 K Asahina A Kambegawa T Higashi Development of a microtiter plate enzyme linked immunosorbent assay for 17a 20B 21 trihydroxy 4 pregnen 3 one a teleost gonadal steroid Fisheri
43. e 2 Phytoestrogens contents in fish diets using LS MS MS analysis Diets Genistein Daidzein Equol Coumestrol Formulated diet FD2 57 6 69 2 4 0 4 0 Trout diet TD2 48 673 2 37 994 7 N D 22 4 Carp diet CD2 128 358 9 100 767 8 N D 42 4 The data of fish diets were represent total ng g diet N D Not Detected less than 4 0 ng g Data represents the mean n 3 146 5 3 2 B hER o B Fig 1 17B E2 Table 3 B FD2 TD2 CD2 hER c Fig 1 A hER B CD2 OF E2 39 91 ng g Fig 1 C B FD2 TD2 CD2 hER a 26 51 ng g 95 91 ng g Fig 1 B FD2 TD2 CD2 hER B
44. es Science Vol 61 491 494 1995 22 M Kobayashi K Aida I Hanyu Annual changes in plasma levels of gonadotropin and steroid hormones in goldfish Bulletin of the Japanese Society of Scientific Fisheries Vol 52 1153 1158 1986 23 S Jobling D Sheahan J A Osborne P Matthiessen J P Sumpter Inhibition of testicular growth in rainbow trout Oncorhynchus mykiss exposed to estrogenic alkylphenolic chemicals Environ Toxicol Chem Vol 15 194 202 1996 24 3 1998 25 C Bennetau Pelissero B B Breton B Bennetau G Corraze F Le Menn B Davail Cuisset C Helou S J Kaushik Effect of genistein enriched diets on the endocrine process of gametogenesis and on reproduction efficiency of the rainbow trout Oncorhynchus mykiss Gen Comp Endocrinol Vol 121 173 187 2001 178 26 D L Villeneuve S A Villalobos T L Keith E M Snyder S D Fitzgerald J P Giesy Effects of waterborne exposure to 4 nonylphenol on plasma sex steroid and vitellogenin concentrations in sexually mature male carp Cyprinus carpio Chemosphere Vol 47 15 28 2002 27 J P Giesy S L Pierens E M Snyder S Miles Richardson V J Kramer S A Snyder K M Nichols D A Villeneuve Effects of 4 nonylphenol on fecundity and biomarkers of estrogenicity in fathead minnows Pimephales promel
45. iomarker for estrogenic contamination of the aquatic environment Environ Health Perspect Vol 103 173 178 1995 7 S S Talmage Environmental and human safety of major surfactants Alcohol ethoxylates and alkylphenol ethoxylates The Soap and Detergent Association 1994 8 H Yokota M Seki M Maeda Y Oshima H Tadokoro T Honjo K Kobayashi Life cycle toxicity of 4 nonylphenol to medaka Oryzias latipes Environ Toxicol Chem Vol 20 2552 2560 2001 9 A G Michelle D M Chris Induction of testis ova in Japanese medaka Oryzias latipes exposed to p nonylphenol Environ Toxicol Chem Vol 16 1082 1086 1997 176 10 N R Adams M R Sanders Persistent infertility in ewes after prolonged exposure to oestradiol 17b J Reprod Fertil Vol 84 373 378 1988 11 J M Obst R F Seamark Plasma hormone levels during pregnancy and parturition in ewes grazing Yarloop clover patures J Reprod Fertil Vol 29 146 147 1972 12 D A Shutt R I Cox Steroid and phyto oestrogen binding to sheep uterine receptors in vitro J Endocrinol Vol 52 299 310 1972 13 M A Thompson B L Lasley B A Rideout L H Kasman Characterization of the estrogenic properties of a nonsteroidal estrogen equol extracted from urine of pregnant macaques Biol Reprod Vol 31 705 713 1984 14 H Adlercreutz K Hockerstedt C Bannwart S Bloigu E Hamalainen T Fotsis A Ollus Effect of dietary components inclu
46. oc Kruskal Wallis Bonferroni adjusement 143 Mann Whitney U 144 5 3 5 3 1 FD2 TD2 CD2 Table 2 CD2 128 358 9 ng g TD2 48 673 2 ng g FD2 57 6 ng g CD2 100 767 8 ng g TD2 37 994 7 ng g FD2 69 2 ng g FD2 4 0 ng g TD2 CD2 CD2 42 4 ng g TD2 22 4 ng g FD2 4 0 ng g 3 CD2 gt TD2 gt FD2 145 Tabl
47. tion of 1 0 ug l of E2 155 Table 4 2 Change in body weight in the FD2 fed group exposed to NP concentrations of 0 1 1 0 10 and 100 ug l for 7 14 21 and 28 days NP treatment Body weight g ug l Day 0 Day 7 Day 14 Day 21 Day 28 Initial 32 3 7 2 Control 320534 325531 2325 31 2339453 0 1 30 1 2 9 30 6424 314421 2322t18 1 0 299 72 30 2474 311t74 317577 10 32 0 7 6 331580 33 6477 342479 100 3 1587 31 7487 323489 33 1492 E2 1 0 312 119 316 116 318 114 32 6 11 7 Data represent the mean and standard deviation The control fish were exposed to the solvent carrier only DMSO 0 01 ml l and the positive control fish were exposed to the nominal concentration of 1 0 ug l of E2 156 Table 5 1 Change in body length in the CD2 fed group exposed to NP concentrations of 0 1 1 0 10 and 100 ug l for 7 14 21 and 28 days JP JQ mmINM NP treatment Body length cm ug l Day 0 Day 7 Day 14 Day 21 Day 28 Initial 9 50 20 67 Control 9 80 0 57 9 86 0 64 9 8640 56 9 94 0 69 0 1 9 64 0 76 9 60 0 63 9 76 0 71 9 76 0 72 1 0 9 68 0 55 9 68 0 72 9 80 0 65 9 68 0 69 10 9 84 1 00 9 96 0 93 10 00 0 92 9 92 0 96 100 9 46 0 54 9 48 0 59 9 32 0 57 9 52 0 58 E2 1 0 9 46 0 78 9 44 0 45 9 42 0 58 9 44 0 48 Data represent the mean and stand
48. y 7 mDay 14 Day 28 a oe 1000 Day 42 a E mDay 63 100 5 Ej 10 A I S N 7 N S 7 0 1 N 7 N Y NZ 0 01 Aa x NZ N Initial FD2 TD2 CD2 Fish diet Fig 4 Plasma vitellogenin levels in adult male goldfish Fish were fed 1 0 body weight volume of one of the three diets TD2 CD2 and FD2 every day for 63 days Columns and bars represent the mean and standard deviation Plasma vitellogenin levels of CD2 fed fish in all feeding period were significant difference compared to that of FD2 fed fish p lt 0 05 Kruskal Wallis test and Mann Whitney s U test Significant difference compared to TD2 fed fish br p lt 0 05 Kruskal Wallis test and Mann Whitney s U test 153 5 3 4 GSi HSI FD2 CD2 1 0 1 1 10 100 ici NP 28 Table 4 1 4 2 5 1 5 2 ANOVA FD2 NP 28 ERRA L 7 XM GSI HSI Fig 5 A FD2 HSI 2 3 NP RREFEN ANOVA GSI iX SURBB ICV 3 4 NP
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