- CC--2.5 2

Magnus Manske

What Dr. Larry Ewing told me 14 years ago: You are the first one to know this!!!!

(dioxin)21075 Polychlorinated dibenzo-P-dioxins135 Polychlorinated dibenzofurans

pg/gppt or ppb pg/kg-bwng-TEQ/Nm3()pg-TEQ/g()

TEQ (Toxicity Equivalency Quantity of 2,3,7,8 TCDD)= TEF (Toxicity Equivalency Factor ) true concentration :8686

10-12pptMagnus ManskeOriginal uploader was Alanmak at en.wikipedia

2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) (1997)Muumi

->->->->-> (gas chromatography electron capture detection) (mass spectrometry)Hey Paul

(in vivo biomarkers) cytochrome P450 1A (CYP1A) (in vivo bioassays) : EROD (ethoxyresorufin -O- deethylase) assayCALUX (chemical-activated luciferase expression) assay

(enzyme-linked immunosorbent assayELISA)

biosensor

17 2,3,7,8PCDD()()()

(AHR) AHR ARNT

TCDD (responsive enhancer) (Cytoplasm) nucleus ()

dioxin response elementsreporter gene

DNA

RAW264.7 (Mouse macrophage )Hepa1C1C7 (Mouse hepatoma )H4IIEC3 (Rat hepatoma )

2,3,7,8-TCDD1nM100pM50pM10pM5pM1pM0.1pMDMSO1ml3.22LM322pg32.2pg16.1pg3.22pg1.61pg322fg32.2fg0TEQ322pg-TEQ32.2pg-TEQ16.1pg-TEQ3.22pg-TEQ1.61pg-TEQ322fg-TEQ32.2fg-TEQ0

RAW264.715hrM4p210pM (3.22ppt) MP---H4IIEC310hrM4p210pM (3.22ppt) MP100pM (32.2ppt)Hepa1C1C-715hrM4p210pM (3.22ppt)MP5pM (1.61ppt)

0.00003 mg/l :1 ppt 1000 pg TEQ/g 0.1~0.5ng-TEQ/Nm3 Functional toxicology

AHR+HSP90 HSP90 AHR

TCDD (Cytoplasm) ( nucleus) (Florescence Resonance Energy Transfer, FRET) (FRET)

AHRHSP90AHRARNT (AHR nucleur translocator)

,

The End

Thanks for your attendance

Huang YW, Lee WS, Chang-Chien GP, Lin CI, Kao CY, Lee H*. Establishment of cellular detection systems for dioxin-like compounds. Chinese Bioscience, 47 (1) : 1-18, 2004CI Lin, CH Hsieh, JT Wu, WS Lee, GP Chang-Chien, CY Pan and H Lee*. Establish of a FRET detection system for dioxin-like compounds. Chinese Bioscience, 50(1):12-25. 2007.CI Lin, CH Hsieh, SSY Lee, WS Lee, GP Chang-Chien, CY Pan and H Lee*. Establish of a FRET based bioassay for dioxin-like compounds. J of Biomed. Sci. 15(6): 833-840. 2008. [Epublished ahead of print, July 5]. (36/81, 2.024, 2007)

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*pcddpcdf2102,3,7,8 TCDD322850

*2,3,7,8 TCDD1 17

() () 1.ng-TEQ/Nm3 2.pg-TEQ/g Nm3273K TEQ(Toxicity Equivalency Quantity of 2,3,7,8-tetrachlorinated dibenzo-p-dioxin)(2,3,7,8-TCDD)I-TEQ(International Toxic Equivalents) () pg/g: ppt(part per trillion): ppb(part per billion): pg/kg-bw:(body weight) *10-12ppt

*,

*20gas chromatographyelectron capture detectionmass spectrometry*bioassays

cytochrome P450 1A (CYP1A)in vivo biomarkersCYP1AAHR pathwayRNACYP1AIn vivo bioassaysIn vitro bioassaysin vitro bioassayAHR-receptor (Hu et.al. 1995)Cell culture bioassaysethoxyresorufin -O- deethylase (EROD) assayCALUX assaydioxin response elementsreporter gene1993Hans Postlind2Human CYP1-Luciferase plasmidCYP1promoter5-flanking sequenceluciferase101LTCDDluciferase0.1~1.0 pMTCDDIn vitro bioassayscell culture bioassaysEROD assayCALUX assayCALUX assay* (enzyme-linked immunosorbent assayELISA)Hapten1pg 2,3,7,8TCDDbiosensor* *(Aryl hydrocarbon receptor pathway) AHR Helix Loop Helix(HLH)chaperoneHeat Shock Protein 90(HSP90)TCDDAHRHSP90AHRAHRAHR nuclear translocator (ARNT)heterodimer,DNAdioxin response elementsCYP1A1 PKCPEPCK *CALUXdioxin response elementsreporter gene1993Hans Postlind2Human CYP1-Luciferase plasmidCYP1promoter5-flanking sequenceluciferase101LTCDDluciferase0.1~1.0 pMTCDDH1L1.1c2 H4IIE*CALUX assaydioxin response elementluciferasereporter vector

*DNAMPCYP1A1AF21090532~1593 (X01681)dioxin response elementspGL3-basic vector12M4P2CYP1A1AF21090529~661dioxin response elementspGL2-promoter vector7 *RAW264.7Hepa1C1C7H4IIEC3

*2,3,7,8-TCDD 10g/mlDMSO3.22l24-wellwell(1ml10) *H4IIEC3M4P22,3,7,8-TCDD3.22l DMSO1010010pM 2,3,7,8-TCDD*H4IIEC3MP2,3,7,8-TCDD3.22l DMSO10100100pM 2,3,7,8-TCDD*Hepa1C1C-7M4P22,3,7,8-TCDD3.22l DMSO151010pM*Hepa1C1C-7MP2,3,7,8-TCDD3.22l DMSO1515pM *RAW264.7M4P22,3,7,8-TCDD3.22l DMSO1510010pM 2,3,7,8-TCDD100pM*RAW264.7MP2,3,7,8-TCDD3.22l DMSO15100*Hepa1C1C-7*1 ppt()5 ppt()5 pg-TEQ/g()()Hepa1C1C-71.61pptH4IIEC3M4P23.22ppt0.00003 g/l0.0000022 g/l0.1ng-TEQ/Nm3Hepa1C1C-71.61pg-TEQH4IIEC33.22pg-TEQ ~32.2pg-TEQ 172,3,7,8-PCDD/FsTEFTEQ17TEQMcLachlan (1993)functional toxicologyTEQDioxinDioxinDioxin in vitroin vivoDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxin *(FRET)AHR(Aryl hydrocarbon receptor) AHRAHRbHLH-PASPAS domainAHRHSP90TCDDAHRAHRAHRAHRHSP90(ligand)AHRHSP90AHRARNT(AHR nuclear translocator)CYP1A1CYP1A2AHR-HSP90*AHRHSP90AHRARNT(AHR nucleur translocator)

*FRETFluorescence Resonance Energy Transfer FRET(dipole-dipole interaction)FRET20~100FRETFRET20~100FRETBFPCFP(Donor-Acceptor Complex) AHRHSP90 DNAAHRHSP90(Endogenous)AHRHSP90AHRHSP90AHR-HSP90AHR-HSP90AHR-HSP90AHR-HSP90FRETAHR-HSP90FRETAHRHSP90AHR-HSP90FRET* Dioxin ,

*DioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinDioxinTEQ